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Journal of Health Science, 57(6) 472–487 (2011)— Review —Analysis of Drugs of Abuse in Biological Specimens
Koichi Saito,∗ Rieko Saito, Yuu Kikuchi, Yusuke Iwasaki, Rie Ito,and Hiroyuki Nakazawa
Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, Hoshi University, 2–4–41 Ebara, Shinagawa-ku, Tokyo
142–8501, Japan(Received September 1, 2011)

Recent reports of the analysis of drugs of abuse in biological specimens are reviewed herein. Different perspectives from reviews so far published and the need for and the background of drug analysis in biological specimens,
and difficulty of drug testing in biological specimens are introduced. Comprehensive biological specimens for the
analysis of drugs of abuse in forensic science, including oral fluid (saliva), hair, umbilical cord, placenta, meconium, cadaver tissue (brain, adipose), sweat, breath, and nail clippings, in addition to the commonly used blood andurine specimens in clinical chemistry, are described along with their outlines, advantages/disadvantages, and actual
examples. Today, liquid chromatography-tandem mass spectrometry (LC-MS/MS) is the method of choice for the
analysis of drugs of abuse. A simultaneous screening method for multiple types of drugs has also become popular
recently. However, because qualitative determination remains important in forensic science, gas chromatographymass spectrometry (GC-MS) is still in use even if it requires complicated specimen preparation and derivatization
procedures. This is because GC-MS is reliable and has been employed ever since for the appraisal of trials.
Key words —— drug of abuse, forensic science, biological specimen, liquid chromatography-tandem mass spectrometry, gas chromatography-mass spectrometry

INTRODUCTION
Drugs are classified into legal drugs and illegal (or illicit) drugs, and the abuse of drugs has become one of the most serious social problems in theworld. In Japan, 32 psychotropic substances werelisted as designated substances by the Pharmaceutical Affairs Law in April 2007 to prevent abuseof those substances. Subsequently, other psychoactive compounds were added and as of July 2009,
40 substances are listed as designated substances.
However, an annual survey has indicated that new
designer drugs still find their way into illegal drug
markets.1)
The Japanese government is looking into specifying the part of a chemical structure that produces
a narcotic effect and regulating drugs having chemical structures similar to those of abused drugs, asnarcotics. However, the problem of designer drugs∗

To whom correspondence should be addressed: Departmentof Analytical Chemistry, Faculty of Pharmaceutical Sciences,
Hoshi University, 2–4–41 Ebara, Shinagawa-ku, Tokyo 142–
8501, Japan. Tel.: +81-3-5498-5764; Fax: +81-3-5498-5764;
E-mail: k-saitok@hoshi.ac.jp

cannot be solved to this day. Clearly, there is an urgent need to develop analytical methods that would
identify such drugs in order to prevent the increasing incidence of drug abuse. Major representative
abused drugs are designated as narcotics or stimulants and the possession of such drugs is regulated.
Numerous publications have described the analysis of amphetamine-like drugs and demonstrated
the significance of improving the methods of analysis of such drugs in biological specimens. In thelast decade, liquid chromatography coupled to mass
spectrometry (LC-MS) has seen rapid development
for forensic and clinical applications.2– 4) LC-MS
offers high sensitivity and specificity and reduces
the specimen preparation time compared with gas
chromatography-mass spectrometry (GC-MS) because relatively non-volatile compounds can be analyzed and no derivatization is necessary.
Many reviews have focused on the drugs ofabuse themselves,5–8) specific biological specimens, such as hair,9, 10) the methodology,11, 12) or the
analytical instruments.2– 4)
In this review, on the basis of reports publishedin the last three years (2008–2010) describing theC 2011 The Pharmaceutical Society of Japan

No. 6analyses of drugs of abuse in biological specimens,
different perspectives, such as the need for and thebackground of drug analysis in biological specimens, and difficulty of drug testing in biological
specimens, are introduced together with some actual examples. Furthermore, comprehensive biological specimens for the analysis of drugs of abuse inforensic science, including oral fluid (saliva), hair,umbilical cord, placenta, meconium, cadaver tissue(brain, adipose), sweat, breath, and nail clippings,
in addition to the commonly used blood and urine
specimens in clinical chemistry, are described alongwith their outlines. At that time, amphetamines, cocaine, opiates, and cannabis were selected as the target drugs because they are the most frequently usedillicit drugs.

NEED FOR AND BACKGROUND OFDRUG ANALYSIS IN BIOLOGICAL
SPECIMENS
Drug analysis in biological specimens, such as
urine, hair, blood, sweat, or oral fluid (saliva), which
is used to determine the presence or absence ofspecified parent drugs or their metabolites, is necessary in forensic science. The background of drug
analysis in biological specimens is shown below.
Testing for Drugs of Abuse in Saliva of Drivers
In forensic science, the study of the detectionof drugs of abuse extends to different fields. In theinterest of road safety, driving under the influenceof drugs has social and legal repercussions and isa cause of grave concern. Drugged drivers pose aserious threat to other drivers as well as to themselves.13) To obtain information about drug abuse indrivers, a comprehensive screening test for such drugs
is necessary, even under normal situations where
the drivers are not involved in any accidents.14)
In Sweden, a person’s driving license is suspended after being convicted for petty drug offense
or driving under the influence of drugs.15) To regain
the license, the person has to prove that he or she
has been drug-free during a specified observation
period. Reliable oral fluid screening devices for onsite screening of drugged drivers are both useful andconvenient for maintaining road safety.13)
Fernández et al.14) screened 632 saliva specimens collected from drivers in Catalonia, Spainfor drugs of abuse through an immunoassay test

473
R
(Cozartkit; Cozart Bioscience Ltd., Abingdon,
UK) and the drugs detected by the screening test
were cocaine, cannabis (Δ9 -tetrahydrocannabinol),
opiates (6-monoacetylmorphine), amphetamine,and metamphetamine. Specimens that were tested
positive were subjected to GC-MS for confirmation.
As a result, more than 80% of the specimens werepositive for a combination of two drugs. The mostcommon combination was cocaine and cannabis.

Workplace Drug Testing
Workplace drug testing is a well-established application of forensic toxicology and its aim is to reduce workplace accidents caused by employees who
are working under the influence of drugs or alcohol.16) Several drugs of abuse may be involved, including alcohol, amphetamines, cannabis, cocaine,
opiates, and prescription drugs, such as benzodiazepines. The use of alternative biological specimens, such as hair, oral fluid or sweat, in workplace drug testing presents several advantages overurinalysis, mainly because specimen collection canbe performed easily without infringing on the workers’ privacy. However, the drugs are usually present
in these alternative specimens at low concentrations
and the amount of specimen available for analysis
is small. In this regard, the use of highly sensitivetechniques is necessary.
Drug Abuse among College Students
Drug abuse among college students is characterized by low academic performance and longterm negative consequences.17) Screening for students who are at a high risk of drug abuse is of primary importance to ensure early identification andappropriate care.
The study of Garcia-Jimenez et al.17) aimed todetermine current or past use of drugs of abuse
through a questionnaire distributed to students at
the Universidad Autónoma del Estado de Morelos. The actual test results of that study showedthat 16% of the specimens were positive for Δ9 tetrahydrocannabinolic acid-A (Δ9 -THCA-A). The
results were not different from those of the National
Questionnaire on Addiction. On the basis of thatfinding, they established a program for detecting
drug abuse in students. This type of study is important in order to implement programs to decrease
the incidence of drug abuse in college students.
Drug-induced Seizures
Approximately 6% of new-onset seizures are474

said to be drug-related. However, there is currently
no reliable way to determine whether a seizure isindeed drug-induced or not. Drees et al.18) investigated whether the results obtained by liquid
chromatography-tandem mass spectrometry (LCMS/MS) from a seizure panel could affect patientcare as LC-MS/MS is a powerful tool that allows
the simultaneous detection of numerous analytes ofdiverse chemical nature in patient specimens. On
analysis of 157 serum or plasma specimens frompatients who underwent seizures, 17 were found to
be positive for a drug of abuse on the seizure panel.
The team of experts determined that the test results
probably or definitely would have affected treatment
in seven of those cases. Therefore, a test that detects
the presence of drugs implicated in drug-induced
seizures can help physicians determine if an unexplained seizure is drug-related, thus leading to potentially better direct patient care.
Body Packing
The illicit transport of cocaine and heroin, either swallowed or inserted into the rectum and/or
vagina of individuals, which is defined as “bodypacking,” is becoming an increasingly common occurrence. The assessment of smuggling by bodypackers through urinalysis has been little reported
and on-site rapid screening methods are essentiallylacking. Marchei et al.19) screened for cocaine andheroin metabolites in the urine specimens of suspected body packers with an on-site immunochromatographic test and confirmed the obtained results
by GC-MS and X-ray examination. From the obtained results, the on-site detection of cocaine andheroin metabolites in the urine specimens of suspected body packers appears to be a reliable screening test to disclose internally concealed drugs andjustify subsequent radiological investigations.

DIFFICULTY OF DRUG TESTING IN
BIOLOGICAL SPECIMENS
When a drug is taken, the drug itself and/or its
metabolites are excreted in the urine. Urinary concentration corresponds to the non-metabolized drug
and its metabolites at the time of specimen collection. Meanwhile, hair is known to reflect the accumulation of drugs. Thus, in the case of an arrest for
drug abuse, “use up to the last minute” can be determined by analyzing urine specimens and chronicVol. 57 (2011)use over the past few months can be determined byanalyzing hair specimens.
In Japan, news of drug abuse among celebrities
has appeared. Some have tried to conceal past useof drugs by cutting their hair. The difficulty of drug
testing in biological specimens arises from not only
the technical problem that the residual level is extremely low, but also the problem of multiple drug
use and thus, the test results may not match the selfreport made by such abusers of the drugs they used.
Discrepancies between simple test methods,
such as biological assays, and the self-report of illicit drug use could undermine epidemiological research findings. Ledgerwood et al.20) examinedthe extent of agreement between self-reported illicit
drug use and the results of hair analysis in a community sample of middle-aged men and tried to identify factors that may be used to predict the discrepancies between self-report and hair analysis. Hair
specimens were analyzed for marijuana, cocaine,
opiates, phencyclidine, and methamphetamine using radioimmunoassay (RIA) and GC-MS techniques. As a result, self-report and the results ofhair analysis generally showed good, but not excellent, agreement.

BIOLOGICAL SPECIMENS COMMONLYUSED IN FORENSIC SCIENCE
Blood and urine are the typical specimens foranalysis in clinical chemistry. However, in addition
to these, a variety of biological specimens are analyzed in forensic science. In this review, as the major specimens used in the analysis of drugs of abuse
for the period of 2008–2010, blood, urine, oral fluid(saliva), hair, umbilical cord, placenta, meconium,
tissue (adipose and brain), sweat, nail clippings, andbreath will be taken up.
Urine
There are a great many studies that use urine
as the biological specimen. As drugs for analysis,
amphetamine-type stimulants21–26) and cocaine andits metabolites27, 28) have been used. Amphetaminetype stimulants, such as amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine(MDA), and 3,4-methylenedioxymethamphetamine(MDMA), are known as central nervous system
stimulants and their abuse has recently increased
throughout the world.

No. 6

It is presumed that the increase in the numberof drug analysis examples is correlated to the increase in the number of criminal cases related todrug abuse. Notable progress in the overall analysis of drugs of abuse in biological specimens would
be the fruit of the development of numerous LCMS (/MS) methods. The successful interface of
LC-MS has brought new light to bioanalytical andforensic sciences as it allows the detection of drugs
and metabolites at concentrations that are difficult
to analyze with commonly used GC-MS based techniques.16)
LC-MS/MS has become very popular and today, it is the leading tool for the simultaneous multicomponent analysis of drugs of abuse. However,in the analysis of amphetamine-type stimulants inurine specimens in particular, a method that uses
GC-MS and requires either acetylation or pentafluorobenzyl (PFB) derivatization is also very much inuse. This is because the method that uses GC-MS isreliable and has been employed ever since in forensic science.
On the other hand, in screening tests for thetarget drug and its metabolites, the LC-MS (/MS)
method is advantageous. More drug metabolites exist in urine than in any other biological specimens.
However, in the case of drug abusers who use multiple similar drugs, it is difficult to determine thetypes of drugs used.
For example, as it is difficult to physicallydistinguish amphetamine-type stimulants, analysts
may not be aware of the drug taken by drug abusers.
Therefore, a rapid and sensitive method that uses
LC-MS/MS was developed for the identificationand classification of amphetamine-type stimulants
used by drug abusers.24) This method detects specific metabolites [such as p-hydroxy amphetamine,p-hydroxy methamphetamine, p-hydroxy N,Ndimethylamphetamine (DMA), and DMA N-oxide]of amphetamine, methamphetamine, and DMA
as biomarkers in drug abusers’ urine specimens.
The determination of p-hydroxy amphetamine,p-hydroxy methamphetamine, amphetamine, methamphetamine, DMA, and DMA N-oxide was accurate and reproducible, with the limit of quantitation being 5 ng/ml in urine. When applied to urine
specimens of suspected amphetamine-type stimulant abusers, the drugs used by methamphetamineand DMA abusers were precisely identified.
The utility of LC-MS/MS in cocaine urinalysis
is also remarkable. Cocaine is a potent central nervous system stimulant that is metabolized to ben-475

zoylecgonine and further to minor metabolites, suchas m-hydroxybenzoylecgonine. Cocaine is also metabolized to norcocaine. Cocaethylene is formed
when cocaine and ethanol are taken simultaneously.
Anhydroecgonine methyl ester is a unique marker incocaine smokers, and anhydroecgonine ethyl ester
is found in cocaine smokers who also use ethanol.
Numerous methods are available for the identification of cocaine or its metabolites in urine.27–29)
Langman et al.28) developed an LC-MS/MS method
for the detection and quantitation of cocaine,benzoylecgonine, norcocaine, cocaethylene, mhydroxybenzoylecgonine, anhydroecgonine methyl
ester, and anhydroecgonine ethyl ester in urine.
Urine specimens (200 individuals) were cleaned bysolid-phase extraction (SPE) and then subjected to
LC-MS/MS measurement and confirmation by GCMS. The results of GC-MS and LC-MS/MS measurements of cocaine and benzoylecgonine showed
good correlation.
Shakleya et al.30) reported that opiates, cocaine,and their metabolites were quantified by LC-MS
in 284 urine specimens collected thrice weekly, tomonitor possible drug relapse in 15 drug-dependent
pregnant women. As a result, 165 urine specimens(58%) from all the 15 subjects were positive forone or more cocaine analytes. Ecgonine methyl ester and/or benzoylecgonine were the major cocaine
biomarkers in 142. Anhydroecgonine methyl ester, a biomarker in cocaine smokers, was positivein six and cocaethylene and/or ecgonine ethyl ester, biomarkers of cocaine and ethanol co-ingestion,
were found in 25.
A simple deproteinization step involves mixing each specimen with an acetonitrile-internal standard mixture.29) The method has excellent precisionacross a wide linear range (i.e., 25–2000 ng/ml) for
each analyte. Clearly, the LC-MS/MS method for
urinalysis is a significant improvement over conventional GC-MS methods. Another point of interestis that different patterns of cocaine biomarkers wereobserved after LC-MS as compared to GC-MS analysis.
As other examples of urinalysis, the 2-(2,5dimethoxy-4-(methylthio)phenyl)ethanamine (2C-T)
series,31) the 2-(2,5-dimethoxy-4-methylsulfanylphenyl)-1-methylethylamine (ALEPH) series,32)
and cannabinoid33) analysis have been reported.
The 2C-T series and the ALEPH series are
psychedelic phenethylamines of the 2C family. 2C
is a general name for the family of psychedelic
phenethylamines containing methoxy groups on

476

positions 2 and 5 of the benzene ring. 2C-T-4
is the 2-carbon homologue of ALEPH-4 and its
full chemical name is 2-4-(isopropylthio)-2,5dimethoxyphenylethanamine. The structural andpharmacodynamic properties of 2C-T-4 are similar
to those of 2C-T-7 and 2C-T-9.
An analytical procedure for the simultaneous determination of four thiophenethylamine designer drugs (2C-T series) in human urine has
been reported. Quantitative analysis was performed by capillary electrophoresis with mass
spectrometry (CE-MS),31) using 2,5-dimethoxy-4methylthiophenethylamine-D4 as the internal standard. In order to minimize interference by matrix
components and preconcentrate the target analytes,
SPE was introduced as a cleanup step. The method
was validated according to international guidelinesand demonstrated to be specific, sensitive, and reliable for the analysis of these derivatives in urine
specimens.
Hidvegi et al.33) tried to identify cannabigerol(CBG), which, in its acid form, is one of themain intermediates in the biosynthesis of cannabinoids in hemp, in urine specimens of confirmed cannabis users. It was shown that enzymatic hydrolysis is necessary for the formation of free neutral cannabinoids from conjugates.
After extraction, derivatization with N-methyl-N(trimethylsilyl)trifluoroacetamide (MSTFA) and/or
bis-trimethylsilyl derivatization, GC-MS was performed for the identification of CBG and its
metabolites in the urine specimens. It was shownthat CBG enters the body through cannabis smoking and is excreted in urine in the conjugated form,
similar to other neutral cannabinoids. The presenceof CBG metabolites, such as its glucuronated form,
was examined by analyzing the chromatograms ofhydrolyzed and trimethylsilylated extracts.
Oral Fluid (Saliva)
Some problems arise when urine specimens arecollected at several occasions. For instance, the riskof manipulation and the risk of false-negative urine
specimens are high. In addition, many suspecteddrug users find it difficult or embarrassing to urinate
while being observed.15)
The collection of oral fluid for drug analysis iseasy and non-invasive. Oral fluid has demonstrated
its usefulness for the analysis of drugs of abuse.
Laboratory tests of oral fluid for drugs of abuse havecontinued to expand in the workplace as well as inlegal and medical settings.34) One of the purposesVol. 57 (2011)

is to evaluate physical alterations in individuals suspected to be under the influence of drugs.
For instance, oral fluid is gaining prominence as
an alternative matrix for monitoring drugs of abusein the workplace, investigating criminal cases, andscreening drivers who may be driving under the influence of drugs. There is a great need for a reliable
and rapid on-site oral fluid test that can be used bypolice authorities to detect drivers suspected of being under the influence of drugs or alcohol. The
European Driving under the Influence of Drugs, Alcohol and Medicines (DRUID) project called for the
analysis of oral fluid specimens collected randomly
and anonymously at the roadside from drivers inDenmark from 2008 through 2009.35)
On the other hand, significant development has
occurred in oral fluid drug analysis in the last few
years. New pharmacokinetic studies have been conducted, optimal cutoffs have been proposed, andnew studies have examined the correlation between
oral fluid drug concentration and impairment.36)
A recent study, e.g., the detection of THCCOOH in oral fluid, can contribute to resolving theissue of false-positive results caused by passive exposure to marijuana.36)
Analytical methods for measuring multiple licit
and illicit drugs and metabolites in oral fluid require
high sensitivity, specificity, and accuracy. As thevolume available for analysis is limited, a comprehensive method is needed for the simultaneous measurement of multiple analytes in a single aliquot.
For instance, Fritch et al.37) reported thevalidation of a semi-automated method forthe simultaneous extraction, identification, andquantitation of 21 analytes in a single oral
fluid aliquot. The target compounds were amphetamine, methamphetamine, MDA, MDMA,
3,4-methylenedioxyethylamphetamine (MDEA),
pseudoephedrine,
cocaine,benzoylecgonine,
codeine, norcodeine, 6-acetylcodeine, morphine,
6-acetylmorphine, hydrocodone, norhydrocodone,
dihydrocodeine, hydromorphone, oxycodone,
noroxycodone, oxymorphone, and phencyclidine.
Oral fluid specimens were collected with theRdevice (OraSure Technologies, Inc.,
Intercept
Bethlehem, PA, USA) and subjected to SPE.
Thirty-nine patients were tested positive for various
combinations of licit and illicit drugs and their
metabolites.
There are many reports of the use of LC-MS/MS
for oral fluid specimens.38– 40) A screening system
to analyze multiple drugs simultaneously has be-

No. 6

come the mainstream. In this regard, matrix effects should be taken into consideration. Although
ion suppression and enhancement were observed forcocaine, opiates, and their metabolites, the performance of the LC-MS/MS method was not influenced by matrix effects when deuterated internal
standards were used.40)
In the case of urinalysis, in addition to LCMS/MS, recently, rapid test kits, such as immunoassay, have been widely used.14, 41) The target drugs ofthe test kits are marijuana, methamphetamine, cocaine, opiates, and so on. However, most of the target drugs require confirmatory tests by LC-MS/MS
and/or GC-MS, in case the immunoassay gives positive results.
So far, urine and saliva are the major specimens
for the analysis of drugs of abuse. However, sometimes, the use of both seems to be best. For example,
tests for sports doping fall under this category.
Stimulants are banned by the World AntiDoping Agency (WADA) if used “in competition.”As the analysis of stimulants is presently carried out
on urine specimens only, for better interpretation ofanalytical data, it may be useful to discriminate between early intake of the stimulant and administration specifically aimed at improving sport performance.
Strano-Rossi et al.42) investigated differences inthe excretion/disappearance of drugs between urine
and oral fluid, a specimen that can reflect plasma
concentration. As a result, both the absolute concentrations and their variations as a function oftime were markedly different between urine andoral fluid; drugs were eliminated much more slowly
from urine than from oral fluid.
In all those cases, the concentration of a drug
metabolite in urine was very low and the parent
compound was not detected at all. It was suggested
that it is indeed impossible to discriminate between
recent administration of small doses and past administration of larger doses, if one relies on only
urine data. In addition, the results also suggested
that the analysis of oral fluid could be used to successfully complement data obtained from urinalysis.
Hair
Urine or saliva sampling is noninvasive and has
been widely used because of ease of collection.
However, the main disadvantage of urinalysis is theshort window of detection. On the other hand, hairis becoming as important as urine and saliva as the477

specimen used in drug analysis.9, 10)Hair has become an important matrix for drug
analysis, complementing blood and urine specimens. The prolonged detection window makes hairanalysis suitable for the detection of exposure to illegal drugs for a period of up to 12 months.43) Hairsampling, which features easy sampling and minimal risk of manipulation, may therefore be a welcome alternative. The prolonged detection window
may also provide more reliable information to thephysician.44)
In order to evaluate the usefulness of hair specimens and investigate practical and interpretive problems or advantages with the use of hair specimens, Kronstrand et al.15) conducted hair analysis
for drugs prior to re-issuing drivers’ licenses as a
Swedish pilot study. Ninety-nine hair specimens
and 198 urine specimens were collected from 84 individuals during the 12-month study period. The
hair specimens were screened for 20 drugs with LCMS/MS and confirmation of positive results was accomplished with GC-MS or LC-MS/MS. As a result, hair analysis identified illegal drugs in sevendifferent occasions, whereas urinalysis failed to
identify any illegal drugs in all occasions. This indicates that the physician must have an understanding
and knowledge of the limitations of the screening
methods used.
An LC-MS/MS method was developed for thedetermination of ketamine (and its metabolite norketamine) and some amphetamines (amphetamine,
methamphetamine, MDA, and MDMA). Hair specimens were washed and pulverized and then extracted with aqueous formic acid solution. Deuterated analogs of the analytes were used as internal
standards for quantification. The analysis by LCMS/MS was simpler and faster than that by conventional GC-MS methods that usually require more laborious extraction procedures and, in most cases, anadditional derivatization process.45)
As hair cocaine concentration is a reliable
marker of exposure to the drug, an original liquid
chromatographic method has been developed forthe determination of cocaine in human hair specimens by HPLC with fluorescence detection.46) The
method was validated and successfully applied tohair specimens collected from cocaine users. Specimen pre-treatment was carried out by incubative
extraction with 0.1 M HCl, followed by SPE with
C2 cartridges. For detection, native cocaine fluorescence was monitored at 315 nm with excitation at
230 nm.

478

Miller et al. used phosphate buffer (pH 5.0) as
the optimum hair incubation medium because of thehigh stability of cocaine and 6-monoacetylmorphinein this buffer. Then, two different SPE methods
R(Agilent Technologies, Inc.,[Bond Elut Certify
R(UnitedSanta Clara, CA, USA) and Clean Screen
Chemical Technologies, Bristol, PA, USA)] were
employed for specimen cleanup before LC-MS/MS
analysis. However, the matrix effect, demonstrating
ion suppression, was observed in most analytes.47)
As regards the solution for extraction and incubation of hair specimens, other than phosphatebuffer, 25 mM formic acid: acetonitrile mixture(5 : 95),43) 0.1 M HCl,48) and methanol49) have beenused. Considering the influence of matrix effects,
deuterated internal standards were usually added
during the specimen extraction procedure.
Cocaine and its metabolites are included in the
proposed United States federal regulations for hairanalysis. The suggested cut-off concentration forthe metabolites is 50 pg/mg, which is difficult toachieve routinely with electron impact GC-MS.50)
This may be due to the inability to derivatize cocaethylene to improve its response and the disturbance in the detection of co-eluted norcocaine
and cocaethylene, or potentially similar ions as thederivatives of norcocaine and benzoylecgonine.
Barroso et al.51) developed and validated ananalytical method for the simultaneous determination of cocaine and its main metabolite, benzoylecgonine, in human hair specimens by GC-MS. After extraction of the hair matrix with a mixture ofmethanol/HCl (2 : 1) at 65◦ C for 3 hr, followed by
specimen cleanup by mixed-mode SPE, the extract
was analyzed by GC-MS after derivatization withMSTFA with 5% chlorotrimethylsilane. The developed method may be useful for the analysis ofcocaine and benzoylecgonine in hair specimens inforensic toxicology laboratories, taking into accountthe speed and sensitivity achieved with a single quadrupole MS that is available in most laboratories.
Unlike cocaine, the analysis of amphetaminesin hair specimens by GC-MS has been widelypracticed. As examples of specimens for analysis
using GC-MS, amphetamine-type stimulants52– 54)
and marijuana55, 56) are mentioned.
The procedure involves liquid-liquid extraction of hydrolyzed hair specimens spiked with deuterated internal standards, followed by derivatization with Nmethyl-bis(trifluoroacetamide) (MBTFA),52) heptafluorobutyric anhydride (HFBA),54) or perfluorooctanoyl chloride.53)

Vol. 57 (2011)

As regards cannabinoid analysis by GC-MS, asimilar alkaline hydrolysis of hair specimens was
employed, and for derivatization of the analytes,
MSTFA was used for silylation.55)
Hair analysis in forensic and clinical toxicology
has focused on drugs of abuse and comprehensive,
drug-class-independent screening methods based on
MS have not been applied to date.
Pelander et al.44) investigated a qualitative basic drug screening method for hair specimens thatuses liquid chromatography coupled to time-offlight mass spectrometry (LC/TOF-MS), which has
been developed and evaluated for forensic toxicological urinalysis. The method included alkaline hydrolysis and purification by mixed-mode SPE priorto LC/TOF-MS analysis. Identification was basedon accurate mass, isotopic pattern fit, and retention
time, if available. The drug classes identified included antidepressants, antipsychotics, antiepileptics, amphetamines, opioids, beta-blockers, a benzodiazepine, a hypnotic, a local anesthetic, anantiemetic, and an antipyretic analgesic.
Analysis of 32 hair specimens from deceased
drug addicts revealed the intake of 35 different
drugs, and the findings were in good agreementwith the findings in blood and urine specimens byother methods. Moreover, information about previous drug use, which is not evident in the analysis ofother matrices, was obtained in the majority of thecases. However, one apparent false-positive result
was identified.
Two points should be noted when conducting
hair analysis. First, discriminative analysis to differentiate the incorporation of abused drugs into hairfrom external contamination of the hair surface isnecessary. Hoelzle et al.57) investigated the utility of discriminative analysis in differentiating between incorporation of cocaine and its congeners
into hair and external contamination, when dealing
with compounds that are administered by sniffing
or inhalation (e.g., cocaine). The statistical discriminant analysis made a prediction for each cocainepositive hair specimen as regards the likelihood of
its belonging to the group of cocaine users or being
contaminated.
As the second point, in hair analysis, particularly cocaine analysis, there is a possibility ofcontamination during sample preparation and hairanalysis. In other words, “hair porosity” should
be considered in evaluating external contamination.
Hill et al.58) applied a method for determining hairporosity to hair specimens, and the porosity charac-

No. 6

teristics of hair are discussed in relation to experimental and actual contamination of hair.
Umbilical Cord, Placenta, and Meconium
As regards biological specimens specific topregnant women, the umbilical cord, placenta, andmeconium will be discussed as specimens for the
analysis of drugs of abuse. The exposure of pregnant women to recreational drugs, that is, in utero
drug exposure, may adversely impact fetal andneonatal development. In this regard, detecting drug
exposure during pregnancy is the most effective way
to improve pre- and post-natal care in the motherand the newborn as it permits implementation of adequate treatment and follow-up.59)
Umbilical Cord: The umbilical cord is considered to be a waste product after childbirth. Nevertheless, the umbilical cord also is suitable foranonymous epidemiological studies. Montgomery
et al.60) suggested that the umbilical cord could be
an alternative to meconium for detecting fetal drug
exposure after demonstrating that comparable results were obtained in those two matrices.
De Castro et al.59) developed and validated an
LC-MS method for the simultaneous quantificationof methadone, cocaine, opiates, and metabolites inumbilical cord tissue. In their method, homogenization of the specimen in acidic conditions and subsequent extraction with mixed-mode cation-exchange
cartridges allowed for adequate sensitivity and selectivity for quantification of the analytes of interest.
The quantification of methadone and its
metabolite 2-ethylidine-1,5-dimethyl-3,3-diphenylpyrrolidine in umbilical cord specimens fromwomen receiving known doses of methadone permits determination of potential correlations between
drug concentration in the umbilical cord and the administered dose, and whether umbilical cord drugconcentration can predict maternal and neonatal
outcomes.
In general, umbilical cord tissue is available inabundance and thus specimen acquisition is not an
issue. Therefore, the simultaneous determination ofother common drugs of abuse will help us understand the usefulness of umbilical cord analysis forthe identification of in utero drug exposure.
Jones et al.61) reported the use of LC-MS/MS
to confirm amphetamines in umbilical cord specimens presumed to be positive for amphetamine andmethamphetamine. The study using matched pairsof meconium and umbilical cord demonstrated a479high degree of agreement.
Placenta: The placenta may serve as an alternative specimen to urine for testing drugs of abuse
during the first trimester of gestation. The advantage of placenta tissue, obtained as discarded material in the event of pregnancy termination, over such
conventional matrices as urine and blood is that thecollection is almost noninvasive and relatively easy
to perform.
Joya et al.62) reported the development and validation of a method for the quantification of drugsof abuse, such as amphetamine, methamphetamine,
MDMA, methadone, cocaine, benzoylecgonine,
cocaethylene, morphine, 11-nor-9-carboxy-delta-9tetrahydrocannabinol, nicotine, and cotinine, in human placenta tissue, using GC-MS. Placenta specimens were stored at −80◦ C until analysis and theanalytes were stable even after three freeze-thaw cycles (stored at −20◦ C). The analytical method wasused to evaluate the effect of the consumption ofdrugs of abuse in women who voluntarily terminated their pregnancy at the 12th week of gestation.
The use of placenta as a non-conventional matrix
showed that it was able to provide information about
early in utero exposure to the drugs of abuse.
In another study, cocaine and its metabolites,
and opiates in placenta have also been analyzed
by LC-MS/MS. A method for the simultaneousquantification of methadone, cocaine, opiates, andmetabolites in human placenta by LC-MS was developed by De Castro et al.63) Specimens were homogenized and subjected to SPE in the conventional manner. The applicability of the method
was demonstrated in the analysis of five placenta
specimens from opioid-dependent women receiving methadone pharmacotherapy, with methadone
doses ranging from 65 to 95 mg on the day of delivery. The detection of drugs of abuse in the placenta
may also improve knowledge of the usefulness of
this matrix for detecting in utero drug exposure andstudying the disposition of drugs in the maternalfetal dyad.
Meconium: The meconium offers several advantages for detecting prenatal drug exposure andhas become the specimen of choice for the past
two decades. Accumulating substances from the12th gestational week until birth, the meconium
acts as a reservoir for exogenous compounds, suchas drugs and metabolites. Drugs are incorporated
into the meconium via fetal biliary excretion or
through ingestion of drug-contaminated amniotic
fluid. Meconium analysis is thought to enable the480

detection of maternal drug use during the second
and third trimesters of pregnancy, whereas neonatal hair and urine analyses indicate more recent exposure. Meconium is usually passed in the first 1
to 3 days of life but may be delayed in prematureinfants. Its complex composition of mucopolysaccharides, water, bile salts, bile acids, epithelial cells,
and other lipids makes drug isolation difficult.64)
Reviews by Gray et al.64) summarize current literature on meconium analysis. A brief overview isprovided as follows: illicit drugs, therapeutic drugs,
ethanol metabolites, pesticides, and other xenobiotics have been detected in meconium using conventional toxicological techniques, including immunoassay, and confirmed by GC-MS or LC-MS.
As regards analytical methodology, because of
the complexity of the meconium matrix, extraction and specimen preparation prior to instrumental
analysis may prove difficult and time-consuming.
Gunn et al.65) described a method for the rapid
extraction and quantification of amphetamine andmethamphetamine from meconium, which uses
SPE (ISOLUTER HM-N supported liquid extraction columns containing a modified form of diatomaceous earth; Biotage Inc., Charlottesville, VA,USA) and GC-MS.
Lopez et al.66) reported the case of a newborn with symptoms of hyperexcitability. Afterit was confirmed that the mother had consumed
drugs during pregnancy using an enzyme multiplied
immunoassay technique, the newborn’s urine andmeconium specimens were analyzed by GC-MS. A
GC-MS method for the simultaneous analysis of cocaine, benzoylecgonine, codeine, morphine, and 6acetylmorphine in meconium is described. Confirmation of urine and meconium results by GC-MS
showed consumption of cocaine and codeine during
pregnancy and the gradual decline of the levels of
those drugs in the newborn, totally disappearing by
the third day of life.
Gunn et al.67) described the simultaneous determination and quantification of cocaine and its major
metabolite, benzoylecgonine, in meconium using
ultra performance liquid chromatography (UPLC)MS/MS. The extraction of cocaine and benzoylecgonine from the homogenized meconium matrix
was achieved with a preliminary protein precipitation or protein “crash” procedure employing coldacetonitrile, followed by mixed-mode SPE. Following elution from the SPE cartridge, the eluents wereconcentrated and injected onto the UPLC-MS/MS
for analysis.

Vol. 57 (2011)

Tissue (Brain, Adipose)
Brain Tissue: Postmortem drug screening incases of presumed overdose as cause of death is routinely carried out in toxicology laboratories. The
most common specimen used for the analysis isblood due to its temporal relation with the effect of adrug on the individual and his/her status at the timeof death. Other specimens, such as vitreous humor,
saliva, and urine, are also analyzed to complement
blood analysis or in cases where blood extraction isdifficult for certain reasons.
There are solid data demonstrating that drugs
and their metabolites distribute mainly in tissues
rich in lipids, including brain and adipose. Braintissue provides excellent information relevant to thestatus of the individual and the possible physical alterations and conduct at the time of death.68) The
analysis of brain tissue specimens may provide suchvaluable information as the type of drug administered when a postmortem investigation is carriedout to determine the potential cause and manner ofdeath.69)
In the analysis of brain tissue specimens, SPEis carried out, followed by the semi-quantitative determination of cocaine levels by GC-MS/MS using
deuterated cocaine as the internal standard.68)
Shakleya et al.69) developed an LC-MS/MS
method for isolating drugs of abuse, such as cocaine, benzoylecgonine, cotinine, ecgonine methyl
ester, 6-acetylmorphine, trans-3-hydroxycotinine,
codeine, and nicotine, in brain tissue specimens.
On the other hand, Reich et al.70) pointed out thedifficulty of detecting drugs in brain tissue, which
typically requires extensive specimen preparation
followed by drug extraction before the extracts are
finally analyzed by LC-MS. Therefore, in order toeliminate any complications introduced by specimen pretreatment, a method for directly analyzing
drugs in intact tissue was developed.
Matrix-assisted laser desorption/ionization tandem mass spectrometry [MALDI-MS(n)] demonstrated the ability to quantify cocaine present inpostmortem brain tissue of a chronic human cocaine
user.70)
Adipose Tissue: Adipose tissue can be used todetect absorbed chemicals in living organisms, provided that the chemicals are highly lipophilic andbind to it.71) In mammals, an increased storage ofchemicals in adipose tissue indicates a longer exposure of the tissue to the chemicals even after the cessation of external exposure; that is, chemicals thatare adsorbed by the body tend to persist in adipose

No. 6tissue on the basis of their lipophilicity. The extent
of chemical accumulation in adipose tissue is correlated with the adipose tissue/blood partition coefficient. For instance, THC is highly soluble in lipids
and rapidly distributed in tissues, including adipose
tissue.
Adipose tissue is also an optimal specimen incases with extended postmortem time lag and whenever body fluids cannot be obtained. Similar to hairspecimens, adipose tissue provides long-term information about an individual’s drug use, which is especially useful when the history of drug abuse is noteasily obtained. Drugs identified in postmortem adipose tissue are accumulated because of antemortem
deposition and not any postmortem changes.71) In
this connection, adipose tissue may be an optimal
matrix in forensic toxicology.
Unfortunately, adipose tissue is a complex
biological matrix that necessitates several preanalytical preparation steps to separate drugs andmetabolites from the lipophilic matrix. Adipose tissue is not considered to be an optimal specimen in
analytical toxicology, primarily because of the difficulties in specimen handling and processing forcleanup. It is for these reasons that few case reports
on drug detection in postmortem adipose tissue havebeen published.
Colucci et al.71) developed a GC-MS method
for the determination of cocaine, its metabolites,
methadone, and morphine in postmortem adipose
tissue. The method involves the aqueous acidextraction of analytes, the alkalinization of theextract, SPE with chloroform, and derivatization
with N,O-bis(trimethylsilyl)trifluoroacetamide before GC-MS analysis. Deuterated compounds wereused as internal standards for the determination andquantification of analytes.
Sweat, Breath, and Nail Clippings
Next, relatively minor specimens for the analysis of drugs of abuse are introduced.
Sweat: Sweat has attracted attention in recent
years as an alternative matrix in forensic toxicology as it can be sampled easily and non-invasively.
Sweat can be collected with patches over a long
period, thereby permitting drug monitoring over alonger period than urine.
Concheiro et al.72) developed an LC-MS/MS
method for the quantification of buprenorphine,
norbuprenorphine, methadone, 2-ethylidene-1,5dimethyl-3,3-diphenylpyrrolidine, cocaine, benzoylecgonine, ecgonine methyl ester, morphine,481

codeine, 6-acetylmorphine, heroin, 6-acetylcodeine,
cotinine, and trans-3 -hydroxycotinine in sweat
specimens. Buprenorphine and methadone are
medications used in the clinical setting to help
opiate-addicted individuals withdraw from and stopusing drugs. Sweat patches were mixed with acetate
buffer at pH 4.5 and the supernatant was subjected
to SPE (Strata-XC-cartridges, Phenomenex, Inc.,
Torrance, CA, USA). The method was applied tosweat patches collected weekly from an opioiddependent buprenorphine-maintained pregnant
woman. As a result, buprenorphine, cocaine,
opiates, and methadone were detected from thesweat specimens.
Breath: Berchtold et al.73) tried to detect suchnarcotics as morphine, fentanyl, norfentanyl, naloxone, cocaine, hydroxybutyrolactone, and nicotine
from breath. They challenged the diagnosis byonline breath analysis using MS, because of the
low concentrations of the pertinent compounds inbreath.
Nail Clippings: Nail clippings were used as analytical specimens for the detection and quantitationof illicit drugs.
Kim et al.74) developed a GC-MS method forthe simultaneous qualification and quantification ofamphetamine, methamphetamine, MDA, MDMA,
ketamine, and norketamine in nail clippings. Similar to hair analysis, methods for the surface decontamination and extraction of the specimens are critical for the analysis. Water and methanol washes,
followed by alkaline hydrolysis and liquid-liquid
extraction with ethyl acetate, were selected for use
in the study. The extracts were evaporated to dryness, derivatized with HFBA at 60◦ C for 30 min,and analyzed by GC-MS. The results indicated thatnail clippings are potentially useful specimens forthe detection of past illicit drug use.
Although it is not a recent report, Lemos et al.75)
demonstrated the analysis of nail clippings for drugsof abuse, i.e., they determined cannabis in the nailclipping specimens by RIA and GC-MS.
Dried Blood Spot
For the last section of biological specimens, wewould like to discuss “blood.” Blood is the mostimportant and versatile specimen for drug analysis
not only in clinical chemistry, but also in forensic
science. In fact, in cases of the analysis of drugsof abuse, blood specimens have often been employed in detecting cocaine,76– 78) amphetamines,79)
and opiates.80)

482

Vol. 57 (2011)

Table 1. Overview List of Analytical Methods for Abused Drugs in Various Biological Specimens
Specimens

Analytes

Urine

amphetamines, 2Cs
amphetamines (TMA series)
amphetamines, ephedrines
amphetamines
amphetamines
amphetamines
cocainescocainescocainescocaines, opiates
2C-T series
ALEPH series
cannabinoids
amphetamines, cocaines, opiates,
cannabinoids, benzodiazepines
amphetamines, opiates
amphetamines, cocaines, opiates
amphetamines, cocaines, opiates,
benzodiazepines
cocaines, opiates
amphetamines, cocaines, opiates,
cannabinoids
amphetamines, cocaines, opiates
cannabinoids
amphetamines, cocaines, opiates,
benzodiazepines, nicotines
amphetamines, opiates,
benzodiazepines, others
amphetamines, opiates
cocaineamphetamines, cocaines, opiates,
benzodiazepines
cocainescocaines, opiates
cocaines
amphetamines
amphetamines
amphetamines, opiates
amphetamines, cocaines, opiates,
cannabinoids
cannabinoids
cocaines
amphetamines
opiates, cocaines
amphetamines, cocaines, opiates,
cannabinoids
amphetamines, cocaines, opiates,
cannabinoids
cocaines, opiates
amphetamines, cocaines, opiates
amphetamines
cocainescocaines, opiates
cocainescocaines, opiates, nicotines
cocainescocaines, opiates
cocaines, opiates, nicotines
cocaines, opiates, nicotines
amphetamines, opiates
cannabinoids
cocaines
amphetamines
amphetamines, opiates
cocainescocaines

Oral fluuid (saliva)

Hair

Unbilical cord

Placenta
Meconium

Tissue (Brain)
Tissue (Adipose)
Sweat
BreathNail clippings
Blood
Dried blood spot

Pre-treatment (extraction,cleanup, and/or derivatization)
SPE
SPE
SPE
filtration
LLE
SPE
SPE
SPE
deproteinaization
no-cleanup (direct injection)
SPESPE
enzymatic hydrolysis, silylation
SPE

Analysis Instruments

References

LC-MS
LC-MS
GC-MS
LC-MS/MS
GC-MS
GC-MS
LC-MS/MS
LC-MS/MS
LC-MS/MS
LC-MS
CE-MS
CE-DAD
GC-MS
LC-MS/MS

21
22
23
24
25
26
27
28
29
30
31
32
33
35

SPE
LLE, SPE
dilution

LC-MS/MS
LC-MS/MS
LC-MS/MS37
38
39

deproteinaization
no-cleanup

LC-MS/MS
immunoassay, GC-MS

40
14

no-cleanup
no-cleanupacid extraction

immunoassay, LC-MS/MS
immunoassay, GC-MS
LC-MS/MS

34
41
43

alkaline hydrolysis, SPE

LC-TOFMS

44

acid extraction
SPE
SPE

LC-MS/MS
LC-FL
LC-MS/MS

45
46
47

SPE
methanol extraction
SPE, sylilaton
alkaline hydrolysis, LLE, acylation
alkaline hydrolysis, LLE, acylation
LLE, acylation
SPME, sylilation

LC-MS/MS
LC-MS/MS
GC-MS
GC-MS
GC-MS
GC-MS
GC-MS

48
49
51
52
53
54
55

SPME
enzymatic digestion, SPESPESPE
no-cleanup

GC-MS/MS
LC-MS/MS
LC-MS/MS
LC-MS
immunoassay

56
58
61
59
60

SPE, sylilaton

GC-MS

62

SPE
SPE
SPE
SPE
no-cleanupSPESPE
no-cleanup
SPE, sylilatonSPEno-cleanup
alkaline hydrolysis, LLE, acylation
alkaline hydrolysis, LLE
SPE
deproteinaization
SPE
SPE
LLE

LC-MS
LC-MS/MS
GC-MS
LC-MS/MS
immunoassay, GC-MS
GC-MS
LC-MS/MS
MALDI-MS(n)GC-MSLC-MS/MSMS
GC-MS
RIA, GC-MS
LC-MS/MS
LC-MS/MSLC-UV, LC-FL
LC-MS/MS
LC-FL

63
64
65
67
66
68
69
70
71
72
73
74
75
78
79
80
76
77

amphetamines: including amphetamine derivatives and metabolites, cocaines: including cacaine derivatives and metabolites, opiates:
including natural and synthetic alkaloids, and their metabolites, cannabinoids: including >9 -THC and synthetic cannabinoids, 2Cs: a familyof psychedelic phenethylamines, 2C-T series: thiophenethylamine desinger drugs, ALEPH series: thioamphetamine desinger drugs, TMA
series: trimethoxy amphetamines, ephedrines: including related homologues, benzodiazepines: including related homologues, nicotines:
includeing metabolites, LLE: liquid-liquid extraction, SPME: solid-phase microextraction, CE-DAD: capillary electrophoresis with diodearray detector, LC-FL: liquid chromatography with fluorescence detector.

483

No. 6

However, blood per se is not interesting as a biological specimen. “Impregnated filter paper blood”
or the so-called “dried blood spots (DBS),” which
increase the shelf life of a drug in blood collectedin the field and facilitate transport and storage, arepresented here as examples of the analysis.
Alfazil et al.76) investigated the stability of benzodiazepines and cocaine in DBS. Previous studies have shown that drug concentrations in bloodcan change during storage especially at room temperature. However, even labile drugs, such as cocaine, may be stable in DBS. A new method has
been developed for the analysis of hydrolytically labile drugs in DBS in order to assess their degradation during a storage period of one month. As thetest drugs, flunitrazepam, temazepam, oxazepam,
lorazepam, nitrazepam, diazepam, and cocaine wereselected. A piece of filter paper was spotted with
100 µl of blood containing the drugs at the concentration of 1000 ng/ml and left overnight to dry atroom temperature. The drugs were then extracted
by suspending the filter paper in phosphate buffer.
The extract was subsequently cleaned up by SPE
and analyzed by LC-MS/MS. In their experiment,
DBS were stored in duplicate at room temperature,
4◦ C, and −20◦ C for up to one month. The degradation of the drugs in DBS at all storage conditions
was minimal compared to the degradation in thecorresponding liquid blood specimens stored undersimilar conditions and more than 80% of each analyte could be recovered from the specimens.
Mercolini et al.77) also employed DBS for the
analysis of cocaine and its two metabolites by liquid
chromatography with fluorescence detection. A solvent extraction technique was implemented for fast
and feasible specimen pre-treatment. The method
was successfully applied to DBS collected fromsome cocaine users, both with and without concomitant ethanol intake. The results were in good
agreement with those obtained from plasma specimens that were subjected to an original SPE procedure on C8 cartridges. The method was suitable formonitoring cocaine/ethanol use by means of DBS or
plasma testing.

CONCLUSION
Various biological specimens have been used in
the analysis of drugs of abuse in the field of forensic
science, as described in this review. Table 1 shows
the overview list of analytical methods for abused

drugs in various biological specimens mentioned inthis review. Although the analytical techniques usedin forensic science are very similar to those usedin clinical chemistry as well as pharmacokinetics
and metabolic monitoring, there are marked differences in the purpose, specimen, and time required
for analysis. In forensic science, because the analytical results greatly affect the outcome of criminal
investigations and court decisions, qualitative ability is thought to be more important than quantitative
ability. Of course, if cutoff values are set for drugsof abuse, high reliability of the quantitatively determined values is required.
Further advances in analytical instrumentation
have paved the way for the development of sophisticated trace analysis methods that enable possible
use of a specimen containing trace amounts of adrug and small amounts of specimen, such as hairand nail clippings. In the analysis of drugs of abuse,
the proliferation of designer drugs has become aserious problem and there is an ongoing cat-andmouse game between the police authorities and drug
abusers. By further advancing analytical methods
for drugs of abuse in the future, it is hoped that drug
abuse would be completely eradicated.

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