Urine Testing

for Drugs

of Abuse

U. S. DEPARTMENT OF HEALTH AND HUMAN SERVICES • Public Health Service • Alcohol, Drug Abuse, and Mental Health Administration
Urine Testing for

Drugs of Abuse

Editors:

Richard L. Hawks, Ph.D.

C. Nora Chiang, Ph.D.

Division of Preclinical Research

National Institute on Drug Abuse

NIDA Research Monograph 73

1986

DEPARTMENT OF HEALTH AND HUMAN SERVICES

Public Health Service

Alcohol, Drug Abuse, and Mental Health Administration

National Institute on Drug Abuse

5600 Fishers Lane

Rockville, Maryland
NIDA Research Monographs are prepared by the research divisions of the

National lnstitute on Drug Abuse and published by its Office of Science. The

primary objective of the series is to provide critical reviews of research problem

areas and techniques, the content of state-of-the-art conferences, and

integrative research reviews. Its dual publication emphasis is rapid and targeted

dissemination to the scientific and professional community.

Editorial Advisors

MARTIN W. ADLER, Ph. D.

Temple University of Medicine

Philadelphia, Pennsylvania

SYDNEY ARCHER, Ph.D.

Rensselaer Polytechnic Institute

Troy, New York

RICHARD E. BELLEVILLE, Ph.D.

NB Associates, Health Sciences

Rockville, Maryland

KARST J. BESTEMAN

Alcohol and Drug Problems Association

of North America

Washington, D.C.

GILBERT J. BOTVIN, Ph.D.

Cornell University Medical College

New York, New York

JOSEPH V. BRADY, Ph. D.

The Johns Hopkins University School of

Medicine

Baltimore, Maryland

THEODORE J. FCICERO, Ph. D.

Washington University School of

Medicine

St. Louis, Missouri

SIDNEY COHEN, M.D.

Los Angeles, California

MARY L. JACOBSON

National Federation of Parents for

Drug Free Youth

Omaha, Nebraska

REESE T. JONES, M.D.

Langley Porter Neuropsychiatric Institute 

San Francisco, California

DENISE KANDEL, Ph.D

College of Physicians and Surgeons of

Columbia University

New York, New York

HERBERT KLEBER, M.D.

Yale University School of Medicine

New Haven, Connecticut

RICHARD RUSSO

New Jersey State Department of Health

Trenton, New Jersey

NIDA Research Monograph Series

CHARLES R. SCHUSTER, Ph.D.

Director, NIDA

JEAN PAUL SMITH, Ph.D.

Acting Associate Director for Science, NIDA

Acting Editor

Parklawn Building, 5600 Fishers Lane, Rockville, Maryland 20857
Urine Testing for

Drugs of Abuse
ACKNOWLEDGMENT

This monograph is the result of a collaborative effort by the scientists noted

as chapter authors, all of whom made extensive contributions to the various

chapters in this monograph in addition to the ones for which they were the

principal author. In addition, several other individuals in the scientific

community reviewed the manuscript in preparation and made valuable

contributions to the final text. These reviewers include Rodger and Ruth

Foltz, Center for Human Toxicology, University of Utah, Salt Lake City;

Bryan Finkle, Genentech Corporation, South San Francisco; Arthur McBay,

Office of the Chief Medical Examiner, University of North Carolina; and

Michael Peat, ChemWest Analytical I.aboratory, Sacramento.

COPYRIGHT STATUS

The National Institute on Drug Abuse has obtained permission from the

copyright holders to reproduce certain previously published material as noted

in the text. Further reproduction of this copyrighted material is permitted

only as part of a reprinting of the entire publication or chapter. For any

other use, the copyright holder’s permission is required. All other material in

this volume except quoted passages from copyrighted sources is in the public

domain and may be used or reproduced without permission from the Institute

or the authors. Citation of the source is appreciated.

Opinions expressed in this volume are those of the authors and do not

necessarily reflect the opinions or official policy of the National Institute on

Drug Abuse or any other part of the U.S. Department of Health and Human

Services.

The U.S. Government does not endorse or favor any specific commercial

product or company. Trade, proprietary, or company names appearing in this

publication are used only because they are considered essential in the context

of the studies reported herein.

DHHS Publication No. (ADM)87-1481

Alcohol, Drug Abuse, and Mental Health Administration

Printed 1986 Reprinted 1987

NIDA Research Monographs are indexed in the Index Medicus. They are

selectively included in the coverage of American Statistics Index,

BioSciences Information Service, Chemical Abstracts, Current Contents,

Psychological Abstracts, and Psychopharmacology Abstracts.

-iv-
Foreword

In the past 5 years, a growing concern over the use of illicit drugs in the

workplace has led to an interest in urinalysis as a way to detect and deter

drug use. Drug testing by urinalysis has been suggested and in many cases

implemented for prospective and current employees in industry; for personnel

of the armed forces; for parolees and bail seekers in civilian court systems;

for workers in the transportation industry; and for individuals who serve as

role models, such as nationally known athletes. Two factors have led to the

widespread use of urinalysis for drugs: technical developments in testing

methods and the growing demand for drug testing. Society is becoming

increasingly aware of the impact of drug use on public safety and of the

financial impact on industry of lost time and productivity. The annual loss of

productivity of employees has been estimated at $100 billion for alcohol and

drug abuse, a third of which is due to drug abuse alone.

Drug and alcohol abuse in the workplace is amenable to carefully planned

prevention programs, however, and urine drug detection provides a powerful

tool for use in such programs. Preemployment urine screening is now

common among “Fortune 500” companies and in several Federal agencies.

As a consequence of drug screening programs, laboratories that were

established to perform urinalysis associated with methadone treatment have

had to greatly expand their capacities; many new laboratories have sprung up

to meet the demands for drug assays; and clinical laboratories associated

with medical centers, under economic pressures in recent years, have begun

to venture into drug testing. However, results from laboratories that are not

subject to any established guidelines for drug testing are sometimes

unreliable. At present few guidelines exist for private laboratories; the

Department of Defense has strict certification requirements for laboratories

testing military personnel, and the Federal Railroad Administration as well as

State agencies in California and New York have quality control standards in

place, the latter for laboratories associated with methadone treatment

programs. Until quality control programs are mandated on a broad scale,

however, employers wishing to establish a drug screening program must rely

on their own initiative to evaluate the reliability of a testing laboratory, so

that no individual will be falsely accused of drug use and at the same time

regular use on the part of any tested employee will not escape detection.

-v-
The National Institute on Drug Abuse (NIDA) is in a unique position to provide

advice on many of the technical issues associated with the role of urinalysis

in the prevention of drug abuse. Since 1972, this Institute has supported

major research efforts to develop analytical methods for detecting and

measuring drugs in biological fluids, with special emphasis on methods for

cannabinoids and other abused drugs. NIDA has supported the development of

many of the major technologies beiug used today for urine drug screening and

confirmation. NIDA has also played a leading rote in the study of behavioral

and pharmacokinetic effects of drugs of abuse--two areas of knowledge that

are critical to the assessment and implementation of effective drug screening

programs.

The purpose of this monograph is to provide informatiou that will assist those

involved in the planning or implementation of drug testing programs in

making informed choices: information such as what urine screening can and

cannot do, how it fits into an overall drug program, and how it can be used

most reliably. It is our hope that this powerful technology which has grown

out of basic research programs will be used to advantage to assist in the

prevention of drug abuse in the United States.

Charles R. Schuster, Ph.D.

Director

National Institute on Drug Abuse

-vi-
Contents

Foreword.......................................... v

List of Contributors ................................. viii

Establishing a Urinalysis Program--Prior Consideration ......... 1

Richard L. Hawks

Drug Testing Programs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Robert E. Willette

Choosing a Laboratory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Robert E. Willette

Proficiency Testing and Quality Control Programs . . . . . . . . . . . . 20

Robert E. Willette

Specimen Collection and Handling. . . . . . . . . . . . . . . . . . . . . . . . 24

Joseph E. Manno

Analytical Methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

Richard L. Hawks

Accuracy in Urinalysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

Robert V. Blanke

Interpretation of Urinalysis Results. . . . . . . . . . . . . . . . . . . . . . . 54

Joseph E. Manno

Implications of Drug Levels in Body Fluids: Basic Concepts . . . . . 62

C. Nora Chiang and Richard L. Hawks

Examples of Specific Drug Assays. . . . . . . . . . . . . . . . . . . . . . . . 84

Richard L. Hawks and C. Nora Chiang

Marijuana/Cannabinoids .........................

Cocaine .....................................

Amphetamine and Methamphetamine ...............

Opiates .....................................

Phencyclidine. ................................

Alcohol .....................................

Lysergic Acid Diethylamide ......................

Methaqualone ................................

Barbiturates .................................

Benzodiazepines. ..............................

85

93

95

98

101

103

105

108

109

111

Glossary .......................................... 113

List of NIDA Research Monographs. ...................... 115

-vii-
List of Contributors

Robert V. Blanke, Ph.D.

Professor of Pathology, Pharmacology and Toxicology

Medical College of Virginia

Richmond, Virginia 23298

C. Nora Chiang, Ph.D.

Research Technology Branch

Division of Preclinical Research

National Institute on Drug Abuse

Rockville, Maryland 20857

Richard L. Hawks, Ph.D.

Chief, Research Technology Branch

Division of Preclinical Research

National Institute on Drug Abuse

Rockville, Maryland 20857

Joseph E. Manno, Ph.D.

Louisiana State University

Medical Center

Section of Toxicology

Shreveport, LA 71130

Robert E. Willette, Ph.D.

President, Duo Research

Annapolis, Maryland 21401

-viii-
Establishing a Urinalysis

Program-Prior Considerations

Richard L. Hawks, Ph.D.

Urinalysis for detection of drug use should be considered in the context of an

overall plan to reduce or prevent the negative impact of drug abuse on an

industry or organization. It would be inadvisable, however, to proceed

without a careful assessment of the group to be affected by testing. Certain

questions should be addressed: What personnel, if any, are affected by drug

use? Which drugs are the major problem? Can the organization shoulder the

economic burden for an appropriate urinalysis and counseling program, and

perhaps the legal and emotional costs associated with such a program?

In some cases, a problem is obviously present, based on incidents of known

drug use or drug dealing within the organization. Or it may be only

suspected, due to higher than normal rates of absenteeism, decreases in

productivity, or an increase accidents or thefts in the workplace. In the

absence of any indicators of drug use, managers might justifiably decide to

pursue the issue no further.

Assessment starts with education. Managers of a potential drug program

should first become knowledgeable themselves about drug abuse and its

indicators. Information is available from State drug and alcohol abuse

organizations, through the National Clearinghouse for Drug Abuse

Information at NIDA (P.O. Box 416, Kensington, MD 20795), through forensic

science centers associated with universities or medical examiners’ offices,

and from a number of expert private consultants.

The presence and extent of a drug problem could be assessed by means of a

survey based on anonymous questionnaires. This approach could include an

anonymous urine drug screen that would not only let the management know

what to expect if a regular urinalysis program were initiated; it would also

constitute a “dry run” with few legal ramifications, and would send a clear

message to employees about the seriousness of concern about the drug

problem and the resolve to remedy it. This could in itself have a deterrent

effect.

Any assessment should include clarification of which substances are

involved. Alcohol is the largest problem in most industries; an estimated 100

million Americans are current users of alcohol. The most recent NIDA

survey estimates that 18 million Americans are current users of marijuana

and 5.8 million are current users of cocaine (based on admitted use at least

once during the month prior to the survey). In some locations, including

Washington, DC, and Los Angeles, use of phencyclidine (PCP) is particularly

acute. These illicit drugs, along with opiates, amphetamines, or hallucinogenic compounds, have a significant presence in our society.

-1-
A more complicated group of drugs to assess are the prescription drugs;

tranquilizers, barbiturates (sleeping pills), and antidepressants are probably

even more prevalent in the workplace than illicit drugs and potentially are

just as likely to impair job performance or to create health problems if used

in excess of prescribed amounts or without adequate medical supervision.

When the “assessment” questions are resolved, establishing an appropriate

drug abuse deterrent program, including urinalysis, becomes feasible. The

care with which the plan is developed and implemented will determine the

success of the program and associated benefits. The plan should include

careful consideration of the organization’s policies, goals, and philosophy.

Finally, the plan should be well documented and available to all employees of

the organization.

The plan should be tailored to the extent of the problem. If no clear

indication of significant drug use at a worksite or in an organization is

apparent, a program beyond a preventive educational effort may not be

warranted. Evidence of heavy psychoactive drug use in job situations where

safety is a sensitive issue should be dealt with more aggressively. The plan

should give paramount consideration to the purpose of the program, the

essence of which is usually the health and safety of employees as well as

economic concerns. Another important element of the plan should be to

guarantee the personal privacy and dignity of the employees as much as

possible.

Various alternatives to a urine testing program may be considered. If the

problem does not warrant a major program, it may be sufficient to maintain

an awareness of drug abuse indicators, to define isolated problems regularly

through the use of personnel data and supervisor reports, and to deal with

them through an employee assistance program.

Most of the urine samples being analyzed in industry today are associated

with preemployment applications. While many of the rights usually accorded

an applicant are not necessarily the same as those of an employee, the same

rights of privacy and accuracy of analysis should be accorded these

individuals. A preemployment screening plan may preclude employment of

individuals with positive urines, but such a plan should include some kind of

counseling, not only to make the individual aware of why the job was denied

but also to offer advice and direction in dealing with the drug problem. A

more enlightened probationary policy might allow the hiring of such an

individual based on other merits, on the condition that counseling be obtained

and drug use cease.

Therapeutic drugs, while potentially problematic when abused, may in fact

improve or enhance workplace safety by decreasing health-related

impairment. Chronic disease states that would be problematic in the

workplace or on the highway without the benefit of drugs include epilepsy and

mental illness. Other conditions such as stress may also contribute to

impaired performance and therefore benefit by drug therapy. Urine drug

programs may identify those individuals who are legitimately taking drugs to

alleviate these disease states. Consideration should be given to the fact that

sufficient discrimination still exists in our society that individuals with these

particular conditions may have been actively hiding them from their

coworkers or employers. This further argues for confidentiality in the

handling of urinalysis results, in the same way an organization would handle

an individual’s medical records.

-2-
Other drug-specific characteristics that might influence policy development

are whether some drugs, because of their particular pharmacological effects,

may be of more concern than others. Alcohol, marijuana, phencyclidine,

heroin, and LSD, for instance, are all drugs of abuse, but they differ in the

time course of their effects on performance and on the time their previous

use can be detected. Marijuana components in some circumstances can be

detected for weeks after the last use; indication of cocaine use can rarely be

detected in urine for more than a day or two. A cocaine-positive urine

therefore indicates recent and possibly dependent use, particularly if the

individual was aware of the pending analysis and still could not abstain. A

similar conclusion cannot be drawn from a single marijuana-positive urine.

Effective programs for the detection of drugs of abuse in human urine

specimens are best accomplished with sensitive testing procedures. Because

of the numerous legally sensitive features of drug detection programs, the

analytical results must be unquestionably reliable and able to withstand

considerable scrutiny. Therefore, the testing laboratory must be experienced

and capable in a number of important functions, including quality control,

documentation, chain of custody, technical expertise, and demonstrated

proficiency over time in urinalysis testing. Most important, the laboratory

must produce data that are secure from false positives and defensible in a

forum where the data may be challenged (e.g., a legal hearing or subsequent

arbitration). A qualified expert should be available to testify as to the

laboratory procedures that were employed, as well as to the accuracy and

reliability of the testing result.

At the moment, private laboratories providing urine drug detection services

for private industry in the United States are generally not compelled to

comply with specific guidelines associated with accuracy of analysis.

Oversight of urinalysis laboratories is essential to ensure quality analyses and

to provide public credibility, either mandated by government regulation or

established within the private sector and coupled with motivation of the

laboratories to participate in such oversight programs. Government and

private industry appear to be interested in moving in this direction. Some

State legislatures are considering bills to regulate drug urinalysis

laboratories, and some organizations and companies are preparing to provide

certification and proficiency-testing services to urinalysis laboratories.

Performance and qualification standards that will be the basis of a NIDA

registry of laboratories engaged in urine drug detection services are currently

under development. The success of such programs will depend on the

motivation of laboratories to participate in them--motivation that will

ultimately come from the laboratories’ clients who will demand the quality

and performance for which they are paying. NIDA officials hope this

monograph will provide sufficient background to enable both clients and

employees to ask the right questions and demand the appropriate quality of

analysis.

Many considerations are important to developing a sound drug program in an

organization. It cannot be overemphasized that the documentation of

well-thought-out policies, developed with input from all organizational

elements, is at the top of the list. An effective program to discourage drug

abuse in an organization must have clearly defined rationales, goals, and

rules. The consequences of a positive urinalysis result must be clearly stated

and not open to arbitrary responses by management. The rights and

sensitivities of the individual should be protected as much as possible.

Results of urine drug assays should be kept confidential. The individual

-3-
should be accorded the benefit of trained counseling, with referral to

treatment programs if necessary. The program should be designed, in other

words, for prevention and rehabilitation rather than for law enforcement.

A technically effective program that generates negative results can be

considered successful, one that is serving to prevent drug use by employees

and simultaneously to provide management (or the public) with assurance that

alcohol and illicit drugs at least are not a factor in performance problems in

the organization. Positive results represent failures of the prevention

system. Nevertheless, as long as they are looked at as diagnostic of a

medical condition or an attitude that is susceptible to appropriate

intervention or treatment strategies, the system will foster hope for

improvement of the drug abuse situation in this country.

The chapters that follow will present examples of methods and procedures to

ensure the quality and accuracy of test results. They will concentrate on the

technical aspects of the methodology used for urinalysis, the means to ensure

accuracy in such analyses, and the background to help in the interpretation of

assay results. Descriptions of appropriately controlled and monitored

urinalysis programs currently in use will be presented, as well as specific

descriptions of what to look for in choosing a laboratory.

Although some parts of the text appear to be geared toward the scientist, the

monograph has been written to inform readers in all phases of program

operation. Some chapters discuss the practical aspects of conducting drug

testing programs, including laboratory selection and quality assurance and

examples of programs already underway. Others relate the technical

concepts and methods involved in employing urinalysis to detect drug use.

The final chapter summarizes testing approaches to selected drugs of abuse

and offers reference lists for further study.

-4-
Drug Testing Programs

Robert E. Willette, Ph.D.

Many government agencies and private employers have initiated drug testing

programs. Although the vast majority have started since 1981, many have

been in place for several years. In order to appreciate the variety and scope

of different approaches to drug testing, several programs will be described.

It can be noted that most of the programs include drug testing under one or

more of the following conditions: (1) before employment or during a

probationary period; (2) under reasonable suspicion of alcohol or drug use,

such as following an accident or bizarre behavior; (3) as part of routine

physicals, often required by Federal regulations; (4) during random testing;

and (5) when monitoring employees during rehabilitation or counseling for

drug use.

MILITARY SERVICES

By far the most extensive programs of drug testing are conducted by the U.S.

military services. Of these, the U.S. Navy program is the most intensive and

successful. The Navy operates five drug testing laboratories, through which

approximately 1.8 million urine specimens are processed each year, each

being tested at the present time for cannabinoids, cocaine metabolite,

phencyclidine, amphetamines, opiates, and barbiturates. The Navy is

currently planning to include testing for LSD. The 1.8 million specimens are

collected, primarily on a truly random basis, at a rate of almost three times

per year per member of the Navy and Marine Corps. Since it is random, some

members may be required to provide specimens several times a year and

some none. This serves as a constant deterrent to drug use.

The Army and Air Force also collect random specimens but at a lower

frequency. AU of the services can collect specimens in the event of a

probable cause situation, usually Limited to cases of suspected drug use. It is

important to note that military standards for probable cause follow the

strictest legal standards and cannot be easily abused. Some amount of

testing is used for service members who have tested positive once and have

completed counseling and/or rehabilitation programs. They are usually

placed on a surveillance program for several weeks following their return to

active service.

All of the military services collect specimens under direct observation. This

is done to eliminate the possibility of specimen substitution or adulteration.

Nevertheless, laboratories occasionally receive specimens that have been

-5-
adulterated. If it can be proven that the provider of the specimen did

adulterate the specimen, the individual will be subject to other charges.

Also, the observer can be disciplined for failing to perform his or her duty. In

this manner, it has been possible for the military to maintain a tight and

effective program.

The individual commanding officer can determine if the initial positive result

is sufficient grounds to discipline or discharge a service member for wrongful

ingestion of an illegal substance. In cases of a good service record and

significant promise for continued useful service, the member can he

retained. This is not very common following a second positive result.

The success of these programs can best be illustrated with the record of the

Navy. Starting in 1981 with an indicated use level of 48 percent for enlisted

personnel under the age of 25, the overall test results for any period after

1984 have been below the 5 percent screened-positive rate. Screening

figures are used, since not all specimens are confirmed due to the presence of

drug concentrations below the confirmation cutoffs. Admitted drug use in

this same population was about 10 percent, as indicated in a questionnaire

survey taken by the Department of Defense in 1985.

Standards for performance are monitored in the military program in several

ways. Testing methods and cutoff levels are established by the Office of the

Assistant Secretary of Defense for Health Affairs. That Office conducts an

annual inspection of each military and contract laboratory. In addition, each

service conducts regular inspections of its own laboratories. The Navy

laboratories are inspected quarterly by the Naval Medical Command, which is

responsible for the operation of the laboratories, and annually by a team of

operations and legal personnel and outside civilian experts. All of the

laboratories are also monitored by a proficiency testing and blind quality

control program, as described in the next chapter.

ADMINISTRATIVE OFFICE OF THE U.S. COURTS

The Administrative Office of the U.S. Courts is responsible for people who

are on probation or parole for Federal crimes. If this involved the use or

possession of drugs, they are provided counseling, if necessary, and are

monitored on a regular basis by means of urine testing. The Office contracts

with commercial laboratories for testing and monitors their performance by

submitting periodic blind quality control samples. Persons found positive for

drug use (usually more than once) have to appear before a Federal judge to

determine if probation or parole has been violated.

FEDERAL BUREAU OF PRISONS

In a manner similar to that of the U.S. Courts, the Federal Bureau of Prisons

maintains a contract laboratory to test specimens collected from Federal

prisoners. These are comprehensive drug screens that include a number of

prescription drugs as well as the more commonly used drugs of abuse. Since

drug administration is closely controlled, it is necessary to monitor for the

use of prescription drugs that are obtained illicitly. It usually requires

repeated offenses before prisoners have to appear before a judge to alter

their sentences or to have certain privileges removed.

-6-
DEPARTMENT OF TRANSPORTATION

The U.S. Department of Transportation has regulatory authority over several

transportation industries, for which it has established certain drug-related

rules. In addition, the following DOT agencies have established drug testing

requirements for their own employees.

Coast Guard

The U.S. Coast Guard is a uniformed service and maintains a drug testing

program similar to that of the military services. Personnel are tested on a

random basis and following probable cause incidents. The Coast Guard uses a

contract laboratory to which specimens are sent under strict chain of

custody. The frequency of testing varies from region to region but is at the

rate of approximately once a year.

The Coast Guard has also issued proposed regulations for shipping fleets

covered under merchant marine laws.

Federal Aviation Administration

It has been announced that, during 1986, the Federal Aviation Administration

will initiate testing of all of its employees at the time of their required

periodic physical examinations. The program will be managed under a

contract that will conduct the testing as well as monitor collection of

specimens by approved air flight surgeons. Appropriate counseling and/or

treatment will be provided for any employee found to be using illegal drugs.

The FAA does not have any specific drug testing requirement for its

regulated industry, commercial aviation. Regulations do lay out

requirements for action following drug-related incidents, although the agency

has announced that no drug-related commercial airplane crash has ever

occurred. The physical examination requirements also preclude pilots who

are diagnosed as being addicted to alcohol or drugs. It is not known if any

airline tests its pilots for drugs, although many have announced that they

screen job applicants for drugs.

Federal Railroad Administration

In 1985, the Federal Railroad Administration (FRA) reissued regulations

governing railroad operations that included specific rules for drug testing.

This “Control of Alcohol and Drug Use in Railroad Operations” rule follows a

10-year history of 48 alcohol- and drug-related train accidents and incidents

that resulted in 37 fatalities, 80 injuries, and $34 million in damages. The

new regulations, which did not take effect until February 1986 following

several court actions, require preemployment drug testing and testing for

alcohol and drugs following certain accidents and in reasonable cause

situations. The regulations also spell out rather specific standards for the

testing, including publication of a field manual to assist the railroads in

developing their own programs.

-7-
National Highway Traffic Safety Administration

Under the Motor Carrier Safety Regulations, the National Highway Traffic

Safety Administration has established physical qualification standards for

drivers engaged in interstate highway activities, i.e., trucks and buses. One

requirement has vacillated from “does not use amphetamine, narcotic, or any

habit forming drug” to “has no current diagnosis of drug addiction.” Although

the regulations do not specifically require drug testing, many regulated

companies have included drug testing as part of the DOT physicals, since this

is the only reliable means of detecting drug use. The regulations also permit

the discharge of a driver who has been found in possession of or “under the

influence” of a drug while on company property or on duty. No standards

have been established, although the regulations include all substances covered

under Schedule I of the Controlled Substances Act.

REGULATED TRANSPORTATION INDUSTRIES

Many companies in the air, rail, bus, and trucking industries have

implemented various drug testing programs, often to comply with the

mentioned regulations or on a voluntary basis. Most common are

preemployment tests, which are the easiest to conduct from all legal

aspects. Only the Federal Railroad Administration has published standards

for testing.

In a rather provocative and forward move, the International Brotherhood of

Teamsters signed an agreement with the major trucking fiis that spells out

specific standards and conditions for drug testing. These include collection

procedures, test criteria, cutoffs for determining violations, and penalties.

The agreement requires that employees receive 30 days’ notice before they

have to provide a urine specimen as part of their physical examination. This

provides an opportunity for the employee to stop using drugs before being

tested. There are also provisions for testing employees under “probable

suspicion” situations, which would include accidents.

Several railroads implemented strict drug testing programs prior to the

effective date of the FRA regulations. One company, Southern Pacific, has

announced that human-factor accidents have gone down each year from 37

percent to 69 percent over the period of 1983 to the first 6 months of 1986

since implementing the testing program.

DEPARTMENT OF JUSTICE

During 1986, the Department of Justice authorized the implementation of

broad drug testing programs for its agencies. Although no indication of drug

use by its employees was seen, it was felt that the Department should set an

example for other government agencies and private employers. Several of its

agencies have tested employees in the past when there has been reasonable

suspicion that the employees were using illegal drugs.

Drug Enforcement Administration

The Drug Enforcement Administration (DEA) is entrusted with enforcing the

Nation’s drug laws and, as such, felt it was important to demonstrate that

-8-
the agency was free of drug use. The program was initiated in 1986 in a

progressive manner. Testing began with trainees and will gradually include

all personnel, adding classes of employees in the more critical positions.

Testing will be done on a random basis, with collection and testing conducted

through a contract. The agency has an employee assistance program that is

available on a voluntary basis prior to detection of drug use. Employees

found to be using drugs will be subject to dismissal.

Federal Bureau of Investigation

The FBI initiated a drug testing program for its trainees in 1986. The

program will extend to special agents and other critical positions on an

incremental basis, similar to that for DEA. Collection and testing of

specimens is combined with that agency’s as well. The full policy for dealing

with employees found to be using drugs has not been announced at this time.

DEPARTMENT OF THE TREASURY

Several of the agencies within the Department of the Treasury are planning

to initiate drug testing within 1986. The first agency to do so was the

Customs Service, which has a direct involvement with the apprehension of

drug smugglers.

U.S. Customs Service

During the spring of 1986, the Customs Service initiated a drug testing

program with its senior staff being the first to be tested. The collection and

testing of specimens is being conducted under a contract. Personnel in

critical positions, such as agents and chemists, will be included in the

program on a random basis.

Secret Service Uniformed Division

Applicants and probationary officers of the Secret Service Uniformed

Division have been tested for a variety of drugs since 1984. The testing is

part of regular probationary physical examinations and is conducted by the

District of Columbia Police and Fire Clinic.

GOVERNMENT SERVICES ADMINISTRATION

The Federal Protection and Safety Division of GSA has recently established

policy and procedural guidelines for urine drug testing of all contract guards

hired by GSA to guard buildings in the Washington, DC, area. This program

includes preemployment, incident-related, and annual physical testing.

Consideration is being given to extending the program to include GSA’s

employees.

CENTRAL INTELLIGENCE AGENCY

Early in 1986, the CIA initiated a trial drug testing program for all applicants.

-9-
NATIONAL AERONAUTICS AND SPACE ADMINISTRATION

NASA conducts drug tests on all astronaut applicant finalists at the Johnson

Space Center. The primary focus of the testing is to detect the chronic use

of several prescription drugs, as well as phencyclidine and amphetamines.

U.S. POSTAL SERVICE

Several offices of the Postal Service test job applicants for illegal drug use.

A general policy is in place to test for drugs in employees whose performance

deteriorates. Plans for testing under other circumstances are being reviewed

in light of protests from the postal workers union.

NUCLEAR REGULATORY COMMISSION--NUCLEAR POWER PLANTS

While the Nuclear Regulatory Commission (NRC) has no current policy

requiring drug testing, NRC licensees (nuclear power plant operators) have

been encouraged, on a voluntary basis, to have mandatory drug testing for all

personnel working in vital areas of the power plants. According to a recent

NRC report, 90 percent of all nuclear plants have some form of drug testing,

and the remaining ones are considering the implementation of programs in

the near future.

Because of the sensitive nature of the work involved in a nuclear power plant,

some operators have developed rather strict programs. All of those who

currently have programs include preemployment tests for drugs. Almost all

others test in probable cause situations. At least one company utilizes a

controversial, anonymous “hot line” for tips on drug dealers and users.

Accusations have been made that the hotline has been used to harass

employees, although it has uncovered many drug dealers. Being caught using

drugs usually results in termination. Many companies provide some level of

counseling or assistance, although this may apply only on a voluntary basis

prior to being detected.

PUBLIC UTILITIES--ELECTRIC AND GAS

Many nonnuclear power companies and gas companies have implemented drug

testing programs. These usually involve preemployment testing and testing

for probable cause. Evidence of growing concern about drug use among

employees is supported by the number of arbitrations conducted on behalf of

public utilities. Typically, a union will file a grievance on behalf of an

employee who has been fired for testing positive for drug use following some

incident. The argument usually questions whether the employee was

“intoxicated” on the job, as opposed to use of the drug off duty. Some

companies have resolved this issue by setting detection or confirmation levels

that serve as “per se” evidence of recent use or that denote a violation of the

rules.

LARGE INDUSTRIAL COMPANIES

A survey taken toward the end of 1984 suggested that nearly 30 percent of

the Fortune 500 companies were conducting preemployment drug testing.

-10-
The number testing employees is unknown. It is known, however, that many

large companies have drug testing programs, some of which include all those

conditions discussed in the first chapter.

The most typical program would be similar to that described for public

utilities, except that many large companies have extensive employee

assistance programs (EAPs). Employees who have alcohol or drug problems

can avail themselves of professional help in the EAP, usually on a completely

confidential basis. They can be referred to the EAP by a supervisor, baaed on

well-defined criteria that would include deteriorating job performance,

repeated absenteeism, and injury. Sometimes the referral is made following

observed behavior, but a growing number of companies use drug testing as a

more objective measure of drug use. It is common for an employee who tests

positive to be given an opportunity to enroll in the EAP and be referred to an

appropriate counseling or treatment service. If employees refuse, they may

then be subject to termination. An employee who fails to satisfactorily

complete counseling or treatment or is detected using drugs a second time is

usually fired.

It is important to note that many companies are now including tests for

alcohol as part of their preemployment screens, in addition to the common

practice of testing for alcohol in accident and other probable cause

situations. Caution must be exercised in the interpretation of test results for

alcohol in urine, since there is significant variability between blood and urine

concentrations. It is strongly recommended that alcohol be included along

with drug tests on individuals in treatment, since multiple substance abuse is

not uncommon.

A few companies have implemented random testing similar to that in the

military. They argue that it is the fairest and most democratic form of drug

testing, because it applies equally to everybody. In order to be fair, the

random selection must be truly random and not a guise for focusing on

certain individuals or classes of employees. In at least one company division,

the employees have endorsed the random program over a probable cause

approach, because it eliminates the potential for supervisor harassment or

being “out to get” someone in particular. Also, a strong possibility exists of

“stigmatizing” individuals who are taken in for testing under a probable cause

condition. This does not happen in random testing because everyone goes.

Whether a company uses random testing usually depends on the nature of the

work and the nature of the drug problem. A program similar to that used by

the Navy offers a strong deterrent to drug use but will still detect the user

who will not stop using drugs. Many legal questions remain about random

testing, however. Although random testing is not specifically precluded by

Federal law, some courts and arbitrators have been holding private employers

to constitutional standards, which ordinarily apply only to government

actions. Therefore, it is very important to have any program, random or

otherwise, meet the constitutional tests for fairness, reasonableness, and due

process.

SMALL COMPANIES

Many small companies are initiating drug testing on preemployment as well

as other conditions. Although large companies can usually obtain volume

discounts for testing services, and thus lower per-specimen costs, the overall

-11-
cost of drug testing has been lowered due to the increase in the volume of

testing. The small company can also obtain a more reasonable price for the

smaller number of specimens submitted to testing. A problem the small

company faces is its inability to maintain an EAP. Several available group

programs can be covered under insurance or benefit plans, but it is not

uncommon for such programs to be too expensive for a small company. For

many of them, firing is the only alternative for employee drug use.

THE PRESIDENT’S DIRECTIVE FOR FEDERAL GOVERNMENT EMPLOYEES

On September 15, 1986, President Reagen issued an Executive Order which

mandated efforts in all agencies to make the Federal workforce a model for

eliminating drug abuse in the national workplace. This includes the use of

urinalysis for drugs of abuse on selected employees of all agencies. The

means of implementing these directives are being rapidly developed and a

government-wide drug detection program is expected to be in place in 1987.

SUMMARY

Many Federal agencies and private companies are conducting drug tests on

job applicants and employees. Although the reasons for testing and the

circumstances under which testing is conducted vary considerably, the main

intent of these programs is to provide a drug-free environment for other

employees and a safe service to the public. The programs that have been

most successful usually include a clear communication to all employees and

applicants as to the nature of the drug program and the consequences of

detected drug use. Also, successful programs usually afford employees some

type of assistance and a second chance. Finally, it is essential for successful

programs to provide a reasonable and fair approach that includes procedures

for due process, that is, a line of review and appeal.

-12-
Choosing a Laboratory

Robert E. Willette, Ph.D.

A number of important considerations need to be reviewed when selecting a

system for drug testing. The first major consideration is to decide if testing

will be done at the place of business (“onsite”) or by an outside laboratory.

Both systems have advantages. The second and more important consideration

is the choice of laboratory used for confirmatory testing and/or all drug

testing.

TESTING ONSITE

Methods available to companies for onsite drug testing provide an effective

and easy-to-use means of conducting urinalysis programs within the

organizational structure. Some of the advantages include simplified chain of

custody, control and a greater sense of confidentiality, and immediate results.

Chain of custody is a critical factor in a valid drug program. Confidence in

the program is derived from knowing that the specimen tested actually

belongs to the person who gave it. Onsite testing reduces the number of

people who handle a specimen, which reduces the potential for mistakes.

Control and a sense of confidentiality may be important factors in some

organizations. Testing at the place of business offers these advantages

although a working relationship with a professional laboratory will, in fact,

provide the same coverage.

Another advantage of onsite testing is that it provides immediate results of

the drug tests, which may be important if action must be taken involving a

member of the organization.

In most testing situations, it is essential that all specimens that give positive

results in the onsite tests be confirmed by an alternate method, i.e., one of

the chromatographic methods such as gas chromatography-mass

spectrometry. An outside laboratory should be used for this procedure. In

some situations, repeated testing of a sample from the original specimen,

coupled with adequate operator training, instrument calibration, and quality

control measures, may provide sufficient evidence of drug use when combined

with other evidence, such as observed behavior or counselor interviews or

admission in treatment programs. Some States have upheld onsite testing

under these rigorous standards for prison populations while others have not.

-13-
One of the most frequently cited disadvantages of onsite testing is the

possibility for taking action on the presumptive positive result before a

confirmation is received. For example, the Department of Defense provides

for temporary removal of individuals in critical positions who test positive;

they must be reinstated with full privileges if the result is not confirmed.

However, concern exists about the “tainting” of the person. It is

recommended that the onsite screening result be kept as confidential as

possible to minimize such exposure.

TESTING BY AN OUTSIDE LABORATORY

The most reliable method of testing depends on a confirmatory test of a

positive result using an alternative method of testing. An outside laboratory

should be used for this procedure.

Using an outside laboratory over onsite testing has advantages. Primarily,

these advantages center on the professional capabilities of a laboratory that

are not available to those who test only on the premises. Inherent in the

decision to use an outside laboratory is the need to ensure accuracy by

selecting a laboratory of the highest caliber.

If all specimens are sent to an outside laboratory, they are handled by a staff

of trained professionals who perform the tests. And an outside laboratory

can perform confirmatory tests immediately following a positive result using

an alternative method of testing, which avoids delays. Carrying out all

testing in the same laboratory minimizes the possibility of sample

mishandling.

Additional benefits from using an outside laboratory often include laboratory

staff who can serve as expert witnesses in legal and labor action and who can

answer technical questions about the drug testing methods. The laboratory

can also use its expertise to help an organization establish sound specimen

collection and storage procedures.

CHOOSING THE BEST APPROACH

The advantages and disadvantages of onsite versus laboratory screening, as

described, suggest some of the factors that must be considered in choosing

the most appropriate arrangement for an organization. It is necessary to

have a technically oriented staff person available to operate the onsite

equipment. The testing area must be able to be secured from unauthorized

entry. It must have a refrigerator and adequate air conditioning in order to

maintain proper temperatures for storage and use of the reagents. The

operator must be reliable and trusted for his or her coufidentiality, although

it is possible to blind the operator from the identity of the specimen provider.

If these criteria can be met, the need for immediate or prompt results would

be a major factor in choosing to screen onsite. A careful evaluation of the

relative costs should be made. If the testing situation presents few positive

results, the onsite operation can be quite cost-effective, since extensive

specimen handling is eliminated. Only the positives need to be shipped to the

laboratory for confirmation.

-14-
SELECTING A LABORATORY

Two major types of laboratories can be used. Large laboratories available

nationally or regionally offer testing for drugs of abuse through a toxicology

service, in addition to routine clinical testing for serum glucose levels,

hematology, and blood enzyme levels. Smaller laboratories often specialize

in drug testing and offer national service through the mail or courier services.

As yet, no official registry of approved or certified laboratories has been

compiled. The National Institute on Drug Abuse is currently developing

standards for proficiency testing and accreditation of laboratories engaged in

drug testing. The standards will be available sometime in 1987, along with a

registry of laboratories meeting them. In the meantime, it is essential to

understand what to look for in selecting the best laboratory available. The

following list of factors will help.

Factors to Consider

Information. Find out from Federal and State agencies if the laboratory has

been licensed in any government programs and how the laboratory

performed. Only California, New York, and Pennsylvania have proficiency

testing programs for drug testing laboratories. Get recommendations from

experts in the field of drug testing programs.

Inspection. Inspect the physical plant. Observe organization and procedures

for processing specimens.

Standard operating procedures. Review the laboratory’s manual for standard

operating procedures. The manual should include a detailed description of

every step for specimen handling and analytical methods. Each page should

be dated and signed to show that it is continually updated as the laboratory

modifies procedures. What is the laboratory doing to ensure that test results

are properly reviewed and recorded?

Chain of custody. Examine documentation on chain-of-custody procedures

from the time the specimens are collected until results are reported. Special

handling procedures should be in effect for employee drug testing specimens.

These specimens should be separated from routine clinical specimens before

sealed containers are opened.

Who has access to stored specimens and why? How are they stored? Positive

specimens should be stored frozen in a secure place, and there should be a

way to identify everyone who has had access to them. The laboratory should

also be able to track exactly where each specimen was from the time it

entered the laboratory until it was stored.

How are records and actual testing data stored? Who has access to them?

They should be filed in an easily retrievable manner.

Handling flawed specimens. An agreement needs to be made with a

laboratory about testing a specimen if there has been a flaw or an error in

the handling of the specimen. In most cases, it is best to obtain a new

specimen that cannot be faulted at a later date. Some laboratories will help

train personnel who collect specimens to avoid such errors.

-15-
Specimen identification. After a container has been inspected, it should be

assigned a special identifying number or accession number. This is the

method for tracking the bottle, the request form, and the test result. Some

laboratories place several copies of the same preprinted number on the

specimen bottle. These extras are used to identify test tubes containing the

specimen or any other container. Bar-coded labels are now in use to further

ensure that accession numbers are not misread or entered incorrectly.

Specimen integrity. Integrity of the specimen is maintained by labeling

specimen and aliquot containers, as mentioned. To maintain chain of

custody, the original specimen container should never leave the secured or

limited-access part of the laboratory.

A completely new aliquot must be obtained from the original container when

doing a confirmatory test. Matching the numbers is essential in order to

avoid mixing up the specimens. Only one specimen container can be open at

a time.

Checking staff credentials. The staff of the laboratory must, at the very

least, meet State requirements. These vary from State to State. The

labaratory should have an internal certification program for each staff

position.

Minimum standards include the following:

The laboratory director or manager should generally have an

advanced degree in chemistry or toxicology, preferably in analytical

chemistry, forensic analysis or forensic toxicology. This person

should be certified or licensed by one of the appropriate boards or

societies.

The technical staff should have formal training as laboratory

technicians or technologists, chemists, or biochemists, or they should

have comparable on-the-job training and experience. Technicians

should be certified by the laboratory and appropriate outside bodies.

Laboratory certification covers each procedure the technician

performs and should include performance on quality control samples.

Find out how staff performance is monitored on a daily basis.

Examine the laboratory’s certification program and when each

technician was last certified.

Quality assurance program. This is a primary requirement for any

laboratory. It should be comprehensive in that it provides constant

surveillance of all aspects of laboratory operations.

QA programs measure accuracy of performance in specimen accessioning,

identification of aliquots, and test results, availability of current

maintenance records for instrument, records of calibration schedules and

general maintenance of good operating conditions; appropriate documentation

and handling of chain of custody procedures; proper level of technical

competence of the laboratory personnel; and generally attention to all

practices which assure accurate laboratory results.

Quality control. This is a significant part of the QA program. QC is intended

to ensure the accuracy of results by including samples with known

-16-
concentrations with every batch of specimens that is analyzed. These

samples are “open,” or known to the operators, allowing them to evaluate the

performance of each batch. In addition, it is strongly recommended that the

laboratory include blind samples in its quality control program.

The laboratory should make these data available for inspection along with

evidence of its performance on proficiency test samples run on a "bind”

basis. These blind samples can be obtained from another laboratory or a

proficiency testing service. Review the quality control records. How are

these samples introduced? Are blind samples truly indistinguishable to the

technicians from regular specimens?

Review of Results. One of the most critical steps in an acceptable testing

process is the final review of all results and reports. The laboratory should

have a senior chemist or the laboratory director certify that the report being

sent to the client is accurate, whether the results are transmitted

electronically, by phone or by mail.

Technical assistance and expert testimony. The laboratory should be able to

provide access to a well-informed staff capable of offering sound advice

about drug testing, selection of appropriate cutoff levels, and interpretation

of results.

In the event of legal or labor action, the laboratory must be able to defend a

drug test in a hearing or in court through expert testimony. An expert

witness who can defend testing methods and the scientific validity of results

usually has a doctoral degree or considerable experience in the field of drug

testing.

Supplies. An important consideration in laboratory selection pertains to the

materials the laboratory will supply. Most will provide all the specimen

containers, request forms, evidence tape or sealers for the bottles, packaging

materials (like plastic bags and boxes), and mailing or freight forms required

for specimen collection. Some laboratories also include overnight courier

service as part of the price.

Reporting results. It is important to understand and contract for specific

turnaround time in the laboratory. Many laboratories provide results within

48 to 72 hours after specimen pickup. If confidential hard copies are needed

immediately, some laboratories will set up an electronic means of

transmitting results, other than by telephone. Telephoned reports should be

avoided, since this method is least secure and most prone to mistakes. If

urgency is not a factor, a mailed envelope, clearly marked confidential and

addressed to the person authorized to receive the results, is adequate.

Equipment maintenance. Examine equipment thoroughly to determine

operation condition. Current maintenance records should be available for

each piece of equipment.

Proficiency testing. The laboratory should be participating in at least one

proficiency testing program. Determine what it is, and review the results.

Contact the agency providing proficiency testing and ask about the

laboratory’s accuracy in the program.

-17-
LABORATORY EVALUATION CHECKLIST

A sample checklist for evaluating proposals from laboratories follows. The

scoring is arbitrary and optional, but its use does provide a simple means of

ranking various choices. An actual site visit should be made to the

top-scoring laboratories to determine if they perform the work as they claim

they do.

DRUG SCREENING--LABORATORY SELECTION

Laboratory Final Score

Quality Of Services (60 points)

Test methods (20 points)

(Consider sensitivity, established reliability)

Screening:

Confirmation:

Score

Internal chain of custody (10 points)

(Consider if description is adequate, methods of

identifying samples, recordkeeping)

Score

Quality assurance program (10 points)

(Consider use of standards, internal blind QC,

certification of standards

Score

Turnaround times, reporting of results (5 points)

(Consider how results are reported, timeliness)

Score

Specimen pickup, shipping (5 points) Score

Provision for frozen storage (5 points) Score

Supplies (5 points) Score

(Consider form design, labeling, security of bottles

and kits, instructions for use)

Services Total Score

-18-
Personnel (30 points)

Laboratory director/manager (15 points)

(Consider who will provide expert testimony)

Management staff (10 points)

Technical staff (5 points)

Score

Score

Score

Personnel Total Score

Experience (10 points)

Current clients (5 points) Score

Court/arbitration experience (5 points) Score

Experience Total Score

General Comments

Reviewer Date

-19-
Proficiency Testing

Control Programs

and Quality

Robert E. Willette, Ph.D.

In a 1985 article, Drs. Hansen, Caudill, and Boone* of the Centers for Disease

Control (CDC) proclaimed a “crisis in drug testing,” based on the results of

studies conducted by CDC on the reliability of drug testing laboratories

during the period of 1973 through 1981. Under funding from NIDA, CDC

conducted a nationwide proficiency testing program wherein 10 samples were

submitted to participating laboratories every 3 months. These “open PT”

samples were used as a measure of how well the laboratory could detect the

absence or presence of specific drugs when they knew they were being

tested. It is interesting to note that not all laboratories could maintain a

passing score, which was set at 80 percent. The “crisis” proclamation was

derived from the results obtained from a limited number of selected

laboratories to which the same samples were submitted on a douhle-blind

basis. Not all laboratories did as well when tested blind.

The article referred to studies conducted in the 1970s on laboratories

providing drug screening services to federally funded drug treatment

programs. Although most of these laboratories did not use the technology

currently available, the study dramatizes the need for adequate external

quality control over laboratory performance. Furthermore, it has focused

attention on the general lack of such programs.

This chapter will describe existing proficiency programs and present some

additional suggestions for monitoring laboratory performance.

DEPARTMENT OF DEFENSE

For more than 10 years, the U.S. Department of Defense has monitored the

performance of drug testing laboratories operated by the military services

and, more recently, laboratories providing drug testing to the military under

contract. The program is conducted by the Armed Forces Institute of

Pathology (AFIP)

At present, the program involves both open proficiency test samples and blind

proficiency testing samples. Twenty-four samples, spiked with varying

concentrations of the six drugs for which the military conducts urine tests,

are sent each month to the nine military laboratories and current contract

laboratories. These samples are analyzed and the quantitative results

*Hansen, H.J.; Caudill, S.P.; and Boone, J. Crisis in drug testing:

Results of CDC blind study. JAMA 253:2382-2387, 1985.

-20-
reported to AFIP along with copies of the screening and confirmation test

records. The results must fall within two standard deviations of the group

mean in order for the laboratory to maintain satisfactory performance.

The blind program requires that AFIP send certified negative and spiked

samples to several field units that submit urine specimens on a regular basis.

Currently, 24 negative and 12 positive samples are submitted each week to

the laboratories. The submitting units must transfer the contents to their

normal bottles and submit the samples along with others using social security

numbers supplied by AFIP. Since these are sent blind to the laboratories,

copies of all results are sent to AFIP for decoding. If any discrepant results

are received, an investigation into the cause is conducted. Laboratories must

maintain a 90 percent score on positive samples and not generate any false

positives. If the latter occurs, the laboratory is not allowed to report out any

further results and must retest all positives that were obtained for a 2-week

period prior to and following the occurrence of the false positive.

Although this level of external proficiency testing seems intense, the military

drug testing laboratories process more than 200,000 specimens per month. In

the last 3 years, no false positive result has been reported out on a quality

control sample.

STATE PROGRAMS

The States of California, Pennsylvania, and New York conduct proficiency

testing programs on certain drug testing laboratories in their jurisdictions.

The California program is now limited to those laboratories conducting tests

for State-approved methadone treatment programs. It is a blind program

wherein several samples per month are submitted to the laboratories through

selected treatment facilities. Pennsylvania sends four open PT samples per

quarter to laboratories that perform drug testing on Pennsylvania urine

specimens, whether in or out of State. The New York program is also open,

with eight samples sent to every laboratory licensed to conduct such testing,

whether in or out of State. Laboratories are tested this way each quarter

unless their score is judged acceptable. Then, they are tested only twice a

year.

COLLEGE OF AMERICAN PATHOLOGISTS

Since the late 1940s, the College of American Pathologists (CAP) has

conducted interlaboratory comparisons designed to assess the state of the art

in clinical laboratory practice. The program includes laboratory inspection,

certification, and proficiency surveys. Starting in 1984, CAP initiated a

survey for urine screening for drugs of abuse. It consists of sending to

subscribing laboratories five urine samples containing five to six drugs each

quarter. The laboratories analyze the samples and report the qualitative and

quantitative (if they offer that service) results back to CAP for review. The

laboratories are provided with an evaluation report.

Laboratories that also conduct analyses of blood samples can subscribe to the

older toxicology surveys that include three or four samples of serum and one

or two samples of urine containing various drugs in toxic concentrations.

-21-
AMERICAN ASSOCIATION OF BIOANALYSTS

The American Association of Bioanalysts has provided a variety of proficiency testing programs since 1949. A urine toxicology program became

available in 1980. This survey provides subscribers with two urine samples

per quarter, each containing 10 of the more commonly abused drugs or their

metabolites.

DUO RESEARCH INC.

A consulting firm specializing in assessing drug testing programs, Duo

Research Inc. provides a blind proficiency testing service to government and

business. The service is similar to that described for the Department of

Defense, in that positive and negative samples are sent to sites that collect

and submit urine samples to a laboratory for drug testing. The number of

samples received by subscribers is tailored to the particular level of testing

being conducted. Monthly status and quarterly summary reports are

provided. An investigation is made in the event of an incorrect result.

INDIVIDUAL PROGRAMS

Some laboratories conduct their own external as well as the mandatory

internal quality control programs. Some laboratories with multiple locations

may submit samples to each laboratory director to insert in a blind fashion

into their routine testing. Others will provide customers with prepared

samples that they can submit back to the laboratory in a blind manner.

Although these are valuable adjuncts to other measures of performance, they

are not as secure as those programs that provide an independent source of

samples and interpretation.

AVAILABILITY OF REFERENCE MATERIALS

One of the difficulties in conducting quality control programs is the limited

availability of suitable reference materials. Experiments with urine

collected from individuals who are known to have taken a specific drug have

shown a significant variability in sample stability. It has thus been favored to

add known amounts of pure drugs and/or metabolites to a pool of drug-free

urine. Collecting sufficient quantities of the latter also presents its

difficulties. Some programs, such as that conducted by AFIP for the

military, use a combination of human urine and a synthetic matrix. This can

pose problems with assay interference and recognizability.

The most reliable proficiency testing and control samples are made from

drug-free human urine where drugs and/or their metabolites have been added

in concentrations consistent with normal detection ranges for the drugs in

question. Also, as is the case with morphine, a mixture of parent drug and its

conjugated metabolite must be added in order to simulate a real positive

specimen. Proper samples for methadone contain at least two of its metabolites in order to be properly identified on thin-layer chromatographic

analysis.

-22-
Few sources exist for some of the pure drug and metabolite standards. The

National Institute on Drug Abuse has maintained a program for preparing

such standards through a contract with the Research Triangle Institute (RTI),

but these materials are available only on a limited basis for research

purposes. Analytical quantities of pure drug standards can be obtained from

Alltech-Applied Science, State College, PA; Supelco Co., Bellefonte, PA; and

RTI, Research Triangle Park, NC. Larger quantities can be purchased

directly from RTI.

To ensure the highest standards of drug testing, every government agency or

private employer testing job applicants or employees must insist that the

laboratory being used participate in at least one, but preferably several, of

the proficiency testing programs described here. This should be a

requirement written into the contract with the laboratory, and test results

should be submitted to the customer as soon as they are received. Open

proficiency tests can indicate the best that a laboratory can do when it knows

it is being tested, but a blind program is most effective in consistently

encouraging maximum laboratory performance.

-23-
Specimen Collection and Handling

Joseph E. Manno, Ph.D.

The urine drug test can be useful and reliable for determining drug use

patterns only if it is performed and interpreted using appropriate procedures.

The validity of the result of a urine drug test requires that consideration be

given to the methods used for the collection, transportation, analysis, and

interpretation of the results.

RESPONSIBILITY

Three different groups are generally associated with the urine drug test

procedure. The collection site is responsible for collecting, labeling, boxing,

and shipping samples, ensuring that collection and storage procedures have

the proper documentation and security methods necessary. The collection

site also must provide all staff with sufficient training to understand the

collection process and the significance of laboratory results.

The courier is responsible for transporting samples to the laboratory and

maintaining appropriate chain-of-custody records for ensuring that samples

are not tampered with during transit. The laboratory is responsible for

receiving samples from the courier and further guaranteeing that the samples

were transported without tampering. The laboratory must also maintain

records to assure that the integrity of the samples is maintained in the

laboratory and that the results are correct. Although it is recognized that

each collection site must develop collection and shipping procedures that

meet its individual needs, the purpose of this discussion is to describe

procedures that will fulfill the necessary criteria in order to guarantee

optimum validity of drug screening results.

RANDOM SAMPLING

Random sampling is a process whereby urine samples are collected in a

manner that the subject cannot predict when the specimen will be requested.

Random sampling is most important when multiple samples will be collected

from the same person over a period of time. Random sampling principles

should also apply when single samples are collected for on-the-job drug

testing programs. For several reasons, random sampling procedures are

important to increase the overall “sensitivity” of the drug detection program.

The laboratory procedures for performing the urine drug screen have defined

sensitivity. Under most circumstances, intermittent drug use (i.e., taking

drugs on less than a daily basis) can be detected within a range of 6 hours to 2

-24-
days or more after the drug was ingested. When individuals take drugs

chronically (on a regular basis, either daily or more than once a day), the

likelihood of detecting the drug in urine increases. However, it is possible for

individuals to tailor their drug use to conform to the pattern of urine

collection used. If they know a collection is forthcoming, they can

discontinue use of the drug long enough prior to the urinalysis so the drug use

will not be detected.

In substance abuse treatment centers, for example, if urines are collected

regularly at specific times, the subject can start using drugs immediately

after the collection and stop a day or two before the next scheduled

collection. With a 2- or 3-day drug-free period before collection, a good

possibility exists that the urine drug screen will be negative.

When urine drug tests are used as part of a preemployment program, if an

individual is told to report for a preemployment physical at a prearranged

tie, drug use could be discontinued prior to the appointment, thereby

increasing the chance for a negative screen. With the proliferation of

Laboratories, subjects may have enough time prior to a scheduled urine

collection to have their urine analyzed for the presence of drugs. If the urine

was found to be positive, they could then make an excuse for not keeping the

appointment. The most effective random sampling schedule for urine

collection is to contact subjects and require that they come immediately for

urine collection.

In substance abuse treatment facilities, random sampling increases the

effectiveness of the drug testing process, because it removes the regularity

needed for patients to schedule their drug use in order to adjust drug taking

so the screen is negative. Subjects are always in the position of not knowing

when the next screen will be taken; therefore, they can never safely take the

drug.

For preemployment drug testing, every attempt should be made to provide

only several hours’ notification before the subject must report for the test.

Ideally, prospective employees can be given a 1 or 2 week “time window”

during which they will report for the test. Then, notification can be given

several hours in advance of the test. As a matter of practice, however, this

may be very difficult to achieve in a preemployment situation.

Random sampling for on-the-job testing is more easily accomplished, since

the employees are readily available. The determination of testing days and

departments should be kept confidential. Employees who are absent on the

day of testing should be tested at a later, unscheduled date.

When a program of “probable cause” or “suspicion” is in force rather than

random sampling, samples are taken when a supervisor determines that the

behavior of the individual is cause for testing. Obviously, in such a case, the

specimen should be taken with as little forewarning as possible.

COLLECTION

The collection process is quite important, and several factors must be

considered in developing a suitable method. The validity of the results of a

urine drug test is dependent on the integrity of the specimen. The urine

container obviously must be clean, unbreakable, and leakproof. Since the

-25-
analysis procedure used by laboratories has defined limitations, anythiug that

might cause the urine to be outside these limitations would produce an invalid

test. The way to achieve many of these limitations is common knowledge to

some individuals who undergo urinalysis. There are many ways for a urine

specimen to be invalidated if the individual has the opportunity.

Individuals have reportedly placed various chemical substances

under their fingernails and released them into the urine sample to

affect the subsequent analysis.

Placing a pinhole in the bottom of the urine container would

result in a leak that would not be detected at the collection site.

During shipping, however, most or all of the urine could leak out.

Ordinary table salt, detergent, or other commonly available

household chemicals can destroy the drugs or affect the assay in

such a manner as to generate a false negative analysis.

Frequently, soap dispensers or cleansers in toilet areas offer the

opportunity to add effective adulterants to the sample.

Use of a fluid-filled bulb placed under the arm, with a tube

leading to the genital area, is another method. The subject can

squeeze the bulb and release water or other substance that would

dilute or contaminate his/her own urine.

The subject can obtain urine from friends not using drugs or save

his/her own urine from drug-free periods. This urine can be

placed in the container during the collection period.

The subject can scoop water from the commode into the

collection container and dilute the urine.

It is important that specimen collection be directly witnessed if at all

possible by a reliable individual to prevent this sort of intentional

adulteration. While direct-observation collections provide the greatest

credibility to a drug deterrent program, the procedure can be embarrassing to

both parties. Where it is determined that privacy of the individual must take

precedent over other considerations (and such was the determination in

President Reagen’s recent Executive Order for urine drug testing in the

Federal workforce), there are means by which adulteration of samples can he

minimized. The temperature of the specimen should be close to body

temperature (37°C)* if the sample has not been diluted with water. This can

be checked by thermometer. Collection facilities can be set up with no soap

dispensers or cleaning agents available that can be used to adulterate the

sample. The water in the toilet can be dyed or the toilet itself can be a

chemical one, eliminating in both cases the availability of water for dilution.

Such a facility should be considered in circumstances where specimens can

not be witnessed as a matter of policy or where the possibility of the

subject’s bringing in something to contaminate or dilute the sample is

unlikely. A patient undergoing a physical, for instance, may not be dressed

and could not likely conceal anything to invalidate a sample by adulteration.

*Judson, B.A.; Himmelberger, D.U.; Goldstein, A. Measurement of

urine temperature as an alternative to observed urination in a narcotic

treatment program. Am J Drug Alcohol Abuse 6:197-205, 1979.

-26-
In most cases, the laboratory is capable of detecting the adulteration of urine

specimens when water has been added by checking the specific gravity of the

urine. A colored water solution or a urine to which a large amount of water

had been added would have a lower than normal specific gravity. A pH check

would indicate an attempt to acidify or alkalinize the specimen to invalidate

screening assays. If adulteration is suspected, the laboratory should be

notified and requested to make such a determination. The laboratory can

also check on the validity of the sample by performing a creatinine analysis.

Creatinine is normally present in a urine sample and will be detected by the

test if the sample is urine. In addition, the concentration of creatinine can

be used to determine if the sample has been diluted (by adulteration or by

drinking excessive quantities of liquid).

Other means of influencing the outcome of a urinalysis that are more

difficult to detect involve the ingestion of large quantities of water before

providing a sample, which in effect dilutes the urine produced. Drinking

large volumes of water or other liquid several hours prior to the urine

collection could easily result in a tenfold dilution of urine. This dilution

could lower the concentration of drug sufficiently so that it could not be

detected by the laboratory analysis.

There is a widespread belief that drinking vinegar can produce negative

urinalysis results. While it is theoretically possible that sufficient vinegar

ingestion could alter urinary pH, it is highly unlikely that such a quantity

could be drunk without toxic consequences. Even if pH were altered slightly,

the effects on different drugs would be variable--the excretion rate of some

might be increased slightly and for others it might be decreased.

SECURITY

The security of samples as well as empty cups, laboratory invoices, cup

labels, and other packing or shipping material is critical. If subjects can

obtain empty cups or other laboratory material, it becomes quite easy to

substitute other urines for their own. Computer-printed labels should be

attached to the cup, rather than to the top, to make it more difficult for

subjects to switch samples.

The specimen donors should not generally be permitted to have any

involvement in the collection, labeling, boxing, packing, or transporting of

samples to the laboratory. It is important, however, that donors witness the

sealing of the bottle and sign or initial the seal. Access to collected urines or

any of the boxes, cups, tape, labels, or other laboratory materials should not

be allowed.

After collection, urine specimens should be stored under locked storage

conditions. If transport of the specimens is inordinately delayed, they should

be kept refrigerated (4°C) if possible.

-27-
DOCUMENTATION

Accurate and complete records of all individuals involved in the urine

collection, storage, and shipping procedures should be maintained. At least

two major documents will be utilized for documentation procedures. These

include a label attached to the urine specimen container and a separate

invoice or other listing of the samples transmitted to the laboratory.

Specimen Label

The specimen label should be affixed to the urine container and not to the

lid. This will prevent accidental or intentional switching of specimens and

identifying labels.

The label should contain the following information:

Name or other identification of the collection site or client

Date and time the sample was collected

Name or identification (social security numbers are frequently used)

of the subject (subjects should initial the label and thereby

acknowledge that the specimen is their urine)

Name or identification of the individual who witnessed the urine

collection (the witness’ initials should also be on the label)

Log number to link the specimen to the transmittal

invoice, although the subject ID number from the label along with

the site code number is generally sufficient for this purpose

Approximate volume of urine collected

If possible, certain information on the labels, such as the name of the

laboratory and/or the identification of the collection, should be preprinted.

This will reduce the chances of subjects’ switching labels or samples. AU

writing on the labels should be in ink that will not run if it becomes wet from

condensation of water or urine spillage. Clear plastic tape over the label

provides an excellent mechanism for preserving the integrity of the

information.

Invoice

A transmittal invoice that accompanies the urines will allow the laboratory

to check the individual urines against the invoice to confirm that all the

specimens collected actually reach the laboratory. The minimum

information the transmittal invoice should contain is:

Collection site or client name or identification number

Subject name or identification code

Accession number of the specimen (if used)

Specimen collection date

Desired tests to be run on the urine specimen (if are not preset)

Name or identification number of the witness and/or persons

responsible for collection, handling, storage, or packing of specimens

at the collection site

-28-
PACKING AND SHIPPING

Sample identifications should be checked against the shipping invoice as they

are placed in the shipping box. The staff member should ensure that the

number of urines shipped and the tests desired correspond between the

invoice and the urine cups.

The shipping container should be sealed at all openings with tape that cannot

be removed. Additional security may be provided by the staff member’s

signing his or her name across the box and tape. In this manner, if the tape is

removed, it will not be possible to reseal the box without detection. The

laboratory should be supplied with a list of acceptable signatures.

Samples should be transported to the laboratory either by the courier or by a

reliable staff member. If a bonded courier transports the samples, a record is

kept which acts as proof of delivery for legal purposes. Courts have upheld

shipment of such samples by U.S. mail, however. If the samples are delivered

by a staff member, a receipt must be issued by the laboratory when the

samples are received. Specimen donors should never be permitted to

transport samples to the laboratory.

If the samples are delivered by courier, the invoice should be checked to

make snre that the invoice accurately states the number of boxes sent. If the

samples are delivered to the laboratory by a staff member, the staff member

should request a hand receipt stating the number of boxes delivered.

If fewer than the number of boxes or samples stated on the invoice are

received by the laboratory, or if a discrepancy is noted between the

information on the container label and the invoice, a report of the situation

discrepancy should be sent by the laboratory to the collection site.

-29-
Analytical Methodology

Richard L. Hawks, Ph.D.

The specific methods chosen by a laboratory will depend on a number of

factors, including cost, workload (number of specimens), turnaround time,

sensitivity required, and reliability. In nearly all applications of urine drug

detection, a confirmation analysis is essential for all specimens screened

positive. A different type of analytical methodology should be used for the

confirmation analysis. Analytical methods used in most laboratories for the

detection of drugs in body fluids can be classified into two main categories--

immunoassays and chromatography--generally used respectively for

screening and confirmation.

Examples of these two types of methodologies are presented, including

sections describing how they are used for screening and confirmation

purposes. A brief discussion of new trends in urine drug detection methods

appears at the end of this chapter.

IMMUNOASSAYS

lmmunoassays are based on the principle of competition between labeled and

unlabeled antigen (drug) for binding sites on a specific antibody. Antibodies

are protein substances with sites on their surfaces to which specific drugs or

drug metabolites will bind. These antibodies are formed by inoculating

animals with appropriate immunogens. Two types of immunoassays are

usually employed in urinalysis at this time--the radioimmunoassay (RIA) and

the enzyme immunoassay (EIA). The difference between these types of

immunoassays is mainly in the indicator that is used. The EIA utilizes an

enzyme as an indicator in the assay (the label), while RIA uses a radioactive

indicator. Because antihodies often cross-react with related drugs, and

sometimes even with unrelated compounds, confirmation of positive

immunoassay results with an independent procedure is imperative for

definitive identification. The Abuscreen RIA manufactured by Roche

Diagnostics in Nutley, NJ, is the RIA system most frequently used for drugs

of abuse in this country (Abuscreen 1983). The EIA of most prevalent use in

the United States is the EMIT manufactured by Syva Company in Palo Alto,

CA.

Radioimmunoassay (RIA)

In radioimmunoassay testing (Chase et al. 1976; Ebert et al. 1981; Cook et al.

1982; Soares et al. 1982; Law et al. 1984; Cross et al. 1985). known amounts

of radioactive-labeled drug are added to a urine sample with knowu amounts

-30-
of antibodies. The mixture is then allowed to incubate, during which time the

labeled drug and unlabeled drug compete for binding sites on the antibody.

After precipitation and centrifugation of antigen-antibody complexes, either

the supernatant fluid or the precipitated antibody is transferred to a gamma

counter (an instrument that determines the level of radioactivity of the

sample). The presence or absence of the drug is indicated by the amount of

radioactivity found, since this is proportional to the amount of antigen

(labeled drug) bound to the antibody. If the supernatant is counted, a positive

specimen is identified when the radioactive counts are equal to or greater

than those of a positive control prepared in the same manner as that of the

unknown urine. Conversely, when the precipitant (bound antigen) is counted,

a positive specimen is identified when the radioactive counts are equal to or

lower than those of a positive control prepared in the same manner as that of

the unknown urine.

RIA can detect very small concentrations of drug with sensitivity ranges on

the order of 1-5 nanograms per milliliter (ng/ml). The required sample

volume is small and sample preparation is minimal. The use of automated

pipetting and counting equipment allows for large-volume, multiple testing.

Some of the disadvantages of this technique are associated with the use of

radioactive substances and the high cost of reagents and instrumentation.

Turnaround time is long--from 1 to 5 hours.

Enzyme Immunoassay (EIA)

Unlike RIA, EIA is a homogeneous enzyme immunoassay technique. That is,

the antigen-antibody complex need not be separated by centrifugation. The

most frequently used EIA method in this country is the EMIT system (Rogers

et al. 1978; DeLaurentis et al. 1982; Law et al. 1982). In the EMIT assay, the

label on the antigen is an enzyme (protein) that produces a chemical reaction

for detection of drugs. This detection is based on the competition between

unlabeled drug or drug metabolite and labeled drug or drug metabolite for

binding sites on the antibody. Urine is mixed with a reagent containing

glucose-g-phosphate (G-6-P) and antibodies to the drug, as well as a second

reagent containing a drug derivative labeled with G-6-P dehydrogenase. The

enzyme-labeled drug when bound to an antibody site is incapable of

interacting with the substrate (G-6-P). If the enzyme-labeled drug does not

bind to the antibody, then it is free to react with the substrate. The drug in

the subject’s urine competes for the limited number of antibody binding sites

and thereby proportionally increases the total enzyme activity. The

enzymatic activity is therefore directly related to the concentration of the

drug present in the urine.

Some of the advantages of EMIT include (1) a short analysis time, (2) an

easily measured nonradioactive endpoint that is simply measured, and (3) no

necessary separation of bound and free fractions as in RIA. The EMIT,

however, is generally less sensitive than RlA but still has moderate to good

sensitivity and specificity (Allen and Stiles 1981). The enzyme/substrate

interaction is somewhat more sensitive to temperature variation and ionic

adulterants (such as salt). This procedure can be automated, allowing for

multiple sample testing and reduced costs. Two systems are currently

marketed--the EMIT-d.a.u. is designed for use in laboratories with large

sample throughput; the EMIT-st is a portable system which can be used

“on-site” and in situations where a small number of samples are analyzed in

any given period.

-31-
CHROMATOGRAPHY METHODS

Chromatography is a method of analysis in which the various components in a

biological specimen can be separated by a partitioning process.

Chromatographic separations to resolve mixtures of drugs and metabolites

require (1) a stationary (fixed) phase, which may be a solid or a liquid on an

inert support having a large surface area, and (2) a mobile (moving) phase of

liquid or gas. With a chromatographic method, substances are carried by the

mobile phase through a column or across a plate, where the stationary phase

interacts with the specimen to cause separation of the various components.

After separation, a detection method distinguishes the components for

identification and measurement.

Separation of the components of biological mixtures containing substances of

various molecular types is based on the time spent by each component in each

phase of the chromatographic system. Several different types of

chromatographic techniques are used in laboratories for urine drug analysis.

These various techniques offer different degrees of resolving power (the

ability to separate one component from another) and often utilized in

combination, depending on the drugs in question. While other

chromatographic techniques are available, thin-layer chromatography (TLC),

gas-liquid chromatography (GLC), and high-pressure liquid chromatography

(HPLC) are the most commonly used ones. The combination of GLC with

mass spectrometry (GC/MS) provides the most specific type of analytical tool

currently used in urinalysis.

Extraction of biological samples is necessary for all chromatographic

techniques for drugs other than alcohol. It is usually not required for the

immunologic methods. Liquid-liquid extraction is the most commonly used

method. This procedure involves the mixing of the sample in water with a

water-insoluble organic solvent. If the drug of interest is more soluble in the

organic phase, most of the drug is extracted from the water phase into the

solvent. The solvent is then evaporated to dryness, and the residue is

redissolved in a small amount of solvent and reserved for further testing.

This is called direct extraction.

Some selectivity can be achieved by an appropriate choice of solvents and pH

adjustment of the aqueous phase. Greater selectivity can also be obtained by

a technique called multiple extraction (or back extraction) when necessary.

Passing the drug back and forth from solvent to aqueous phase with pH

adjustment tends to isolate it from nondrug or other interfering substances.

Separation of classes of drugs can also be attained by this technique.

Liquid-solid extraction with resin or charcoal, and more recently other

solid-phase extraction techniques, provide the necessary isolation of drug or

drug metabolite from the biological sample and provide a relatively clean

sample for analysis by GLC, HPLC, TLC, or related procedures.

Thin-Layer Chromatography (TLC)

Of the chromatographic techniques, TLC is one of the oldest methods but is

still utilized as a practical technique for many large-scale multiple drug

screening programs (Michaud and Jones 1980).

-32-
In TLC, an absorbent (stationary phase), such as silica gel, alumina, cellulose,

or ion exchange resin, is uniformly applied to a glass plate or plastic film.

Mixtures of known drug compounds (standards) or residues from an extraction

of drugs from urine are applied as spots to prepared plates, which are then

placed in a closed container with just enough solvent (mobiie phase) to wet

the bottom of the plate. The solvent is allowed to flow across the stationary

phase by capillary action, usually in an ascending fashion, allowing the

substances to separate. The separated substances can then be identified by

spraying the plate with reagents that produce characteristic color reactions.

Drugs visualized in this way are identified on the basis of (a) reference values

(ratio between the distance the mobile phase moves up the plate and the

distance the compound moves from the point of application), (b) metabolic

patterns (parent drug and characteristic metabolite), and (c) functional group

analysis (chemical characteristics as defined by the color reaction with the

spray reagent).

Advantages of using TLC are (1) low cost of equipment, (2) rapid analysis,

and (3) ability to detect more than one drug or metabolite per analysis.

Relatively small amounts of drugs can be detected, usually as low as of

0.5-1.0 micrograms per milliliter. The recent development of highperformance thin-layer chromatography (HPTLC) plates has enhanced the

capability of thin-layer chromatography. In HPTLC, silica gel particle size

and the thickness of the layer on the plate are reduced, allowing for the

separation of drugs in much shorter distances. Increased sensitivity is also

gained because the applied spots are smaller than those for regular TLC. A

small spot in combination with the short migration distance results in high

drug concentration at the migrated spot. The net effects are increased

sensitivity for the detection of drugs and a shorter analysis time. References

to TLC methods for a variety of drugs can be found in the last chapter of this

monograph.

Some of the disadvantages of TLC are that it provides only fair specificity

and sensitivity and results that are highly dependent on the technician’s skill.

It does require practice to recognize patterns of drugs and/or their

metabolites by the visualized colored spots. These problems are minimized

by commercial systems that attempt to standardize the elements of

extraction, application, and visualization. One such system, manufactured by

Analytical Systems, Laguna Hills, CA, is called the Toxi-Lab system. TLC

also is a highly labor-intensive technique and sometimes requires extensive

sample preparation.

Gas-Liquid Chromatography (GLC)

Gas-liquid chromatography (interchangeably referred to as gas chromatography or GC) is widely used in drug analysis as a confirmation method as

well as a primary screening method under some conditions (ElSohly et al.

1984; Woodworth et al. 1984). It utilizes an inert gas, such as nitrogen or

helium, as the moving phase to transport a vaporized sample of a drug

through a glass column containing a stationary liquid phase. The drug is

identified and quantified by a detector at the far end of the column.

The column’s capability to separate and identify drugs is optimized by

altering the types and amounts of a liquid (stationary phase) absorbed on

solid-phase substances such as silica compounds. Typically, columns used in

many GC methods are 3-6 feet in length and a few millimeters in diameter.

-33-
There has been great technical development in capillary columns, which offer

an increase in separation power over the conventionally packed column.

Capillary columns (0.2-0.7 millimeters in diameter and 5-100 meters in

length) are made of fused silica and are available in several forms. With one

type, the Liquid phase (organic compound) is present as a thin film on the

capillary column walls. Bonded phases are widely used and offer several

advantages over nonbonded phases, notably (1) on-column injections and (2)

better stability and resistance to thermal shock. Capillary columns coupled

with GC provide superior resolution of compounds, give greater sensitivity,

and are generally recommended for applications in urine drug assays.

Either a conventional or a capillary column is placed inside an oven with

precise temperature control; the sample is injected into a heated port and is

carried through the column by the inert gas at a controlled flow rate. The

column material has the ability to absorb substances as they are being moved

from injector port to the detector. Different drug molecules tend to be

differentially adsorbed by the liquid phase and the gas vapor phase. The

equilibrium between these two phases as the drug passes through the column

creates the characteristic column retention time for that drug. This

retention time is the parameter of identification associated with GLC

procedures.

Several types of detectors are available to provide the selectivity and

sensitivity needed to properly detect and identify drugs of interest as they

emerge from the column. Popular detectors are the electron capture

detector (ECD), the flame ionization detector (FID), and the nitrogenphosphorous detector (NPD). Each of these detectors has its own

characteristics of sensitivity and specificity. The NPD detector is

particularly suitable for nitrogen containing compounds such as phencyclidine

or cocaine. The FID is of more general applicability, but is less sensitive

than the NPD or the ECD detector.

Gas-Liquid chromatography is a sensitive technique, and small amounts of

drugs can easily be detected and identified by determining their respective

retention times as compared with known drug standards under optimum

instrument conditions. Some of the limiting factors are (1) the slowness of

analysis, since only a single sample can be processed at one time, (2) the

expertise required in conducting the tests, and (3) the sample preparation

time, since many drugs or their metabolites must be derivatized before they

can become sufficiently volatile to move through the column. Additionally,

GLC as well as all other chromatographic methods suffers from the

deficiency that the retention time, which provides only a single parameter,

cannot be used as an unequivocal identification in many cases. Certain drugs

or conditions may require that other methods be used to provide rigorous

proof of identity.

High-Performance Liquid Chromatography (HPLC)

This method is sometimes used in urine and blood analysis (ElSohly et al.

1983; Law et al. 1984a; Dye et al. 1984). It employs a column through which

the drug passes while undergoing equilibration between two liquid phases,

rather than a gas and liquid phase as in the case of GLC. Again, the

characteristic of the drug molecule that is measured is the time it takes for

the drug to traverse the column at a given solvent flow rate. Detectors are

-34-
ultraviolet, fluorescent, or electrochemical in nature. HPLC has the

advantage that polar drugs requiring derivatization on GLC systems can be

assayed directly on HPLC. Its disadvantages are similar to those of GLC,

although specimen preparation may be simpler. Some laboratories take

advantage of HPLC’s chromatographic capabilities and the superior

sensitivity of RIA methods by using RIA as the HPLC detector. In this case,

fractions from the HPLC column are sequentially analyzed by RIA to provide

a highly specific and sensitive system of analysis (Law et al. 1984a). This

technique is useful in many forensic applications but generally is too

cumbersome for routine urinalysis.

Gas Chromatography/Mass Spectrometry (GC/MS)

The analytical technique of gas chromatography/mass spectrometry (GC/MS)

combines the efficient separating power of gas chromatography with the high

sensitivity and specificity of mass spectrometric detection. GC/MS is

generally considered to be the moat conclusive method of confirming the

presence of a drug in urine. The major factors that have limited the use of

GC/MS have been its comparatively high cost and complexity. Fortunately,

GC/MS instrument manufacturers have recently introduced lower Priced

systems that are easier to operate and this should result in significantly lower

fees for GC/MS analyses.

In spite of the remarkable potential capabilities of GC/MS, it should not be

assumed that the results of all drug confirmations performed using GC/MS

are conclusive. There are many different modes of operating a GC/MS. It

can be operated in the “full scan” mode which provides a complete mass

spectrum for each component of the urine extract that passes through the gas

chromatograph. Since a complete mass spectrum represents a “fingerprint”

pattern that is unique for each drug, this mode of operation will give the

most conclusive identification if there is a sufficiently high concentration of

the drug to provide a good quality mass spectrum.

Alternatively, the GC/MS can be operated in the selected ion monitoring

mode in which the mass spectrometer monitors the ion currents at only a few

masses which are characteristic of a specific drug. This mode of operation

affords far higher sensitivity, but provides a less specific pattern for

identification. Other choices of modes of operation include the method by

which the drug molecules are ionized. Electron impact (EI) ionization is the

technique most widely used. Mass spectra obtained using EI ionization are

typically quite complex and therefore very suitable for obtaining a

“fingerprint” identification of a drug. The technique of chemical ionization

(CI) is an alternative method of ionizing molecules for mass spectral

analysis. CI mass spectra are typically less complex than EI mass spectra of

the same drug, and therefore are less unique. However, because CI is often

more sensitive than EI ionization, it can be used to detect and measure lower

concentrations of the drug. Also, some methods of chemical ionization are

relatively selective as to what compounds are ionized, as a result detection

of a drug by CI mass spectrometry is often less subject to potential

interferences from co-eluting components of the urine extract.

The choice of which modes of operation are to be used (full scan or selected

ion monitoring, CI or EI ionization, etc.) depends on what drugs are to be

detected, the minimum concentration of the drug that constitutes a positive

-35-
identification, and whether or not the concentration of the drug is to be

quantitatively determined. The reliabitity of a GC/MS assay is also

dependent on the skill and experience of the operator, as well as on the

method used for extraction of the drug(s) from the urine and for preparation

of the extract for injection into the GC/MS. An extensive knowledge of

GC/MS technology is often needed to adequately evaluate the reliability of a

specific assay, but if appropriate methods are used by well-trained analysts,

a GC/MS analysis will ensure that a suspected drug is identified properly, and

conversely, that no one will be falsely accused of drug use.

SCREENING PROCEDURES

The principle of screening by immunoassay, a highly sensitive technique, is to

minimize the possibility of false negatives. By selecting highly sensitive

techniques for broad classes of drugs, however, absolute specificity is

lessened such that some false-positive results may be produced from

cross-reacting substances having a similar chemical structure present in the

specimen. For this reason, positive results from the screening procedure

should be considered only presumptive; they must be confirmed by a second

and distinctly different analytical technique.

Alternatively, thin-layer chromatography is often used as an initial screen

when the ability to screen inexpensively for a large number of drugs is more

important than the degree of sensitivity. Due to the level of subjectivity

involved in the interpretation of TLC assays, however, it is important to

confirm any presumptive positives with a highly specific method such as

GC/MS.

SENSITIVITY AND ASSAY “CUTOFF”

The ability of any assay to detect low levels of drugs has an inherent limit.

The concentration of drug in the urine sample below which the assay can no

longer be considered reliable is the “sensitivity” limit. It is sometimes called

the “detection limit” and is expressed as a concentration of the analyte in the

specimen. The “cutoff” point is the concentration limit that will actually be

used to assay samples. It is a value serving as an administrative breakpoint

for labeling a urine result positive or negative. Manufacturers of commercial

urine screening systems set cutoff limits to their assays well above the

sensitivity limits of the assay to minimize the possibility of a sample that is

truly negative giving a (fake) positive result. For example, although most

immunoassays for detection of marijuana are sufficiently sensitive to detect

drug metabolites at levels below 20 ng/ml, the assays are usually set for

cutoff levels of 20 to 100 ng/ml. This decreases the possibility of a false

positive that could result from operating the assay too close to its level of

sensitivity.

The cutoff levels selected should be reasonable concentrations reflecting

realistic urinary elimination of drugs. In immunoassay screening procedures,

a cutoff standard is selected along with control standards at other

concentrations. Any sample that contains the drug of interest at

concentration levels equal to or greater than the designated cutoff is

reported positive, and any sample that is less than the cutoff level is reported

negative. Setting screening cutoffs too low would allow for longer detection

-36-
time after drug administration, but the results might be difficult to confirm

reliably. If the confirmatory procedures are not sensitive enough, the

screened positive may not be confirmed and the result would appear as a

false positive.

The distinction between an unconfirmed positive and a “true” false positive is

sometimes confusing. A sample determined positive by one method and

negative by a confirmation (different) method could be a situation where a

false result occurred in the first analysis and the drug was not in fact

present, or, alternatively, the drug might in fact be present but was not

detected in the second assay due to differences in the sensitivity of the two

assays. On the other hand, setting high cutoff levels for the screening

procedures will generate false negatives because drugs may be present in

significant concentration but below the designated cutoff and would

therefore be reported negative. Each urine sample must undergo a separate

immunoassay for each of the selected drug groups. Depending on the

laboratory and the technique employed, cutoff levels may differ. Therefore,

negative screening reports should contain a statement with reference to

established cutoff levels only, without providing the specific numbers leading

to the negative evaluation. Urine specimens testing negative do not require a

confirmatory test.

ASSAY SPECIFICITY

Specificity or selectivity of an assay method refers to the ability of the assay

to identify a single chemical component in a mixture of chemicals and

biological materials. This characteristic is a function of one or all of the

processes of isolation, separation, and detection of a particular product in a

biological matrix. A highly selective detector on an HPLC or GC can

compensate for a complex mixture, while the same compensation may be

achieved by an unusually efficient separation technique such as capillary

column chromatography.

The most specific types of assay methods optimize all these factors. GC/MS,

for instance, with a capillary column permits highly efficient separation of

components on the column, followed by extremely selective detection in the

mass spectrometer. It therefore achieves the most specific results of all

assay methods.

CONFIRMATION PROCEDURE

The principle of confirmation procedures is to use a highly specific and

alternate chemical technique to ensure that false-positive results do not

occur at the selected or established cutoff levels. For most assays, the

sensitivities for the confirmation procedures should be lower than the cutoff

of the screening procedures used, so that the number of false negatives or

positives due to concentration differences is minimized. In some cases (e.g.,

cannabinoids), the confirmation cutoff is generally set at a much lower level

than the screening cutoff, because the immunoassay reacts additively with

several metabolites from THC and the more specific confirmation methods

are directed at only one. A selective liquid-liquid or liquid-solid extraction

procedure prior to the confirmatory test is used to isolate the drug and/or

metabolite from the urine. The purified extraction product may require

further treatment (e.g., derivatization) before it is analyzed.

-37-
Prior to extraction, a known amount of an internal standard is added to each

standard control and to each sample to ensure extraction integrity and to

serve as a basis for quantifying the drug in question. The internal standard is

a chemical compound having chemical and physical properties similar to the

drug being tested. A distinct advantage for GC/MS is the ability to use a

deuterium-labeled internal standard. In this case, the internal standard is

virtually identical to the drug being tested, but it can be measured separately

due to the different mass. A calibration standard containing the drug at the

cutoff level is included in each analysis, as are blanks and positive and

negative controls. (The quantitative analytical data are compared with

calibration curves for the analyte with a known quantity of the internal

standard.) AU quality control materials being run should give results within

acceptable deviations from the true mean (generally ±2 standard deviations).

RECOMMENDED ANALYTICAL APPROACH TO DRUG TESTING

Because of the potential impact of the results of a urinalysis on an individual,

only the most rigorous and conclusive procedures should be used. It is

essential to incorporate both a screening and a confirmation step in any urine

drug detection program where the consequences of such an analysis will he

the basis of actions taken against the individual who supplied the sample. ln

this regard, the confirmation techniques chosen by the laboratory should

provide the most accurate and unequivocal results possible. While

confirmation techniques other than GC/MS may be adequate for some drugs

of abuse, GC/MS is generally accepted as a rigorous confirmation technique

for all drugs, since it provides the best level of confidence in the result. Put

another way, the appropriate use of GC/MS as a confirmation technique is

least likely to become a topic of debate between expert witnesses at a legal

proceeding. Major Federal drug screening programs, such as the one used by

the Department of Defense, mandate GC/MS as the confirmatory method for

all drugs.

Thus, an effective analytical system for the detection of drugs of abuse in

urine should consist of (1) a sensitive, drug-class-selective technique such as

EMIT or RIA, employed as the initial screening process to identify negative

specimens and to select presumptive positive specimens, and (2) a highly

specific technique such as GC/MS, used for confirmation of the presumptive

positive results.

NEW DIRECTIONS IN URINE DRUG SCREENING

Because of the tremendous interest in drug screening today, efforts are being

made in many areas to develop new techniques that would be applicable to

this question. Some of these efforts are directed at developing new

analytical systems that will be simpler and perhaps less expensive than

conventional systems for drug screening or confirmation. In some cases, the

goal is portability, such as in efforts to produce dipstick-type assays.

Different biological media are being explored as alternatives to urine.

Efforts are also being directed at developing noninvasive means of diagnosis,

such as equipment to determine drug effects in the individual based on

certain electrical outputs from the brain. Other attempts are aimed at

developing methods or devices to better analyze impaired performance as an

indicator of a drug effect, a phenomenon not measured by urinalysis.

-38-
Developments in screening systems are underway in the area of

immunoassays. Because fluorescent systems have very high sensitivity and do

not require the use of radiolabeled material as does RIA, fluorescent labels

have become an attractive idea. Problems with background fluorescence

have prevented fluorescence spectrometry from becoming highly useful in

drug analysis in spite of the high sensitivity, but in recent years, systems

have been developed that combine highly specific separation systems, based

on antibodies with fluorescent detectors, to produce highly sensitive and

specific fluorescent immunoassays. One example is the widely used TDx

System manufactured by Abbott Laboratories. Abbott has recently marketed

a new system called the TDx Abuse Drug Assay, which is designed to detect

classes of drugs of abuse. It is to be expected that other systems based on

immunoassay and fluorescent labels will be forthcoming in the near future.

Other research groups are investigating various ways to incorporate

immunoassay specificity into systems that would give rapid readouts of

presumptive drug presence based on a dipstick analysis of a urine, blood, or

saliva specimen.

Work continues in the area of mass spectrometry to make this highly specific

method more economical. The specificity and sensitivity of GC/MS are

already sufficient for most applications of drug screening. A disadvantage in

GC/MS analyses is the time required for the drug to traverse the

chromatography column and for the analyst to prepare the sample for

injection into the instrument, although ways to reduce this time element are

being explored with an eye to using GC/MS as a screening as well as

confirmation tool.

A liquid chromatograph can be connected to a mass spectrometer (LC/MS) in

much the same way as the gas chromatograph is connected in GC/MS. As

discussed in the previous section on HPLC, derivatization is usually not

necessary and sample workup time is greatly decreased. While this type of

instrumentation is not generally in use now in urine drug assays, it may have

applicability in situations where confirmation analyses without derivatization

are desirable.

Another type of new MS technique that may have application to urinalysis is

called tandem mass spectrometry (MS/MS); this is particularly attractive

because it can often eliminate the need for a chromatography column. It also

offers the possibility of increased sensitivity and specificity over that of

conventional GC/MS. An extremely sensitive assay for THC in rabbit plasma

using this technique has been reported (Harvey et al. 1982). MS/MS is a

technique that couples two mass spectrometers together, so that one acts as

the sample cleanup system and the second as the ultimate analyzer. This

approach theoretically would allow a relatively crude extract to be

introduced directly into the first MS, eliminating the time-consuming

chromatography step, while at the same time providing increased sensitivity,

which may be necessary when drugs such as LSD and some fentanyl

derivatives which are extensively metabolized and appear in extremely low

concentrations in urine become the subject of urine screens.

OTHER BIOLOGICAL SAMPLES

Other types of biological samples, including blood, hair, and saliva, have been

proposed as alternatives to urine for drug screening. Although studies on

-39-
analysis of blood for drugs have been extensive and such tests potentially

provide a more specific indication of drug impairment, blood analysis

generally requires more sophisticated techniques of analysis than urine. It

also is considered a more invasive sample to obtain, and requires trained

personnel to do so. Saliva is perhaps the most easily obtained sample. It has

been established that many drugs can be detected in saliva (Caddy 1984).

THC, for instance, is sequestered in the mucous membranes and can be

detected (Norton and Garriott 1983; Gross et al. 1985; Cook 1986) for several

hours after marijuana is smoked. Saliva testing is not generally used, because

the methods of analysis are frequently more difficult than for urine. Further,

the time period during which drugs can be detected in saliva after use is

usually only a few hours, often paralleling plasma levels.

The analysis of hair has also been proposed as a way of detecting past drug

use (Baumgartner et al. 1981, 1982). It has been shown that certain drugs can

be detected in hair samples by means of sensitive RIA or GC/MS techniques,

but the methods have not yet been sufficiently validated in clinical studies in

comparison with urinalysis to adequately assess their suitability for general

drug screening (Puschel et al. 1983). This type of screen also suffers from

the fact that the extremely small sample (20-30 hairs) is frequently

consumed in the screening assay, leaving insufficient material for

confirmation by any but the most sensitive GC/MS techniques.

REFERENCES

Allen, L., Jr., and Stiles, M.L. Specificity of the EMIT drug abuse urine

assay methods. Clin Toxicol 18:1043-1065, 1981.

Abuscreen cannabinoid test. Package insert. Nutley, NJ.: Roche Diagnostic

Systems, January 1983.

Baumgartner, W.A.; Jones, P.F.; and Black, C.T. Detection of phencyclidine

in hair. J Forensic Sci 26:576-581, 1981.

Baumgartner, W.A.; Black, C.T.; Jones, P.F.; and Blahd, W.H. Radioimmunoassay of cocaine in hair: Concise communication. J Nucl Med 23:790-792,

1982.

Caddy, B. Saliva as a specimen for drug analysis. In: Baselt, R., ed.

Advances in Forensic Toxicology. Foster City, CA: Biomedical Publications,

1984. pp. 198-254.

Chase, A.R.; Kelley, P.R.; Tauton-Rigby, A.; Jones, R.T.; and Hardwood, T.

Quantitation of cannabinoids in biological fluids by radioimmunoassay. In:

Willette, R.E., ed. Cannabinoid Assays in Humans. NIDA Research

Monograph No 7. DHEW Pub. No. (ADM)78-39. Washington, DC: Supt. of

Docs., U.S. Govt. Print. Off., 1976. pp. 1-9.

Cook, C.E.; Seltzman, H.H.; Schindler, V.H.; Tallent, C.R.; Chin, K.M.; and

Pitt, C.G. Radioimmunoassays for cannabinoids. In: Hawks, R.L., ed. The

Analysis of Cannabinoids in Biological Fluids. NIDA Research Monograph No.

42. DHHS Pub. No. (ADM)82-1212. Washington, DC: Supt. of Docs., U.S.

Govt. Print. Off., 1982. pp. 19-32.

-40-
Cook, C.E. Analytical methodology for delta-9-tetrahydrocannabinol and its

metabolites. In: Moscovitz, H., ed. Alcohol, Drugs and Driving Abstracts and

Reviews, in press, 1986.

Dye, D.; East, T.; and Bayne, W.F. High performance liquid chromatographic

method for post-column, in-line derivatization with o-phthalaldehyde and

fluorometric detection of phenylpropanolamine in human urine. J

Chromatogr 284:457-461, 1984.

DeLaurentis, M.J.; McNeil, K.; Mann, A.J.; Clark, S.; and Greenwood, H.M.

An EMIT assay for cannabinoid metabolites in urine. In: Hawks, R.L., ed. The

Analysis of Cannabinoids in Biological Fluids. NIDA Research Monograph No.

42. DHHS Pub. No. (ADM) 82-1212. Washington, DC: Supt. of Docs., U.S.

Govt. Print. Off., 1982. pp. 69-84.

Ebert, M.H.; Van Vunakis, H.; and Hawks, R.L. Radioimmunoassays for

psychotropic drugs. In: Usdin, E., ed. Clinical Pharmacology in Psychiatry.

New York: Elsevier North Holland, Inc., 1981. pp. 73-94.

ElSohly, M.A.; Arafatt, E.S.; and Jones, A.B. Analysis of the major

metabolite of delta-9-tetrahydrocannabinol in urine. III. A GC/ECD

procedure. J Anal Toxicol 8:7-9, 1984.

ElSohly, M.A.; ElSohly, H.N.; Jones, A.B.; Dimson, P.A.; and Wells, K.E.

Analysis of the major metabolite of delta-9-tetrahydrocannabinol in urine.

II. A HPLC procedure. J Anal Toxicol 7:262-264, 1983.

Foltz, R.L.; Fentiman, A.F.; and Foltz, R.B. GC/MS Assays for Abused Drugs

in Body Fluids, NIDA Research Monograph No. 32. DHHS Pub. No.

(ADM)80-104. Washington, DC: Supt. of Docs., U.S. Govt. Print. Off., 1980.

pp. 62-89.

Foltz, R.L.; McGinnis, K.M.; and Chinn, D.M. Quantitative measurement of

delta-9-tetrahydrocannabinol and two major metabolites in physiological

specimens using capillary column gas chromatography in negative chemical

ionization mass spectrometry. Biomed Mass Spectrom 10:316-323, 1983.

Foltz, R.L. Analysis of cannabinoids in physiological specimens by gas

chromatography/mass spectrometry. In: Baselt, R., ed. Advances in

Analytical Toxicology. Volume 1. Foster City, CA: Biomedical Publications,

1984. pp. 125-157.

Gross, S.J.; Worthy, T.E.; Nerder, L.; Zimmermann, E.G.; Soares, J.R.; and

Lomax, P. Detection of recent cannabis use by saliva delta-9-THC

radioimmunoassay. J Anal Toxicol 9:1-5, 1985.

Harvey, D.J.; Leuschner, J.T.A.; and Paton, W.D.M. Gas chromatographic

and mass spectrometric studies on the metabolism and pharmacokinetics of

delta-1-tetrahydrocannabinol in the rabbit. J Chromatogr 239:243-250, 1982.

Law, B.; Mason, P.A.; Moffat, A.C.; and King, L.J. Confirmation of cannabis

use by the analysis of delta-9-tetrahydrocannabinol metabolites in blood and

urine by combined HPLC and RIA. J Anal Toxicol 8:19-21, 1984a.

-41-
Law, B.; Mason, P.A.; Moffat, A.C.; and King, L.J. A novel

125radioimmunoassay for the analysis of delta-9-tetrahydrocannabinol and its

metabolites in human body fluids. J Anal Toxicol 8:14-18, 1984b.

Law, B.; Pocok, K.; and Moffat, A.C. An evaluation of homogeneous enzyme

immunoassay (EMIT) for cannabinoid detection in biological fluids. J

Forensic Sci Soc 22:275-281, 1982.

Michaud, J.D., and Jones, D.W. Thin layer chromatography for broad

spectrum drug detection. Am Lab 12:104-107, 1980.

Norton, L.E., and Garriott, J.C. Detection of marijuana use by GC/MS

analysis of mouth swabs. Am J Forensic Med Pathology 4:185-188, 1983.

Puschel, K.; Thomasch, P.; and Arnold, W. Opiate levels in hair. Forensic Sci

Int 21:181-186, 1983.

Rogers, R.; Crowl C.P.; Eimstad, W.M.; et al. Homogeneous enzyme

immunoassay for cannabinoids in urine. Clin Chem 24:95-100, 1978.

Soares, J.R.; Grant, J.D.; and Gross, S.J. Significant developments in

radioimmune methods applied to delta-9-THC and its 9-substituted

metabolites. In: Hawks, R.L., ed. The Analysis of Cannabinoids in Biological

Fluids. NIDA Research Monograph No. 42. DHHS Pub. No. (ADM)82-1212.

Washington, DC: Supt. of Docs., U.S. Govt. Print. Off., 1982. pp. 44-55.

Woodworth, J.R.; Mayersohn, M.; and Owens, S.M. Quantitative analysis of

phencyclidine and metabolites by capillary column gas chromatography. J

Anal Toxicol 8:2-6. 1984.

-42-
Accuracy in Urinalysis

Robert V. Blanke, Ph.D.

Accuracy is the absolutely essential ingredient of laboratory analysis. The

public perception of scientific measurements is that they are indisputable. If

a laboratory reports the presence of a quantity of drug in a specimen, this

ruling is judged to be correct, regardless of protestations to the contrary by

the subject. Users of laboratory tests assume the results are reliably

accurate; otherwise, the cost of these tests is wasted. But what is

“accuracy,” and how can it be estimated?

Accuracy can be viewed as the closeness with which test results agree with

the “true” quantity of a drug in urine. In an unknown urine specimen, the

“true” result, the center of the proverbial bull’s eye, obviously is not known.

Judging whether a specific test result is accurate can be done only by

evaluating a laboratory’s quality assurance program (Annino 1978).

Quality assurance (QA) is a term used, to represent those practices carried

out to assure that specified quality goals are achieved (Eilers 1975). Once

the quality goals have been defined, quality control (QC) procedures can be

designed to monitor performance parameters. Thus, it is important that the

testing laboratory and the user of the test results clearly understand the goal

of the testing program.

Urine drug testing programs can demonstrate the presence of certain drugs or

drug metabolites in urine. These results alone cannot be used to determine

dosage or the time of drug administration or the extent of any drug effects in

the subject, but urine drug testing programs can indicate drug use. They may

also provide presumptive evidence that certain behavioral changes or

decrements in performance observed in the subject may be associated with

drug use. Thus, the goal of urine drug testing may be stated as the reliable

demonstration of the presence, or absence, of specified drugs or metabolites

in the specimen--that is, the production of a valid positive or negative result.

FACTORS CONTRIBUTING TO ACCURACY

Generally, when large numbers of specimens are processed by a laboratory,

screening tests are used to separate the greater number of negative

specimens from those producing positive results. Since screening tests are

subject to interferences, all specimens that test positive must be confirmed

by an independent, more specific procedure. If a relatively high

concentration of drug is present in the specimen, screening and confirmatory

-43-
tests are more reliable. As the drug concentration approaches the limit of

the test to detect a drug, more uncertainty is associated with the test result.

This involves another aspect of lesting procedures known as “sensitivity.”

Sensitivity is the lowest concentration of a drug that can he reliably detected

by a particular test procedure. The key word here is “reliable.” A test may

be considered very sensitive when measuring a pure drug, but the test’s

reliability may be poor when the drug is in a complex matrix such as urine.

Most laboratory instruments are devices that measure drug concentrations by

means of a detector. The detector may be a gamma counter in

radioimmunoassay, an optical device in fluorescence or enzyme immunoassay

techniques, or more complex in nature for some chromatographic methods.

The detector, in turn, generates an electronic signal, which drives a printer

or recorder to permit the analyst to visualize the intensity of the detector

response and thereby estimate the amount of drug present. All detectors

produce a certain amount of background “noise.” When tests are run near the

limit of sensitivity to a drug, it is increasingly difficult to discriminate

between the noise and a true signal in response to a small amount of drug.

In order to compensate for sensitivity problems, and possibly minimize the

incidence of positive results from passive drug exposure, frequently a cutoff

concentration is selected. Above this level, a test is considered positive, and

below it is considered negative. A disadvantage of establishing cutoff

concentrations is that some individuals with actual drug present in the urine,

albeit at low concentrations, will be deemed negative. These false-negative

tests are generally tolerable in order to avoid false-positive results due to

sensitivity variability.

Cutoff levels, when adopted, require consideration of another parameter of

drug testing methods known as precision. Precision is the degree of

agreement between repeated measurements. Good precision of a testing

method increases confidence that the test can discriminate between drug

concentrations above or below the cutoff level. Precision can be measured

by the standard deviation (s) or coefficient of variation (c.v.) of a method.

These are indicators of random error.

Consider the example in table 1. A cutoff level of 100 ng/ml has been set for

a test for a metabolite of THC. That is, a test showing greater than 100

ng/ml of this metabolite will be called positive; below 100 ng/ml the test is

negative. In order to determine the precision of this test, a specimen is

divided into 10 portions and each portion is assayed.

One result, test number 6, appears to be unusually different from the rest.

This result can be examined statistically to estimate the probability that it is

an extreme value and can be rejected. When this is done, it is found that the

probability that result number 6 is an “outlier” is greater than 995 times in

1,000. An outlier is a test result far removed from most of the other test

results on the same specimen. Thus, it can be rejected.

-44-
Note that the “true” value is not known, but repetitive testing can result in a

valid estimate of the true value. When the outlier is included, the mean is

102.1 ng/ml (interpreted as positive), but when the outlier is excluded, the

mean is 99 ng/ml (interpreted as negative).

Table I

Test No. Result (ng/ml) Result (ng/ml)

1-

2-

3-

4-

5-

6-

7-

8-

9-

10-

98

101

95

99

103

(130)

100

97

96

102

98

101

95

99

103

100

97

96

102

Mean (x)

Standard deviation (s)

Coefficient of variation (c.v.)

102.1 99.0

±10.14 ±2.74

9.93 % 2.77 %

This example is chosen to illustrate two aspects of analytical testing:

1. The necessity for excellent precision to discriminate between

positive and negative when the concentration of the analyte is near

the cutoff (or limits of sensitivity) of the test

2. The troublesome problem of outliers

At times, a careful review of procedures used can explain why the unusual

result occurred, but frequently no obvious explanation can be found. When

repetitive analyses are done, outliers can he recognized and tested

statistically. Unfortunately, if a single test is done on an unknown specimen,

it is not possible to know whether the result obtained is an outlier or an

estimate of the true value within the standard deviation of the test. It has

been reported that, in intralaboratory testing, 5 percent to 15 percent of the

results may be statistical outliers (Horwitz 1982). Only repetitive analyses of

the same specimen can identify these discrepant results.

Tests for urinalysis should be precise and accurate and generally accepted by

the scientific eommunity. Such tests are of two types, screening and

confirmatory. Screening tests must reliably identify negative specimens,

although it is recognized that some weakly positive specimens will be

interpreted as negative due to sensitivity or precision limitations of screening

procedures or due to negative outliers. Since negative results by screening

tests are generally of little concern, confirmation of these is not necessary.

-45-
Positive results of screening, on the other hand, should be repeated. If still

positive, the test must be confirmed by independent procedures of greater

specificity and precision. This is to better characterize the drug, confirm its

presence at concentrations above any cutoff level, and identify outliers. It is

also important to carefully document and follow specific criteria that defii

a positive versus a negative sample with any particular analytical

methodology.

Implementing these procedures, monitoring their performance, and

identifying and correcting defects in the testing procedure require continuous

attention to a quality assurance program.

QUALITY ASSURANCE

A quality assurance program includes all of the practices carried out by the

testing laboratory to assure that the goal of reliably identifying urine

specimens containing defiied quantities of specified drugs or drug

metabolites is achieved (Tietz 1986). All of the parameters of drug testing

(such as accuracy, precision, detection of outliers, etc.) are monitored by

quality control procedures. QA involves all aspects of the testing

laboratory. Specimen acquisition, processing, testing, and reporting of test

results must all be as error free as possible in order to achieve the goals of

urine drug testing.

Dedication to quality testing must be of paramount importance to the

laboratory management. Not only must laboratory directors and supervisors

be committed to these goals, but administrative support is essential to

provide a budgetary basis for space, equipment, staff, and all other resources

necessary for quality testing. The cost involved in maintaining QA must be

recognized at the outset.

PERSONNEL

The qualifications of personnel are important in achieving quality results in

urine drug testing. Different tasks require different degrees of training and

experience; all require attention to details and a disciplined approach to

carrying out procedures. Graduation from an accredited school of medical

technology with certification in clinical chemistry qualifies most individuals

to operate automated instruments, carry out laboratory recordkeeping

procedures, and implement quality control, all under supervision. Analysts

operating chromatographic instruments require additional training and

experience in these techniques. An individual holding a baccalaureate degree

in chemistry from an accredited university should understand the necessary

basic theory of instrumental analysis to permit the recognition of aberrant

results and the ability to “troubleshoot” an instrument that does not meet

specifications.

Supervisors and laboratory directors must possess these qualifications as a

minimum. In addition, experience and advanced training in analytical

methods and pharmacology or toxicology are required to understand the

strengths and weaknesses of the methods used to identify drugs, discriminate

between real knowledge of drugs and their effects as opposed to conjecture,

and interpret the test results reported by the laboratory.

-46-
Certification in clinical chemistry (by the American Board of Clinical

Chemistry) or forensic toxicology (by the American Board of Forensic

Toxicology) is highly desirable. These individuals must be sufficiently skilled

and knowledgeable to defend and interpret the laboratory results in court, if

necessary (Forney 1978).

SPECIMEN ACQUISlTION

Drug testing laboratories are involved only rarely in actually obtaining the

specimen being examined. Obviously, collecting a valid, uncontaminated

specimen uniquely identified as from a specific individual is a key issue in

urine drug testing. Details relating to the specifics of specimen acquisition

are discussed in a previous chapter.

Once the specimen is collected, it is marked in a unique way traceable to the

individual giving the specimen. A name is normally not sufficient. Rather, a

unique number such as a social security number, employee number, etc., is

preferred. A record of the name, unique identifying number, time and date

of collection, and the identity of the individual receiving the specimen is

made. The specimens are packaged in a secure manner, marked “Urine Drug

Screen” (or flagged in a less obvious manner), and sent promptly to the

laboratory by courier, U.S. mail, or other secure means.

Upon receipt by the laboratory, the title “Urine Drug Screen” alerts the

receiving clerk to forward the unopened package to the individual assigned to

receive this type of specimen. A log is kept, listing name of the subject,

unique identifying number, laboratory accession number, time and date

received, condition of the specimen, and name of the individual carrying out

the task of logging in the specimen. The laboratory accession number is

firmly affixed to the specimen container, and it is stored securely at 4°C

until analyzed.

This process, termed “chain of custody,” must be followed in handling

specimens of legal significance. Records of all individuals, both in and

outside the laboratory, having access to the specimen must be kept, and these

records documented and preserved for future reference.

Errors can occur in this phase of specimen processing. Transcription errors,

misspelling, and even confusion of specimens can occur. Quality control can

be exercised by proper training and motivation of personnel involved and by

computer tracking of the specimen, together with computer printing of labels

and accession numbers. Further, artificially prepared specimens with known

errors can he introduced into the system. Technicians receiving the

specimens are made aware of this check but are not informed as to the

frequency of the checks. In a similar fashion, other steps in specimen

processing can be monitored.

CONTROL OF ANALYTICAL VARIABLES

A number of factors relating to the environment, water quality, power

sources, and other general aspects of a laboratory may affect analytical

testing and must he monitored. Measuring devices of all types--balances,

pipets, thermometers--must be monitored frequently to determine if they

-47-
meet prescribed tolerances and reliably indicate true values. lnstruments of

all types must be checked routinely to maintain calibration and confirm that

they are performing according to specifications. All of these monitoring

procedures must be documented and reviewed regularly. When properly

implemented, review processes not only ensure that monitoring procedures

are conducted but identify emerging problems before they become critical

(Blanke 1978).

Analytical Methodology

The choice of a valid analytical procedure is an obvious and important factor

in achieving results of high quality. The specificity, sensitivity, and

reproducibility of the method must be known in order to achieve the quality

goats selected previously. Facility in the application of the method to real

samples by routine personnel must be demonstrated. The method must be

described in detail in a procedure manual. Any deviations from the method

must he approved by an authorized supervisor and documented. Improvement

in methodology is a continuous process and should be encouraged, provided all

improvements are validated. New and novel procedures should not be used in

routine testing until accepted by the scientific community.

Standards

The standards by which a method is calibrated must be selected with care.

Generally, drug standards can be obtained from Supelco Inc., Bellefonte, PA;

Alltech-Applied Science, State College, PA; Sigma Chemical Co., St. Louis,

MO, or the United States Pharmacopeia, Rockville, MD. Other sources such

as pharmaceutical companies or chemical suppliers may be used. In all cases,

efforts to assess the purity of the standard by chromatography or by

measuring a physical constant should be carried out frequently, since some

drugs degrade with time. Accounting for the degree of hydration of

crystalline substances can be particularly troublesome. Obviously, any

contaminant, whether water or another substance, is weighed out as the drug

when a standard solution is prepared. Unless the magnitude of the

contamination is known and corrected for, an error is easily introduced

(Blanke 1978).

Controls

An important aspect of any quality control procedure is the control material

or control specimen. This is a urine specimen containing a known quantity of

the drug(s) being tested that is run along with the subject’s specimen.

Controls can be purchased commercially or prepared by the laboratory.

Ideally, they are specimens containing the drug(s) in a physiological state,

i.e., present in the same matrix and environment as it would be in a patient.

Metabolites, conjugated forms of drugs or metabolites, interferences,

endogenous substances, all are present in the control. In practice, control

material can be collected by pooling excess specimens. The well-mixed

material is aliquoted into small volumes and frozen until used. By repetitive

assays, a mean concentration of the analyte is measured with a calculated

standard deviation. Thereafter, for an assay procedure to be “in control,” the

control result must agree with the known mean value within ±2 standard

deviations. A result outside of this range is deemed “out of control,” and no

-48-
results are reported until the cause of the discrepancy is identified and the

assay again meets acceptable criteria.

Control material for screening procedures should contain analytes at or near

the cutoff or sensitivity limit of the test. A negative control with the

analyte below the cutoff and a positive control with the analyte slightly

elevated above the cutoff will permit the analyst to determine when the teat

is performing properly.

Control results are documented and recorded on a chart or in a computer

program such that trends in control results may be visualized to enable a

trained analyst to discover problems in an assay before they become critical

In addition, users of laboratory results should inspect these control charts

regularly and make sure that this documentation is retained if required in

cases that may be challenged in court.

EXTERNAL QUALITY CONTROL PROCEDURES

The QC procedures described earlier are designed to detect changes in

performance during routine operations, as compared with the careful setup

procedures used when the method was initiated. However, if the method was

not set up accurately (e.g., impure standard, miscalibration of a key

measuring device, etc.), or if other methods perform more acceptably,

internal QA procedures do not suffice.

Extemal QC procedures permit a laboratory to be compared with other

laboratories. External QC may be of two types: open proficiency testing

(PT) or blind proficiency testing. These two types of QC procedures have

been reviewed in a series of papers that appeared in the May/June 1977 issue

of the Journal of Analytical Toxicology.

In open PT, a sponsoring group prepares a large quantity of control material

and sends a portion to each participating laboratory. The laboratories are

aware of this program, test the material when it arrives, and send their

reports to the sponsoring group. When all the results are received, the

sponsoring group calculates the mean result reported by all participating

laboratories and reports back to the participants their performance by means

of a standard deviation index (SDI). This is an indication of the participating

Laboratory’s performance in terms of standard deviations from the mean

result of all laboratories. An SDI greater than ±2 indicates that the

Laboratory does not agree well with most of the others testing the same

specimen.

As its name suggests, a blind proficiency test is identical to the open test,

except that the participating laboratory is unaware of the test. The

proficiency specimen arrives at the laboratory exactly like other subject

specimens and is processed, tested, and results reported in a routine fashion.

The surrogate subject then reports results back to the sponsoring group,

which compares and scores results as before. Unfortunately, laboratories

that do well in open proficiency testing sometimes perform poorly in blind

testing (Hansen et al. 1985). Laboratory users should be aware of this

difference in proficiency testing programs and attempt to identify

laboratories willing to share their performance on blind PT programs before

contracting for their services.

-49-
In order for. QA programs to work for the purpose for which they are

designed, a continuous review process must be carried out. This keeps the

laboratory director and supervisors constantly alert to the many variables

that influence the results of chemical testing. In addition, it enables

appropriate action to be carried out before errors are reported.

Excellent QA is costly, since personnel time, equipment, and supplies must be

committed to it. It is essential, however, when test results may be used in

decisions affecting employment, reputation, or even imprisonment of the

subjects being tested.

SOURCES OF ERRORS

Scientists recognize that errors may occur during any type of scientific

measurement. It is for this very reason that sound quality assurance

programs must be implemented and carried out. In this way, random errors

can be identified and corrected before reporting an analytical result.

Responsible critics have identified those aspects of urine drug testing that

are vulnerable (McBay 1966; Hanson 1986). This discussion centers on human

errors, errors in methodology, and alternate interpretations.

Human Errors

Errors of omission as well as commission occur in all human activities.

Fatigue, poor health, and boredom arising from the tedium of routine tasks

all contribute to high error rates. Providing good working conditions,

effective rest periods, and rotation of workers through different tasks can

help to alleviate these problems. Automation can minimize human error,

provided automated steps arc monitored by an effective QC program, which

is more easily accomplished in a laboratory with a high workload than in a

small laboratory.

Inappropriate training or experience for the task being carried out can also

lead to errors. It is for this reason that personnel qualifications, discussed

earlier, must become part of the total QA program.

The most difficult errors to control are administrative ones. Labeling errors,

spelling errors, transposition of numbers, all can lead to a correct test result

being assigned to the wrong subject. In fact, most laboratories have learned

by participating in external PT programs that these occur more frequently

than errors in testing procedures. For this reason, the steps involved in

specimen acquisition, transport, and processing (chain of custody) must be

part of the complete QA program. Drug testing laboratories must practice

good personnel policies like any other well-run enterprise.

Errors of Methodology

These types of errors have been discussed earlier relative to QC procedures.

Instrument malfunction, outdated reageuts, measurement errors, and many

other factors must be controlled by an effective QA program.

-50-
It is important to emphasize that testing procedures cannot be forced to yield

results for which they are not designed. In particular, screening tests must

be confirmed.

Chromatographic procedures may not resolve substances sufficiently for

clear identification, while immunoassay procedures may use antihodies that

cross-react with a variety of substances. Recently, it was reported that an

immunoassay procedure, EMIT-d.a.u., erroneously gave a false-positive result

for amphetamine in the presence of the antihypertensive agent lahetalol.

Similarly, a commercial TLC screening procedure, TOXI-LAB A, confused

the same antihypertensive agent with amphetamines or trimethoprim (Apple

et al. 1985). Although the TONI-LAB manufacturer has indicated that

resolution of these substances can be made (Martel et al. 1986), the incident

illustrates the necessity for confirming all screening tests.

Other examples of false-positive results due to cross-reaction of

immunoassays have been reported. The popular antihistamine diphenhydramine can react with the EMIT-d.a.u. antibody for methadone (Kelner

1984). Recently, Syva notified users of the EMIT kits for marijuana that the

analgesic ibuprofen (available over the counter), as well as some other

nonsteroidal anti-inflammatory agents, can interfere with the EMlT test for

marijuana (at the 20 ng/ml cutoff level), although the company has reportedly

taken steps to eliminate the problem by alteration of the enzyme reagent in

the system. Other interferences have been reported by Allen and Stiles (1981).

These types of false-positive testing results are not due to deficiencies in

methodology. Rather, they are the result of using screening tests without

adequate confirmation. In each case cited, effective confirmation

procedures will prevent the erroneous reporting of false-positive results.

RETENTION OF POSITIVE SPECIMENS

Since the possibility of error exists, although this is unlikely when appropriate

confirmation is done, excess urine from all positive specimens should be

retained until the case has been resolved. The remaining specimen in its

properly labeled, original container should be stored by freezing (-20°C or

lower). This will permit reanalysis by the original laboratory or a different

laboratory in the event the positive results are challenged by the subject.

The time between the original testing and ultimate resolution of the matter

can be quite variable, depending on the circumstances. For this reason,

clients should inform the laboratory as to when ultimate disposal of the

specimen can occur.

DIFFERENCES IN INTERPRETATION

Finally, the testing procedure may produce results that accurately indicate

the presence of drugs or their metabolites in a urine specimen, but

explanations other than drug abuse may be invoked. These are not truly

errors but rather alternate interpretations of the accurate test results.

Frequently, these interpretations are invoked in the event that a charge is

brought against a subject whose urine test has been reported as positive.

-51-
Many examples can be mentioned, most of which are related to marijuana.

Accidental or inadvertent ingestion of marijuana as salad greens or

components of food has been offered as an explanation. While experimental

evidence is not available to support this claim for marijuana in uncooked

foods, studies have demonstrated the possibility of this occurrence in baked

goods (Cone and Johnson 1986). Passive inhalation of marijuana smoke is

more frequently raised as a defense. The latter phenomenon has been

investigated. Although passive inhalation can give rise to metabolites of

THC being excreted and measured in urine, it would be extremely unlikely

that high concentrations can occur by this means of dosage (Perez-Reyes et

al. 1983).

Recently, cocaine has been identified in certain herbal teas (Siegel et al.

1986). It is possible, therefore, that a positive test for cocaine may be

obtained in urine from a subject who ingests large amounts of this material.

Note, however, that such teas are technically illegal and have recently been

removed from the market by Federal authorities.

Other alternative explanations of positive drug findings are based on claims

of errors arising from screening tests. These have already been discussed.

Effective testing procedures in which positive screening results are

confirmed by valid tests will refute these claims. The presence of a specific

drug or metabolite will be documented, although how or why the substance

was absorbed by the subject may not be answered.

REFERENCES

Allen, L.V., Jr., and Stiles, M.L. Specificity of the EMIT drug abuse urine

assay methods. Clin Toxicol 18:1043-1065, 1981.

Annino, J.S. What does laboratory “quality control” really control? N Engl J

Med 299:1130-1131, 1978.

Apple, F.S.; Googins, M.K.; Kastner, S.; Nevala, K.; Edmonson, S.; and Kloss,

J. Labetalol: False positive indices by EMIT d.a.u. assay and TOXI-LAB A

urine screen. Clin Chem 31:1250-1251, 1985.

Blanke, R.V. Quality control in the toxicology laboratory. Clin Toxicol

13:141-151, 1978.

Cone, E., and Johnson, E., NIDA Addiction Research Center, Personal

communication.

Eilers, R.J. Quality assurance in health care: Missions, goals, activities. Clin

Chem 21:1357-1367, 1975.

Fomey, R.B. What constitutes qualified personnel? J Anal Toxicol

2:155-158.1978.

Hanson, D.J. Drug abuse testing programs gaining acceptance in workplace.

Chem Eng News 64:7-14, 1986.

Hansen, H.J.; Caudill, S.P.; and Boone, J. Crisis in drug testing: Results of

CDC blind study. JAMA 253:2382-2387, 1985.

-52-
Horwitz, W. Evaluation of analytical methods used for regulation of foods

and drugs. Anal Chem 54:67A-76A, 1982.

J Anal Toxicol 1:105-150, May/June, 1977.

Kelner, M. J. Positive diphenhydramine interference in the EMIT-d.a.u.

assay. Clin Chem 30:1430, 1984.

Martel, P.A.; Adams, D.; Jones, D.W.; and O’Donnell, C.M. Labetalol

analysis with the TOXI-LAB A drug-detection system. Clin Chem 32:915,

1986.

McBay, A.J. Problems in testing for abused drugs. JAMA 255:39-40, 1986.

Perez-Reyes, M.; Guiseppi, S.D.; Mason, A.P.; and Davis, K.H. Passive

inhalation of marijuana smoke and urinary excretion of cannabinoids. Clin

Pharmacol Ther 34:36-41, 1983.

Siegel, R.K.; ElSohly, M.A.; Plowman, T.; Rury, P.M.; and Jones, R.T.

Cocaine in herbal tea. JAMA 255:40, 1986.

Tietz, N. Acquisition, management and application of laboratory data.

Textbook of Clinical Chemistry. Philadelphia: Saunders, 1986.

-53-
Interpretation of Urinalysis

Results

Joseph E. Manno, Ph.D

Urine drug testing has been used for several decades to monitor human drug

use. This type of urinalysis procedure was first used for patients who had

been using drugs on a regular basis, and positive results were an indication of

continued drug abuse. The methodology most commonly used for performing

the tests, thin-layer chromatography, was satisfactory for detecting a wide

variety of drugs in common use at that time.

Over the years, both the population being tested and the laboratory

methodology have changed dramatically. Today, the use of urine drug tests

has been extended outside the substance abuse treatment facility and is

currently used as part of preemployment physicals and for monitoring drug

use in employees in a variety of industries and government agencies.

From an analytical standpoint, the development of immunoassay procedures

has provided inexpensive and very sensitive procedures for analyzing for

classes of drugs in urine. The analytically powerful technique of gas

chromatography-mass spectrometry has become more commonly available

and is currently the method of choice for the analysis of drugs in a sensitive

and specific manner and for confirming the presence of drugs found in an

initial screen. Other methodologies such as gas-liquid chromatography and

high-performance liquid chromatography also play a role in the analysis of

drugs in urine. The variety of methodology available permits inexpensive

mass screening and also allows the specificity needed to provide accurate and

sensitive confirmation of drugs found in the initial screening test. The wide

variety of methodology now in use, as well as the variety of drug use

patterns, makes the interpretation of urine drug assays more complex.

Although the vast majority of all drug tests will be negative, the user must

understand that both negative and positive test results have meaning. In

order to understand how to properly interpret the results of a drug assay, one

must first determine what information is desired. In addition to finding a

reputable laboratory with adequate professional staff to assist them,

consumers must define their individual goals for starting the program. In

particular, they must determine the consequences that will befall individuals

who test positive. Since no uniform required standards exist for urine drug

testing, the use of expert consultants to assist in the development of a

program will be beneficial.

Before embarking on any urine drug testing program, the consumer must first

determine the program goals.

-54-
Some questions that might be asked about the meaning of a drug screen

follow. If an analysis is positive, does it mean that:

the subject is using the drug chronically?

the subject is using the drug intermittently?

the subject is addicted to the drug?

the subject is taking the drug under a physician’s order?

the subject was under the influence of the drug when the urine was

collected?

If, on the other hand, the analysis was negative, does it mean that:

the subject has never used the drug?

the subject may use the drug intermittently but has not used the drug

recently?

the subject knew that the urine would be screened, and stopped

taking the drug long enough for the urine specimen to be negative?

the subject diluted the urine at the time of collection, or prior to

collection, by drinking large amounts of fluids prior to sample

collection and thereby rendered it negative?

the subject adulterated the urine by adding water or another

substance or switched it with urine from another individual during

the confusion inevitable when large numbers of persons are involved

in a process like urine collection?

All these possibilities must be considered as part of the process of deciding

the relevance of urine drug test results.

The end user of the results of a urine drug analysis must also be familiar with

proper specimen collection and handling procedures, have a general

understanding of laboratory methodology used to perform the analysis, and

have some understanding of drug kinetics. This information will permit

correct interpretation of laboratory results and allow for reconciliation of

these data with the subject history.

The purpose of this chapter is to provide the reader with information that

will help in such interpretation.

DRUG FACTORS THAT DETERMINE WHETHER A TEST

IS POSITIVE OR NEGATIVE

Dose

The higher the dose of drug taken, the more likely that the drug will be

detected in urine. As an example, a dose of 30 mg codeine might be

detected for 1 to 6 hours after use by a particular method; a 60 mg dose by

the same method might be detected for 1-10 hours.

Frequency of Use

Drugs remain in the body for varying lengths of time. Drugs like cocaine are

eliminated from the body relatively rapidly. Depending on the assay

-55-
procedure, a single dose of cocaine, for example, may only be detectable in

urine for 1 day or less. Continued use on a daily basis may cause the drug to

be detectable for 2 or 3 days after cessation of use. Smoking a single

marijuana cigarette may result in a positive urine test for cannabinoids for 1

or 2 days with some methods and for 3 to 5 days with more sensitive

methods. Continued use of marijuana on a daily basis however, may result in

a positive urine for 3 or more weeks after drug use has stopped. As a general

rule, most drugs tend to accumulate in the body if they are taken on a regular

basis. The more frequently a drug is ingested, the more likely that it will be

detected on a urine drug test.

Time From Drug Use to Urine Collection

Different drugs leave the body over various time intervals depending on the

drug, the dose administered, and the frequency of drug use. For drugs like

cocaine that leave the body very rapidly, it is necessary to collect the urine

very close to the time of drug use to get a positive sample. With drugs like

marijuana that are eliminated more slowly, the urine collection could occur

up to several days after use and still be positive.

Sensitivity of the Urinalysis Test Used

Laboratory tests used to detect drugs in urine have different sensitivities for

various drugs. Immunoassays, for example, generally can detect smaller

quantities of drug in urine than can thin-layer chromatography, If the assay

is not sensitive, drugs will not readily be detected in the urine specimen. If

tbe assay is extremely sensitive, it may detect drugs in urine for days or

weeks after their use. By understanding the sensitivity of the assay, the

client will be able to relate the assay result to a “drug use window” and

thereby determine approximately when the drug could have been used.

The sensitivity of the first urine drug test, often referred to as a preliminary

screen, is important for determining whether a urine is positive or negative.

The most commonly used laboratory procedures for the initial screen are

thin-layer chromatography (TLC), radioimmunoassay (RIA), the EMIT test,

and the newly introduced TDx Abused Drug Detection Assays (TDx).

Although the RIA, EMIT, and TDx tests use methodologies that are

significantly different from each other, they have in common several

features. Each of the tests assays for a specific drug or for drugs in a single

class (e.g., methadone, barbiturate class, amphetamine class, cannabinoids,

etc.). The tests are very sensitive and will generally detect drug use for 1 or

more days after a single use. The TLC system has the advantage that it can

detect a wide variety of drugs, but it has less sensitivity than the

immunoassay tests. Ideally, a combination of the TLC and immunoassay tests

could be used for optimum sensitivity and versatility. If costs are a

significant factor, then the number and variety of different tests will be

limited.

As an example, initial screening could include a TLC assay and immunoassays

for drugs that are not detected well (or at all) by the TLC screen, but are

known to be commonly abused. A typical test battery might, include the

combination of a TLC screen, a cannabinoid immunoassay test, and a cocaine

immunoassay test.

-56-
In addition to TLC and the immunoassay procedures, other laboratory tests

that can be utilized include gas-liquid chromatography, high-performance

liquid chromatography, and gas chromatography-mass spectrometry. These

tests are rarely used as initial screens. They have the advantage that they

can detect a wide variety of drugs and are generally more sensitive than

TLC. Their primary disadvantages include relatively high cost and long assay

times. For these reasons, they are used primarily for specific drug analyses

and for confirmation (particularly GC/MS) of presumed positives from initial

screens.

If the results of the preliminary screen are negative, no further chemical

testing of the urine specimen is usually performed. If the preliminary screen

is positive for one or more drugs, however, a second laboratory test is then

necessary to verify the presence of the detected drugs. This “confirmatory”

test must use different technology, must be as sensitive as or preferably

more sensitive than the preliminary screen, and must be specific for the drug

or drugs detected in the initial screen. The confirmatory test is usually

performed by gas-liquid chromatography, high-performance liquid

chromatography, or ideally by gas chromatography-mass spectrometry.

RELATING DRUG FACTORS TO INTERPRETATION OF

THE URINALYSIS TEST

Negative Test Result

Several interpretations are possible:

The subject is not using a drug that can be detected by the test.

The subject may be taking one of the drugs detected by the test but

-is not taking a large enough dose to be detected

-is not taking it frequently enough to be detected

-the urine is being collected too long after the drug was ingested

-the urine sample was diluted or otherwise tampered with or

switched with another sample by the subject.

The subject may be taking the drug but the assay used was not

sufficiently sensitive to detect the drug in urine.

Positive Test Result

If the test was properly performed and the results from the laboratory are

valid, the result means that the drug indicated was present in the urine. A

positive result does not mean that the individual tested was under the

influence of the drug at the time the specimen was collected. An expert

would be able to provide some general information regarding the timeframe of

the drug use. Even experts will vary in their opinions, however. The

significance of a single positive urine drug test can be increased by repeat

testing at regular intervals. Multiple positives over a period of time reinforce

that an individual may be a regular user of the detected substance. Although

multiple negatives do not completely negate drug use, they do provide

evidence that the individual may not be using on a regular basis.

-57-
Prolonged Monitoring

In some cases, such as treatment situations or probationary periods for an

employee, it may be advisable to collect sequential specimens over time to

assess continued drug abstinence.

Collection of multiple urine specimens over a prolonged period of time

requires that special attention be given to subjects who have ingested drugs

such as marijuana and certain benzodiazepine antianxiety agents that are

excreted in urine for long periods of time. If the subject had used one of

these drugs chronically prior to the start of urine drug testing, it is possible

that the test could be positive for several weeks or months after he or she

stopped using the drug.

Since the results of urine drug tests are normally reported as either positive

or negative, it is not possible to determine if a positive result is due to

continued use of the drug or continued excretion of the drug from previous

use. In order to differentiate between recent drug use and continuing urine

drug excretion from previous use, it is necessary to request that the laboratory perform a semiquantitative analysis for the drug in the urine. If the

subject has stopped using the drug, the concentration of drug in urine would

be expected to decrease each time the urine is assayed. If the concentration

of drug increases, continued use of the drug should be considered.

Monitoring this decreasing drug level is complicated by variations in urine

water content. Greater than normal intake of water can dilute the amount of

drug in the urine, and, conversely, a limited intake particularly in conjunction

with dehydrating exercise can lead to an abnormally concentrated urine

specimen.

Figure 1 illustrates a series of samples taken every 2 days from one subject.

The solid line is urine concentration for cannabinoids. The concentrations are

seen to decrease over the 3-week period with the exception of an increase on

day 6, which might imply a smoking incident.

When using the semiquantitative analysis procedure, it is imperative that the

concentration of drug found in the urine be adjusted for the changes in the

urine water content that occur throughout the day. The adjustment can be

made by performing a urine creatinine analysis and dividing the drug

concentration (ng/ml of urine) by the creatinine concentration (mg creatinine/ml of urine). This allows the drug concentration to be expressed as ng

drug/mg creatinine. After drug use has stopped, measurement of ng/mg

creatinine will show a continuous decrease on sequential sample analysis,

while sequential concentration measures (ng/ml urine) may show significant

fluctuation. Because creatinine is excreted at a relatively constant rate, the

use of the creatinine analysis compensates for (or corrects) the hour-to-hour

concentration or dilution of urine that normally occurs.

The need for this adjustment can be seen in the example in figure 1. The

dashed line shows the excretion levels in terms of ng/mg creatinine. As can

be seen, the apparent increase in excreted cannabinoids at day 6 is, in fact, a

more concentrated urine specimen and not an indication of further marijuana

use. On the other hand, the creatinine-corrected levels show a clear increase

in excreted cannabinoid at day 10, indicating that the subject probably used

marijuana again. This was not apparent in the ng/ml analysis. Apparently at

-58-
day 10, the urine was relatively dilute (perhaps in an attempt by the subject

to mask the drug use), but the creatinine analysis provided the compensation

for an appropriate interpret at ion.

Figure 1. Urinary cannabinoid levels of specimens taken on alternate

days after last marijuana use. concentration of THC metabolite in

ng/ml urine. THC metabolite concentration divided by the

creatinine concentration expressed in ng metabolite/mg creatinine.

In this hypothetical example, several benefits were obtained by performing

semiquantitative analyses and adjusting by the use of creatinine levels. The

semiquantitative results allowed more careful monitoring than the positive

or negative (concentration analysis) results. In the example shown, adjusting

the cannabinoid concentration by means of the creatinine analysis protected

the subject on day 6 by substantiating his story about sweating (which could

have led to dehydration and subsequent concentration of the urine). On day

10, it allowed the detection of smoking and the subject’s attempted coverup

by sample dilution.

When used attentively, semiquantitative urine drug analysis and adjustment

with creatinine can provide a valuable adjunct to the interpretation of

multiple urine drug screens taken from healthy individuals who have abused

drugs that are excreted slowly from the body.

-59-
METHODS OF URINALYSIS

It is important to remember that no single laboratory test is capable of

detecting all drugs in urine. The commonly used term “drug screen” is

somewhat inaccurate in that it implies that all drugs are detected. Quite

frequently, laboratories will provide lists of drugs that can be detected by

their particular drug screen. Even with such a list, the consumer must still

be cautious unless the list specifically details which drugs are detected in

urine. For purposes of this discussion, the term “comprehensive drug screen”

will be used to describe tests that can detect a variety of drugs in urine.

Other laboratory tests that can detect individual or classes of drugs will be

referred to as special or specific tests.

Comprehensive Drug Screen

The comprehensive drug screen can be performed by a variety of laboratory

techniques. While gas-liquid chromatography, gas chromatography-mass

spectrometry, and high-performance liquid chromatography are sometimes

used for this purpose, thin-layer chromatography remains the most

commonly utilized technique for the detection of a large number of drugs at

low cost. The other chromatographic techniques are used to a much more

limited extent because of higher cost per assay. They are generally

employed for specific drug assays and for confirmation purposes.

It must be understood that considerable variation does exist between

laboratories in the performance of this test, a factor that will influence both

the types of drugs detectable and the time interval during which they can be

detected. Detection times are also affected by the dose of the drug, the

frequency of use, the time from drug use to urine collection, and the specific

laboratory procedure used for performing the test.

The following drugs can be detected by comprehensive drug screens based on

TLC procedures for periods of 24 hours or longer after use:

Amphetamines such as amphetamine, methamphetamine,

pseudoephedrine, phenylpropanolamine, and ephedrine

Benzodiazepines such as chlordiazepoxide (Librium), diazepam

(Valium), and flurazepam (Dalmane)

Barbiturates such as phenobarbital, secobarbital, and amobarbital

Methadone

Propoxyphene (Darvon)

Tricyclic antidepressants such as imipramine, desipramine, etc.

Nicotine

Drugs that can be detected for shorter periods of time after use, usually 3 to

12 hours, include:

Opiates such as morphine, codeine, etc.

Pentazocine (Talwin)

Cocaine

-60-
Drugs that cannot normally be detected in urine by a TLC comprehensive

drug screen are:

Cannabinoids (marijuana)

Phencyclidine (PCP)

LSD and other hallucinogens

Specific Tests

Specific tests include EMIT, RIA, TDx, GC, HPLC, and GC/MS. The specific

urine tests that are available are usually more sensitive than thin-layer

chromatography, but they can generally be used to test for only one drug or

class of drugs in a single assay experiment. Generally, a subject will be

positive with one of the specific urine tests for 1 to 3 days after drug use.

These tests are most commonly performed using immunoassay procedures.

Because they are restricted to individual drugs or classes of drugs, desired

tests must be specifically ordered. The option includes tests for:

Alcohol (ethanol)

Amphetamine

Barbiturate

Benzodiazepines [detects various benzodiazepines, including

chlordiazepoxide (Librium), diazepam (Valium), and flurazepam

(Dalmane)]

Cocaine

Methaqualone (Quaalude)

Opiates (detects several of the opiate drugs, including morphine,

codeine, and hydrocodone [Dilaudid] for a day or more after use)

Methadone

Phencyclidine (PCP)

Propoxyphene (Darvon)

Cannabinoids (THC metabolite--detects marijuana use)

DRUG TESTING STRATEGY

It can be seen that a urinalysis can include a single test or a battery of

different tests. In order to develop a drug testing program that serves the

needs of the client at a reasonable cost, individualized test batteries must be

carefully selected. In some cases, a general or comprehensive screen with

followup confirmation testing may be appropriate where the client or

employer is willing to sacrifice some sensitivity in order to screen for a wide

variety of drugs. In other cases, an analysis program aimed at three or four

drugs that are known to be problematic in a given work situation may be

indicated. Increased sensitivity for a smaller number of drugs increases the

likelihood of detecting these substances and decreases the chance of false

negatives.

-61-
Implications of Drug Levels in

Body Fluids: Basic Concepts

C. Nora Chiang, Ph.D., and Richard L. Hawks, Ph.D.

Urine drug analysis aimed at the detection of drug users generally is focused

on the detection of illicit drugs such as marijuana, cocaine, amphetamines,

etc., or their metabolites To effectively use the results that can be provided

by current technology for the analysis of biological fluids, a basic

understanding of drug pharmacokinetics and pharmacodynamics is important

(Rowland and Tozer 1980; Holford and Sheiner 1981; Gibaldi and Perrier 1982;

Goodman and Gilman 1985). Such an understanding provides the basis for

attempts to estimate the time and extent of drug use and possibly the extent

to which the drug level is predictive of impairment or pharmacologic effect.

Such knowledge is equally important in understanding the limits of

interpretation of drug levels in specific instances.

Pharmacodynamics describes the relationship between the concentration and

the effect of a drug. Drug effects result from the presence of appropriate

drug concentrations at the site of drug action To reach the site of drug

action, except for a few drugs that act topically, a drug must enter the blood

circulation from the site of administration. Through blood circulation, the

drug distributes to the site of drug action and simultaneously to other tissues

where it is stored, metabolized, and excreted. The fate of a drug in the body

is schematically represented in figure 1. The concentration of most drugs at

the site of action is therefore a function of the drug concentration in the

blood. The concentration or amount of a drug in other body fluids such as

saliva and urine can, in some cases, be related to drug concentration in the

blood. Pharmacokinetics is a science that quantitatively describes these

processes--absorption, distribution, metabolism, and excretion--that a drug

undergoes in the body.

This chapter describes basic principles of pharmacokinetics and pharmacodynamics and the application of this information to the understanding of the

significance of drug concentrations in terms of prediction of time of drug

exposure or degree of drug effects. Marijuana and cocaine will be used as

specific examples to illustrate these principles.

PHARMACODYNAMICS

The effects of most drugs result from interactions with their receptors at the

site of drug action. The time course of most responses, determined by the

effective concentrations of the drug at these receptor sites, depends on the

processes that a drug undergoes in the body. As plasma concentrations

generally reflect drug concentrations at the site of drug action, the intensity

of the effects for most drugs can be related to plasma concentrations,

although this relationship is usually not a simple linear one.

-62-
Figure 1. Schematic representation of the fate of a drug in the body.

A poor correlation is often found between effects and plasma drug concentrations. Factors that may contribute to the poor correlation are the

presence of active metabolites, time delays in the drug response, or the

development of tolerance. The relationship of specific drug concentrations

to effects for a general population is further complicated by individual

differences in the pharmacological response and the pharmacokinetics of the

drug.

Most abused drugs act on the central nervous system and produce effects on

mood, perception, behavior, and performance (Goodman and Gilman 1985).

The relationship of a perceived subjective “high” and performance decrements with plasma concentrations has been a subject of many studies.

Psychological "High" Effects and Drug Concentrations

Pleasurable effects have been associated with the self-administration of

psychoactive drugs such as cocaine and marijuana. The time course of the

effects produced by cocaine and plasma levels after administration of

cocaine by the intravenous (20 mg), intranasal (95 mg), and smoking (50 mg)

routes are shown in figure 2 (Cook et al. 1985). In spite of the twofold

difference in the dose for intravenous and smoking routes of administration,

the plasma profiles of cocaine are very similar, as are the profiles for the

self-reported “high” effects. Following the intranasal route of administration, the plasma cocaine levels peaked more slowly and were prolonged longer

as compared with those following the intravenous and smoking routes. The

high effects reported by the subjects were also perceived at a later time

point for the intranasal dose. This result indicates that the high effect is

-63-
Figure 2. Comparison of cocaine plasma concentrations (upper panel)

and psychological self-reported “high” (lower panel) after administration of cocaine by intravenous, intranasal, and smoking routes (From

Cook et al. 1985. Copyright 1985, PSG Publishing Company, Inc.).

better related to plasma cocaine concentrations than to the dose

administered. The decline of the high effect is more rapid than the decline

of plasma cocaine levels. Tolerance to the subjective high effects has been

suggested (Fischman et al. 1985).

In the case of the psychoactive component in marijuana, delta-9-tetrahydrocannabinol (THC), the pattern of the high effect showed a slower rise and a

slower decline than the plasma level of THC (figure 3) (Hollister et al., 1981;

Chiang and Barnett 1984; Ohlsson et al. 1985). This delay of the high effect

suggests that the site of action of THC is not easily accessible to the blood.

When this was taken into consideration in a pharmacokinetic and pharmacodynamic compartmental model analysis, the effects were able to be well

correlated with plasma THC levels (Chiang and Barnett 1984). However, the

estimation of the degree of intoxication from a single value of THC plasma

level is very difficult (Hollister et al. 1981), due not only to this time delay

but also to large individual variations in both effects and plasma levels.

Performance Decrement and Plasma THC Concentrations

Marijuana use has been associated with decrements in behavior and cognitive

performance. The incidence of detectable levels of THC in fatally injured

drivers ranges from 3.7 percent to 37 percent (Mason and McBay 1984;

Williams et al. 1985). While a direct causal effect of marijuana is difficult to

establish from such studies because of the generally high incidence of alcohol

-64-
Figure 3. Plasma THC concentration-time and subjective high-time

after smoking one 2.5 percent THC cigarette (mean ± SE; n=6)

Solid curves are computer fits to the data (From Chiang and Barnett

1984. Copyright 1984, The C.V. Mosby Co.)

in the subjects as well as lack of control groups, the indication is clear that a

performance-impairing drug (THC) is present and is therefore likely to

contribute to the factors leading to the accident.

In laboratory investigations, it has been reported that THC may affect

complex performance skills up to 24 hours after drug ingestion. One study

reports a “hangover” effect on the morning following a dose of marijuana (9

-65-
hours after smoking a marijuana cigarette of 3 percent THC) (Chait et al.

1985). The mean performance of pilots in a flight simulator showed a trend

toward impairment at 24 hours after smoking a cigarette of 2 percent THC

(Yesavage et al. 1985). These results have implications for THC effects on

the performance of complex tasks the day after smoking when blood

concentrations are very low.

A recent study demonstrated that a mathematical correlation could be

established between certain laboratory tasks and THC plasma concentrations

(Barnett et al. 1985). This correlation was shown for the “critical tracking

breakpoint” task for up to 7 hours after smoking and for plasma THC

concentrations as low as 2 ng/ml. This and similar tasks are widely used to

evaluate factors related to driving; however, the precise predictive validity is

unknown. Although impairment on a laboratory task can be related to plasma

THC levels, the extent to which it predicts driving impairment from smoking

marijuana is unclear.

Presumptive impairment levels for THC are difficult to establish, although

the impairment for alcohol has been reasonably correlated with blood alcohol

levels. Data currently available indicate that wide ranges of drug concentrations for different individuals may be present at equal levels of

impairment (Reeve et al. 1983). Conversely, evidence of impairment is often

lacking in some subjects at drug concentrations that are associated with

impairment in others. A general consensus of forensic toxicologists stated

that the blood concentrations associated with impairment after smoking

marijuana and after use of many other drugs has not been sufficiently

established to provide a basis for legal testimony in cases concerning driving

under the influence (Consensus Report 1985).

PHARMACOKINETICS

Pharmacokinetics, which characterizes the relationship of drug concentrations with time, is important for the understanding of drug actions. It is a

quantitative description of the complex processes that a drug undergoes in

the body, as shown in figure 1. From the site of administration, a drug is

absorbed into the systemic circulation. Through circulation, the drug is

distributed into various tissues. At the site of action, it exerts its pharmacological effects. It may also accumulate in tissues. It is eliminated from

the body through metabolism or excretion by the eliminating organs. (The

liver is the primary organ for metabolism and the kidney the most important

organ for excretion for most psychoactive drugs.) The concentration of a

drug in the body (pharmacokinetic profile) is a function of these processes--

absorption, distribution, metabolism, and excretion (Rowland and Tozer 1980;

Gibaldi and Perrier 1982; Goodman and Gilman 1985).

Absorption

Absorption describes the transfer of a drug from the site of administration

into the systemic circulation (figure 1). Drugs can be administered by various

routes, which are classified as intravascular and extravascular.

-66-
Intravascular administration refers to a direct and instant input of a drug into

the systemic circulation and is usually accomplished by intravenous

administration of a bolus dose or by an infusion. The intravenous route of

administration is commonly used for heroin, methamphetamine, and cocaine.

For a given dose, the shorter the time of infusion, the earlier and higher is

the maximum concentration in the blood. The onset of the pharmacological

response is also more rapid, and the intensity is greater. This increases the

risk of toxic effects of the drug.

By extravascular routes of administration such as smoking, intranasal

administration, or oral ingestion, the drug gets into the systemic circulation

through absorption processes. The rate and extent of the absorption of the

drug from different routes may be quite different, depending on the

physicochemical properties of the drug as well as the physiological and

anatomical factors involved in absorption.

From the smoking route, the drug is inhaled and absorbed from the lung or

the respiratory tract into the circulation. The amount absorbed may be quite

variable, as part of the dose is lost from pyrolysis and from side-stream

smoke. The amount absorbed is affected by the burning characteristics of

the cigarette, the depth of inhalation, and the puff duration. However, the

rate of absorption from this route is in general very rapid. Peak plasma

concentrations are rapidly reached, and the plasma profile is very similar to

that of an intravenous dose. This route of administration is generally used

for marijuana, phencyclidine (PCP), cocaine, and occasionally for heroin and

methamphetamine.

The intranasal route of administration, in which the drug is absorbed from the

nasal mucous membrane, is commonly used for cocaine and occasionally for

heroin. A plasma concentration time curve for cocaine is shown in figure 2.

As shown here, the intranasal route also provides a relatively quick and

sustained level of drug in the plasma.

Oral administration, the most commonly used route for therapeutic drugs, is

a relatively less popular route for abused drugs except for the hallucinogens,

such as lysergic acid diethylamide (LSD), 3,4-methylenedioxymethamphetamine (MDMA or “Ecstasy”), and mescaline. The drug so taken is absorbed

from the gastrointestinal (GI) tract. The absorption rate depends on the

physicochemical properties of the drug, and it may not be completely

absorbed. The drug may be degraded by gastric juice or the digestive

enzymes, or it may also be metabolized by intestinal flora in the GI tract or

the enzymes in the liver, which result in less drug entering the systemic

circulation. Absorption may be affected by the presence of food or other

drugs.

Figure 4 illustrates the plasma profiles of THC after administration by

intravenous (IV), oral, and smoking (marijuana) routes. Plasma THC profiles

for IV and smoking are similar, with peak levels reached rapidly and declining

rapidly. After oral administration, plasma levels for THC increase slowly and

the peak level is lower. This indicates that smoking provides an efficient

method of delivering drug into the systemic circulation. However, the

amount absorbed from smoking is quite varied, ranging from 2 percent to 56

percent of the amount of THC in the cigarette, depending on the efficiency

of the smoker (Lindgren et al. 1981). Experienced smokers may inhale more

efficiently than naive ones. The fraction of dose absorbed after the oral

-67-
Figure 4. Plasma concentrations (means and standard deviations) of THC after

intravenous, smoking, and oral routes of administration (From Ohlsson et al. 1980.

Copyright 1980, The C.V. Mosby Co.).
route ranged from 6 percent to 20 percent (Ohlsson et al. 1980; Wall et al.

1983). Because THC is extensively metabolized by the liver, part of the drug

may be degraded to metabolites before reaching the systemic circulation.

Distribution

Once the drug is absorbed into the systemic circulation, it is distributed into

different tissues, into the site of action to elicit pharmacological effects,

into organs where metabolism or excretion occurs, and into tissue reservoirs

in which it may accumulate and serve as a storage depot of the drug. The

rate and extent of distribution in tissue are determined by the blood flow to

the tissue, the ability of the drug to pass through the tissue membrane, and

the relative binding affinity of the drug for the tissue and plasma proteins.

Storage of drugs in the tissue by tissue binding or dissolution in the adipose

tissue may prolong the time in which drugs are detected in the plasma. Drugs

such as THC are very lipophilic and distribute into tissues, including the

adipose (fatty) tissue, leaving a small amount in the blood. The slow release

of THC from the sequestered tissue including adipose tissue into the plasma

is suggested for the reason for the slow elimination of the drug from the body

and therefore the long detection times of previous marijuana use in urinalysis.

Metabolism

Drugs are eliminated from the body through metabolism and excretion. Liver

is the primary metabolizing organ, although lung, kidney, and other tissues

also have metabolic activities. The drug is metabolized by oxidation,

reduction, hydrolysis, and conjugation reactions to more polar forms

(metabolites) to facilitate the excretion process. Metabolism also provides a

mechanism to terminate drug effects, as most metabolites are devoid of

pharmacological activities. Some metabolites, however, rnay have

pharmacological activities that are similar to or different from those of the

parent drug.

Most psychoactive drugs are extensively metabolized. Over twenty

metabolites of THC have been identified in urine and feces, including various

carboxylated (acidic) and hydroxytated metabolites as well as glucuronic acid

conjugates (Wall et al. 1981, Widman et al. 1985). Still, a significant portion

of the dose in both urine and feces has not been identified. Metabolites in

the urine are primarily the carboxylated products. The major metabotite is

11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid (9-carboxy-THC), a

non-psychoactive metabolite. The metabolites, 11-hydroxy-delta-9-

tetrahydrocannabinol (11-OH-THC) and 8-beta-hydroxy-delta-9-tetrahydrocannabinol are reportedly psychoactive (Lemberger et al. 1972; Perez-Reyes

et al. 1972). However, they are found only in very low (frequently

undetectable) concentrations in plasma after smoking or intravenous

administration and are unlikely to make a significant contribution to the

effects of THC. Their concentrations in urine as well as that of THC itself

are frequently undetectable.

Cocaine undergoes primarily metabolic hydrolysis by the esterases present in

liver and in plasma (Cook et al. 1985). The major metabolites identified in

urine are benzoylecgonine, ecgonine methyl ester, and ecgonine. These

metabolites are not psychoactive.

-69-
Excretion

Drugs are eliminated from the body as unchanged drug or as metabolites

through urine, bile, sweat, saliva, and expired air. Renal excretion is a

common and important route for drug elimination, making kidney the most

important organ for drug excretion. For many drugs, biliary excretion also

plays an important role. Through bile, drugs enter into the gastrointestinal

tract, where they may be either excreted in feces or reabsorbed and excreted

through urine or feces. Other routes such as saliva or sweat do not in general

contribute significantly to the excretion of most drugs. They may be of

forensic importance for the detection of drugs, however.

For THC, about 80 percent to 90 percent of the dose is excreted during the

first 5 days following a dose. Approximately 65 percent of the dose is

excreted in the feces and 20 percent excreted in urine (Hunt and Jones 1980;

Wall et al. 1983). Cocaine is excreted almost exclusively in the urine. Only 4

percent to 6 percent is excreted in the feces (Cook et al. 1985). Both THC

and cocaine, as well as most other drugs, are extensively metabolized. The

urinary as well as fecally excreted compounds are primarily metabolites with

only a minor fraction of unchanged drugs. Phencyclidine, on the other hand,

is excreted unchanged to the extent of 10 percent (Cook et al. 1982).

Salivary excretion-- The excretion of a drug into saliva depends on the ability

of the drug to pass through the epithelial cells of the salivary glands into the

saliva. The concentration of a drug in saliva could be higher, such as

amphetamine (Wan et al. 1978), or lower, such as methaqualone (Sharp et al.

1983), than that in plasma. THC or its metabolites are not detectable in

saliva following an intravenous dose (Perez-Reyes, personal communications). THC is detectable after smoking marijuana, however, due to the

absorption of THC by the oral mucosa during smoking. This illustrates the

need for caution in the interpretation of saliva concentrations of drugs

shortly after an oral or inhalation dose. Nevertheless, the THC in saliva

after smoking is reported to be indirectly correlated with recent use of

marijuana (Gross et al. 1985).

Saliva is perhaps the most easily obtained sample, and it has been established

that some drugs can be easily detected in saliva (Danhof and Breimer 1978).

It has not come to be generally used, however, because salivary concentrations for many drugs are so low that they cannot be detected. In addition,

the methods of analysis for saliva samples are frequently more difficult than

for urine samples. Furthermore, the time period during which drugs can be

detected after use is usually shorter than that in the urine.

Renal excretion-- Most drugs are extensively metabolized in the body and

excreted in the urine as metabolites. The processes involved with the

excretion of a drug via the kidney are filtration, secretion, and reabsorption.

In the kidney, drugs that are in the “plasma water” (not bound to plasma

proteins) pass through the glomerulus, which functions to filter the water as

welt as small molecules and leave the protein material of the blood behind

(along with any drug bound to it). This water filtrate then passes through the

tubules of the kidney, where water is reabsorbed and thereby the filtrate

become more concentrated. This “concentrated” filtrate leaves the kidney to

the bladder as urine. In the kidney, acidic or basic drugs (or metabolites) can

be added to the filtrate (urine) by the process of secretion through the

-70-
tubular membrane. Drugs or metabolites in the filtrate (urine) may diffuse

back into the blood by reabsorption through the tubular membrane.

The filtration process occurs with all drugs and metabolites, but secretion

and reabsorption do not occur with all drugs or metabolites. Since only

unbound drugs and metabolites are filtered in the kidney, the extent to which

a drug is protein-bound influences directly the rate at which it will be

excreted in the renal system. The reabsorption process is affected by urinary

pH as well as urinary flow, as are the excretion rates of some drugs and

metabolites. For most drugs that are not actively secreted or whose

concentration is far below the saturation concentration for secretion, urinary

excretion rates are directly proportional to drug plasma concentrations.

Plots of the urinary excretion rate and the plasma concentration of cocaine

and benzoylecgonine are shown in figure 5 (Ambre 1985). The excretion rates

of cocaine and benzoylecgonine are parallel to the respective plasma

concentrations.

As the urinary flow rate and pH may change from time to time, urinary

excretion rates could also be affected by these changes and may fluctuate.

In some cases, a plot of excretion rates may not parallel that of plasma

concentrations.

Urine drug analysis--Urinalysis quantitates the concentration of a drug in the

urine. A change in the urinary flow rate may change the concentration of a

drug in the urine, even if the renal excretion rate remains the same. ln

general, drug in the urine is more concentrated than that in the plasma. For

example, if a drug is neither secreted nor reabsorbed by the kidney, the

concentration of the drug in the urine will be about 100 times that of the

unbound drug in plasma. This is due to the fact that about 99 percent of the

plasma water filtered into the tubular lumen is reabsorbed, and only 1-2

ml/min of water leaves the kidney as urine. Ingesting a large volume of

liquid or using diuretics may increase the urine volume, and drug in that urine

is therefore diluted. Creatinine is an endogenous substance that is neither

secreted nor reabsorbed and can therefore be used as a “marker” to correct

the fluctuation of urine flow. In some laboratories engaged in urinalysis, drug

urine levels are reported as nanograms or micrograms of drug per milligram

of creatinine (see the previous chapter).

PHARMACOKINETIC PARAMETERS

Pharmacokinetics is a mathematical description of the time course of a drug

in the body. The important parameters are bioavailability, clearance, volume

of distribution, and half-life. Bioavailability refers to the fraction of a dose

that enters into the systemic (blood) circulation. Clearance indicates the

ability of an organ to clear drug from the systemic circulation. The volume

of distribution indicates the amount of drug in the body in relation to the

drug concentration in the plasma or blood after equilibrium has been

achieved. Half-life is the time required to reduce the plasma concentration

by 50 percent.

Generally, the half-life is determined from plasma concentration measurements during the terminal or elimination phase (after distribution processes

have equilibrated). As the clearance of a drug by an eliminating organ in

-71-
Cocaine and Benzoyl Ecgonine

Figure 5. A similar plot of plasma levels and urinary excretion rates of

cocaine and benzoylecgonine (From Ambre 1985. Copyright 1985,

Preston Publications, Inc.).

general is constant, the rate of elimination of a drug is a function of the

concentration of a drug in plasma. Plasma drug concentrations decline

following a first-order rate constant--a constant fraction of the drug is

eliminated per unit time. A half-life is the time unit for removing 50

percent of the drug from the body by either metabolism or excretion. A five

half-life period is required to eliminate approximately 97 percent of the drug

in the body.

Examples of pharmacokinetic analysis for cocaine (one-compartment model)

and THC (multiple-compartment model) follow.

-72-
One-Compartment Model

Cocaine exemplifies a one-compartment pharmacokinetic model (figure 6).

This model considers the body as a single homogeneous compartment and

assumes that the drug which is introduced into the plasma is instantaneously

in equilibrium with other tissues. Although the drug concentration in other

tissues may not necessarily be the same as that in the plasma, any changes in

the tissue are reflected by changes in plasma--the central compartment.

The elimination of the drug in the body follows first-order kinetics (the rate

constant k), and the elimination rate of the drug is proportional to plasma

drug concentration.

Figure 6. Plasma levels of cocaine (o) following an intravenous dose of

20 mg cocaine. Solid curve is a computer fit of the data to a

one-compartment model (data from Cook et al. 1985). The inset is a

schematic representation of a one-compartment model.

-73-
The plasma concentration (C) at a certain time (t) after an intravenous bolus

dose is described by the following equations.

C = Coexp(-kt) (Eq. 1)

V = Dose/Co (Eq. 2)

t1/2 = 0.693/k (Eq. 3)

Where Co is the plasma concentration extrapolated to time zero, k is the

elimination rate constant, V is volume of distribution, and t1/2 is

half-life.

A plasma concentration-time curve for an intravenous dose of 20 mg cocaine

in a subject is illustrated in figure 6 (data from Cook et al. 1985). The solid

line is the least square fitted curve of the data using equation 1. Co is

estimated to be 102 ng/ml, and the elimination rate constant (k) is 0.46/hour.

The volume of distribution (V) estimated by Eq (2) is 196 liters, which indicates

that the majority of cocaine in the body is distributed in the tissue. The

half-life from Eq (3) is 1.5 hour.

The half-life is a very important parameter for estimating the time required

to eliminate the drug in the body. It takes one half-life for plasma levels to

fall to half of their original level. In the case of cocaine (fig. 6), it takes 1.5

hour for cocaine plasma levels to fall from 102 ng/ml to 51 ng/ml. This is the

same time that it takes for concentrations to fall from 51 ng/ml to 25.5

ng/ml. By five half-lives (7.5 hours) the plasma concentration of cocaine

decreased from 102 ng/ml to 3.1 ng/ml, which is 3 percent of the original drug

in the body. Almost all the drug (97 percent) will be eliminated by five

half-lives. For reaching a certain drug level, an additional half-life will be

required if the dose is doubled. For example, if cocaine is given at a dose of

40 mg, it will take two half-lives to reach the level of 51 ng/ml and six

half-lives to reach the level of 3.1 ng/ml.

Mu1ticompartment Model

The disposition of most drugs, including THC, is described by a multicompartment model. Here, the body consists of a central compartment interacting

with several peripheral compartments Drugs entering the systemic

circulation require some time to distribute fully throughout the body. In the

“postdistributive” phase, where a pseudoequilibrium has been reached between

the central compartment (plasma) and the peripheral compartments (other

tissues), any changes in the central compartment reflect changes in the

peripheral compartments. In this phase, the loss of drug in the central

compartment (plasma) or peripheral compartments (other tissues) can be

described by a monoexponential process as for the one-compartment model.

The half-life for this terminal or postdistributive phase, as for that in the

one-compartment, is the time required to eliminate half of the drug from the

body.

In the multicompartment model for THC, concentrations in plasma (see figure

7) decrease rapidly, due to distribution of the drug to different tissues. The

plasma concentration follows a multiexponential decay, where each disposition

phase has a characteristic half-life. As four exponentials are required for the

description of the plasma levels of THC, a four-compartment model best

-74-
describes the pharmacokinetics of THC (Hunt and Jones 1980). This is

indicative of a minimum of four composites of the body compartment into

which the drug has variable rates of permeation. The volume of distribution

of 600-750 liters, after equilibrium is reached between blood and tissues, is

about 200 times that of the plasma volume, indicating that the majority of

the drug is accumulated in other tissues. The slow return of THC from

sequestered tissues (including adipose or fatty tissue) to plasma is suggested

as the reason for the long terminal half-life of THC--about 18 hours. It

takes at least 3-4 days to eliminate about 90 percent of THC remaining in

the body.

Metabolite Kinetics

The concentrations of metabolites in the body depend on the rate of generation of metabolites from the parent compound and the rate of elimination of

the metabolites. After the drug administration, the metabolite concentration

will increase until the formation rate of the metabolite is equal to its

elimination rate, and then the metabolite level will decrease. When the

elimination of the metabolite is very fast, whatever metabolite formed is

almost immediately eliminated, and the decline of the metabolite concentration is therefore at the same rate as that of the parent drug. If the elimination rate for metabolite is not significantly faster than the formation rate of

the metabolite, the metabolite might build up in the body and decline at a

rate slower than that for the parent drug. Tbe elimination half-life in this

case is consequently longer than that for the parent drug. The urinary

excretion of the metabolite is subsequently a function of how fast the

metabolites are formed and excreted.

This latter case is illustrated by a graphic presentation of cocaine and

metabolite (benzoylecgonine) levels after the intravenous injection of

cocaine, as shown in figure 5. The urinary excretion rates of cocaine and

benzoylecgonine are also shown. The terminal half-lives in both plasma and

urine for benzoylecgonine are longer than those of cocaine, and consequently

the detection of benzoylecgonine after a dose of cocaine is easier than for

the parent drug.

Dose-Dependent Kinetics

“Dose-dependent kinetics” refers to a situation where the kinetics of a drug

change with the dose administered, an increase in dose resulting in a disproportional increase in plasma concentration. This may be caused by changes in

the absorption, distribution, or elimination of a drug with changes in dose.

Cocaine, for example, has been reported to be dose-dependent in the dose

range of 1 mg/kg to 3 mg/kg (Barnett et al. 1981). The half-life increased

from about 40 minutes to 80 minutes, and the clearance decreased from 1.95

liters/hour/kg to 0.6 liters/hour/kg. This means that increasing the administered dose of cocaine 2-3 times may in fact increase the blood concentration (and associated adverse effects) significantly more than 2-3 times.

-75-
Figure 7. A semilogarithmic plot of average THC plasma concentrations vs. time

following a 2-min infusion of 2 mg THC in six subjects. Each point is the group average

and each bar represents one standard deviation. The inset is a continuation of the data on

a large time scale. (From Hunt and Jones 1980, copyright 1980, American Society for

Pharmacology and Experimental Therapeutics). The far right-hand figure is a compartment model consistent with THC plasma kinetics. C is the concentration of THC in the

central or plasma compartment; T2, T3, and T4 are the hypothetical tissue compartments.
FACTORS TO BE CONSIDERED IN THE INTERPRETATION OF DRUG

CONCENTRATIONS

The interpretation of a drug concentration in biological fluids (blood or urine)

to estimate the time of drug administration requires knowledge about the

disposition kinetics of the drug. In addition, it requires some knowledge of

the route of drug administration, the dose given, and the pattern or frequency

of the drug use. This information is very rarely available in a forensic or

urine screening case. It is sometimes available in treatment situations when

a series of samples over time gives an indication of use patterns along with

self-reports (see the preceding chapter).

Disposition Kinetics

The decline of plasma concentrations of a drug depends on the disposition

(distribution, metabolism, and elimination) of the drug in the body. The

disposition kinetics of a drug for each individual may be different, as each

individual may handle the drug differently.

The drug concentration in urine is more variable than that in blood or plasma,

as the urine volume and the urinary pH (which affects drug elimination) may

change considerably. All the factors that may affect the urine concentration

discussed in the previous section on renal excretion have to be considered in

the use of urine data. For single samples, the variables involved create a

sufficiently great range of possible interpretations to render any specific

interpretation questionable other than that drug was probably used in the

immediate past (days) by the individual.

Doses

Drug levels in the body depend on the dose given; a higher dose in general

produces higher drug concentrations in plasma and urine, etc. Figure 8 shows

simulated plasma concentration-time curves of THC when marijuana

cigarettes of two different potencies (the high-potency one containing 4

times the THC of the low-potency one) were given to a subject. For the

purpose of the illustration, it is assumed that similar kinetics were followed

for these two doses, although it has been suggested that a subject may titrate

THC intake during smoking (Perez-Reyes 1985). It is seen that the plasma

concentration of 2 ng/ml is reached about 1 hour after smoking a

low-potency cigarette, while this concentration is not reached until 9 hours

after the high-potency cigarette.

It would therefore be very difficult to predict a time of administration from

the plasma concentration, even in this idealized situation, if the exact dose

were not known. It would be more difficult, if not equal, for the estimation

of the time of drug administration without the knowledge of the exact dose.

Route of Administration

As explained in the section on absorption, plasma profiles may be quite

different with different routes of administration (see figures 2 and 4).

-77-
Figure 8. A simulated plasma THC concentrations vs. time curve following smoking

marijuana cigarettes of two different potencies of THC content, the high-potency one

(broken line) containing 4 times the THC of the low-potency one (solid line), using the

pharmacokinetic parameters of THC reported by Hunt and Jones (1980).
Figure 9. A simulated plasma THC concentration vs. time curve following smoking

marijuana at four times a day (at 12, 6, 7, and 8 pm) for 4 days, using the pharmacokinetic

parameters reported by Hunt and Jones (1980).
Consequently, the urinary excretion patterns may also be different, although

it is difficult to characterize urine excretion patterns as predictive of route

of administration.

Chronic Dose vs. Single Dose

When a drug is taken over a period of time, accumulation of the drug or its

metabolites may occur if elimination of the drug or metabolites is not

complete in the interval between doses. This is illustrated by the simulated

curves in figure 9, which compares plasma levels of THC following a single

marijuana cigarette (single dose) and four cigarettes a day for 5 days (chronic

dose). The THC plasma levels at 24 hours after the last cigarette for the

chronic dose is 3 ng/ml, which is about 4 times the level at 24 hours after a

single marijuana cigarette. It will take more than 2 days for THC plasma

levels to decline to 1 ng/ml after the chronic user (the subject who has taken

four cigarettes for 5 days) stops smoking, while it takes about 15 hours for

the subject who takes only a single cigarette.

For chronic and heavy marijuana users (cannabis use daily or more often for

periods of several months), it has been reported that the marijuana

metabolites were detectable in the urine with EMIT-d.a.u. at 20 ng/ml for an

average of 31 days with a range of 4-77 days of abstinence (Ellis et al. 1985),

while for light users (cannabis use weekly or less often), it is detectable for

an average of 13 days with a range of 3-29 days. Due to the individual

variations, substantial overlap occurs in the ranges observed. As explained in

the multicompartment model for THC, THC is sequestered in tissues (deep

compartment), and the rate of elimination of THC from the body is limited

by the slow release of THC from this “tissue compartment.” As substantial

amounts of THC can be accumulated in tissues for chronic users, considerably

long times are required to eliminate it. Consequently, a positive analysis for

THC metabolite in urine at these concentration levels may indicate last time

marijuana use of a few hours to as much as a few weeks previously.

SUMMARY

Drug concentrations in biological fluids are affected by the dose, route of

administration, pattern of drug use, and the dispositional kinetics

(distribution, metabolism, and excretion) of the drug. As most drugs are

distributed to the site of action by blood, drug concentration measurement in

this body fluid provides the best information as to the potential effect on

behavior such as driving impairment or on psychological high. Due to wide

individual variations in the pharmacokinetics and pharmacodynamics of drugs,

however, the use of plasma drug concentrations for the estimation of

impairment has not been established for most drugs.

As for urinalysis, drug concentrations in the urine are further complicated by

other factors such as urine flow and pH. Even if a specific method is used for

the quantitation of a specific drug (the active species, not the inactive

metabolite), interpretation in forensic samples to predict time of drug use or

impairment is not possible, except within broad time periods, because of the

variations in urine drug concentration as well as the limited knowledge

available about the dose or the route of administration.

-80-
REFERENCES

Ambre, J. The urinary excretion of cocaine and metabolites in humans: A

kinetic analysis of published data. J Anal Toxicol 9:241-245, 1985.

Barnett, G.; Hawks, R.; and Resnick, R. Cocaine pharmacokinetics in

humans. J Ethnopharm 3:353-366, 1981.

Barnett, G.; Licko, V.; and Thompson, T. Behavioral pharmacokinetics of

marijuana. Psychopharmacology 85:51-56, 1985.

Chait, L.D.; Fischman, M.W.; and Schuster, C.R. “Hangover” effects the

morning after marijuana smoking. Drug Alcohol Depend 15:229-238, 1985.

Chiang, C.N., and Barnett, G. Marijuana effect and delta-9-tetrahydrocannabinol plasma level. Clin Pharmacol Ther 36:234-238, 1984.

Consensus Report: Drug concentrations and driving impairment. JAMA

254:2618-2621, 1985.

Cook, C.E.; Jeffcoat, A.R.; and Brine, D.R. Cocaine Final Report, NIDA

Contract 271-80-3705, 1984. pp. 54-58.

Cook, C.E.; Jeffcoat, A.R.; and Perez-Reyes, M. Pharmacokinetic studies of

cocaine and phencyclidine in man. In: Barnett, G., and Chiang, C.N., eds.

Pharmacokinetics and Pharmacodynamics of Psychoactive Drugs. Foster

City, CA: Biomedical Publications, 1985. pp. 48-74.

Cook, C.E.; Brine, D.R.; Quin, G.D.; Perez-Reyes, M.; and Di Guiseppi, S.R.

Phencyclidine and phenylcyclohexene disposition after smoking

phencyclidine. Clin Pharmacol Ther 31:635-641, 1982.

Danhof, M., and Breimer, D.D. Therapeutic drug monitoring in saliva Clin

Pharmacokinetics 3:39-57, 1978.

Ellis, G.M., Jr.; Mann, M.A.; Judson, B.A.; Schramm, N.T.; and Tashchian, A.

Excretion patterns of cannabinoid metabolites after last use in a group of

chronic users. Clin Pharmacol Ther 38:572-578, 1985.

Fischman, M.W.; Schuster, C.R.; Javaid, J.; Hatano, Y.; and Davis, J. Acute

tolerance development to the cardiovascular and subjective effects of

cocaine. J Pharmacol Exp Ther 235:677-682, 1985.

Gibaldi, M., and Perrier, D. Pharmacokinetics. New York and Basel: Marcel

Dekker, Inc., 1982.

Goodman, L.S., and Gilman, A. Pharmacological Basis of Therapeutics New

York: McMillan Co., 1985.

Gross, S.J.; Worthy, T.E.; Nerder, L.; Zimmermann, E.G.; Soares, J.R.; and

Lomax, P. Detection of recent cannabis use by saliva delta-9-THC

radioimmunoassay. J Anal Toxicol 9:1-5, 1985.

-81-
Hollister, L.E.; Gillespie, H.K.; Ohlsson, A.; Lindgren, J-E.; Wahlen, A; and

Agurell, S. Do plasma concentrations of delta-9-THC reflect the degree of

intoxication? J Clin Pharmacol 21:171s-177s, 1981.

Holford, N.H.C., and Sheiner, L.B. Understanding the dose-effect relationship: Clinical application of pharmacokinetic-pharmacodynamic models.

Clin Pharmacokinetics 6:429-453, 1981.

Hunt, C.A., and Jones, R.T. Tolerance and disposition of tetrahydrocannabinol in man. J Pharmacol Exp Ther 215:35-44, 1980.

Lemberger, L.; Crabtree, R.E.; and Rowe, H.M. 11-hydroxy-delta-9-tetrahydrocannabinol: Pharmacology, disposition, and metabolism of a major

metabolite of marijuana in man. Science 177:62-64, 1972.

Lindgren, J-E.; Ohlsson, A.; Agurell, S.; Hollister, L.E.; and Gillespie, H.

Clinical effects and plasma levels of delta-9-tetrahydrocannabinol in heavy

and light users of cannabis. Psychopharmacology 74:208-212, 1981.

Mason, A.P., and McBay, A.J. Ethanol, marijuana, and other drug use in 600

drivers killed in single-vehicle crashes in North Carolina, 1978-1981. J

Forensic Sci 29:987-1026, 1984.

Ohlsson, A.; Agurell, S.; Lindgren, J-E.; Gillespie, H.K.; and Hollister, L.E.

Pharmacokinetic studies of delta-1-tetrahydrocannabinol in man. In:

Barnett, G., and Chiang, C.N., eds. Pharmacokinetics and Pharmacodynamics

of Psychoactive Drugs. Foster City, CA: Biomedical Publications, 1985. pp.

75-92.

Ohlsson, A.; Lindgren, J-E.; Wahlen, A.; Agurell, S.; Hollister, L.E.; and

Gillespie, H.K. Plasma delta-9-tetrahydrocannabinol concentrations and

clinical effects after oral and intravenous administration and smoking. Clin

Pharmacol Ther 28:409-416, 1980.

Perez-Reyes, M. Personal communication.

Perez-Reyes, M.; Timmons, M.C.; Lipton, M.A.; Davis, K.H.; and Wall, M.E.

Intravenous injection in man of delta-9-tetrahydrocannabinol and 11-OHdelta-9-tetrahydrocannabinol. Science 177:633-635, 1972.

Perez-Reyes, M. Pharmacodynamics of certain drugs of abuse. Barnett, G.

and Chiang, C.N., eds. Pharmacokinetics and Pharmacodynamics of

Psychoactive Drugs. Foster City, CA: Biomedial Publications, 1985. pp.

287-310.

Reeve, V.C.; Robertson, W.B.; Grant, J.; Soares, J.R.; Zimmermann, E.G.;

Gillespie, H.K.; and Hollister, L.E. Hemolyzed blood and serum levels of

delta-9-THC: Effects on the performance of roadside sobriety tests. J

Forensic Sci 28:963-971, 1983.

Rowland, M., and Tozer, T.N. Clinical Pharmacokinetics Concepts and

Applications. Philadelphia: Lea & Febiger, 1980.

-82-
Sharp, M.E.; Wallace, S.M.; Hindmarsh, K.W.; and Peel, H.W. Monitoring

saliva concentrations of methaqualone, codeine, secobarbital, diphenhydramine and diazepam after single oral doses. J Anal Toxicol 7:11-14, 1983.

Wall, M.E., and Perez-Reyes, M. The metabolism of delta-9-tetrahydrocannabinol and related cannabinoids in man. J Clin Pharmacol 21:178s-189s,

1981.

Wall, M.E.; Sadler, B.M.; Brine, D.; Harold, T.; and Perez-Reyes, M. Metabolism, disposition, and kinetics of delta-9-tetrahydrocannabinol in men

and women. Clin Pharmacol Ther 34:352-363, 1983.

Wan, S.H.; Martin, S.B.; and Azarnoff, D.L. Kinetics, salivary excretion of

amphetamine isomers, and effect of urinary pH. Clin Pharmacol Ther

23:585-590, 1978.

Widman, M.; Halldin, M.M.; and Agurell, S. Metabolism of delta-1-tetrahydrocannabinol in man. In: Barnett, G., and Chiang, C.N, eds. Pharmacokinetics and Pharmacodynamics of Psychoactive Drugs. Foster City, CA:

Biomedical Publications, 1985. pp. 415-426.

Williams, A.F.; Peat, M.A.; Crouch, D.J.; Wells, J.K.; and Finkle, B.S. Drugs

in fatally injured young male drivers. Public Health Rep 100:19-25, 1985.

Yesavage, J.A.; Leirer, V.O.; Denari, M.; and Hollister, L.E. Carry-over

effects of marijuana intoxication on aircraft pilot performance: A

preliminary report. Am J Psychiatry 142:1325-1329, 1985.

-83-
Examples of

Specific Drug Assays

Richard L. Hawks, Ph.D., and C. Nora Chiang, Ph.D.

The following protocols and summary information will illustrate typical

combined screening and confirmation techniques for a selected group of

abused drugs. Reference to a particular assay or technique does not

necessarily indicate a preference for that system. More detailed descriptions

of the specific analytical methods mentioned in this chapter can be found in

the previous chapter on analytical methodology. For more details about

pharmacology, toxicology, and metabolism of drugs, several textbooks on

these subjects are available (Baselt 1982, 1984; Goodman and Gilman 1985;

Clarke 1986).

The chapter sections are arranged by drug. Each section concludes with a list

of references for further information. Drugs discussed are:

Marijuana/Cannabinoids

Cocaine

Amphetamine and Methamphetamine

Opiates (narcotics)

Phencyclidine (PCP)

Alcohol

Lysergic Acid Diethylamide (LSD)

Methaqualone

Barbiturates

Benzodiazepines

GENERAL REFERENCES

Baselt, R.C. Disposition of Toxic Drugs and Chemicals in Man. Foster City,

CA: Biomedical Publications, 1982.

Baselt, R.C., ed. Advances in Analytical Toxicology. Vol. I. Foster City, CA:

Biomedical Publications, 1984.

Clarke, E.G.C. Clarke’s Isolation and Identification of Drugs. London: The

Pharmaceutical Press, 1986.

Goodman, L.S., and Gilman, A. The Pharmacologic Basis of Therapeutics.

London: MacMillan Co., 1985.

-84-
MARIJUANA/CANNABINOIDS

Delta-9-tetrahydrocannabinol (THC) is the primary psychoactive ingredient

present in the leaves and flowering tops of cannabis plants, sold on the street

as marijuana. Confiscated marijuana analyzed over the past 10 years shows a

steadily increasing potency as defined as the percentage THC by weight

found in the plant material. In the mid seventies, this potency averaged

about 1 percent. Now it averages 4 percent; a special preparation called

sinsemilla averages almost 7 percent. Marijuana is commonly abused by

smoking and occasionally by oral ingestion of the plant material.

How Cannabinoids Are Handled by the Body

THC enters the bloodstream rapidly by the smoking route (in minutes) and

more slowly by the oral route (1.5-3 hours) and is rapidly transformed by liver

enzymes to several metabolites, the primary one being 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid (9-carboxy-THC). THC itself is detectable for a few hours in blood, but because it is so rapidly metabolized and

distributed into body tissues, practically no THC is excreted in the urine.

THC tends to be stored in fatty tissue and can therefore accumulate faster

than it can be eliminated in chronic (repetitive) smokers. This accumulation

leads to much longer detection times in urinalysis for the chronic marijuana

smoker than for the occasional smoker.

The concentrations of THC metabolites found in urine are influenced by the

amount of THC (the dose) absorbed into the bloodstream, by frequency of

prior use, by the timing of collection of the urine specimen with respect to

the last exposure to marijuana, and by the rate of release of stored

cannabinoids from adipose tissue. In addition, the quantity of liquids ingested

prior to the time of sampling also affects cannabinoid concentrations in urine

(see previous chapters on implications of drug levels and on interpretations of

urinalysis results).

Methods of Analysis for Cannabinoids in Urine

Marijuana components and their metabolites have been the subject of many

analytical methods. A useful review of recent methods has been published by

Cook (in press). Cannabinoids present particularly difficult analytical

problems because of their high lipid solubility and low concentrations in body

fluids. Lipid solubility increases the difficulty of separating cannabinoids

from the biological matrix for analysis.

The immunoassays (EMIT, RIA, and FIA) detect the major metabolite of THC

(9-carboxy-THC) in urine, along with probable cross-reactivity to many of

the other metabolites of THC and their glucuronide conjugates known to be

excreted in urine. These are generally the methods of choice for the initial

screening assay. TLC is sometimes used as a screening method, although it is

a more labor-intensive approach.

The chromatography methods in current use (GLC, HPLC, TLC, GC/MS) can

separate and detect more specifically 9-carboxy-THC itself.

GLC, HPLC, and TLC have all been reported as confirmation methods for 9-

carboxy-THC, although they all are subject to interference from co-eluting

-85-
substances. Some published methods do not appear to have adequate

specificity for use as confirmation methods, while others seem to be

adequately validated (see Irving et al. 1984).

Gas chromatography-mass spectrometry (GC/MS) is clearly the most reliable

confirmatory method. Both the electron impact (EI) and the chemical

ionization (CI) modes have been applied to the quantitation of 9-carboxyTHC. The El spectrum provides generally three ions in a particular ratio

characteristic of 9-carboxy-THC, and the CI modes (positive ion CI and

negative ion CI) provide a single ion. While the EI spectrum provides more

information (three ions), it can be argued that the CI modes are less prone to

producing interfering ion fragments and are therefore inherently more

specific. The point to be made here is that, regardless of the type of mass

spectrometry used, it provides much more specific information and therefore

certainty of analysis than other chromatography methods. While other

confirmation methods can be adequately validated in some situations, GC/MS

will provide the best assurance against legal challenge.

Special Assay Considerations

Acute or occasional (less than twice a week) smoking. Assuming that

screening assays of 50 to 100 ng/ml cutoff and confirmation assays for

9-carboxy-THC of about 10-15 ng/ml are used, urine samples will generally

be positive for 1 to 3 days.

Chronic (daily) smoking. An individual who smokes regularly even as few as

two or three times a week will generally have marijuana-positive urines most

of the time. A heavy (daily for months at least), chronic smoker who stops

smoking may continue to produce positive samples for longer than a month

(depending on the assay cutoff) because of the amount of THC accumulated

in the body. It becomes difficult, therefore, to distinguish between the

chronic smoker who may in fact have stopped smoking weeks before from the

smoker who has not.

Oral administration (ingestion) of marijuana. For the first few hours after a

dose, the metabolic profile (relative concentrations of THC and its

metabolites) of THC in blood samples, is quite different following a dose

taken orally versus the one taken from smoking. However, metabolic profiles

in urine samples cannot generally differentiate between a dose taken orally

and one taken by smoking.

Passive inhalation of marijuana. Marijuana smoke can be inhaled passively

and can result in detectable body fluid levels of THC and 9-carboxy-THC.

However, the studies cited generally involve several smokers in a small room

or car and one or more nonsmokers in the room for at least an hour with no

ventilation. Other studies have used smoking machines to generate the

smoke. The probability of this type of exposure leading to a positive urine is,

of course, dependent on the sensitivity of the analytical method used, but the

screening methods in use today generally incorporate assay cutoffs high

enough to make such a possibility highly improbable.

What the Results Mean

A positive urine analysis for THC metabolite(s) indicates that the individual

has consumed marijuana or marijuana derivatives within 1 hour to as much as

-86-
several weeks before the specimen was collected. While some disagreement

exists about what concentration is indicative of “recent” use, it is generally

accepted that total cannabinoid concentrations of less than 50 ng/ml by

immunoassay in urine may be consistent with usage beyond 36 hours, or from

long-term excretion in the chronic user. Without other knowledge of the

individual’s habits, more specific interpretatiou than this is not usually

feasible.

Multiple sampling of urine can frequently help make interpretations more

specific. For instance, if several weekly samples are taken from an

individual whose fit sample was positive, there should be a different pattern

of positives depending on the circumstances of use. If the individual is an

occasional or “one time” user, the second or third sample should be negative,

along with any subsequent ones. If the individual is a previously heavy

chronic smoker who has in fact stopped, samples may be positive for 3 or

more weeks, but the concentrations should show a generally decreasing trend,

eventually becoming negative for an extended period (2 weeks). If, however,

the individual continues to smoke, the samples will continue to be positive for

several weeks with no particular indication of a decreasing concentration

trend. There has been a recent report by McBurney of a “non-acid”

metabolite of THC (8-beta-11-hydroxy-delta-9-THC) which is only

detectable in urine for a few hours after the dose. This metabolite may be

useful as an indicator of recent use.

A single positive urine test does not mean that the person was under the

influence of marijuana at the time the urine specimen was collected. A

true-positive urine test means only that the person providing the specimen

used marijuana in the recent past, which could be hours, days, or weeks

depending on the specific use pattern.

References--Marijuana/Cannabinoids

Reviews

Cook, C.E. Analytical methodology for delta-9-tetrahydrocannabinol and its

metabolites. In: Alcohol, Drugs and Driving: Abstr Rev, in press.

Hawks, R.L., ed. The Analysis of Cannabinoids in Biological Fluids. NIDA

Research Monograph No. 42. DHHS Pub. No. (ADM) 82-1212. Washington,

DC: Supt. of Docs., U.S. Govt. Print. Off., 1982.

Hawks, R.L. Developments in cannabinoid analyses of body fluids:

Implications for forensic applications. In: Agurell, S.; Dewey, W.L.; and

WiIlette, R.E., eds. The Cannabinoids: Chemical, Pharmacologic, and

Therapeutic Aspects. Orlando, FL: Academic Press, 1984. pp. 123-134.

Harvey, D.L. Advances in methods for detection and measurement of the

cannabinoids. In: Harvey, D.J., ed. Marihuana ‘84, Proceedings of the

Oxford Symposium on Cannabis. Oxford, England: IRL Press Ltd., 1984. pp.

121-136.

Lindgren, J.-E. Quantification of delta-1-tetrahydrocannabinol in tissues and

body fluids. Arch Toxicol [Suppl] 6:74-80, 1983.

-87-
Thin-layer chromatography (TLC)

Kaistha., K.K., and Tadrus, R. Semi-quantitative thin-layer mass-screening

detection of 11-nor-delta-9-THC-9-carboxylic acid in human urine. J

Chromatogr 235:528-533, 1982.

Kanter, S.L.; Hollister, L.E.; and Zamora, J.U. Marijuana metabolites in

man. XI. Detection of unconjugated and conjugated delta-9-THC-11-oic acid

by thin layer chromatography. J Chromatogr 235:507-512, 1982.

Kogan, M.J.; Newman, E.; and Wilson, N.J. Detection of the marijuana

metabolite 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid in human

urine by bonded-phase absorption and thin-layer chromatography. J

Chromatogr 303:441-443, 1984.

Sutheimer, C.A.; Yarborough, R.; Hepler, B.R.; and Sunshine, I. Detection

and confirmation of urinary cannabinoids. J Anal Toxicol 9:156-160, 1985.

Verebey, K.; Jukofsky, D.; and Mule, S.J. Evaluation of a new TLC

confirmation technique for positive EMIT cannabinoid urine samples. Res

Commun in Substances Abuse 6:1-9, 1985.

Vinson, J.A., and Lopatofsky, D.J. A semi-automated extraction and

spotting system for drug analysis by TLC. I. Procedure for analysis of the

major metabolite of delta-9-tetrahydrocannabinol in urine. J Anal Toxicol

9:156-160, 1985.

Vinson, J.A., and Patel, A.H. Detection and quantification of

tetrahydrocannabinol in serum using thin layer chromatography and

fluorometry. J Chromatogr 307:493-499, 1984.

High-performance liquid chromatography (HPLC)

Borys, H.K., and Karler, R. Post-column derivatization procedure for the

colorimetric analysis of tissue cannabinoids separated by high-performance

liquid chromatography. J Chromatogr 205:303-323, 1981.

ElSohly, M.A.; ElSohly, H.N.; Jones, A.B.; Dimson, P.A.; and Wells, K.E.

Analysis of the major metabolite of delta-9-tetrahydrocannabinol in urine.

II: A HPLC procedure. J Anal Toxicol 7:262-264, 1983.

Law, B.; Mason, P.A.; Moffat, A.C.; and King, L.J. Confirmation of cannabis

use by the analysis of delta-9-tetrahydrocannabinol metabolites in blood and

urine by combined HPLC and RIA. J Anal Toxicol 8:19-21, 1984.

Posey, B.L., and Kimble, S.N. Quantitative determination of 11-nordelta-9-tetrahydrocannabinol-9-carhoxylic acid in urine by HPLC. J Anal

Toxicol 8:234-238, 1984.

Gas-liquid chromatography (GC or GLC)

ElSohly, M.A.; Arafatt, E.S.; and Jones, A.B. Analysis of the major

metabolite of delta-9-tetrahydrocannabinol in urine. III. A GC/ECD

procedure. J Anal Toxicol 8:7-9, 1984.

-88-
Fenimore, D.C.; Freeman, R.R.; and Loy, P.R. Determination of

delta-9-THC in blood by electron capture GLC. Anal Chem 45:2331-2335,

1973.

Garrett, E.R., and Hunt, C.A. Picogram analysis of THC and application to

biological fluids. J Pharm Sci 62:1211-1214, 1973.

Karlsson, L., and Roos, C. Combination of liquid chromatography with

ultraviolet detection and gas chromatography with electron-capture

detection for the determination of delta-9-tetrahydrocannabinol-11-oic acid

in urine. J Chromatogr 306: 183-189, 1984.

McCallum, N.K.; Cairns, E.R.; Ferry, D.G.; and Wong, R.J. A simple gas

chromatographic method for routine delta-1-tetrahydrocannabinol analysis of

blood and brain. J Anal Toxicol 2:89-93, 1978.

Gas chromatomphy-mass spectrometry (GC/MS)

Baker, T.S.; Harry, J.V.; Russell, J.W.; and Myers, R.L. Rapid method for the

GC/MS confirmation of 11-nor-9-carboxy-delta-9-tetrahydrocannabinol in

urine. J Anal Toxicol 8:255-259, 1984.

Foltz, R.L.; McGinnis, K.M.; and Chinn, D.M. Quantitative measurement of

delta-9-tetrahydrocannabinol and two major metabolites in physiological

specimens using capillary column gas chromatography in negative chemical

ionization mass spectrometry. Biomed Mass Spectrom 10:316-323, 1983.

Foltz, R.L. Analysis of cannabinoids in physiological specimens by gas

chromatography/mass spectrometry. In: Baselt, R., ed. Advances in

Analytical Toxicology. Vol. 1. Foster City, CA: Biomedical Publications,

1984. pp. 125-157.

Karlsson, L.; Jonsson, H.; Aberg, K.; and Roos, C. Determination of

delta-9-tetrahydrocannabinol-11-oic acid in urine as its pentafluoropropylpentafluoropropionyl derivative by GC/MS utilizing negative ion chemical

ionization. J Anal Toxicol 7:198-202, 1983.

Rosenfeld, J.M.; McLeod, R.A.; and Foltz, R.L. Solid-supported reagents in

the determination of cannabinoids in plasma. Anal Chem 58:716-721, 1986.

Wall, M.E.; Brine, D.R.; Bursey, J.T.; and Rosenthal, D. Detection and

quantitation of tetrahydrocannabinol in physiological fluids. In: Vinson, J.A.,

ed. ACS Symposium Series 98, Cannabinoid Analysis in Physiological Fluids.

Washington, DC: American Chemical Society, 1979. pp. 39-57.

Immunoassays--RIA

Cook, C.E. Radioimmunoassay of cannabinoid compounds. In: Vinson, J.A.,

ed. ACS Symposium Series 98, Cannabinoid Analysis in Physiological Fluids.

Washington, DC: American Chemical Society, 1979. pp. 137-154.

Jones, A.B.; ElSohly, H.N.; and ElSohly, M.A. Analysis of the major

metaholite of delta-9-tetrahydrocannabinol in urine. V. Cross-reactivity of

selected compounds in a radioimmunoassay. J Anal Toxicol 8:252-254, 1984.

-89-
Yeager, E.P.; Goebelsmann, U.; Soares, J.R.; Grant, J.D.; and Gross, S.J.

Delta-9-tetrahydrocannabinol by GLC-MS validated radioimmunoassays of

hemolyzed blood or serum. J Anal Toxicol 5:81-84, 1981.

Cook, C.E.; Seltzman, H.H.; Schindler, F.H.; Tallent, C.R.; Chin, K.M.; and

Pitt, C.G. Radioimmunoassays for cannabinoids. In: Hawks, R.L., ed. The

Analysis of Cannabinoids in Biological Fluids. NIDA Research Monograph No.

42. DHHS Pub. No. (ADM) 82-1212. Washington, DC: Supt. of Docs., U.S.

Govt. Print. Off., 1982. pp. 19-32.

Jones, A.B.; ElSohly, H.N.; and ElSohly, M.A. Analysis of the major

metabolite of delta-9-tetrahydrocannabinol in urine: V. Cross-reactivity of

selected compounds in a radioimmunoassay. J Anal Toxicol 8:252-254, 1984.

Law, B.; Mason, P.A.; Moffat, A.C.; and King, L.J. A novel 125radioimmunoassay for the analysis of delta-9-tetrahydrocannabinol and its

metabolites in human body fluids. J Anal Toxicol 8:14-18, 1984.

Owens, S.M.; McBay, A.J.; Reisner, H.M.; and Perez-Reyes, M. 125I

Radioimmunoassay of delta-9-tetrahydrocannabinol in blood and plasma with

a solid-phase second-antibody separation method. Clin Chem 27:619-624,

1981.

Peat, M. The analysis of delta-9-tetrahydrocannabinol and its metabolites by

immunoassay. in: Baselt, R., ed. Advances in Analytical Toxicology.

Volume 1. Foster City, CA: Biomedical Publications, 1984. pp. 59-80.

Soares, J.R.; Grant, J.D.; and Gross S.J. Significant developments in

radioimmune methods applied to delta-9-THC and its 9-substituted

metabolites. In: Hawks, R.L., ed. The Analysis of Cannabinoids in Biological

Fluids. NIDA Research Monograph No. 42. DHHS Pub. No. (ADM)82-1212.

Washington, DC: Supt. of Docs., U.S. Govt. Print. Off., 1982. pp. 44-55.

Immunoassays--EIA

DeLaurentis, M.J.; McNeil, K.; Mann, A.J.; Clark, S.; and Greenwood, H.M.

An EMIT assay for cannabiioid metabolites in urine. In: Hawks, R.L.. ed.

The Analysis of Cannabinoids in Biological Fluids. NIDA Research Monograph

No. 42. DHHS Pub. No. (ADM)82-1212. Washington, DC: Supt. of Docs., U.S.

Govt. Print. Off., 1982. pp. 69-84.

Deutsch, A.; LeGrand, C.; and Spinner, H. Generation of monoclonal

antibodies to cannabinoids and their use in an EIA. Clin Chem 32:1052

(abstr.), 1986.

Law, B.; Pocok, K.; and Moffat, A.C. An evaluation of homogeneous enzyme

immunoassay (EMIT) for cannabinoid detection in biological fluids. J

Forensic Sci Soc 22:275-281, 1982.

Rogers, R.; Crowl, C.P.; Eimstad, W.M.; et al. Homogeneous enzyme

immunoassay for cannabinoids in urine. Clin Chem 24:95-100, 1978.

-90-
Comparisons of methods

Frederick, D.L.; Green, J.; and Fowler, M.W. Comparison of six cannabinoid

metabolite assays. J Anal Toxicol 9:116-120, 1985.

Hanson, V.W.; Buonarati, M.H.; Baselt, R.C.; Wade, N.A.; Yap, C.; Biasotti,

A.A.; Reeve, V.C.; Wong, A.S.; and Orbanowsky, M.W. Comparison of 3H- and

125I-Radioimmunoassay and gas chromatography-mass spectrometry for the

determination of delta-9-tetrahydrocannabinol and cannabinoids in blood and

serum. J Anal Toxicol 7:96-102, 1983.

Irving, J.; Leeb, B.; Foltz, R.L.; Cook, C.E.; Bursey, J.T.; and Willette, R.E.

Evaluation of immunoassays for cannabinoids in urine. J Anal Toxicol

8:192-196. 1984.

Jones, A.B.; ElSohly, H.N.; Aragat, E.S.; and ElSobly, M.A. Analysis of the

major metabolite of delta-9-tetrahydrocannabinol in urine. IV. A

comparison of five methods. J Anal Toxicol 8:249-251, 1984.

O’Connor, J.E., and Rejent, T.A. EMIT cannabinoid assay for urinary

metabolites and confirmation by alternate techniques. Clin Chem 27:1104,

1981.

O’Connor, J.E., and Rejent, T.A. EMIT cannabinoid assay: Confirmation by

RlA and GC/MS. J Anal Toxicol 5:168-173, 1981.

Rosenthal, D.; Harvey, T.M.; Bursey, J.T.; Brine, D.R.; and Wall, M.E.

Comparison of gas chromatography mass spectrometry methods for the

determination of delta-9-tetrahydrocannabinol in plasma. Biomed Mass

Spectrom 5:312-316, 1978.

Metabolism and kinetics

Agurell, S.; Gillespie, H.; Halldin, M.; Holliiter, L.E.; Johansson, E.; Lindgren,

J.E.; Ohlsson, A.; Szirmai, M.; and Widman, M. A review of recent studies on

the pharmacokinetics and metabolism of delta-1-tetrahydrocannabinol,

cannabidiol and cannabinol in man. In: Harvey, D.J., ed. Marihuana. ‘84,

Proceedings of the Oxford Symposium on Cannabis. Oxford, England: IRL

Press Ltd., 1984. pp. 49-62.

Halldin, M.M.; Carlsson, S.; Kanter, S.L.; Widman, M.; and Agurell, S.

Urinary metabolites of delta-1-tetrahydrocannabinol in man.

Arzneim-Forsch/Drug Res 32:764-768, 1982.

Halldin, M.M.; Andersson, L.K.R.; Widman, M.; and Holliiter, L.E. Further

urinary metabolites of delta-1-tetrahydrocannabinol in man. Arzneim -

Forsch/Drug Res 329:1135-1138, 1982.

Hunt, C. A., and Jones, R.T. Tolerance and disposition of tetrahydrocannabinol in man. J Pharmacol Exp Ther 215:35-44, 1980.

McBurney, L.J.; Bobble, B.A.; and Sepp, L.A. GC/MS and EMIT analyses for

delta-9-tetrahydrocannabinol metabolites in plasma and urine of human

subjects. J Anal Toxicol 10:56-64, 1986.

-9l-
Peat, M.A.; Deyman, M.E.; and Johnson, J.R. High performance liquid

chromatography-immunoassay of delta-9-tetrahydrocannabinol and its

metabolites in urine. J Forensic Sci 29: 110-119, 1984.

Wall, M.E., and Perez-Rcyes, M. The metabolism of delta-9-tetrahydrocannabinol and related compounds in man. J Clin Pharmacol 21:1785-1795, 1980.

Wall, M.E.; Sadler, B.M.; Brine, D.; Taylor, H.; and Perez-Reyes, M.

Metabolism, disposition, and kinetics of delta-9-tetrahydrocannabinol in men

and women. Clin Pharmacol Ther 34:352-363, 1983.

Wall, M.E.; Brine, D. R.; and Perez-Reycs, M. Metabolism of cannabinoids in

man. In: Braude, M.C., and Szara, S., eds. The Pharmacology of Marijuana.

Vol. 1. New York: Raven Press, 1976. pp. 93-116.

Passive inhalation

Cone, E.J., and Johnson, R.E. Contact highs and urinary cannabinoid

excretion after passive exposure to marijuana smoke. Clin Pharmacol Ther

40:247-256, 1986.

Ferslew, K.E.; Manno, J.E.; and Manno, B.R. Determination of urinary

cannabinoid metabolites following incidental exposure to marijuana smoke.

Res Commun in Substance Abuse 4:287-300, 1983.

Law, B.; Mason, P.A.; Moffat, C.A.; King, L.J.; and Marks, V. Passive

inhalation of cannabis smoke. J Pharm Pharmacol 36:578-581, 1984.

Mason, A.P.; Perez-Reyes, M.; McBay, A.J.; and Foltz, R.L. Cannabinoids in

plasma after passive inhalation of marijuana smoke. J Anal Toxicol

7:172-174, 1983.

Morland, J.; Bugge, A.; Skuterud, B.; Steen, A.; Wethe, G.H.; and Kjeldsen,

T. Cannabinoids in blood and urine after passive inhalation of cannabis

smoke. J Forensic Sci 30:997-1002, 1985.

Perez-Reyes, M.; Giuseppi, S.D.; Mason, A.P.; and Davis, K.H. Passive

inhalation of marijuana smoke and urinary excretion of cannabinoids. Clin

Pharmacol Ther 34:36-41, 1983.

Waterhouse, G.A.W.; Pence, P.J.C.; and Forney, R.B. Positive urine

cannabinoid levels produced in individuals passively exposed to marijuana

smoke. Abstracts of the 35th Annual Meeting of the American Academy of

Forensic Sciences 1983. p. 90.

Urinary excretion

Ellis, M.G.; Mann, M.A.; Judson, B.A.; Schramm, N.T.; and Taschian, A.

Excretion patterns of cannabinoid metabolites after last use in a group of

chronic users. Clin Pharmacol Ther 38:572-598, 1985.

Dackis, C.A.; Pottash, A.L.C.; Annitto, W.; and Gold, M. Persistence of

urinary marijuana levels after supervised abstinence. Am J Psychiatry

139:1196-1198, 1982.

-92-
COCAINE

Cocaine, a central nervous system stimulant prepared from an extract of the

coca plant, is self-administered in a variety of ways: snorting, smoking

(“freebasing”), and intravenous injection. It is considered one of the most

highly reinforcing drugs abused today. Its smoked form (freebase or “crack”)

appears to be extremely addicting because of the rapid onset and

disappearance of its sought-after effect.

How Cocaine Is Handled by the Body

Cocaine is rapidly absorbed (the maximum plasma concentration occurring at

about 5 minutes) after smoking. Plasma profiles after smoking are almost

equivalent to those following an intravenous dose. There are significant

temporal differences in plasma cocaine profiles between the intranasal route

and the intravenous or smoking routes. Maximum cocaine concentrations are

reached at around 30-40 minutes and persist longer after intranasal

inhalation than via intravenous or smoke. The terminal half-life (1.5 hours)

of cocaine is the same for all the routes of administration. The fraction of a

cocaine dose that enters the circulatory system after the intranasal and

smoked routes is estimated at 80 percent and 46 percent, respectively,

although large variations occur among individuals.

Cocaine is extensively metabolized, primarily by liver and plasma esterases,

and only 1 percent of the dose is excreted in the urine unchanged.

Approximately 70 percent of the dose can be recovered in the urine over a

period of 3 days. Benzoylecgonine (25-40 percent of the dose) is the major

metabolite found in the urine. About 18-22 percent of the dose is excreted as

ecgonine methyl ester and 2-3 percent as ecgonine.

Examples of Analytical Methods

lmmunochemical techniques such as EMIT and RIA designed to detect

benzoylecgonine are widely used. Unchanged cocaine can sometimes be

detected by chromatographic methods for up to 24 hours after a given dose,

while benzoylecgonine can generally be detected by immunoassays for 24-48

hours.

Gas-liquid chromatography (GLC), HPLC, and GC/MS are probably the most

generally useful techniques for confirmation of cocaine and its metabolite(s)

in urine. GC/MS provides the most specific and unchallengeable confirmation.

Special Assay Considerations

Benzoylecgonine can generally be detected in urine up to 2 days after cocaine

use. A positive cocaine metabolite assay therefore indicates use within thii

period. Depending on the frequency of urine testing, a negative assay may

not be a clear indication of lack of chronic use. With this and other drugs

that clear the body relatively rapidly, short detection times in urinalysis

procedures mean that individuals using such drugs will be less likely to be

detected than those using drugs like marijuana or PCP, which have longer

detection times.

-93-
References--Cocaine

Ambre, J.J. The urinary excretion of cocaine and metabolites in humans: A

kinetic analysis of published data. J Anal Toxicol 9:241-245, 1985.

Ambre, J.; Fischman, M.; and Ruo, T.I.. Urinary excretion of ecgonine methyl

ester, a major metabolite of cocaine in humans. J Anal Toxicol 8:23-25, 1984.

Allen, L.V., and Stiles, M.L. Specificity of the EMIT drug abuse urine assay

methods. Clin Toxicol 18:1043-l065, 1981.

Burnett, G.; Hawks, R.; and Resnick, R. Cocaine pharmacokinetics in

humans. J Ethnopharm 3:353-366. 1981.

Chinn, D.M.; Crouch, D.J.; Peat, M.A.; Finkle, B.S.; and Jennison, T.A. Gas

chromatography-chemical ionization mass spectrometry of cocaine aud its

metabolites in biological fluids. J Anal Toxicol 4:37-42, 1988.

Cook, C.E.; Jeffcoat, A.R.; and Perez-Reyes, M. Pharmacokinetic studies of

cocaine and phencyclidine. In: Bamett, G., and Chiang, C.N., eds.

Pharmacokinetics and Pharmacodynamics of Psychoactive Drugs. Foster

City, CA: Biomedical Publications, 1985. pp. 48-74.

Falk, P.M., and Harrison, B.C. Use of DB-1 capillary columns in the GC/FID

analysis of benzoylecgonine. J Anal Toxicol 9:273-274, 1985.

Froldi, R.; Gambaro, V.; Mariani, R.; Pisoni, F.; and Procaccianti, P. Rapid

quantitation of cocaine and benzoylecgonine in urine by “on column” capillary

gas chromatography after Extrelut extraction. Farmaco 39:417-423,1984.

Jacob, P.; Elias-Baker, B.A.; Jones, R.T.; and Benowitz, N.L. Determination

of cocaine in plasma by automated gas chromatography. J Chromatogr

306:173-181, 1984.

Javaid, J.I.; Musa, M.N.; Fischman, M.; and Schuster, C.R. Kinetics of

cocaine in humans after intravenous and intranasal administration. Biophys

Drug Dispos 4:9-18, 1983.

Kogan, M.J.; Verebey, K.G; DePace, A.C.; Resnick, R.B.; and Mule, S.J.

Quantitative determination of benzoylecgonine and cocaine in human

biofluids by gas-liquid chromatography. Anal Chem 49:1965-1969, 1977.

Matsubara, K.; Kasawa, M.; and Fukui, Y. In vivo and in vitro studies on

cocaine metabolism: Ecgonine methyl ester as a major metabolite of

cocaine. Forensic Sci Int 26:169-180, 1984.

Peel, H. W.; Perriso, B.J.; and Mikhael, N.Z. Detection of drugs in saliva of

impaired drivers. J Forensic Sci 29:185-189, 1984.

-94-
AMPHETAMINE AND METHAMPHETAMINE

Amphetamine and methamphetamine (the N-methyl derivative of

amphetamine) are central nervous system stimulants usually taken orally,

intravenously, or by snorting. Recent reports cite increased use of smoking

as a preferred route for methamphetamine by some individuals.

Amphetamines increase the heart rate and blood pressure and curb the

appetite. In large doses, they cause irritability and anxiety. Tolerance has

been observed in amphetamine abusers, and the possibility of developing

psychological dependence on the drug is quite significant. Studies have also

suggested that chronic abuse may lead to permanent neuronal damage to

certain essential nerve structures in the brain.

How Amphetamines Are Handled by the Body

When methamphetamine is administered, some of the drug is metabolized

into amphetamine, its major active metaholite, and both of these drugs will

appear in the urine. Amphetamine is metabolized to deaminated (hippuric

and benzoic acids) and hydroxylated metabolites.

The fraction of a dose of amphetamine excreted unchanged varies with the

pH of the urine, with a range of 2 percent (alkaline pH) to 68 percent (acidic

pH). In 24 hours, about 79 percent of the dose is excreted in acidic urine and

about 45 percent in alkaline urine. Typically, 20-30 percent is excreted as

unchanged amphetamine and 25 percent as benzoic acid and its conjugate

(hippuric acid).

Methamphetamine is excreted primarily unchanged (44 percent) with a small

fraction as amphetamine (6 percent). Its urinary excretion also fluctuates

with urinary pH. Under acidic urine conditions, the excretion of unchanged

methamphetamine is increased.

Methods of Analysis for Amphetamines in Urine

RIA assays such as the Roche Abuscreen will detect only amphetamine, while

Syva’s EMIT is able to detect both amphetamine and methamphetamine.

Although RIA does not react significantly with methamphetamine, sufficient

amphetamine is often produced by metabolism to cause a positive response.

A more specific Abuscreen amphetamine assay is also available and is

sometimes used as a second screen. The new Abbott TDx Drug Detection

System is reported to detect both methamphetamine and amphetamine with

little or no cross-reactivity to ephedrine and phenylpropanolamine.

Special Assay Considerations

Unchanged amphetamine has been detected in the urine up to 29 hours after a

single oral dose of 5 mg amphetamine. Unchanged methamphetamine also

has been identified up to 23 hours following a single oral dose.

After chronic intravenous administration, methamphetamine abusers have

shown methamphetamine concentrations of 25-300 ug/ml and amphetamine

-95-
concentrations of 1-90 ug/ml in urine. Several over-the-counter preparations used as decongestants and diet aids contain ephedrine and phenylpropanolamine, which are capable of producing positive EMIT and RIA tests if

present in the urine in significant concentrations. Several prescription drugs,

such as benzphetamine, fenfluramine, mephentermine, phenmetrazine, and

phenter- mine, can also produce positive immunoassay results.

A positive amphetamine analysis indicates previous use of amphetamine or

methamphetamine, generally within the previous 24-48 hours.

Because of the high prevalence of phenylpropanolamine and ephedrine in use

for dietary aid and cold remedies and the high probability of their crossreactivity in immunoassays, it is important to perform careful confirmatory

tests for samples screened presumptively positive by immunoassay tests.

Gas-liquid chromatography with a nitrogen-phosphorus detector (GLC/NPD)

and with appropriate columns for resolving derivatized and underivatized

phenylethylamines, and GC/MS with capillary column capability, provide

excellent sensitivity and specificity for resolution of presumptive positives by

immunoassay tests.

References--Amphetamines

Beckett, A., and Rowland, M. Urinary excretion kinetics of amphetamine in

man. J Pharm Pharmacol 17:628-639, 1965.

Beckett, A.H.; Salmon, J.A.; and Mitchard, M. Relationship between blood

levels and urinary excretion of amphetamine under controlled acidic and

under fluctuating urinary pH value using amphetamine- 1 4C. J Pharm

Pharmacol 21:251-258, 1969.

Budd, R.D. Amphetamine EMIT--structure versus reactivity. Clin Toxicol

18:91- 110, 1981.

Budd, R.D. Amphetamine radioimmunoassay--structure versus reactivity.

Clin Toxicol 18:299-316, 1981.

Caldwell, J., and Sever, P.A. The biochemical pharmacology of abused drugs,

amphetamine, cocaine and LSD. Clin Pharmacol Ther 16:625-638, 1974.

Caldwell, J. The metabolism of amphetamines in mammals. Drug Metab Rev

5:219-280, 1976.

Davis, J.M.; Kopin, E.J.; Lemberger, L.; and Axelrod, I. Effects of urinary

pH on amphetamine metabolism. Ann NY Acad Sci 179:493, 1971.

Dye, D.; East, T.; and Bayne, W.F. High performance liquid chromatographic

method for post-column, in-line derivatization with o-phthalaldehyde and

fluorometric detection of phenylpropanolamine in human urine. J Chromatogr 284:457-461, 1984.

Farrell, B.M., and Jefferies, T.M. An investigation of high-performance

liquid chromatographic methods for the analysis of amphetamines. J Chromatogr 272:111-128, 1983.

-96-
Foltz, R.L.; Fentiman, A.F.; and Foltz, R.B. GC/MS Assays for Abused Drugs

in Body Fluids. NIDA Research Monograph No. 32. DHHS Pub. No. (ADM)

80-104. Washington, DC: Supt. of Docs., U.S. Govt. Print. Off., 1980. pp.

150-178.

Gunne, L.M., and Anggard, E. Pharmacokinetic studies with amphetaminesrelationship to neuropsychiatric disorders. J Pharm Biopharm I:481-495, 1973.

Javaid, J.I., and Davis, J.M. GLC analysis of phenylalkyl amines using

nitrogen detector. J Pharm Sci 70:813-815, 1981.

Martinez, D., and Gimenez, M.P. Determination of amphetamine and

methylamphetamine by gas-liquid chromatography (head-space). Hum

Toxicol 2:391-393, 1983.

Mason, P.A.; Bal, T.S.; Law, B.; and Moffat, A.C. Development and

evaluation of a radioimmunoassay for the detection of amphetamine and

related compounds in biological fluids. Analyst 108:603-607, 1983.

Narasimhachari, N., and Friedel, R.D. Quantitation of biologically important

primary amines as their isothiocyanate derivatives by gas chromatography

using nitrogen detector and validation by selected ion monitoring. Clin Chim

Acta 110:235-243, 1981.

O’Brien, B.A.; Bonicamp, J.M.; and Jones, D.W. Differentiation of

amphetamine and its major hallucinogenic derivatives using thin-layer

chromatography. J Anal Toxicol 6:143-147, 1982.

Suzuki, S.; Inoue, T.; and Niwaguchi, T. Rapid screening method for

methamphetamine in urine by color reaction in a Sep-Pak C18 cartridge. J

Chromatogr 267:381-387, 1983.

-97-
OPIATES

Morphine, codeine, and semisynthetic derivatives of morphine belong to the

class of drugs called opiates. Both morphine and codeine (methylmorphine)

are naturally occurring alkaloids from opium, the dried milk juice of the

unripe seed pod of the opium poppy Papaver somniferum. An opiate exerts

its effects on the central nervous system. Heroin, a semisynthetic derivative

(diacetylmorphine) of morphine, is more potent than morphine and is strictly

a drug of abuse. Codeine, commonly used in analgesics and cough medicine,

and morphine, used in analgesics, are prescription drugs.

Heroin users may take the drug by insufflation (snorting), by subcutaneous

injection (“skin-popping”), by intravenous injection (“mainlining”), or by

smoking.

How Opiates Are Handled by the Body

Morphine is rapidly absorbed from an oral dose (peak plasma levels at 15-60

minutes) and from intramuscular and subcutaneous injection (peaks at 15

minutes). It is metabolized extensively, with only 2-12 percent excreted as

unchanged morphine in the urine. Large amounts (60-80 percent) of the

conjugated metabolites (glucuronides) are excreted in the urine, with small

amounts (5-14 percent) being excreted in the feces. The quantitatively most

important metabolite is morphine-3-glucuronide, which is excreted in the

urine to an extent of 67-70 percent of the given dose in 48 hours. The

half-life for morphine has been reported in the range of 1.7-4.5 hours.

Heroin is rapidly broken down first to monoacetylmorphine, which is then

metabolized to morphine in the body. Both heroin and monoacetylmorphine

disappear rapidly from the blood (half-life for heroin is 3 minutes, and that

for monoacetylmorphine is somewhat longer), while morphine levels rise

slowly, persist longer, and decline slowly. The pattern of urinary excretion of

heroin is similar to that of morphine: unchanged morphine (7 percent) and

conjugated morphine (glucuronides, 50-60 percent). However, additional

trace amounts of 6-acetylmorphine are detectable in the urine.

Codeine is rapidly absorbed from an oral dose; maximum concentrations

occur at 1 hour after ingestion. It is extensively metabolized, primarily to

conjugated 6-codeine-glucuronide, while 10-15 percent of the dose is

demethylated to form morphine and norcodeine, principally in the form of

conjugates. Therefore, codeine, norcodeine, and morphine in free and

conjugated form appear in the urine after codeine ingestion.

Methods of Analysis for Opiates in Urine

Both EMIT and the Abuscreen RIA detect codeine and morphine in free and

conjugated forms but do not distinguish between them. Morphine from heroin

use may be detected 2-4 days after the last dose by the immunoassays when

these are used at cutoffs of 300 ng/ml. Other narcotics detected by the

immunoassays for morphine include dihydrocodeine, dihydromorphine, and

hydromorphone. Acid or enzyme hydrolysis of the urine sample is necessary

when testing techniques such as TLC, HPLC, and GLC are employed, since

approximately 90 percent of codeine and morphine are found in urine in the

-98-
conjugated form (glucuronide). Because morphine can come from either

heroin or codeine administration, it is important that (1) a confirmatory

procedure such as GC/MS, HPLC, or GLC be selected, and (2) the sample be

hydrolyzed prior to analysis.

Special Assay Considerations

A screening assay that is positive for opiates could be the result of several

different circumstances of drug administration. Since immunoassays do not

distinguish between codeine, morphine, or their glucuronide conjugates, a

confirmation test that is specific for morphine and/or codeine is necessary.

The presence of morphine alone or its conjugate can indicate either clinical

morphine use or illicit morphine or heroin use (within the previous 1-2 days).

The presence of both morphine and codeine in urine is consistent with

ingestion of codeine alone, when the codeine concentration is high and

greater than that of morphine, which can be produced as a metabolite in

urine. Prescribed use of codeine must be ascertained in this case. Generally,

ingestion of a therapeutic dose of codeine (30 mg) will lead to detectable

levels of the free morphine or codeine for only a few hours, although other

metabolites may be detectable for 2-3 days by immunoassay.

Street heroin also contains acetylcodeine, which metabolizes to codeine.

Therefore, in cases of low morphine and codeine concentrations in urine, it is

not possible to determine whether the subject has taken heroin, codeine, or

morphine. Although codeine presence in urine may indicate illicit drug use,

its presence in many antitussive or analgesic prescription preparations makes

such a conclusion questionable.

The ingestion of a large quantity of poppy seeds can result in positive opiate

findings in urine samples by immunoassay methods up to 60 hours after

ingestion. This is apparently due to trace amounts of morphine in

commercial poppy seeds. A recent report by Fehn and Megges suggests that

an analysis by GC/MS for 6-O-acetylmorphine, a metabolite of heroin can be

used to distinguish between an opiate positive urine resulting from poppy seed

ingestion versus heroin use (1985).

References--Opiates

Bjerver, K.; Jonsson, J.; Nilsson, A.; Schuberth, J.; and Schuberth, J.

Morphine intake from poppy seed food. J Pharm Pharmacol 34:798-801, 1982.

Brunk, S.F., and Delle, M. Morphine metabolism in man. Clin Pharmacol

Ther 16:51-57, 1974.

Dahlstrom, B. Pharmacokinetics of morphine in relation to the analgesic

effect. In: Barnett, G., and Chiang, C.N., eds. Pharmacokinetics and

Pharmacodynamics of Psychoactive Drugs. Foster City, CA: Biomedical

Publications, 1985.

Fehn, J., and Megges, G. Detection of 0-6-monoacetylmorphine in urine

samples by GC-MS as evidence for heroin use. J Anal Toxicol 9:134-138,

1985.

-99-
Fritschi, G., and Prescott, W.R. Morphine levels in urine subsequent to poppy

seed consumption. Forensic Sci Int 27:111-117, 1985.

Inturrisi, C.E.; Max, M.B.; Foley, K.M.; Schultz, M.; Shin, S.; and Honde,

R.W. The pharmacokinetics of heroin in patients with chronic pain. N Engl J

Med 310:1213-1217, 1984.

Misra, A.L. Metabolism of opiates. In: Adler, M.C.; Manara, L.; and

Samanin, R., eds. In: Factors Affecting the Action of Narcotics. New York:

Raven Press, 1978. pp. 297-343.

Posey, B.L., and Kimble, S.N. High-performance liquid chromatographic

study of codeine, norcodeine, and morphine as indicators of codeine

ingestion. J Anal Toxicol 8:68-73, 1984.

Sawe, J.; Dahlstrom, B.; Paalzow, L.; and Rane, A. Pharmacokinetics of

morphine in cancer patients. Clin Pharmacol Ther 30:629-635, 1981.

Svensson, J.-O.; Rane, A.; Sawe, J.; and Sjoquist, F. Determination of

morphine, morphine-3-glucuronide and (tentatively) morphine-6-glucuronide

in plasma and urine using ion-pair high performance liquid chromatography.

J Chromatogr 230:427-432, 1982.

Yeh, S.Y.; Gorodetsky, C.W.; and McQuinn, R.L. Urinary secretion of heroin

and its metabolites in man. J Pharmacol Exp Ther 196:249-256, 1976.

-100-
PHENCYCLIDINE

Phencyclidine (PCP, or angel dust) is commonly taken orally, by inhalation

(smoked), by insufflation, or intravenously. The drug can be added to parsley,

mint, oregano, or other leaves and smoked in the form of a cigarette. In

liquified form, it can be swallowed, injected, or applied to smoking material.

Several PCP derivatives produce similar pharmacological effects.

How PCP Is Handled by the Body

Phencyclidine is well absorbed following all routes of administration.

Maximum plasma PCP concentrations are observed 5-15 minutes after

smoking. In one study, approximately 40 percent of PCP spiked on a

cigarette entered into smoker’s bloodstream as PCP and 30 percent as

phencyclohexene, a decomposition product of PCP. Oral absorption of PCP is

slower; the maximum plasma concentration is observed at 2 hours after the

dose. About 72 percent of the oral dose is absorbed. The terminal half-life

for PCP varies considerably, with a range of 8-55 hours and average of 18

hours.

PCP undergoes oxidation and conjugation in the body. Unchanged PCP is

excreted in the urine in moderate amounts (10 percent of the dose).

Metabolites identified are primarily conjugates of hydroxylated PCP. About

40 percent of the material in the urine has not been identified. Excretion of

PCP is increased in acidic urine, but excretion of polar metabolites is

unaffected by urinary pH. PCP may be detectable in urine for several days to

several weeks. Some PCP is secreted in the saliva.

Examples of Analytical Methods

Immunochemical methods are relatively specific for PCP, its metabolites,

and some of the closely related analogs. Methods for confirmation include

gas-liquid chromatography with nitrogen-phosphorus detection (GLC/NPD),

with differential columns to rule out interference from other drugs, or gas

chromatography-mass spectrometry (GC/MS), which will provide the most

specific and unchallengeable assay for PCP and its analogs.

Special Assay Considerations

A positive urine assay for PCP generally indicates drug use within the

previous week. There have been reports that sufficient PCP can be absorbed

through skin to lead to a positive urinalysis. The use of saliva has been

reported to correlate well with blood as a means of detection of recent PCP

use. Hair analysis for the detection of PCP has also been reported.

False positives in immunochemical assays for PCP have been reported with

the administration of thioridazine (Mellaril), dextromethorphan, and

chlorpromazine (Thorazine). This supports the need for specific confirmation

of the screening analysis.

-101-
References--Phencyclidine

Budd, R.D., and Leung, W.J. Mass screening and confirmation of

phencyclidine (PCP) in urine by radioimmunoassay/TLC. Clin Toxicol

18:85-90, 1981.

Budd, R.D. Comparison of methods of analysis for phencyclidine. J

Chromatogr 20:492-496, 1984.

Cone, E.J.; Buchwald, W.; and Yousefnejad, D. Simultaneous determination

of phencyclidine and monohydroxylated metabolites in urine of man by gas

chromatography-mass fragmentography with methane chemical ionization. J

Chromatogr Biomed Appl 223:331-339, 1981.

Cook, C.E.; Brine, D.R.; Jeffcoat, A.R.; Hill, J.M.; Wall, M.E.; et al.

Phencyclidine disposition after intravenous and oral doses. Clin Pharmacol

Ther 31:625-634, 1982.

Cook, C.E.; Jeffcoat, A.R.; and Perez-Reyes, M. Pharmacokinetic studies of

cocaine and phencyclidine in man. In: Bamett, G., and Chiang, C.N., eds.

Pharmacokinetics and Pharmacodynamics of Psychoactive Drugs. Foster

City, CA: Biomedical Publications, 1985. pp. 48-74.

Lewy, R. Preemployment qualitative urine toxicology screening. J Occup

Med 25:579-580, 1983.

McCarron, M.M.; Walberg, C.B.; Soares, J.R.; Gross, S.J.; and Baselt, R.C.

Detection of phencyclidine usage by radioimmunoassay of saliva. J Anal

Toxicol 8:197-201, 1984.

Perez-Reyes, M.; DiGiuseppi, S.; Brine, D.R.; Smith, H.; and Cook, C.E.

Urine pH and phencyclidine excretion. Clin Pharmacol Ther 32:635-641, 1982.

Simpson, G.M.; Khajawali, A.M.; Alstorre, E.; and Staples, F.R. Urinary

phencyclidine excretion in chronic abusers. J Toxicol Clin Toxicol

19:1051-1059, 1982-83.

Sramek, J.J.; Baumgartner, W.A.; Tallos, J.A.; Ahrens, T.N.; Heiser, J.F.;

and Blahd, W.H. Hair analysis for detection of phencyclidine in newly

admitted psychiatric patients. Am J Psychiatry 142:950-953, 1985.

Walberg, C.B.; McCarron, M.M.; and Schuize, B.N. Quantitation of

phencyclidine in serum by enzyme immunoassay: Results in 405 patients. J

Anal Toxicol 7:106-110, 1983.

Woodworth, J.R.; Mayersohn, M.; and Owens, S.M. Quantitative analysis of

phencyclidine and metabolites by capillary column gas chromatography. J

Anal Toxicol 8:2-6, 1984.

-102-
ALCOHOL

With the possible exception of caffeine, ethyl alcohol is the most widely used

drug in our society. The determination of alcohol in bodily fluids is perhaps

the most commonly performed drug analysis.

How Alcohol Is Handled by the Body

Alcohol is used orally, and its concentration in the blood at any given time is

influenced by such factors as a person’s weight, the rate at which the

individual drinks, whether food is taken with the “dose,” and tolerance. Blood

alcohol concentrations (BACs) are usually expressed as “percent” (g/deciliter)

or as “mg percent” (mg/dl). Drinking 1.5 oz of 86 proof alcohol in a short

period of time will result in a BAC of approximately 0.03 percent (or 30 mg

percent) in a 160-pound individual.

The liver metabolizes 95 percent of the ingested alcohol at a relatively

constant rate. A normal liver will metabolize alcohol at approximately .015

g/dl per hour. This means it takes the body about 2 hours to metabolize 3/4

oz of pure alcohol. This is equivalent to a 5-oz glass of wine, a 12-oz can of

beer, or a 1.5 oz glass of 88 proof liquor. If a person drinks at a rate faster

than 1.5 oz per hour, the blood concentration (and the effects) will

accumulate.

If a 160-pound individual took four drinks each containing 1.5 oz of 100 proof

liquor within 4 hours, a BAC of about 0.08 percent (80 mg percent) would be

reached. The same number of drinks or a six-pack of beer imbibed over a

l-hour lunch period might render the individual above the legal limit for the

rest of the workday.

Examples of Analytical Methods

Methods for analysis are classified as chemical, biochemical, and gas

chromatographic. Chemical methods utilizing acid potassium dichromate

solutions are not specific for ethyl alcohol but can be used to provide rapid

screening for volatile substances. If negative, no further testing is required.

If positive, a confirmation test is necessary to ensure the presence of ethyl

alcohol.

Biochemical methods for alcohol analysis employ an enzyme. The enzyme

alcohol dehydrogenase (ADH) is combined with other reagents in a kit form

and is available from several manufacturers. The enzyme method is reliable

and accurate when used by experienced analysts. The enzyme is fairly

specific in that it does not react with methanol or acetone, but it does react

to some degree with isopropyl and long chain alcohols. Although these higher

molecular weight alcohols are rarely encountered, positive identification for

ethyl alcohol is required in legal situations.

Gas chromatographic methods offer excellent proof for the identification of

ethyl alcohol and are the most widely used. These methods can

simultaneously detect other alcohols, ketones (acetone from diabetics), and

other volatile substances. Of the three types of methods, gas

chromatographic procedures are the methods of choice.

-103-
A variety of techniques involve extraction, distillation, or direct injection.

All are reliable when an internal standard is incorporated into the procedure

for quantitation purposes. More recently, automated head space analyses are

being used; they offer excellent precision and accuracy, in addition to

providing rapid turnaround time.

What the Results Mean

Unlike most of the other drugs discussed in this monograph, alcohol is a legal

drug and its presence in urine does not indicate illicit activity. An alcohol

analysis is always aimed at determining the BAC in order to relate this to a

particular level of impairment or at least to a legally defined definition of

impairment. In practice, evidence or suspicion of alcohol abuse is best

confirmed by breath or blood analyses to determine the concentration

present. If this concentration is more than (or close to) the legally accepted

presumptive concentration for impairment (100 mg/dl or 0.1 percent in most

States), grounds exist for an assumption of impairment.

References--Alcohol

Caplan, Y.H., and Levine, B. The analysis of ethanol in serum, blood, and

urine: A comparison of the TDx REA ethanol assay with gas chromatography.

J Anal Toxicol 10:49-52, 1986.

Cravey, R.H., and Jain, N.C. Current studies of blood alcohol methods. J

Chromatogr Sci 12:209-213, 1974.

Dubowski, K.M. Alcohol determination in the clinical laboratory. Am J Clin

Pathol 74:747-750, 1980.

Dubowski, K.M. Recent developments in alcohol analysis. Alcohol, Drugs

and Driving 2:13-46, 1986.

Jain, N.C., and Cravey, R.H. A review of breath alcohol methods. J

Chromatogr Sci 12:214-218, 1974.

Jones, A.W. Electrochemical measurement of breath-alcohol concentrations

precision and accuracy in relation to blood levels. Clin Chim Acta

146:175-183. 1985.

Wilkinson, P.K., Pharmacokinetics of ethanol: A review. Alcoholism 4:6-21,

1980.

-104-
LYSERGIC ACID DIETHYLAMIDE

Lysergic acid diethylamide (LSD) is an extremely potent drug capable of

producing altered mental states at doses as low as 25 micrograms

(approximately one-millionth of an ounce). The drug is relatively easily

prepared from natural products. Although LSD was most widely used during

the 1960s. it continues to be available through illicit channels, and there is

evidence that its use is again increasing. The drug is not physically addictive,

and there are no known. examples of deaths directly attributable to the

pharmacological effects of LSD. While under the influence of the drug,

however, a person’s ability to perform complex mental and physical tasks is

severely affected. Furthermore, use of the drug is known to sometimes cause

bizarre behavior, which can lead to fatal accidents and suicides.

How LSD Is Handled by the Body

Following oral ingestion of LSD, the effects are perceived within a few

minutes and usually last for about 12 hours. Recurrence of the drug’s effects

long after its use (“flashbacks”) has been reported, but the actual cause of

this phenomenon is not understood. Studies with laboratory animals have

shown that LSD is rapidly metabolized and only a small proportion of the dose

is excreted in the urine as the parent drug. Few reports have been published

regarding the concentrations of LSD and its metabolites in urine following

ingestion of the drug by man. However, because only small amounts of LSD

are ingested and because it is likely that the drug is rapidly metabolized in

man just as it is in laboratory animals, concentrations of the drug in the urine

of a user are unlikely to exceed a few nanograms per milliliter. In a recent

study, after administration of LSD to two volunteers at a dose of 1 microgram

of drug per kilogram of body weight, the concentration of the parent drug in

the urine reached a maximum of 1 to 2 ng/ml between 2 and 8 hours, and then

decreased to less than 0.1 ng/ml within 20 hours. A radioimmunoassay (RIA)

with a cutoff of 0.5 ng/ml for LSD-reactive substances gave a positive test

for the urine collected from the same subjects for up to 30 hours after

administration. Two metabolites of LSD could be detected by a highly

sensitive GC/MS assay in the urine specimens collected for up to 72 hours

after administration.

Methods for Analysis of LSD in Urine

Most published assays for LSD are intended for identification of the drug in

illicit preparations and do not offer the sensitivity and specificity required

for detection of LSD in urine specimens from users. A radioimmunoassay for

LSD was recently introduced commercially by Roche Diagnostic Systems,

Nutley, NJ, and appears to offer an effective means of detecting very recent

use of the drug. However, confirmation of the presence of LSD or its

metabolites remains a difficult task. High-performance liquid

chromatography combined with fluorescence detection has been used to

detect LSD at urinary concentrations as low as 0.5 ng/ml. A method

employing the combination of capillary column gas chromatography and

electron ionization mass spectrometry has been developed and is also capable

of measuring LSD concentrations as low as 0.5 ng/ml. However, neither of

these assays is useful for detection of LSD in urine for more than about 12

hours after ingestion. A GC/MS assay for the N-demethyl and 13-hydroxy

metabolites of LSD in urine has been developed and shown to permit

-105-
detection of LSD use for more than 2 days after ingestion. Other analytical

techniques that have been evaluated for detection of LSD in urine include the

combination of liquid chromatography and mass spectrometry (LC/MS)

(Kidwell, personal communication) and tandem analyzer mass spectrometry

(MS/MS).

Special Assay Considerations

The task of developing an assay with sufficient sensitivity to detect LSD or

its metabolites in urine from users is made more difficult by the drug’s

sensitivity to both light and heat. However, two studies have shown that LSD

is stable in urine for more than a month when stored at or below room

temperature and protected from direct sunlight or other sources of

ultraviolet radiation. LSD analyses based upon gas chromatography require a

well-deactivated capillary column in order to avoid severe adsorptive loss of

the drug. Conversion of LSD to its N-trimethylsilyl or N-trifluoroacetyl

derivative results in improved gas chromatographic behavior and permits

higher sensitivities to be achieved.

References--LSD

Christi, J.; White, M.W.; and Wiles, J.M. A chromatographic method for the

detection of LSD in biological fluids. J Chromatogr 120:496-501, 1976.

Foltz, R.L.; Lim, H.-K.; and Francom, P. Center for Human Toxicology,

University of Utah, Salt Lake City, UT, and Jones, R.T., University of

California, San Francisco. Unpublished data.

Foltz, R.L.; Francom, P.F.; Lim, H.-K.; Bridges, R.; and Andrenyak, D. “A

GC/MS Method for Measurement of LSD in Urine.” Paper presented at the

Annual Meeting of the American Academy of Forensic Sciences, New

Orleans, LA, February 10-15,1986.

Freedman, D.X. The psychopharmacology of hallucinogenic agents. Annu

Review Med 20:409-418, 1969.

Kidwell, D., Naval Research Laboratories, Washington, DC. Personal

communication.

Lim, H.-K., and Foltz, R.L., Center for Human Toxicology, University of

Utah, Salt Lake City, UT. Private communication.

Peel, H.W., and Boynton, A.L. Analysis of LSD in urine using

radioimmunoassay-excretion and storage effects. Can Soc J Forensic Sci

13:23-28, 1980.

Roche Diagnostics Systems, Inc., Nutley, NJ. Package insert.

Siddik, Z.H.; Barnes, R.D.; Dring, L.C.; Smith, R.L.; and Williams, R.T. The

fate of LSD in the rat, guinea pig and rhesus monkey and of iso-LSD in rat.

Biochem Pharmacol 28:3093-3101, 1979.

-106-
Sullivan, A.T.; Twitchett P.J.; Fletcher, S.M.; and Moffat, A.C. The fate of

LSD in the body: Forensic considerations. J Forensic Sci Soc 18:89-98, 1978.

Twitchett, P.J.; Fletcher, S.M.; Sullivan, A.T.; and Moffat, A.C. Analysis of

LSD in human body fluids by HPLC, fluorescence spectroscopy and RIA. J

Chromatogr 150:73-84, 1978.

Yost, R.A. University of Florida, Gainesville, FL. Personal communication.

-107-
METHAQUALONE

Methaqualone (Quaalude) is a sedative-hypnotic drug taken orally in doses of

75-300 mg. Far higher doses may be taken in nontherapeutic abuse

situations. Chronic use in tolerant individuals of over 3 g per day has been

reported.

How Methaqualone Is Handled by the Body

The absorption of methaqualone is rapid, with peak plasma concentration

reached in 1.5-2 hours. The drug is extensively metabolized in man; less than

1 percent of the dose is excreted as unchanged methaqualone in the urine.

Approximately 30 percent of the dose is excreted in the urine in 24 hours, 25

percent as hydroxylated metabolites (12 have been identified).

After the ingestion of a therapeutic dose (300 mg) of methaqualone,

methaqualone is negligible (usually less than 1 ng/ml) in the urine, while

metabolites may still be detectable for more than a week by sensitive GC/MS

methods.

Examples of Analytical Methods

EMlT and RIA not only detect unchanged drug but can significantly detect

various metabolites of the drug, thus providing longer detection periods after

last ingestion. Confirmatory methods for the detection of unchanged

methaqualone and conjugated (bound) metabolites after hydrolysis of the

sample include GLC and GC/MS.

References--Methaqualone

Alvan, C.; Lindgren, J.-E.; Bogentoft, C.; and Ericsson, O. Plasma kinetics

of methaqualone in man after single oral doses. Eur J Clin Pharmacol

6:187-190, 1973.

Bonnichsen, R.; Marde, Y.; and Ryahge, R. Identification of free and

conjugated metabolites of methaqualone by gas chromatography-mass

spectrometry. Clin Chem 20:230-235, 1974.

Evenson, M.A., and Lensmeyer, G.L. Qualitative and quantitative

determination of methaqualone in serum by gas chromatography. Clin Chem

20:249-254, 1974.

Peat, M.A., and Finkle, B.S. Determination of methaqualone and its major

metabolite in plasma and saliva after single oral doses. J Anal Toxicol

4:114-118, 1980.

Permisohn, R.C.; Hillpert, L.R.; and Kazyak, L. Determination of methaqualone in urine by metabolite detection via gas chromatography. J

Forensic Sci 21:98-107, 1976.

Sleeman, H.K.; Cella., J.A.; Harvey, J.L.; and Beach, D.J. Thin-layer

chromatographic detection and identification of methaqualone metabolites in

urine. Clin Chem 21:76-80, 1975.

-108-
BARBITURATES

Barbiturates are central nervous system depressants and used as

hypnotics/sedatives. They are classified as ultra-short, short-,

intermediate-, and long-acting. The duration of action of barbiturates is

quite variable, ranging from 15 minutes for ultra-short-acting drugs to a day

or more for long-acting drugs. The most commonly abused barbiturates are

short- and intermediate-acting agents such as pentobarbital (Nembutal),

secobarbital (Seconal), and amobarbital (Amytal). Long-acting agents such as

phenobarbital are rarely subject to abuse.

How Barbiturates Are Handled by the Body

Barbiturate derivatives are excreted into the urine in varying amounts of

unchanged drug and metabolites. Long-acting barbiturates like phenobarbital

are excreted with a higher percentage of unchanged drug in the urine, while

short-acting barbiturates, secobarbital and amobarbital, are extensively

metabolized and excreted in the urine with a smaller percentage of

unchanged drugs.

Examples of Analytical Methods

EMIT and RIA are designed to detect unchanged secobarbital in the urine;

however, both assays will detect other commonly encountered barbiturates,

depending on the concentration of drug present in the sample. Phenobarbital

positives have been noted in chronic users up to several weeks after cessation

of use. With standard single doses of secobarbital, pentobarbital, or

amobarbital, RIA and GLC identified drug presenee for up to 52 hours and 76

hours, respectively, utilizing a 100 ng/ml cutoff. TLC, less sensitive than

RIA, demonstrated the presence of barbiturate for approximately 30 hours

under the same conditions. With high dosages and/or chronic daily doses,

EMIT and RIA can be used effectively for screening. Gas-liquid

chromatography/flame ionization (GLC/FID) after derivatization, GLC/NPD

and GC/MS are reliable methods used for confirmation of the various

barbiturates.

References--Barbiturates

Colbert, D.L.; Smith, D.S.; Landon, J.; and Sidki, A.M. Single-reagent

polarization fluoroimmunoassay for barbiturates in urine. Clin Chem

30:1765-1779, 1984.

Gill, R.; Stead, A.H.; and Moffat, A.C. Analytical aspects of barbiturate

abuse: Identification of drugs by the effective combination of gas-liquid,

high performance liquid and thin-layer chromatographic techniques. J

Chromatogr 204:275-284, 1981.

Hall, R.C., and Risk, C.A. Rapid and selective determination of free

barbiturates by gas chromatography using the electrolytic conductivity

detector. J Chromatogr Sci 13:519-524, 1975.

Jain, N.C. Mass screening and confirmation of barbiturates in urine by

RIA/gas chromatography. Clin Toxicol 9:221-233, 1976.

-109-
Roerig, D.L.; Lewand, D.L.; Mueller, M.A.; and Wang, R.I. Comparison of

radioimmunoassay with thin-layer chromatographic and gas-liquid

chromatographic methods of barbiturate detection in human urine. Clin

Chem 21:672-675, 1975.

Pillai, D.N., and Dilli, S. Analysis of barbiturates by gas chromatography. J

Chromatogr 25:253-274, 1981.

Saferstein, R.; Manura, J.J.; and De, P.K. Drug detection in urine by

chemical ionization mass spectrometry. J Forensic Sci 23:29-36, 1978.

-110-
BENZODlAZEPlNES

The benzodiazepines are considered by many as the most prescribed drugs in

the United States. They are primarily used as antianxiety and sedativehypnotic drugs and also have broad therapeutic use as anticonvulsants and

muscle relaxants. The benzodiazepines have a low order of acute and chronic

toxicity when used in a medically supervised manner. Chronic use does result

in some moderate dependence and tolerance to the drug. More than a dozen

benzodiazepines are in clinical use today. The best known benzodiazepine

drugs are Valium (diazepam) and Librium (chlordiazepoxide).

How Benzodiazepines Are Handled by the Body

These drugs are well absorbed when administered orally, the most common

route of administration. Most benzodiazepines are extensively metabolized

in the liver and excreted in the urine as metabolites. Nonconjugated

metabolites may possess pharmacological activity that may account for the

“next day” effects for some benzodiazepines. Many of the new

benzodiazepines are metabolites and derivatives of the old drugs. For

example, oxazepam is a common urinary metabolite of many benzodiazepines

and is also a marketed drug (Serax).

The duration of action and elimination half-lives of the different

benzodiazepines vary widely. The half-lives for major benzodiazepines are:

chlordiazepoxide, 5-10 hours; diazepam, 30-60 hours; oxazepam (Setax), 5-10

hours; flurazepam (Dalmane), 2-3 hours for the parent drug and 50-100 hours

for active metabolites.

Because of the long elimination time for the benzodiazepines, an individual

who has been using a drug for months or years may maintain detectable

urinary concentrations of the drug for weeks to months after discontinuation

of its use.

Examples of Analytical Methods

A broad spectrum of analytical methods, GC, HPLC, TLC, GLC/MS, RIA, and

EMIT, has been reported for the analysis of the benzodiazepines, or their

metabolites, or of their acid hydrolysis products, e.g., benzophenones.

Because many benzodiazepines have common metabolites, it is not always

possible to determine the drug taken through the use of urine testing. The

EMIT screening procedure is rapid and specific for benzodiazepiues, as it

detects thii class of drugs by recognizing oxazepam, a common metabolite of

many benzodiazepines, and also many cross-reacting benzodiazepine drugs or

metabolites. Other screening procedures used to detect benzodiazepines

alone or in combination with major drugs of abuse in urine screening

programs can be achieved by TLC and HPLC. Radioimmunoassay methods

possess the necessary sensitivity for the determination of diazepam directly

in microsamples of blood/plasma (10 ul) and saliva (100 ul) for up to 16 hours

following oral administration of a single 5 mg dose of the drug.

Confirmation of positive results from tests performed with immunoassay or

thin-layer chromatography, which detect many of the metabolites, may be

-111-
difficult using more specific techniques such as GLC and GC/MS. Care must

be taken to ensure that the metabolites detected with TLC or immunoassay

will chromatograph when using GLC and GUMS techniques.

References--Benzodiazepines

Budd, R.D., and Walkin, E. Mass screening and confirmation of diazepam in

urine by EMIT-thin layer chromatography. Clin Toxicol 16:201-207, 1980.

Budd, R.D. Benzodiazepine structure versus reactivity with EMIT oxazepam

antibody. Clin Toxicol 18:643-656, 198l.

Poklis, A. An evaluation of EMIT d.a.u. benzodiazepine metabolite assay for

urine drug screening. J Anal Toxicol 5:174-176, 1981.

Rutterford, M.G., and Smith, R.N. Radioimmunoassay of diazepam in

samples of forensic interest. J Pharm Pharmacol 32:449-452, 1980.

Slightom, E.L.; Cagle, J.C.; McCurdy, H.H.; and Castagna, F. Direct and

indirect homogeneous enzyme immunoassay of benzodiazepines in biological

fluids and tissues. J Anal Toxicol 6:22-25, 1982.

Verebey, K.; Jukofsky, D.; and Mule, S.J. Confirmation of EMIT

benzodiazepine assay with GLC/NPD. J Anal Toxicol 6:305-308, 1982.

Violon, C.; Pessemier, L.; and Vercruysse, A. High-performance liquid

chromatography of benzophenone derivatives for the determination of

benzodiazepines in clinical emergencies. J Chromatogr 236:157-168, 1982.

-112-
Glossary

Accuracy--Ability to get the correct (or true) result.

Analyte--Substance to be measured.

Blank--Biological specimen with no detectable drugs added, routinely

analyzed to ensure that no false-positive results are obtained.

Blind sample--Control material submitted to the analyst (unknown to him or

her) as a routine specimen.

Chain of custody--Handling samples in a way that supports legal testimony to

prove that the sample integrity and identification of the sample have not

been violated, as well as the documentation describing these procedures.

Concentration--Amount of a drug in a unit volume of biological fluid,

expressed as weight/volume. Urine concentrations are usually expressed

either as nanograms per milliliter (ng/ml), as micrograms per milliliter

(ug/ml), or milligrams per liter (mg/l). (There are 28,000,000 micrograms in

an ounce, and 1,000 nanograms in a microgram.)

Confirmation--A second test by an alternate chemical method to positively

identify a drug or metabolite. Confirmations are carried out on presumptive

positives from initial screens.

Creatinine--An endogenous substance appearing in the urine.

Cross-reacting substances--In immunoassays, refers to substances that react

with antiserum produced specifically for other substances.

Cutoff level (threshold)--Value serving as an administrative breakpoint (or

cutoff point) for labeling a urine result positive or negative.

Detection limit--Lowest concentration of a drug that can reliably be

detected.

False negative--An erroneous result in an assay that indicates the absence of

a drug that is actually present.

False positive--An erroneous result in an assay that indicates the presence of

a drug that is actually not present.

-113-
Interfering substances--Substances other than the analyte that give a similar

analytical response or alter the analytical result.

Metabolite--A compound produced from chemical changes of a drug in the

body.

Pharmacokinetics--The study of the time course of the processes (absorption,

distribution, metabolism, and excretion) a drug undergoes in the body.

Pharmacodynamics--The study of the relationship of drug concentration to

drug effects.

Precision--Ability to get the same result between repeated measurements.

Presumptive positive--Sample which has been flagged as positive by

screening but which has not been confirmed by an equally sensitive

alternative chemical method.

Proficiency-testing specimen--A specimen whose expected results are

unknown to anyone in the laboratory, known only by an external agency, and

later revealed to the laboratory as an aid to laboratory improvement and/or a

condition of licensure.

Quality Assurance (QA)--Practices that assure accurate laboratory results.

Quality Control (QC)--Those techniques used to monitor errors which can

cause a deterioration in the quality of laboratory results. Control material

most often refers to a specimen, the expected results of which are known to

the analyst, that is routinely analyzed to ensure that the expected results are

obtained.

Qualitative test--Chemical analysis to identify the components of a mixture.

Quantitative test--Chemical analysis to determine the amounts or

proportions of a mixture.

Screen--A series of initial tests designed to separate samples with drugs at

the particular minimum concentration from those below that minimum

concentration (positive versus negative).

Sensitivity--The detection limit, expressed as a concentration of the analyte

in the specimen.

Specificity--Quality of an analytical technique that tends to exclude all

substances but the analyte from affecting the result.

Split specimen--Laboratory specimen that is divided and submitted to the

analyst, unknown to him or her, as two different specimens with different

identifications.

Standard--Authentic sample of the analyte of known purity, or a solution of

the analyte of a known concentration.

-114-
monograph series

While limited supplies last, single copies of the monographs may be

obtained free of charge from the National Clearinghouse for Drug

Abuse Information (NCDAI). Please contact NCDAI also for information about availability of coming issues and other publications of

the National Institute on Drug Abuse relevant to drug abuse research.

Additional copies may be purchased from the U.S. Government Printing

Office (GPO) and/or the National Technical Information Service

(NTIS) as indicated. NTIS prices are for paper copy. Microfiche

copies, at $6.50, are also available from NTIS. Prices from either

source are subject to change.

Addresses are:

NCADI

National Clearinghouse for Alcohol and Drug Information

P.O. Box 2345

Rockville, Maryland 20852

GPO

Superintendent of Documents

U.S. Government Printing Office

Washington, D.C. 20402

NTIS

National Technical Information

Service

U.S. Department of Commerce

Springfield, Virginia 22161

1 FINDINGS OF DRUG ABUSE RESEARCH. NCDAI out of stock

Vol. 1: GPO out of stock NTIS PB #272 867/AS $36.95

Vol. 2: GPO out of stock NTIS PB #272 868/AS $30.95

2 OPERATIONAL DEFINITIONS IN SOCIO-BEHAVIORAL DRUG USE RESEARCH

1975. Jack Elinson, Ph.D., and David Nurco, Ph.D., eds.

NCDAI out of stock

GPO out of stock NTIS PB #246 338/AS $18.95

3 AMINERGIC HYPOTHESES OF BEHAVIOR: REALITY OR CLICHE? Bruce J.

Bernard, Ph.D., ed. NCDAI out of stock

GPO out of stock NTIS PB #246 687/AS $18.95

4 NARCOTIC ANTAGONISTS: THE SEARCH FOR LONG-ACTING PREPARATIONS.

Robert Willette. Ph.D., ed. NCDAI out of stock

GPO out of stock NTIS PB #247 096/AS $11.95

115
5 YOUNG MEN AND DRUGS: A NATIONWIDE SURVEY. John A. O'Donnell,

Ph.D., et al. NCDAI out of stock

GPO out of stock NTIS PB #247 446/AS $18.95

6 EFFECTS OF LABELING THE "DRUG ABUSER": AN INQUIRY. Jay R.

Williams, Ph.D. NCDAI out of stock

GPO out of stock NTIS PB #249 092/AS $11.95

7 CANNABINOID ASSAYS IN HUMANS. Robert Willette, Ph.D., ed.

GPO out of stock NCDAI out of stock

NTIS PB #251 905/AS $18.95

8 Rx: 3x/WEEK LAAM - ALTERNATIVE TO METHADONE. Jack Blaine, M.D.,

and Pierre Renault, M.D., eds. NCDAI out of stock

Not available from GPO NTIS PB #253 763/AS $18.95

9 NARCOTIC ANTAGONISTS: NALTREXONE PROGRESS REPORT. Demetrios

Julius, M.D., and Pierre Renault, M.D., eds.

GPO out of stock NCDAI out of stock

NTIS PB #255 833/AS $18.95

10 EPIDEMIOLOGY OF DRUG ABUSE: CURRENT ISSUES. Louise G. Richards,

Ph.D., and Louise B. Blevens, eds. NCDAI out of stock

GPO out of stock NTIS PB #266 691/AS $24.95

11 DRUGS AND DRIVING. Robert Willette, Ph.D., ed.

NCDAI out of stock

GPO Stock #017-024-00576-2 $5.50 NTIS PB #269 602/AS $18.95

12 PSYCHODYNAMICS OF DRUG DEPENDENCE. Jack D. Blaine, M.D., and

Demetrios A. Julius. M.D., eds. NCDAI out of stock

GPO out of stock NTIS PB #276 084/AS $18.95

13 COCAINE: 1977. Robert C. Petersen, Ph.D., and Richard C.

Stillman. M.D., eds. NCDAI out of stock

GPO Stock #017-024-00592-4 $6 NTIS PB #269 175/AS $24.95

14 MARIHUANA RESEARCH FINDINGS: 1976. Robert C. Petersen, Ph.D.,

ed. NCDAI out of stock

GPO out of stock NTIS PB #271 279/AS $24.95

15 REVIEW OF INHALANTS: EUPHORIA TO DYSFUNCTION. Charles Wm. Sharp,

Ph.D., and Mary Lee Brehm, Ph.D., eds. NCDAI out of stock

GPO out of stock NTIS PB #275 798/AS $30.95

16 THE EPIDEMIOLOGY OF HEROIN AND OTHER NARCOTICS. Joan Dunne

Rittenhouse. Ph.D., ed. NCDAI out of stock

GPO out of stock NTIS PB #276 357/AS $24.95

17 RESEARCH ON SMOKING BEHAVIOR. Murray E. Jarvik, M.D., Ph.D., et

al., eds. NCDAI out of stock

GPO out of stock NTIS PB #276 353/AS $30.95

116
18 BEHAVIORAL TOLERANCE: RESEARCH AND TREATMENT IMPLICATIONS.

Norman A. Krasnegor, Ph.D., ed. NCDAI out of stock

GPO out of stock NTIS PB #276 337/AS $18.95

19 THE INTERNATIONAL CHALLENGE OF DRUG ABUSE. Robert C. Petersen,

Ph.D., ed. NCDAI out of stock

GPO out of stock NTIS PB #293 807/AS $30.95

20 SELF-ADMINISTRATION OF ABUSED SUBSTANCES: METHODS FOR STUDY.

Norman A. Krasnegor, Ph.D., ed. NCDAI out of stock

GPO out of stock NTIS PB #288 471/AS $24.95

21 PHENCYCLIDINE (PCP) ABUSE: AN APPRAISAL. Robert C. Petersen,

Ph.D., and Richard C. Stillman, M.D., eds. NCDAI out of stock

GPO Stock #017-024-00785-4 $7 NTIS PB #288 472/AS $30.95

22 QUASAR: QUANTITATIVE STRUCTURE ACTIVITY RELATIONSHIPS OF

ANALGESICS, NARCOTIC ANTAGONISTS, AND HALLUCINOGENS. Gene Barnett.

Ph.D.; Milan Trsic. Ph.D.; and Robert Willette, Ph.D.; eds.

GPO out of stock NCDAI out of stock

NTIS PB #292 265/AS $36.95

23 CIGARETTE SMOKING AS A DEPENDENCE PROCESS. Norman A. Krasnegor,

Ph.D., ed. NCDAI out of stock

GPO Stock #017-024-00895-8 $6 NTIS PB #297 721/AS $24.95

24 SYNTHETIC ESTIMATES FOR SMALL AREAS: STATISTICAL WORKSHOP PAPERS

AND DISCUSSION. Jos. Steinberg, ed. NCDAI out of stock

GPO out of stock NTIS PB #299 009/AS $24.95

25 BEHAVIORAL ANALYSIS AND TREATMENT OF SUBSTANCE ABUSE. Norman A.

Krasnegor, Ph.D., ed. NCDAI out of stock

GPO out of stock NTIS PB #80-112428 $24.95

26 THE BEHAVIORAL ASPECTS OF SMOKING. Norman A. Krasnegor, Ph.D.,

ed. (Reprint from 1979 Surgeon General's Report on Smoking and

Health.)

GPO out of stock NTIS PB #80-118755 S18.95

27 PROBLEMS OF DRUG DEPENDENCE, 1979: PROCEEDINGS OF THE 41ST

ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS OF DRUG

DEPENDENCE, INC. L.S. Harris, Ph.D., ed. NCDAI out of stock

GPO Stock #017-024-00981-4 $9 NTIS PB #80-175482 S42.95

28 NARCOTIC ANTAGONISTS: NALTREXONE PHARMACOCHEMISTRY AND

SUSTAINED-RELEASE PREPARATIONS. Robert Willette, Ph.D., and

Gene Barnett. Ph.D., eds. NCDAI out of stock

GPO out of stock NTIS PB #8l-238875 $24.95

29 DRUG ABUSE DEATHS IN NINE CITIES: A SURVEY REPORT. Louis A.

Gottschalk, M.D., et al. NCDAI out of stock

GPO out of stock NTIS PB #80-178882 $18.95

117
30 THEORIES ON DRUG ABUSE: SELECTED CONTEMPORARY PERSPECTIVES. Dan

J. Lettieri, Ph.D.; Mollle Sayers; and Helen W. Pearson, eds.

NCDAI out of stock

GPO Stock #017-024-00997-1 $1O Not available from NTIS

31 MARIJUANA RESEARCH FINDINGS: 1980. Robert C. Petersen. Ph.D.,

ed.

GPO out of stock NTIS PB #80-215171 $24.95

32 GC/MS ASSAYS FOR ABUSED DRUGS IN BODY FLUIDS. Rodger L. Foltz.

Ph.D.; Allison F. Fentlman. Jr., Ph.D.; and Ruth B. Foltz.

GPO out of stock NTIS PB #81-133746 $24.95

33 BENZODIAZEPINES: A REVIEW OF RESEARCH RESULTS, 1980. Stephen I.

Szara. M.D., D.Sc., and Jacqueline P. Ludford, M.S., eds.

GPO Stock #017-024-01108-8 $5 NTIS PB #82-139106 S18.95

34 PROBLEMS OF DRUG DEPENDENCE, 1980: PROCEEDINGS OF THE 42ND

ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS OF DRUG

DEPENDENCE, INC. Louis S. Harris, Ph.D., ed. NCDAI out of stock

GPO Stock #017-024-01061-8 $8 NTIS PB #81-194847 $36.95

35 DEMOGRAPHIC TRENDS AND DRUG ABUSE, 1980-1995. Louise G.

Richards, Ph.D., ed. NCDAI out of stock

GPO out of stock NTIS PB #82-103417 $18.95

36 NEW APPROACHES TO TREATMENT OF CHRONIC PAIN: A REVIEW OF

MULTIDISCIPLINARY PAIN CLINICS AND PAIN CENTERS. Lorenz K.Y.

Nq, M.D., ed.

GPO out of stock NTIS PB #81-240913 $24.95

37 BEHAVIORAL PHARMACOLOGY OF HUMAN DRUG DEPENDENCE. Travis

Thompson, Ph.D., and Chris E. Johanson, Ph.D., eds.

NCDAI out of stock

GPO Stock #017-024-01109-6 $7 NTIS PB #82-136961 $30.95

38 DRUG ABUSE AND THE AMERICAN ADOLESCENT. Dan J. Lettieri, Ph.D.,

and Jacqueline P. Ludford, M.S., eds. A RAUS Review Report.

GPO Stock #017-024-01107-0 $4.50 NCDAI out of stock

NTIS PB #82-148198 $18.95

39 YOUNG MEN AND DRUGS IN MANHATTAN: A CAUSAL ANALYSIS.

Richard R. Clayton, Ph.D., and Harwin L. Voss, Ph.D.

GPO Stock #017-024-01097-9 $5.50 NCDAI out of stock

NTIS PB #82-147372 $24.95

40 ADOLESCENT MARIJUANA ABUSERS AND THEIR FAMILIES. Herbert

Hendln, M.D., Ann Pollinger, Ph.D., Richard Ulman. Ph.D., and

Arthur Carr. Ph.D. NCDAI out of stock

GPO out of stock NTIS PB #82-133117 $18.95

41 PROBLEMS OF DRUG DEPENDENCE, 1981: PROCEEDINGS OF THE 43RD

ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS OF DRUG

DEPENDENCE, INC. Louis S. Harris, Ph.D., ed. NCDAI out of stock

Not available from GPO NTIS PB #82-190760 $42.95

118
42 THE ANALYSIS OF CANNABINOIOS IN BIOLOGICAL FLUIDS. Richard L.

Hawks, Ph.D., ed.

GPO Stock #017-024-01151-7 $5 NTIS PB #83-136044 $18.95

43 PROBLEMS OF DRUG DEPENDENCE, 1982: PROCEEDINGS OF THE 44TH

ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS OF DRUG

DEPENDENCE, INC. Louis S. Harris, Ph.D., ed. NCDAI out of stock

GPO out of stock NTIS PB #83-252-692/AS $42.95

44 MARIJUANA EFFECTS ON THE ENDOCRINE AND REPRODUCTIVE SYSTEMS.

Monique C. Braude. Ph.D., and Jacqueline P. Ludford, M.S., eds. A

RAUS Review Report. NCDAI out of stock

GPO Stock #017-024-01202-5 $4 NTIS PB #85-150563/AS $18.95

45 CONTEMPORARY RESEARCH IN PAIN AND ANALGESIA, 1983. Roger M.

Brown, Ph.D.; Theodore M. Pinkert, M.D., J.D.; and Jacqueline P.

Ludford, M.S., eds. A RAUS Review Report. NCDAI out of stock

GPO Stock #017-024-01191-6 $2.75 NTIS PB #84-184670/AS $13.95

46 BEHAVIORAL INTERVENTION TECHNIQUES IN DRUG ABUSE TREATMENT. John

Grabowski, Ph.D .; Maxine L. Stitzer. Ph.D.. and Jack E.

Henningfleld, Ph.D.. eds.

GPO Stock #0l7-024-01192-4 $4.25 NTIS PB #84-184688/AS $18.95

47 PREVENTING ADOLESCENT DRUG ABUSE: INTERVENTION STRATEGIES.

Thomas J. Glynn, Ph.D .; Carl G. Leukefeld, D.S.W.; and

Jacqueline P. Ludford, M.S.. eds. A RAUS Review Report.

GPO Stock #0l7-024-01180-l $5.50 NTIS PB #85-159663/AS $24.95

48 MEASUREMENT IN THE ANALYSIS AND TREATMENT OF SMOKING BEHAVIOR.

John Grabowski, Ph.D.. and Catherine S. Bell, M.S., eds.

GPO Stock #017-024-01181-9 $4.50 NCDAI out of stock

NTIS PB 84-145-184 $18.95

49 PROBLEMS OF DRUG DEPENDENCE, 1983: PROCEEDINGS OF THE 45TH

ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS OF DRUG

DEPENDENCE, INC. Louis S. Harris, Ph.D., ed. NCDAI out of stock

GPO Stock #017-024-01198-3 $12 NTIS PB 85-151553/AS $36.95

50 COCAINE: PHARMACOLOGY, EFFECTS, AND TREATMENT OF ABUSE. John

Grabowski. Ph.D., ed.

GPO Stock #0l7-024-01214-9 $4.50 NTIS PB 85-150381/AS $18.95

51 DRUG ABUSE TREATMENT EVALUATION: STRATEGIES, PROGRESS, AND

PROSPECTS. Frank M. Tims, Ph.D., ed.

GPO Stock #017-024-01218-1 $4.50 NTIS PB 85-150365/AS $18.95

52 TESTING DRUGS FOR PHYSICAL DEPENDENCE POTENTIAL AND ABUSE

LIABILITY. Joseph V. Brady, Ph.D., and Scott E. Lukas, Ph.D., eds.

GPO Stock #017-024-0204-1 $4.25 NTIS PB 85-150373/AS $18.95

53 PHARMACOLOGICAL ADJUNCTS IN SMOKING CESSATION. John Grabowski,

Ph.D., and Sharon M. Hall, Ph.D., eds.

GPO Stock #017-024-01266-1 $3.50 NCDAI out of stock

119
54 MECHANISMS OF TOLERANCE AND DEPENDENCE. Charles Wm. Sharp,

Ph.D.. ed. NCDAI out of stock

GPO Stock #017-024-01213-1 $8.50

55 PROBLEMS OF DRUG DEPENDENCE, 1984. PROCEEDINGS OF THE 46TH

ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS OF DRUG

DEPENDENCE, INC. Louis S. Harris, Ph.D., ed. NCDAI out of stock

GPO Stock #017-024-01242-4 $9.50

56 ETIOLOGY OF DRUG ABUSE: IMPLICATIONS FOR PREVENTION. Coryl LaRue

Jones, Ph.D., and Robert J. Battjes, D.S.W., eds.

GPO Stock #0l7-024-01250-5 $6.50

57 SELF-REPORT METHODS OF ESTIMATING DRUG USE: MEETING CURRENT

CHALLENGES TO VALIDITY. Beatrice A. Rouse, Ph.D., Nicholas J.

Kozel. M.S.. and Louise G. Richards. Ph.D., eds.

GPO Stock #017-024-01246-7 $4.25 NCDAI out of stock

58 PROGRESS IN THE DEVELOPMENT OF COST-EFFECTIVE TREATMENT FOR DRUG

ABUSERS. Rebecca S. Ashery, D.S.W.. ed.

GPO Stock #017-024-01247-5 $4.25 NCDAI out of stock

59 CURRENT RESEARCH ON THE CONSEQUENCES OF MATERNAL DRUG ABUSE.

Theodore M. Pinkert, M.D., J.D., ed.

GPO Stock #017-024-01249-1 $2.50

60 PRENATAL DRUG EXPOSURE: KINETICS AND DYNAMICS. C. Nora Chiang.

Ph.D., and Charles C. Lee, Ph.D., eds.

GPO Stock #017-024-01257-2 $3.50

61 COCAINE USE IN AMERICA: EPIDEMIOLOGIC AND CLINICAL PERSPECTIVES.

Nicholas J. Kozel. M.S., and Edgar H. Adams, M.S., eds.

GPO Stock #017-024-01258-1 $5

62 NEUROSCIENCE METHODS IN DRUG ABUSE RESEARCH. Roger M. Brown,

Ph.D., and David P. Friedman, Ph.D., eds.

GPO Stock #017-024-01260-2 $3.50

63 PREVENTION RESEARCH: DETERRING DRUG ABUSE AMONG CHILDREN AND

ADOLESCENTS. Catherine S. Bell, M.S., and Robert Battjes.

D.S.W.. eds.

GPO Stock #017-024-01263-7 $5.50

64 PHENCYCLIDINE: AN UPDATE. Doris H. Clouet, Ph.D., ed.

GPO Stock #017-024-01281-5 $6.50

65 WOMEN AND DRUGS: A NEW ERA FOR RESEARCH. Barbara A. Ray, Ph.D.,

and Monique C. Braude, Ph.D., eds.

GPO Stock #017-024-01283-1 $3.25

120
66 GENETIC AND BIOLOGICAL MARKERS IN DRUG ABUSE AND ALCOHOLISM.

Monique C. Braude, Ph.D., and Helen M. Chao, Ph.D. eds.

GPO Stock #017-024-01291-2 $3.50 NCDAI out of stock

67 PROBLEMS OF DRUG DEPENDENCE, 1985. PROCEEDINGS OF THE 47TH

ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS OF DRUG

DEPENDENCE, INC. Louis S. Harris, Ph.D., ed. NCDAI out of stock

GPO Stock #017-024-01292-1 $18

68 STRATEGIES FOR RESEARCH ON THE INTERACTIONS OF DRUGS OF ABUSE.

Monique C. Braude. Ph.D., and Harold M. Ginzburg. M.D., J.D., M.P.H.,

eds.

GPO Stock #0l7-024-01296-3 $6.50 NCDAI out of stock

69 OPIOID PEPTIDES: MEDICINAL CHEMISTRY. Rao S. Rapaka, Ph.D.;

Gene Barnett, Ph.D .; and Richard L. Hawks, Ph.D., eds.

GPO Stock #017-024-1297-1 $11

70 OPIOID PEPTIDES: MOLECULAR PHARMACOLOGY, BIOSYNTHESIS, AND

ANALYSIS. Rao S. Rapaka, Ph.D., and Richard L. Hawks, Ph.D., eds.

GPO Stock #017-024-1298-0 $12

71 OPIATE RECEPTOR SUBTYPES AND BRAIN FUNCTION. Roger M. Brown,

Ph.D.; Doris H. Clouet. Ph.D .; and David P. Friedman, Ph.D., eds.

GPO Stock #017-024-01303-0 $6

72 RELAPSE AND RECOVERY IN DRUG ABUSE. Frank M. Tims, Ph.D., and

Carl G. Leukefeld, D.S.W.. eds.

GPO Stock #017-024-01302-1 $6

73 URINE TESTING FOR DRUGS OF ABUSE. Richard L. Hawks, Ph.D., and

C. Nora Chiang. Ph.D., eds.

GPO Stock #017-024-1313-7 $3.75

74 NEUROBIOLOGY OF BEHAVIORAL CONTROL IN DRUG ABUSE. Stephen I.

Szara. M.D., D.Sc.. ed.

GPO Stock #017-024-1314-5 $3.75

75 PROGRESS IN OPIOID RESEARCH. PROCEEDINGS OF THE 1986 INTERNATIONAL NARCOTICS RESEARCH CONFERENCE. John W. Holaday, Ph.D.;

Ping-Yee Law, Ph.D.; and Albert Herz, M.D., eds.

GPO Stock #017-024-01315-3 $21

76 PROBLEMS OF DRUG DEPENDENCE, 1986. PROCEEDINGS OF THE 48TH

ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS OF DRUG

DEPENDENCE, INC. Louis S. Harris, Ph.D., ed.

GPO Stock #017-024-1316-1 $16

IN PRESS

77 ADOLESCENT DRUG ABUSE: ANALYSES OF TREATMENT RESEARCH.

Elizabeth R. Rahdert, Ph.D. and John Grabowski, Ph.D., eds.

121
DHHS Publication No. (ADM)87-1481

Printed 1986 Reprinted 1987

Alcohol, Drug Abuse and Mental Health Administration