*

- REPORT NO. DOT-TSC-NHTSA-76-4 HS 802 208

MANUAL FOR ANALYSIS OF

ETHANOL I N BIOLOGICAL LIQUIDS

Kurt M. Dubowski

University of Oklahoma Health Sciences Center

College of Medicine

P.O. Box * 26901

Oklahoma City OK 73190

fat OF TRAAs

4 -

o ^^ z

^e Z

irfQ S44TES Ot P^^E

JANUARY 1977

FINAL REPORT

DOCUMENT IS AVAILABLE TO THE U.S. PUBLIC

THROUGH THE NATIONAL TECHNICAL

INFORMATION SERVICE, SPRINGFIELD,

VIRGINIA 22161

Prepared for

U.S. DEPARTMENT OF TRANSPORTATION

NATIONAL HIGHWAY TRAFFIC SAFETY ADMINISTRATION

RESEARCH AND DEVELOPMENT

Office of Driver and Pedestrian Research

Washington DC 20590

r.

*
 *

NOTICE

This document is disseminated under the sponsorship

of the Department of Transportation in the interest

of information exchange. The United States Government assumes no liability for its contents or use

thereof.

NOTICE

The United States Government does not endorse products or manufacturers. Trade or manufacturers'

names appear herein solely because they are considered essential to the object of this report.

 * 
It Technical Report Documentation Page 

.,s 1. Report No. 2. Government Accession No. 3. Recipient's Catalog No. 

r^ 

U HS 802 208 

4 4. Title and Subtitle S. Report Date 

January 1977

MANUAL FOR ANALYSIS OF ETHANOL IN BIOLOGICAL LIQUIDS 6. Performing Organization Code

8. Performing Organization Report No. 

7. AutFhorr s) 

DOT-TSC-NHTSA-76-4 Kurt M. Dubowski 

9. Performing Organization Name and Address 10. Work Unit No. (TRAIS) 

University of Oklahoma Health Sciences Center* HS602/R7403 

College of Medicine 11. Contract or Grant No. 

P.O. Box 26901 DOT-TSC-472

Oklahoma City OK 73190 13. Type of Report and Period Covered

12. ons ring Agency Name and Address 

U. S. department Final Report of Transportation 

National Highway Traffic Safety Administration July 1972 - February 1974 

Research and Development 

Office of Driver and Pedestrian Research 14. Sponsoring Agency Code 

Washington DC 20590 

15. Supplementary Notes U.S. Department of T ransportation 

*Under contract to: Transportation Systems Center

Kendall Square

Cambridge MA 02142

16. Abstract 

This manual covers selected aspects of the analysis of ethanol in biological

liquids and the interpretation of the results of such analyses. Recommendations

are made concerning the selection, collection, identification, and preservation

of suitable biological liquid specimens from living and dead subjects for traffic

law enforcement and related purposes. Procedural details are given for analysis

of ethanol in such biological liquids by both automated and manual versions of

gas chromatography of headspace vapors, with and without internal standards; and

the analytical performance characteristics of the method are set forth. Brief

consideration is also given to the interpretation of the results of alcohol an­

alysis in blood and other biological liquids.

17. Key Words 18. Distribution Statement 

Alcohol Analysis Gas Chromatography PUBLIC

B io l o g ical L i q uids Heads pace Analysi s THROUGH THE NATIONAL TECHNICAL

CAL

Blood-Alcohol INFORMATION SERVICE, SPRINGFIELD,

V r_ Ethanol VIRGINIA 22161

19. Security Classif. (of this report) 20. Security Classif. (of this page) 21. No. of Pages 22. Price 

Unclassified Unclassified 126 

Form DOT F 1700.7 (8-72) Reproduction of completed page authorized 
PREFACE 

This manual was prepared for the U. S. Department of Transportation as a contribution to efforts by the Department and practicing forensic scientists toward improvement of the state of alcohol analysis for traffic law enforcement 

and related purposes. Accordingly, it covers pertinent aspects of the selection, 

procurement, and retention of biological liquid specimens, of their analysis for 

alcohol, and of the interpretation of the findings. Details are given for a 

highly reliable and practical method of gas chromatographic headspace analysis 

for alcohol, the performance characteristics of which make it suitable for use 

as a reference or referee method as well as for routine applications. 

It is hoped that this information will assist forensic scientists everywhere 

in their important functions of performing valid and reliable analyses for 

alcohol in biological liquids and interpreting the results of such analyses 

for traffic law enforcement purposes, 

iii 
 *

METRIC CONVERSION FACTORS

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TABLE OF CONTENTS 

Section

Page 

1 INTRODUCTION ......................................... 1

2 SAMPLES AND SAMPLING FOR ALCOHOL ANALYSIS ............ 3

2.1

Selection of Specimens .......................... 3

-2.1.1 Living Subjects .......................... 4

2.1.2

Cadavers ................................. 7

2.2

Collection of Specimens ......................... 10

2.3

Identification of Specimens ................... 17

2.4

Preservation and Storage Specimens .............. 20

2.5

Preparation for Analysis ........................ 27

3 ANALYSIS OF BIOLOGICAL LIQUIDS FOR ETHANOL ........... 29

3.1

Principles ... ............................... 29

3.2

Automated Method ................................ 33

3.2.1

Materials: Apparatus, Reagents,

Supplies ................................. 33

3.2.2

Sumrhary of Operation ... ......... 34

3.2.3

Instrument and Analysis Conditions ....... 36

3.2.4

Procedure - Direct Analysis .............. 37

3.2.5

Procedure - Internal Standard Analysis... 39

3.3

Manual Method ................................... 41

3.3.1

Materials: Apparatus, Reagents,

Supplies .. ... ...................... 41

3.3.2

Summary of Operations .......... 43

3.3.3

Instrument and Analysis Conditions ....... 43

3.3.4

Procedure - Direct Analysis .. ... 44

3.3.5

Procedure - Internal Standard Analysis ... 46

3.4

Procedural Notes and Comments ................... 49

3.5

Analytical and Performance Characteristics of

the Method ...................................... 65

4 QUALITY ASSURANCE: SELECTED ASPECTS ................. 77

4.1

Reference Materials and Their Validation ........ 77

4.2

Performance Monitoring .. .. .................. 83

4.3

Other Quality Assurance Practices ............... 86

v 
TABLE OF-CONTENTS (CONTINUED)

Section

Page 

5' INTERPRETATION OF RESULTS .............................. 90

5.1

Blood-Alcohol Concentrations.... ................. 91

5.2Alcohol Concentrations in Other pecimen

Materials .... ................................. 99

5.3

Decomposed Specimens .............................. 102

APPENDIX - REPORT OF INVENTIONS ........................ 107

REFERENCES ............................................. 109

LIST OF ILLUSTRATIONS 

Figure

Page 

1

SPECIMEN IDENTIFICATION AND EXAMINATION REQUEST ........ 19

2

IN-VITRO ETHANOL PARTITION RATIOS FOR AIR/BLOOD

AND AIR/WATER SYSTEMS .................................. 32

3

PRINCIPLE OF HEADSPACE SAMPLING WITH THE MULTIFRACT

F-40 AUTOMATED GAS CHROMATOGRAPH ....................... 35

4STAMP FOR RECORDING PARTICULARS OF GAS CHROMATOGRAPHIC

ANALYSIS ............................................... 37

5

TIME COURSE OF GC DETECTOR RESPONSES TO ETHANOL AND

ACETONITRILE AND THEIR RATIO, SHOWING TIME REQUIRED

FOR EQUILIBRATION OF HEADSPACE VAPOR AT 65°C........... 55

6

TIME COURSE OF GC DETECTOR RESPONSES TO ETHANOL AND

ACETONITRILE, AND THEIR RATIO, SHOWING TIME REQUIRED

FOR EQUILIBRATION OF HEADSPACE VAPOR AT 38°C,.......... 55

7

EFFECT OF SAMPLE VOLUME IN 24-ML SEPTUM VIAL ON

DETECTOR RESPONSE TO ETHANOL WITH MULTIFRACT F-40

AUTOMATED GAS CHROMATOGRAPH ............................ 58

8

TYPICAL HEADSPACE CHROMATOGRAM OBTAINED WITH

MULTIFRACT F-40 AUTOMATED GAS CHROMATOGRAPH ............ 61

vi 
C LIST OF ILLUSTRATIONS (CONTINUED) 

s 

Figure Page 

9 TYPICAL HEADSPACE CHROMATOGRAM OBTAINED WITH 

MULTIFRACT F-40 AUTOMATED GAS CHROMATOGRAPH ............. 66 

10 HEADSPACE CHROMATOGRAMS OBTAINED WITH MANUAL METHOD.... 67 

11 TYPICAL CALIBRATION CURVES FOR ETHANOL WITH AUTOMATED 

GAS CHROMATOGRAPHIC HEADSPACE METHOD .................... 66 

12 LINEARITY OF DETECTOR RESPONSE TO ETHANOL WITH 

AUTOMATED GAS CHROMATOGRAPHIC HEADSPACE METHOD .......... 69 

13 CHROMATOGRAM ILLUSTRATING TYPICAL PRECISION OF 

REPLICATE ANALYSES FOR ETHANOL IN BLOOD WITH 

AUTOMATED GAS CHROMATOGRAPHIC HEADSPACE METHOD 

(DIRECT PROCEDURE) ..................................... 71 

14 CHROMATOGRAM ILLUSTRATING TYPICAL PRECISION OF 

REPLICATE ANALYSES FOR ETHANOL IN BLOOD WITH 

AUTOMATED GAS CHOMATOGRAPHIC HEADSPACE METHOD 

(INTERNAL STANDARD PROCEDURE) .......................... 71 

15 EMBOSSED AUTHENTICATION FOR TOXICOLOGY LABORATORY 

REPORTS, EMPLOYED IN AUTHOR'S LABORATORY ................ 89 

16 SCHEMATIC FLOWCHART ILLUSTRATING ROLE OF INTERPRETATION 

OF BLOOD ALCOHOL FINDINGS IN TYPICAL CRIMINAL AND 

CIVIL JUSTICE PROCEEDINGS ............................... 92 

LIST OF TABLES 

Table Page 

1 GAS CHROMATOGRAPHIC COLUMN CHARACTERISTICS FOR 

ETHANOL UNDER THE STATED GC CONDITIONS .................. 50 

2 TYPICAL RELATIVE RETENTION TIMES OF VOLATILE 

SUBSTANCES ON PORAPAK S AND CARBOWAX 1500 ON 

CARBOPACK A COLUMNS ..................................... 51 

vii 
rLIST OF TABLES (CONTINUED) 

Table Page I 

3 TYPICAL ABSOLUTE RETENTION TIMES OF VARIOUS 

SUBSTANCES OBSERVED ON THREE COLUMNS .................... 52 

4 PRECISION OF AUTOMATED GC HEADSPACE ANALYSIS FOR 

ETHANOL WITH MULTIFRACT F-40 INSTRUMENT IN REPLICATE 

DETERMINATIONS BY DIRECT PROCEDURE ........ .............. 70 

5 PRECISION OF AUTOMATED GC HEADSPACE ANALYSIS FOR 

ETHANOL WITH MULTIFRACT F-40 INSTRUMENT IN REPLICATE 

DETERMINATIONS BY INTERNAL STANDARD PROCEDURE ........... 70 

6 RESULTS OF INDEPENDENT ROUTINE ANALYSES FOR ETHANOL 

ON IDENTICAL BLOOD SPECIMENS, IN TWO SEPARATE 

LABORATORIES, BY AUTOMATED GC HEADSPACE ANALYSIS ........ 73 

7 

8 

TYPICAL PERFORMANCE OF THE AUTOMATED GC HEADSPACE 

ANALYSIS IN "DOUBLE-BLIND" ANALYSES FOR ETHANOL 

IN A STATE PROFICIENCY SURVEY ........................... 

TYPICAL PERFORMANCE OF THE AUTOMATED GC HEADSPACE 

ANALYSIS IN "DOUBLE-BLIND" ANALYSES FOR ETHANOL 

IN A NATIONAL PROFICIENCY SURVEY ........................ 

75 

76 

t 

9 STAGES OF ACUTE ALCOHOLIC INFLUENCE/INTOXICATION ........ 94 

viii 
1. INTRODUCTION 

i 

Analysis of biological specimens for alcohol is the most commonly performed 

forensic science procedure and has occupied that position for many decades. 

More than a dozen analytical principles have been applied to this task, and 

hundreds of methodological modifications have been developed and published 

during the past one hundred years for the analysis of alcohol in blood and 

other biological liquids. 

As can be anticipated, many of the published procedures vary greatly from 

each other in their significant analytical characteristics, practicability 

features, and consequent applicability, acceptability, and utility for forensic-scientific purposes. This diversity has undoubtedly played a prominent role, together with other factors, in the undesirably and unacceptably wide range of reported results of alcohol analyses upon supposedly 

identical specimens which has been repeatedly demonstrated by participating laboratories in proficiency surveys (1). The elucidation and elimination of the causes of such inter-laboratory variations in the analysis of 

biological liquids for alcohol, which presumably also affect the routine 

workload of many laboratories, would be much facilitated by the availability 

and use of an analytical method for alcohol in biological specimens whose 

procedural details and performance characteristics were precisely and comprehensively established, widely known and appreciated, and capable of being 

commonly if not universally employed as a referee or reference method. 

Preparation of a manual containing the details of such a method for alcohol 

analysis, together with other relevent information, was among the recommendations made be a widely representative group which met in Washington on 

May 10, 1973, under the auspices of the U. S. Department of Transportation, 

to inquire into various aspects of the standardization and improvement of 

1 

In this report, the unmodified term alcohol refers to ethanol; BAC refers 

to blood-alcohol concentration 

1 

c 
forensic alcohol analysis (2). 

A 

* 

The author accepted responsibility for preparation of the manual, and this 

report constitutes the resultant manual. Its contents are intended and 

should be considered to be recommendations. No inference is intended, and 

none should be drawn, that other methods, techniques, and procedures are 

necessarily inferior to those described herein. 

F 

Kurt M. Dubowski, Ph.D., Professor of Medicine and Director of Toxicology 

Laboratories, College of Medicine, University of Oklahoma 

-2­
2. SAMPLES AND SAMPLING FOR ALCOHOL ANALYSIS 

2 

2.1 SELECTION OF SPECIMENS 

Various factors enter into the decision concerning the selection of appropriate biological specimens for alcohol analysis, either as a matter of general 

policy or in a given instance. In most jurisdictions, statutory provisions 

govern specimen procurement from living subjects, in relation to alcohol-related traffic offenses. Leaving aside consideration of breath, some jurisdictions provide that only blood may be collected for analysis for alcohol 

intended to be introduced into evidence at the trial of alcohol-related traf 

fic offenses, others vest choice of specimen in the subject to be tested, and 

many impose specific requirements for or limitations upon persons authorized 

to obtain specified specimen materials such as blood under implied consent 

statutes or other applicable laws. The respective statutory provisions, 

when they exist, of course govern the situation in living subjects. 

Specimen selection in dead persons who are the subjects of official investigations or inquiries into the death is generally placed, statutorily or by 

regulation of comparable import, within the discretion of the investigating 

medicolegal authority, e.g., the medical examiner or coroner system. 

Because of these and other practical considerations, it is convenient to consider samples and sampling separately for living and dead persons, and the 

following subject matter is so arranged. 

2 

In this report, the term specimen is generally intended to apply to the entire 

quantity of a biological liquid obtained for analysis, while the term scnnple 

generally applies to that portion of a specimen which is subjected to analysis. 

3 

Although breath is a suitable specimen for alcohol analysis in law enforcement, 

the subject of breath-alcohol analysis is not treated in this report. 

-3­
2.1.1 Living Subjects 

Blood. Blood has been the traditional and most commonly obtained biological 

liquid for forensic alcohol analysis. It has many actual and potential limitations and shortcomings for that purpose (3,4), especially when venous whole 

blood is the specimen collected, as is almost universally the case. In most 

jurisdictions, however, the evidential import of the body burden of alcohol, 

with respect to alcohol-related traffic offenses such as operation of a 

motor vehicle under the influence of alcohol, is stipulated exclusively in 

terms of concentration of alcohol in the blood. All existing laws provide for admissibility into evidence of information concerning the alleged 4 

blood-alcohol concentration of the accused at the material time. 

It is usually appropriate, therefore, to select blood as the specimen for 

analysis in order to obviate the often difficult problem of translating the 

results of alcohol analyses of other body fluids into corresponding BAC's. 

When a choice is possible, the physiologically and parmacokinetically most 

logical and useful "blood" specimen for alcohol analysis is capillary plasma, I 

obtained by centrifugation of whole blood collected, from a suitably deep 

skin puncture, into an anticoagulant-treated micro blood-collection tube. 

Plasma obtained from anticoagulant-treated whole venous blood is the next 

choice for a specimen, followed by serum promptly separated from untreated 

whole venous blood after coagulation and anticoagulant-treated venous 

whole blood, in that order. As a practical matter, however, leaving theoretical considerations aside, venous whole blood is generally the most appropriate blood specimen to be collected for alcohol analysis in Zaw-enforcement practice at the time of preparation of this report. 

The usual site of collection for venous blood specimens is an antecubital 

4 

The material time is not necessarily the time at which the specimen was ob­

tained and frequently is not; the controlling factors may be established

by statute and/or case law and are often highly complex (see Section 4.1).

4

9 
vein unless special considerations (e.g., injuries) dictate otherwise. Other 

suitable sites are equally acceptable, it being understood that the concentration of alcohol in a blood specimen obtained from any given site is not necessarily identical to that of blood in the contemporaneous peripheral circulation at another site, nor necessarily fully representative of the concentration of alcohol in the blood circulation as a whole. 

Cerebrospinal fluid. This is not a specimen material of practical value for 

alcohol analysis for law enforcement purposes. Its only applicability would 

presumably arise in the unique circumstance of its sole availability (as a 

medically-obtained specimen for clinical diagnostic purposes) to the exclusion of other biological liquids. 

Saliva. Because of various practical constraints such as the substantial 

subject cooperation required for its collection, saliva has not been employed 

as a specimen material for alcohol analysis in law-enforcement practice recently. Parotid saliva is generally considered to be in alcohol equilibrium 

with the peripheral blood circulation, at the time of secretion. It could 

thus, in theory, be used to estimate the coexisting blood-alcohol concentration, and it possesses certain practicability advantages such as the relatively non-invasive nature of the feasible collection techniques. However, 

its suitability for that purpose under current conditions has not been adequately evaluated, nor have possible differences between parotid, sublingual, 

submaxillary, and pooled saliva been adequately investigated. 

In view of the lack of documented suitability and of other information needed 

for proper interpretation, saliva is not at present considered a suitable 

specimen for law-enforcement applications of alcohol analysis. 

Urine. The only urine specimen which could, in theory, be confidently em­

­ ployed for alcohol analysis for the purpose of estimating the coexisting BAC 

is ureteral urine - clearly an impracticable specimen for law-enforcement 

5 

.ol
purposes. The hypothetically-acceptable expedient of collecting vesical 

urine after a specified time interval following voluntary emptying of the 

bladder by micturition often does not achieve the desired purpose of eliminating the effects of pooled bladder urine upon the alcohol content of the 

specimen. The various, numerous difficulties in collecting appropriate 

urine specimens for alcohol analysis and in validly interpreting the results 

of such analyses for medicolegal purposes - including, especially, estimation of the coexisting BAC - have been extensively documented (5). The 

1968 declaration (6) of an ad hoc committee of the National Safety Council's 

Committee on Alcohol and Drugs, in this respect, subsequently approved by 

the Committee on Alcohol and Drugs, is still pertinent and applicable: 

"Because of various problems in the interpretation of the

results of analyses of urine specimens for alcohol which cannot

readily be overcome in law enforcement practice, analysis of

urine for the purpose of determining blood alcohol concentra­

tion is to be discouraged except under the strictly controlled

conditions employed in determining renal solute clearances."

s 

Additional practical constraints upon urine specimens or their collection 

further limit their suitability for alcohol analysis for law-enforcement 

purposes, such as the invasion of privacy required to eliminate subsequent 

legal questions regarding the unequivocal origin of such specimens and the 

absence of tampering (e.g., dilution with water) with them. 

Occurrence of fermentable carbohydrates, especially glucose, in urine is 

very common, as is contamination with yeast spores and other microorganisms. 

These and other adventitious factors can lead to alcohol neoformation in 

voided urine specimens. Unless statutorily mandated, therefore, use should 

not be made of urine specimens for alcohol analysis for law-enforcement 

purposes, except in the unique circumstance of sole availability of such a 

specimen to the exclusion of more suitable specimens; in any event, it should 

be used with full realization of the inherent, unavoidable strictures and 
rz 

limitations of urine as a specimen material for forensic alcohol determinat­ tion and the attendant uncertainties necessarily engendered in the valid 

interpretation of the significance of the alcohol analysis results. Urine 

is not a recommended specimen material. 

In summary, in the living human subject, only blood, saliva, and urine are 

potentially usable specimens in law-enforcement practice; of these 

only blood is sufficiently free of forensically-disabling disadvantages 

and contraindications to be a practical specimen material for routine 

applications. (While breath is also a suitable specimen, it is not considered here.) 

2.1.2 Cadavers 

Although a variety of biological specimens (including tissues and body fluids) 

can be obtained postmortem for alcohol analysis, examination of tissues is a 

technique generally limited to specialized forensic toxicology laboratories 

and thus is not further considered here. In dead subjects, particularly those 

subjected to autopsy, the functional restraints upon invasion in living subjects are, of course, absent and do not limit selection of specimen materials. 

However, other and often more complex and binding considerations are always 

present. Admixture of various body liquids and both internal and external 

contamination are factors which must be minimized; it is often difficult 

or impossible to ascertain whether and to what extent postmortem biological 

specimens have been diluted, contaminated, or otherwise altered from their 

immediate postmortem or agonal state. In persons whose death was preceded 

by even brief hospitalization or by emergency medical treatment at the scene 

of injury or during subsequent transportation from the scene, the composition 

of body fluids may be substantially altered by routine trauma care such as 

intravenous fluid therapy, blood transfusions, etc., as well as by significant blood loss. The postmortem composition of even uncontaminated, carefully collected specimens, therefore, may not be even remotely representative 

-7­
a 

of.the pretrauma subject state. Alcohol metabolism and excretion during any 

substantial period of survival after a subsequently fatal injury is also a 

commonly encountered factor, even when uncomplicated by medical therapy or 

clinical factors such as shock which can further complicate it. 

These considerations, among others, make it highly desirable, if not imperative, to select two or more different specimen materials for alcohol analysis in many cases and/or to collect such specimens from two or more completely dissociated sites, whenever possible. 

Blood. Blood is the postmortem specimen of first choice in undecomposed bodies when available and properly selected (with regard to collection site) and 

collected. Large blood vessels in the extremities (e.g., femoral artery or 

vein) are the recommended sampling site in unautopsied bodies, in order to 

obviate such problems as internal specimen contamination through unrecognized 

organ rupture or by needle penetration into the gastrointestinal tract during attempted blood withdrawal from the heart or great vessels in the thorax. 

A substantial literature has developed on the alleged contamination of blood 

in the heart and thoracic great vessels by alcohol diffusion from gastrointestinal contents and on related matters (7-17). Many of these publications are further complicated by confusion between the effects of biological 

factors (e.g., inter-tissue or inter-organ diffusion, or decomposition), 

artefactual difficulties created by the collection process or technique 

(e.g., needle penetration of adjacent hollow organs or external microbial 

contamination), and the characteristics and limitations of analytical methods (e.g., nonspecificity of chemical oxidation-reduction procedures resulting in unrecognized effects of nonethanol substances). 

Postmortem liquid specimens submitted as "blood" frequently bear little resemblance to the circulating whole blood of living subjects. They commonly 

display all manner and degree of alteration from the in-vivo state in such 

respects as presence of clots and lipids, hemolysis varying from slight to 

- 8 ­

s 

h 

i 
t 

t 

-complete, dilution with water or biological liquids, and many, often extreme biochemical changes. Several of these factors result from collection 

of "blood" from pooled liquids in the pericardial or pleural cavities or 

even from liquid pools in the thoracic or abdominal cavities at autopsy. 

The contents of these cavities should not be collected and submitted as 

"blood." When collection of suitable quantities of blood from large peripheral vessels is infeasible, blood properly taken from the intact chambers of the heart is usually an acceptable specimen. 

Cerebrospinal Fluid. Because of its distant location from the gastroin­

testinal tract and its relatively protected position with respect to ex­

ternal contamination, as well as its intimate association during life with

the brain, CSF is a most suitable postmortem specimen for forensic alcohol

determination (but not for estimation of the BAC).

Urine. The limitations outlined above for urine as a specimen material in 

living subjects apply fully in the postmortem situation, recognizing that 

only pooled bladder urine present, if any, can constitute the specimen. 

When collected with due care to avoid contamination with blood, water, or 

other substances it may be a functional specimen for analyses of admittedly 

limited significance or validity. (A common reason for collection of postmortem urine is to examine it for drugs other than alcohol or their metabolites.) It has been reported that alcohol diffusion through the bladder 

mucosa can occur both antemortem and postmortem (18). 

Vitreous Humor. Because of its specially protected location, normal sterility during life, low protein contents, and other useful characteristics, 

vitreous fluid (or "humor") is a postmortem specimen of choice for alcohol 

analysis. As with other specimens, due caution is required in the interpretation of the results of alcohol determinations on this material, especially 

in respect to estimation of the coexisting BAC or antemortem intoxication 

-9­

J 
states. In decomposing or decomposed cadavers, it will often be significantly less affected than other available specimen materials and is usually 

accessible without dissection, even in bodies of persons who have died in 

aviation crashes or which have been partially incinerated. A number of recent studies have described use of vitreous humor as a postmortem specimen 

for alcohol analysis (or other chemical studies) and the interpretation of 

the results (19-26). 

2.2 COLLECTION OF SPECIMENS 

The collection of specimens for forensic alcohol analysis is subject to a 

combination of either pertinent and relevant or controlling factors of medical, legal, scientific, and technical nature or combination thereof. Several are discussed below, but others of equal pertinence cannot be considered 

here. 

Ideally, the collection procedure and its ancillary processes should accomplish the following: 

a.­ Provide any and all necessary and desirable specimens, in

ample quantities and of suitable quality

b.­ Maintain and secure the integrity of each specimen material ­

i.e., its original unique composition at the time of collection ­

by avoiding any adventitious change resulting from the collection technique or other factors and by assuring the continued 

maintenance of that integrity and original composition 

without significant alteration(s) from biological processes or 

handling/storage effects or attempted interference, as by tampering 

c.­ Establish and maintain the identity of the person from whom the 

specimen(s) were collected, as well as that of the specimen(s) 

- 10 ­

I 
IV 

themselves with respect to their nature, origin, and signifit cant time factors (place, date, time of collection) 

d.­ Establish and maintain a readily traceable, demonstrably unbroken chain of custody and possession of the specimen(s), 

with appropriate indication of the identity of all persons 

associated with each specimen in any capacity from original 

collector to the last analyst and of the location, date, 

and time of any transfer(s). 

Some individual features of recommended collection procedures and safeguards 

are set forth by specimen material category below. 

Blood. In living subjects, various legal considerations arising from local 

statutory provisions or applicable case law apply to collection of blood 

specimens in the law-enforcement context. Two decisions by the U. S. Supreme Court include relevant elements. 

In Breithaupt vs. Abram (27), the U. S. Supreme Court sanctioned the withdrawal of blood from an unconscious subject, without his consent, and the 

subsequent admission of evidence concerning the results of analysis of that 

blood specimen for alcohol, over the defendant's timely objection at his 

trial on a charge of motor vehicle homicide. Relevant excerpts from the 

majority opinion are: 

"...there is nothing 'brutal' or 'offensive' in taking of a 

sample of blood when done, as in this case, under the protective 

eye of a physician..." (Emphasis added.) 

"...the individual's right to immunity from such invasion of the 

body as is involved in a properly safeguarded blood test is far 

outweighed by the value of its deterrent effect..." (Emphasis 

added.) 
VS 

In Schmerber vs. California (28), the U. S. Supreme Court held that the 

collection of blood for subsequent evidential alcohol analysis may be carried out upon a lawfully arrested subject without warrant and without his 

consent, under stipulated conditions set forth in the following relevant excerpt from the majority opinion: 

"...Finally, the record shows that the test was performed in a 

reasonable manner. Petitioner's blood was taken by a physician 

in a hospital environment according to accepted medical practices. 

We are not thus presented with the serious questions which would 

arise if a search involving use of medical technique, even of the 

most rudimentary sort, were made by other than medical personnel 

or in other than a medical environment - for example, if it were 

administered by police in the privacy of the stationhouse. To 

tolerate searches under these conditions might be to invite an 

unjustified element of personal risk of infection and pain..." 

(Emphasis added.) 

F 

The Supreme Court, in Schmerber, also reaffirmed the decision in Breithaupt 

that it is not a violation of the fourteenth amendment due process for the 

state to order withdrawal of a blood specimen from an unconscious, nonconsenting person "by a physician in a simple, medically acceptable manner in 

a hospital environment." (29) (Emphasis added.) 

It should be recognized that collection of blood from living subjects, 

whether by phlebotomy, skin puncture, or otherwise is a professional activity within the scope of clinical laboratory technology, clinical laboratory 

sciences, and the appropriate healing arts. For full discussion of the several relevant procedural aspects, standard texts and appropriate manuals 

should be consulted; the recommendations and comments relating to this subject here are not intended as a substitute for such references. 

Medical considerations include avoidance of infection of the puncture site, the 

- 12 ­
necessity for completely aseptic technique, and various other factors beyond 

the scope of this report. Skin preparation and disinfection before penetration by needle or lancet should be accomplished by means of a medically 

acceptable and clinically effective nonalcoholic (and preferably nonvolatile) 

preparation, in order to obviate later fruitless arguments over the possible 

contribution of an alcoholic skin disinfectant to any alcohol concentration 

found in the blood. Aqueous solutions of benzalkonium chloride and of thimerosal, among others, have been successfully used for this purpose, as has 

an aqueous solution of povidone-iodine. Unduly long application of tourniquets should be avoided during venipuncture both on medical grounds and because the induced stasis can induce hemoconcentration and thus alter the 

original physiological alcohol distribution between blood at the sampling 

site and the surrounding tissues as well as between the liquid and formed 

element portions of the intravascular blood. The blood collection technique 

should conform, in all significant respects, to currently accepted clinical 

standards and good practice. 

All instruments employed in skin penetration or blood withdrawal must be 

sterile and pyrogen free at time of use, preferably through prior heat sterilization. The blood containers should likewise be pyrogen-free, sterile, 

and dry. Employment of individual-use, disposable equipment and supplies of 

reputable manufacture, accompanied by appropriate manufacturers' affidavits, is to be greatly preferred over use of locally prepared expedients. 

Containers for blood specimens and any closure device should be chemically 

inert, chemically clean, dry, and of suitable durability and dimensions. It 

follows that glass containers of proper construction are to be preferred over 

most other materials. Closure devices such as screw caps or stoppers should 

have only chemically clean and inert surfaces exposed to. the blood specimen 

and should be so designed or employed as to have the required mechanical security against displacement and specimen loss or accessibility. Suitable 

seals for securing specimen containers such as vials or tubes and outer con­

- 13 ­

a 

a 
tainers such as mailing tubes and cartons are readily available commercially. 

They should have non-recurring serial identification numbers, whether factory 

or locally affixed. 

When venous blood is collected from living subjects, the collected volume 

should be 5 or 10 ml for convenience in collection and handling to allow 

replicate analyses or partial sharing of the specimen if necessary at a 

later time. It is difficult to obtain more than 200 to 500 microliters of 

blood from a single skin puncture, and that quantity is adequate for modern 

analysis methods. In cadavers it is possible and desirable to collect 

larger quantities of blood (e.g., 30 ml) since additional laboratory procedures may be required to validate the blood-alcohol analysis results or 

to prepare a suitable sample aliquot for analysis. (These recommendations 

pertain solely to analysis for alcohol and do not comtemplate use of the 

specimen for other examinations.) 

In collection of blood from cadavers, rigorous precautions should be exercised against the various contaminations referred to in Section 2.1 above. 

In particular, the specimen should be obtained with due regard for the 

separation of blood components which occurs postmortem by sedimentation and 

clotting; appropriate mixing or withdrawal of all portions of a specimen 

pool are desirable. Obviously, blood specimens should be withdrawn prior to 

embalming, whether by intravascular or cavity injection (30), and in particular, blood should not be obtained by displacement drainage or aspiration 

during vascular injection of embalming fluid. Embalming fluids and their 

components, expecially formaldehyde, are ubiquitous in mortuaries, cadaver 

preparation rooms, and autopsy suites. As a necessary precaution against 

specimen contamination with these materials, blood (and other specimens) 

should be collected exclusively with clean, individually packaged, initially 

sealed instruments and containers of commercial or comparable local manufacture. 

- 14 ­
i 

It 

.It is good practice to minimize trauma to and alterations in composition of 

blood specimens (e.g., hemolysis) during the collection process by use of 

the largest gauge needles compatible with the medical situation and by removing needles from syringes prior to ejection of the blood into storage containers. In collection of blood by skin puncture, the puncture site should 

be aseptically dried after disinfection to permit "beading" of the blood and 

to avoid its dilution. The puncture must be sufficiently deep to allow ready 

and adequate blood flow. Undue stasis during collection (e.g., "milking" of 

a digit) should be avoided. The usual skin puncture site in adults is a digit; when the extremities or ear lobes are collection sites, it is good practice to warm the site prior to skin penetration to promote "arteriolization" 

of the capillary blood. 

Cerebrospinal Fluid. The specimen must be collected from cadavers by an 

appropriately professionally qualified person, normally a pathologist. Precautions should be observed against specimen dilution by water or contamination with blood or other substances. A specimen volume of 10-30 ml is 

convenient and desirable. 

Urine. Since use of this specimen material is not recommended in living subjects, no recommendations are given with respect to collection techniques. 

If urine is, however, collected, the above precautionary notes should be 

observed with respect to unequivocal identification and positive establishment of absence of dilution with water or other tampering. Urine from cadavers should be collected with due regard for avoidance of dilution or contamination. A specimen volume of 10-30 ml is convenient and desirable. 

Vitreous Humor. Collection should be carried out from cadavers by appropriately professionally qualified persons, with due precautions against specimen 

dilution or contamination. Withdrawal of all available vitreous humor (typically 2-3.5 ml) is recommended. If vitreous humor is collected from both 

- 15 ­

A
01 

4 

eyes, the specimens should be collected and kept separately and identified 

as to their respective origin. 

Commercially-available CoZZection "Kits" and Containers. Availability of 

such items is subject to demand, marketing, and other economic considerations, 

and there has consequently been considerable fluctuation in both availability and cost of commercial materials for blood collection for forensic alcohol analysis-in assembled "kit" form in recent years. At the time of this 

writing (June 1976) the following items intended for collection and forwarding of blood specimens for forensic alcohol analysis were understood by the 

author to be commercially available:. 

a. Blood Alcohol Specimen Kit 

Reorder No. KT-IOIAK 

Manufacturer: Kimble-Terumo, Inc., P. 0. Box 605, Elkton, 

Maryland 21921. 

b. Vacutainer Brand Blood Collection Kit for Blood Alcohol Determinations 

Order No. 4990 

Manufacturer: Protective Products Division of Becton, Dickinson and 

Co., P. 0. Box 291, Grand Prairie,Texas 75050. 

c. Vacutainer Brand Blood Collection Kit for Post Mortem Blood 

Alcohol Determinations 

Order No. 4991 

Manufacturer: Protective Products Division of Becton, Dickinson and 

Co., P. 0. Box 291, Grand Prairie, Texas 75050. 

The specifications of these items, including that of the components and the 

"kit" contents, were not necessarily in agreement with the recommendations 

given here. While these specimen collection "kits" were intended for use in 

the collection of blood specimens for forensic alcohol analysis, they may 

also be suitable for use with other specimen materials. 

- 16 ­
ezl 

Individual components, such as blood collection tubes, syringes, needles, 

lancets, nonalcoholic antiseptic pads, etc., for local assembly of collection 

t­ sets are widely available through the clinical laboratory and healing arts 

supply industry. In many instances, such items as blood collection tubes 

can be purchased from commercial sources prepared according to local proprietary formulation (e.g., with respect to anticoagulant and preservative formulation, closure, or labeling). 

It is necessary to assure absence of tampering with specimen containers and 

associated collection paraphernalia both before and after they have been 

employed in collecting specimen materials. Original sealed commercial items 

serve that purpose where provided with separate, distinctive re-sealing devices. When specimen containers, syringe holders, etc., are locally prepared, 

it is recommended that the respective glass, plastic, or other containers be 

sealed, after preparation, with shrink-fit cellulose sealing bands, or caps, 

as is common practice in the alcoholic beverage, pharmaceutical, and cosmetic 

industries.­ When quantities used are sufficiently great, these can be obtained with appropriate proprietary legends, logos, or other designs. 

. i 

2.3­ IDENTIFICATION OF SPECIMENS 

Information should be obtained and recorded, appropriately, at the time any 

specimen is obtained with respect to at least the following items: 

a.­ Full identity of the subject from whom specimen was obtained 

(complete name, address if known, age, sex, race, whether subject was living or dead when specimen was obtained) 

b.­ Nature of specimen (e.g., blood, vitreous humor, etc.) and 

anatomical location from which obtained 

5 

These bands are available through the pharmaceutical and cosmetics industry. 

For commercial sources for this and comparable items, consult the current 

edition of THOMAS REGISTER (31) 

- 17 ­
4P 

J 

c. Nature and quantity of any additive, preservative, or adjuvant 

added to the specimen or initially present in the specimen container 

d. Geographical location, date, and exact time the specimen was 

obtained 

e. Name and other identifying data (e.g., profession, title, and 

affiliation) of the person(s) who collected the specimen and 6 

established its identity (the simple principle recommended 

here is the same as underlies the ritual attest every notary 

public provides with each notarization to establish the identity and authority of the official performing the act) 

f. Names and affiliation of any witnesses to the specimen procurement 

g. Full identity and other particulars needed (e.g., mailing address) 

of the official or agency submitting the specimen and/or ordering 

its collection. 

Many of these information items can be provided readily and conveniently by 

means of a suitable form. Figure 1 illustrates a form which has satisfactorily served this purpose in connection with the submission to the author's 

laboratory of blood specimens for forensic alcohol analysis. A unique, nonreplicating numbering system for specimen containers should be established, 

and all information forms used should bear the same number as the corresponding specimen container. Reliance on color-coding or other pre-use identification for nature of specimens (e.g., blood) should be scrupulously avoided 

since ample experience has established that the intended guidelines for use 

of such containers are frequently disregarded. 

6Failure or inability to provide this information (e.g., affirmatively to 

establish that a person who withdrew a blood specimen from a lawfully arrested subject for subsequent forensic alcohol analysis met the statutory 

qualifications for such an act) by acceptable means may result at any 

subsequent trial in suppression of the evidence or reversal of a conviction relying thereon (32). 

- 18 ­
(.SAME AND ADDRESS OF AGENCY And/or 1ADt1RATURY) 

y 

SPECIMEN IDENTIFICATION a REQUEST 

See Reverse Side For Directions DIRECTION FOR COLLECTION AND 

PLEASE PRINT ALL INFORMATION SUBMISSION OF SAMPLE 

t 

soul pcr u as eht 

Thumb Print 

SUBJECT: 

[met Nsa. First Intl (El 

SUBJECT'S. 1. This bottle contains sodium fluoride as a preservative 

ADDRESS and sodium heparin as an anticoagulant; and is suitable 

SPECIMEN: for Submission of blood, urine. Cerebrospinal fluid or

sample site other liquid specimens. Do not add anything other than 

SPECIMEN. _ M. , 19-by 

OBTAINED Time oat. Col lector 

the sample to the container. 

2. Use only a sterile, dry, clean syringe and needle for 

EXAMINE FOR: q Ethyl Alcohol Content obtString blood specimens and, where necessary, use only 

a non-alcoholic, non-volatile skin disinfectant. Place 

q Other: approximately 10 al. of whole blood or other liquid 

specimen into the bottle, cap tightly, and gently shake 

CIII.CK IF q Container Unit Seal was Intact before use for about I minute to dissolve the chemicals. 

APPLICABLE: q yon-alcoholic, non-volatile Skin disinfectant 

( ) was used. 3. Replace the specimen bottle and this completed form into

the container; then seal unit with the wire aid numbered 

REQUESTED BY: seal furnished. If possible, refrigerate the entire

container until mailed or delivered to the laboratory. 

REPORT TO:_ 4. The specimen in its sealed Container n ay be delivered to 

REMANKS:_- the ('livE• u", 1.room ln•:.1 

Toxicology Laboratories by messenger or sent 

by U. S. Mail, preferably by 'Certified Mail - Return 

FOR LABORATORY USE Receipt Requested.' 

CASE N0'. SEAL NO 

AGENCY: 

RECEIVED AT y ty

LAROHATOHY Tim o.te

FROM: 

i 

FIGURE 1. SPECIMEN IDENTIFICATION AND EXAMINATION REQUEST 

(OBVERSE AND REVERSE SIDES SHOWN) 

Provision must be made for establishment and documentation of the requisite 

physical security of all specimens from the moment of procurement and for 

the customary chain of custody and possession as the specimen changes location and/or hands. Discussion of further details is beyond the scope of 

this report. 
2.4 PRESERVATION AND STORAGE OF SPECIMENS J 

The purpose and function of preservatives or other additives used to treat 

biological specimens is basically to prevent or retard normal biological 

changes (such as coagulation of blood), to prevent or retard decomposition 

of the specimens, and to assist in maintaining the original chemical composition of such specimens by preventing loss of any component or constituent 

initially present and generation of any neoformed substances or compounds. 

Toward these goals, use of additives is often, desirably, combined with 

appropriate physical measures such as hermetic closure of containers and 

low-temperature storage. 

No single set of treatments or conditions has been found equally suitable 

and efficacious for all possible uses, and the recommendations made here 

are addressed specifically only to the task of facilitating maintenance of 

the integrity and initial composition of biological liquids intended for 

forensic alcohol analysis. Ideally, no additives should be employed, and 

sole reliance should be placed on physical measures such as storage at 

sufficiently low temperatures. However, freshly shed blood from living 

subjects will promptly coagulate, and chemical treatment will be required 

to prevent such coagulation since unclotted whole blood specimens are 

easier to analyze. Further, specimens are often collected remote from the 

site of analysis, and transportation over long distances and times under 

often extreme conditions of heat is a common requirement. 

Accordingly, chemical treatment of biological liquids by means of additives, 

is a common, if not universal practice. Although the following procedures 

have been found satisfactory, no implication is intended that others are not 

equally effective and useful. 
Blood. Freshly drawn blood from living subjects requires immediate treat- a 

ment with anticoagulants to prevent coagulation under the routine conditions 

here contemplated, and such anticoagulant treatment can be efficiently combined with use of preservatives to retard specimen decomposition and alcohol 

loss or neoformation. While most postmortem blood specimens will not further 

coagulate in vitro, it is more convenient to treat blood from both living 

and dead subjects alike. 

Suitable anticoagulants are citrate, edetate, oxalate salts, and heparin. 

Fluoride salts have both anticoagulant and preservative action, and various 

mercury salts and azide salts are also suitable preservatives. A combination of sodium citrate or potassium oxalate (5 mg/ml blood) with sodium 

fluoride (1 mg/ml blood) has been commonly employed. 

The author has for some years successfully employed the combination of 0.5 

mg heparin sodium and 1.5 mg sodium fluoride per ml blood as an anticoagulant-preservative mixture for freshly-drawn sterile blood from living subjects. 

It preserves the original alcohol contents of such specimens without detectible changes for at least 18 days at ordinary room temperature, for more than 

1 month when the blood is refrigerated at 5°C., and apparently indefinitely 

when the specimen is stored at -20°C. (33). The mixture is conveniently dispensed, in liquid form, into specimen containers in quantities which will 

yield at least the stated concentrations when the containers are filled to 

capacity, and the additives are then deposited in film form on the container 

walls by low-heat (below 90°C) evaporation of the water. Disodium edetate 

(1 mg/ml blood) can be substituted for the heparin. 

Postmortem blood specimens, with their inherent risk of microbial contamination 
0 

b 

before sampling require higher concentrations of preservatives to prevent 

bacterial alcohol production and significant enzymatic changes. Use of 

sodium fluoride in a concentration of 1% w/v (10 mg per ml blood) was 

found effective by Blackmore (17), and by Glendening and Waugh (34) who 

found no significant changes from the original alcohol contents of blood 

specimens, thus treated, after 2 months storage at room temperature or 

after 10 months storage at 1°C. Plueckhahn (35) found that a sodium fluoride concentration of 0.2% w/v did not consistently prevent alcohol neoformation in autopsy blood specimens contaminated with microbial inoculates but that 1% w/v of sodium fluoride was an adequate preservative for 

autopsy blood specimens. Likewise, Blume and Lakatua (36) reported that 

addition of 10 mg sodium fluoride per ml of blood (i.e., 1% w/v) prevented production of ethanol in bank blood after 24 hours of incubation 

at 37°C despite initial deliberate microbial contamination, except for 

specimens inoculated with C. albicans. Bradford (37) has proposed use 

of 5 mg sodium citrate and 0.1 mg mercuric chloride per ml of blood 

as the anticoagulant-preservative mixture of choice, reporting that blood 

specimens containing alcohol and treated with mercuric chloride (1:10,000) 

were stable at room temperature for periods of six months and longer. 

Christopoulos et al. (15) found that when autopsy blood specimens were 

treated with 1% w/v of sodium fluoride, no alcohol was produced after refrigerated storage of up to 45 days in originally alcohol-free specimens, 

while those with initially elevated concentrations of alcohol or alcohol 

and glucose demonstrated only slight changes (predominantly insignificant 

alcohol decreases) after refrigerated storage of up to 45 days. 

The stability of ethanol in stored blood specimens was extensively investigated by Smalldon, Brown, and associates (38-40), who found that human erythrocytes could oxidize ethanol to acetaldehyde in such specimens, that the 

mechanism for alcohol loss which was linked with the-oxyhemoglobin-methemoglobin redox system was strongly temperature-dependent, and that these 
s 

losses were not inhibited by 1% w/v sodium fluoride. Sodium azide, dithionite, and sodium nitrite were found to inhibit alcohol oxidation significantly 

during storage of blood specimens for 16 weeks at 20°C. The combination of 

at least .1% w/v sodium fluoride and 0.5% w/v of sodium nitrite added to blood 

was recommended to inhibit both alcohol neoformation and alcohol loss. Marbach et al. (41) investigated glycolysis in specimens of whole blood and 

serum, and found that sodium iodoacetate (0.05 to 0.2% w/v) and sodium fluoride (0.25% w/v) both satisfactorily inhibited glycolysis in such specimens 

at room temperature for periods of up to 24 hours. 

High concentrations of both inorganic anticoagulants such as sodium oxalate 

and preservatives such as sodium fluoride lead to hemolysis of the blood 

specimen as well as to alteration of the concentrations of plasma components 

through erythrocyte shrinkage and water displacement. These effects can be 

disregarded when whole blood is the sample subsequently analyzed for its 

alcohol content, the specimen is well mixed before withdrawal of the analyzed aliquot, and the latter is completely hemolyzed prior to analysis. 

However, these effects cannot be tolerated if the desired, retained specimen is blood plasma; in that case, heparin sodium or disodium edetate or 

dipotassium edetate should be employed as the anticoagulant, followed by 

prompt separation of the supernatant plasma from the formed blood elements 

by centrifugation, and further preservation of the plasma with low concentrations of sodium or potassium fluoride (i.e., 0.5-1 mg per ml of blood). 

High concentrations of additives, especially inorganic salts, also alter the 

density and solids content of whole blood substantially, a factor that must 

be taken into consideration in analysis methods which involve the weighing 

of samples or the use of headspace gases above untreated samples. (The 

analysis methods described herein are not affected adversely by this factor.) 

The several combined anticoagulant-preservatives mixtures can be placed into 

specimen containers in powder form, as well as deposited as dried films therein. In the former situation, a warning should accompany the container against 

- 23 ­
discarding the additive as a presumed contaminant, and in the latter a warning against rinsing of the container prior to collection of the specimen is 

appropriate. When blood is collected with capillary micro-collection tubes, 

the anticoagulant-preservative mixture must be deposited on the walls as 

dried films. Thorough mixing of the specimen with the additives is required. 

In addition to chemical treatment, as outlined above, the blood specimen 

should be stored at reduced temperature until after analysis. When 

whole blood is to be analyzed, such storage should preferably be at -20°C 

or lower temperature, which permits long-term storage without significant 

changes in original alcohol contents of the specimen. The hemolytic effect 

of freezing is an automatic preparation step for subsequent analysis, but 

care must be exercised upon removal from low-temperature storage properly 

to reconstitute the specimen by effective mixing after thawing. Storage 

at ordinary refrigerator temperature (about 3-5°C) will suffice for shortterm storage (up to 4 weeks) of blood specimens which have been properly 

chemically preserved, as outlined above, or when immediate separation of 

supernatant plasma or serum from cellular components of blood cannot be a 

accomplished. Refrigeration is now widely available in transportation 

facilities, and blood specimens for alcohol analysis should not be left at 

room or higher temperatures for periods of hours or longer when avoidable. 

Storage in glass containers with adequate mechanical closures (screw-caps 

secured with tape) is recommended since many polymeric substances (plastics). 

are not impervious to gaseous or vapor state substances, including ethanol, 

over prolonged periods, and since leaching of plasticizers from such container materials into the specimens is an undesirable event. 

Although alcohol loss from liquid specimens into the airspace above them in 

It is noted, parenthetically, that centrifugal separation of blood components

should be carried out in sealed containers since the substantial air movements

thus generated will cause loss of alcohol and water from open specimen con­

tainers, thus substantially altering the original specimen composition.

- 24 ­

7 
i 

G 

properly-sealed containers is negligible at room or lower temperatures because of the favorable partition ratio, container volume should be suitably 

proportional to anticipated specimen volume. 

Cerebrospinal Fluid. For all practical purposes in preservation and storage, 

CSF specimens can be treated like whole blood. Since they will generally be 

of autopsy origin, they should be considered microbially contaminated, and 

low temperature storage, preferably at -20°C., should be routinely employed. 

In other respects, the recommendations and comments given under Blood, supra, 

apply. 

Saliva. Saliva specimens can be treated like blood with respect to preservation and storage, although less intensive chemical treatment will suffice 

to assure stability of their alcohol content because of the absence of many 

of the blood components. Addition of picric acid (0.1-0.5% w/v) has been 

recommended (42). Friedemann,et al. (43) employed sodium fluoride (2 mg 

per ml of saliva) for preservation, combined with refrigeration, and 

Nanikawa and Kotoku (44) treated saliva with zinc sulfate (2 mg per ml 

of saliva), or cupric sulfate (20 mg per ml of saliva), or mercuric chloride and hydrochloric acid (0.4 mg./ml and 1 ml of N/10 per ml of saliva, 

respectively). Low-temperature storage of saliva specimens is indicated. 

Urine. These will generally be specimens obtained at autopsy and must be 

assumed to be microbially contaminated. The author has effectively employed 

preservation with 15 mg of sodium fluoride per ml of urine (in dried film 

form pre-deposited on container walls with allowance for the maximum contents) 

combined with low-temperature storage; saturatiDn of the urine specimen with 

excess benzoic acid (approx. 5 mg per ml of urine) was equally effective 

(33). Phenylmercuric nitrate has been commonly employed as a urine preservative, usually added to specimen containers in tablet or powder form to 

provide sufficient preservative for a saturated solution (about 1:1000). 

Christopoulos et al. (15) reported slight decreases from original alcohol 

- 25 ­
contents of urine specimens obtained at.autopsy and stored for periods up 

to 30 days at room temperature after treatment with phenylmercuric nitrate; 

none demonstrated any significant neoformation of alcohol in originally 

alcohol-free urine specimens. Refrigeration, with and without preservative, 

of autopsy-derived urine specimens yielded comparable results Reduction 

of the pH of urine specimens will delay and minimize decomposition with 

formation of ammonia when combined with refrigeration; hydrochloric acid 

is a satisfactory additive for this purpose to yield a pH of 1-3. In clinical practice, numerous volatile organic compounds or substances have been 

employed, alone or in combination, to retard decomposition of urine (camphor, 

formalin, thymol, etc.); all of these should be avoided in preserving urine 

specimens for alcohol analysis. Urine specimens can generally be treated 

like blood specimens with respect to chemical preservation with due allowance for the specimen volume concerned. 

Urine specimens should be stored at low temperature, before and after analysis. 

Freezing of such specimens at -20°C. or lower temperature is most satisfactory; 

care is indicated in subsequent thawing to mix the specimen well since layering occurs during freezing. The specimens should be stored in glass containers; transfer from any plastic or cardboard containers used in collection 

or clinical examinations should be carried out as soon as possible. 

Vitreous Humor. For all practical purposes, these specimens can be treated 

like blood, with respect to preservation and storage. Being of autopsy origin, they should be considered to be microbially contaminated, and accordingly 

should be stored at low temperature until analyzed. 

G 

- 26 ­
2.5 PREPARATION FOR ANALYSIS 

Biological specimens tend to be nonhomogeneous systems in which various 

changes occur with time, including biochemical, chemical, and physical alterations. Some of these are of spontaneous, natural origin, while others 

are storage-induced artefacts such as the hemolysis of blood specimens induced by freezing. Good laboratory practice therefore requires examination of biological specimens prior to analysis, especially when they have 

been subjected to prior storage and after reconditioning or rehabilitation 

of such specimens prior to removal of aliquot samples, and appropriate preparation to counteract any such effects. 

Preliminarz/ Observations and Measurements. Visual observations should be 

made and recorded of such specimen characteristics as appearance, clarity, 

color, apparent homogeneity or inhomogeneity, and viscosity, as appropriate. In blood specimens, color, evidence of hemolysis, presence of clots, 

s­ and odor should be especially noted; in urine specimens, the foregoing observations and preliminary pH measurement are useful. Measurement or estimation of initial specimen volume or quantity is useful. Occasionally, 

repeating the same observations after mixing or other preliminary specimen 

treatment is desirable. 

Reconditioning or Rehabilitation of Specimens. All biological liquids should 

be vigorously mixed, in their original containers, to restore the maximum 

feasible specimen homogeneity. Thereafter, depending on nature of the specimen, it may be desirable to employ homogenization technique, or alternatively, 

to employ separational procedures such as centrifugation or filtration to obtain the most suitable sample for analysis. If a blood fraction, such as 

plasma or serum is the desired specimen, centrifugation in closed vessels to 

obtain a supernatant liquid free of formed blood elements, cellular debris, 

and other particulate matter is the initial step, followed by prompt and 
careful removal of the supernatant phase. When whole blood is to be analyzed, 

thorough mixing to restore the original liquid/cell distribution is indicated; if clots are present, it is desirable to homogenize the entire specimen 

by mechanical or ultrasonic techniques.8 The incidental cell disruption and 

hemolysis is a helpful side effect of effective homogenization. For other 

well-mixed liquids, such as cerebrospinal fluid, urine, and vitreous humor 

in which alcohol distribution can be safely assumed to be uniform, the aim 

is to eliminate precipitated proteins, debris, and other particulate and solid 

matter from the aliquot analyzed. Centrifugation, filtration, or occasionally 

simple sedimentation can be advantageously employed for this purpose. 

Recording of Particulars. In addition to and apart from the routine but careful noting and recording of identification data concerning the specimen and 

its container(s) as well as the usual data concerning location, date and time 

of the examination, apparent absence of tampering with the specimen (if applicable), and identification of the examiner, an initial contemporaneous, 

permanent record should be made of all steps of the examination including I 

significant omissions. If a prescribed, written protocol for preparation 

and analysis of specimens is available and used, a notation to that effect 

suffices, giving sufficient details to identify the protocol and any discretionary actions. In the absence of such complete protocol, all observations, 

manipulations, procedural steps, computations, and other component parts of 

the analysis should be contemporaneously entered into a permanent notebook 

and authenticated by the analyst. 

8 

For occasional use or small specimen volumes, tissue grinders with Teflon

pestles are convenient and efficient; e.g., Cat. No. 3431-E04 through

3431-E25, Arthur H. Thomas Co., P. 0. Box 779, Philadelphia PA 19105
3. ANALYSIS OF BIOLOGICAL LIQUIDS FOR ETHANOL 

This section contains the operational and procedural details of the method 

for alcohol analysis in biological liquids which has been selected for presentation in this report, together with information concerning the performance characteristics of the method. 

The basic method presented here is analysis of alcohol in the equilibrated 

headspace above liquid specimens by means of gas chromatography. Four versions of the basic method are described: 

1)­ Automated gas chromatographic analysis of headspace vapor without 

use of internal standards 

2)­ Automated gas chromatographic analysis of headspace vapor with 

use of internal standards 

3)­ Manual gas chromatographic analysis of headspace vapor without s use of internal standards 

4)­ Manual gas chromatographic analysis of headspace vapor with 

use of internal standards. 

The method described is based on that of Machata (45-47). For convenience 

in reference, the automated and manual procedures are described separately, 

although they are in fact modifications of the same method. 

3.1 PRINCIPLES 

From the general gas law PV = nRT and Dalton's law of partial pressures, it 

follows that the partial pressure of a gas or conforming vapor, for dilute 

solutions, is directly related to the mole fraction (or concentration) of 

that component in the mixture and to the absolute temperature of the system. Further, Raoult's law states that the partial vapor pressure of a 

constituent is proportional to its mole fraction in the liquid at all compositions; Henry's law can be expressed 

- 29 ­
R 

Concentration of gas or vapor in gaseous phase 

= constant 

Concentration of gas or vapor in liquid phase 

at definite temperature, independent of pressure (48). At isothermal equilibrium of a dilute solution of volatile substances and the adjacent gas phase 

in a closed system, therefore, the concentration in the liquid phase of the 

component of interest can be determined from experimental measurement of the 

concentration of that component in . the gas phase. In the methods considered 

here, the "headspace" above the sample constitutes such a gas phase. 

The functional relations can be expressed as follows: 

Palc kalc x talc 

P = E Pi 

P Palc + PH20 + Px + 

where Palc = Partial Pressure of Ethanol 

Ostwald Partition Coefficient for Ethanol kalc­ = 

Concentration of Ethanol in the solution (or liquid mixture) talc = 

P­ = Total Pressure 

Pi­ = Partial Pressure of any Compound Present. 

Further, gases and vapors are generally less soluble in aqueous solutions 

of electrolytes than in pure water, a phenomenon known as the salting-out 

effect (49). 

The basic method thus utilizes the principle of isothermal equilibrium partition of ethanol and other volatile substances between liquid samples and 

the "headspace" gas in a closed system, and employs gas chromatographic 

- 30 ­
I 

analysis to separate the components of the headspace vapor, allowing individual quantitation of their respective concentrations by means of a 

flame ionization detector.9 Addition of sodium chloride to the liquid 

sample in sufficient quantity to assure saturation eliminates differences 

between blood and other biological liquids and aqueous alcohol solutions 

in their respective ethanol partition into air, and thus allows use of 

aqueous reference standards. Quantitation of the detector signal response 

is accomplished by manual measurement of the resultant trace peak heights 

on a potentiometric recorder chart, or by mechanical or electronic integration of the peak areas, or by other measurement of the electrical detector signal as a function of time. These data are translated into 

corresponding concentrations of ethanol and other substances of interest 

in the sample by comparison with suitable contemporaneous calibration 

standards. Identification of ethanol and other components of interest 

is established by comparison of their relative retention times with those 

of contemporaneous reference samples. 

The several physico-chemical relationships are fixed. For consistent, 

reproducible conditions the functional constants for a given system can be 

experimentally determined by suitable calibration techniques and then applied to the experimental determination of the liquid phase composition. 

The ratio of ethanol concentration in the gas and liquid phases (also termed partition coefficient or partition ratio) is temperature dependent. The 

thermodynamic theory for the mixtures of interest indicates that the logarithms of the partition coefficients should vary linearly with the reciprocal of the absolute temperature; for narrow ranges the linear relation 

should also hold for absolute temperature or system temperature expressed 

as °C. This relation is illustrated in Figure 2 for the data of Harger 

et al. (50). The higher partition ratio, at a given temperature, for the 

9 

Presumably, other detectors can be used, provided they have sufficient sensitivity and linearity for the volatiles involved and low response to water. 

- 31 ­
 *

air/blood system compared to the air/water system is presumably the result

of'the difference in water content and the salting-out effect. These differences can be abolished by saturation of the liquid phase with soluble

inorganic salts, which also substantially increases the absolute value of

the partition ratios.

W

D

Data of Harger et al. •

J. Blol. Chem. 183197(1950) /

•

•

o

•

AIR/BLOOD

0

•

AIR/WATER

15 20 25 30 35 40 45 5C

TEMPERATURE, °C

FIGURE 2. IN-VITRO ETHANOL PARTITION RATIOS FOR AIR/BLOOD AND

AIR/WATER SYSTEMS (DATA OF HARGER ET AL. (50))

 * 

*
-3.2

AUTOMATED METHOD 

3.2.1 Materials: Apparatus, Reagents, Supplies 

Apparatus 

1. Automated Gas Chromatographic Headspace Analyzer (Model F-40 lu 

Multifract, Perkin-Elmer Corp., Norwalk, Conn. 06856) 

2.

Potentiometric Strip-Chart Recorder, 250 mm.(Model 194; Honeywell, 

Inc., Ft. Washington, Pa. 19034); equipped with Mechanical 

Trace Integrator (Model 252-A, Disc Instruments Inc., Santa 

Ana, Calif. 92705) and with Automatic Printing Integrator 

(Model 610, Disc Instruments, Inc., Santa Ana, Calif. 92705) 

3.

Automatic Diluter (Cat. No. 2112, York Instrument Corp., 

Berkeley, Calif. 95710) 

4.Hydrogen Generator, Elhygen (Model ER-150, Milton Roy Co., St. 

Petersburg, Fla. 33733). 

Reagents 

1. Calibration Reference Materials (Calibrators) 

a.

Ethanol, 0.10, 0.20, 0.40% w/v (1.00, 2.00, 4.00 g/Liter) 

Aqueous Solutions 

b.

Mixed Calibrator, "Low" (Acetone 1.00 g/Liter; Ethanol 2.00 

g/Liter; Isopropanol 1.00 g/Liter; Methanol 1.00 g/Liter 

Aqueous Solution) 

c.

Mixed Calibrator, "High" (Acetone 1.00 g/Liter; Ethanol 4.00 

g/Liter, Isopropanol 1.00 g/Liter; Methanol 1.00 g/Liter 

Aqueous Solution) 

10 

The Model F-42 instrument can also be used. 
2.­ Internal Standard Solution

Acetonitrile, A.R., Nanograde, 0.15% v/v

(1.50 ml. per Liter Aqueous Solution) 

3. Sodium Chloride, A.R., granular 

Supplies 

Glass Vials, 24 ml., with polymer septum stoppers and aluminum 

seals (Cat. No. 105-0118, Perkin-Elmer Corp., Norwalk, Conn. 06856) 

3.2.2 Summary of Operation 

The liquid sample (10 microliters to 1 ml) is placed into a glass septum 

vial (after dilution in fixed proportion with the internal standard solution when that procedural modification is used) together with sufficient 

sodium chloride to assure saturation. Reference samples are similarly 

treated, and all vials are sealed with a polymeric septum stopper and a 

crimped aluminum cap and inserted into the thermostated instrument turntable, which has a capacity of 30 vials. After a 45-minute equilibration 

period, the septum vials are sequentially presented to the chromatograph 

in accordance with a preset instrument control program. A dosing capillary 

penetrates the septum cap of the vial, and pressurization of the vial with 

N2 occurs. A highly reproducible quantity of the headspace vapor is then 

pneumatically transferred to the separating column, and the analysis proceeds 

for an adjustable, preselected analysis time. The response of the flame ionization detector is recorded, as a function of time, on a potentiometric stripchart recorder and/or monitored at a separate detector signal integrator output. Each sample is identified on the recorder chart by the relative length 

of an identification trace which precedes the respective chromatogram. 

11 

The author has successfully substituted food-grade salt (Kraft Salt, N1 g. 

packet, Kraft Foods Div., Kraftco Corp., Chicago, Ill. 60611) for the reagent 

material. 

- 34 ­
 *

The sequence of these steps in the automated headspace analysis is illustrated in Figure 3.

CG

 * 

* *

II IIL

I

FIGURE 3. PRINCIPLE OF HEADSPACE SAMPLING WITH THE MULTIFRACT F-40 AUTOMATED

GAS CHROMATOGRAPH (STEPS SHOWN ARE INITIAL NORMAL STATE (I),

PRESSURIZATION (II), AND HEADSPACE SAMPLING (III).

B = SAMPLE VIAL; C = CG COLUMN; CG = CARRIER GAS; M = SOLENOID

VALVE; MC = MOVABLE DOSING HEAD CYLINDER; N = DOSING NEEDLE;

S1 = SAMPLE VIAL SEPTUM; S2 = MOVABLE CYLINDER SEPTUM)

7
3.2.3 Instrument and Analysis Conditions 

The following are typical instrument and analysis conditions which have been 

found satisfactory in the author's laboratory. Numerous modifications of 

these conditions are also acceptable, or occasionally preferable in special 

circumstances. 

Column: _Carbowax 1500 (0.4%) on 60/80 mesh Carbopack A (51, 53) 

1.8 M x 3.2 mm.O.D. (6 ft x 1/8 in ) stainless steel column 

Carrier Gas: Nitrogen; inlet pressure 20 psig 

2 

manometer 1.1 kp/cm (117.7 kPa) 

flow rate 35 ml/min. 

Temperatures: Heating bath (turntable) 65°C ± 0.03°C 

Dosing line 175°C 

Injection block 150°C 

Column oven 80°C 

FID: Hydrogen; inlet pressure 20 psig 

2 

manometer 1.1 kp/cm (107.9 kPa) 

flow rate 35 ml/min. 

Air; inlet pressure 28 psig 

manometer 1.2 kp/cm2 (117.7 kPa) 

flow rate 300 ml/min. 

Program: Pressurization 60 seconds 

Injection 3 seconds 

Analysis 300 seconds 

Flushing (Purge) 12 seconds 

Stabilization 12 seconds 

Analyses/vial 1 

Electrometer: Range 10 

Attenuation x 128 

Baseline 

Correction: Automatic 

Recorder: Input Range 1 my 

Chart Speed 5 mm/minute • 

- 36 ­
i 

y 

3.2.4 Procedure - Direct Analysis 

1. A well-mixed aliquot of the blood specimen,12 1 ml in volume , 

is delivered into each of two clean, dry, numbered septum vials which contain approximately 1 gram of crystalline sodium chloride. The vials are 

closed with a polymeric stopper and sealed with a crimped aluminum cap, and 

the contents are thoroughly mixed by horizontal rotation. 

2. Ethanol or mixed-volatiles reference solutions (normally the "Low" 

and "High" mixed Calibrators) are similarly treated, and all sealed vials 

are inserted into the preheated turntable water bath at 65°C and allowed to 

equilibrate for at least 45 minutes. Calibrators are placed at the beginning, middle, and end of a run. 

3. Automatic gas chromatographic analysis of headspace vapor from each 

vial is initiated after the minimum equilibration period, and the potentiometric strip-chart recorder and peak-area integrator-printer (and/or other 

signal conditioner and recorder) are activated. 

4. The analysis record is appropriately identified (we routinely employ 

a date/time stamp imprint also naming the laboratory), and a stamp with the 

chief particulars of the analysis conditions (Figure 4) is affixed. 

Ot crator Date

Column Detector

Length Range 

Dia. Atten 

Liquid Fhase_- Flow Ratts. ml'min 

Wt- 776 Hydrog.-:n Air 

Support Scaver a 

hie=h.___ Split 

C,rnar Gds Temperature, °C 

Rutameler Det., Inj 

Inlet Press paig Column In-ti at 

Rate_ mi/min Final 

CHART SPEED Rate

SAMPLE Solvent

Size Concn.

FIGURE 4. STAMP FOR RECORDING PARTICULARS OF GAS CHROMATOGRAPHIC ANALYSIS 

12For simplicity and brevity the procedural directions were limited to blood. 

Other biological or nonbiological liquids are treated identically except for 

possible preparation (e.g., dilution of alcoholic beverages). 

13In this modification of the method, the sample volume is -not a critical 

variable, and use of pipettes calibrated "to contain" is not required; 

however, see Section 3.4.and Figure 7 below. 

- 37 ­
x 

5. Results 14 are computed as follows: 

a. By Peak Height Measurements 

PHunknown x BACunknown Conc•EtOH, calibrator PH calibrator 

where PH = Peak Height of the Ethanol Detector Response Recording. 

b. By Peak Area Measurements 

PAunknown BAC Conc• unknown x EtOH, calibrator PAcalibrator 

where PA = Peak Area of the Ethanol Detector Response Recording. 

6. Ethanol and other volatile substances in the sample, if present, are 

identified by their relative retention time(s) in comparison with those of the 

contemporaneous reference solutions. 

7. Micro-Sample Modification: If indicated because of restricted specimen 

volume, the above procedure is modified as follows, without other changes in 

preparation or analysis of samples or computation of results: A well-mixed 

100 ul specimen aliquot is delivered into each of two clean, dry numbered 

septum vials which contain 0.1 gram of crystalline sodium chloride. Calibrators are treated identically. 

14 

Concentration of ethanol in the unknown and reference samples can be expressed 

in any desired units, arbitrary or otherwise. In recognition of common current 

practices, it is recommended that ethanol concentration of blood and other biological specimens be reported in percent w/v units (% w/v) to two decimal 

places, truncated (i.e., with the third or additional digits dropped without 

rounding), or in grams per liter (g/liter) to two decimal places, truncated. 

- 38 ­

i 
8. Ultra-Micro Scnnple Modification: 

15 

An aliquot of precisely 40.0 .tl 

-of well-mixed specimen is diluted with fully quantitative technique (using 

pipettes calibrated "to contain") with 0.9% w/v aqueous sodium chloride to a 

total volume of 200 p1. A 100 p1 aliquot of that diluted sample is then 

placed into each of two clean, dry numbered septum vials which contain 0.1 

gram of crystalline sodium chloride. Calibrators are treated identically. 

Appropriate change in electrometer range and/or attenuation settings may be 

needed to achieve suitably large recorder response. 

3.2.5 Procedure - Internal Standard Analysis 

16 

1. A well-mixed aliquot of the blood specimen is mixed with an identical 

volume of the acetonitrile internal standard solution, using fully quantitative 

technique (including pipettes calibrated "to contain" when an automatic diluter 

is not used). Normally, the respective volumes are both 500 p1. The sample 

measurement and internal standard solution measurement and mixing are carried 

out with an automatic diluter (appropriately preset, with calibration verified 

by weighing of sampled and dispensed quantities), and the sample-internal 

standard mixture is delivered directly into one clean, dry numbered septum 

vial containing 1 gram of crystalline sodium chloride. 

15 

For the analytical system and procedure described, the gas chromatograph 

electrometer is normally operated at 1/1280 or 1/640 of maximum sensitivity. It is thus feasible to detect and quantitate ethanol in sample volumes varying from the routine sample volume by at least these factors. 

Additional sensitivity can be gained by increases in equilibration temperature and/or use of inorganic salts other than sodium chloride 

16Provided a simple fixed ratio of blood/internal standard solution is maintained for both unknown specimens and calibrators within a given analytical 

run, the exact volumes and proportion are not critical variables. However, 

for reproducibility of detector response, etc., between runs, it is recommended that the fixed volumes and proportion given be uniformly employed. 
}

2. Ethanol or mixed-volatiles reference solutions (normally the "Low" 

and "High" mixed Calibrators) are treated identically. 

3. The remainder of the procedure for analysis is identical with 

that described in Section 3.2.4. It may be desirable to employ an 

electrometer attenuation setting 0.5x that normally used with the 

Direct Analysis procedure, in order to achieve sutably large recorder 

responses to the detector signal. 

4. Results are computed as follows: 

a. By Peak Height Measurements 

R1 

R2 x Conc BAC unknown­ .EtOH, calibrator 

where PH­ Peak Height of the Indicated Detector Response 

Recording (e.g., PHis = Peak Height of the Acetonitrile Internal Standard Response) 

PHEtOH R1­ for the unknown sample-internal standard PH is mixture 

PHEtOH R2­ for the calibrator-internal std. mixture. 

PHis 

b. By Peak Area Measurements 

R1 

BAC x C unknown R2 onc.EtOH, calibrator 

where PA =­ Peak Area of the Indicated Detector Response Recording (e.g., PAis = Peak Area of the Acetonitrile Internal Standard Response) 

R 1 = PAEtOH for the unknown samp le-internal standard 

is mixture 

PAEtOH fnr tha ralihratnr-internal std . mixture ­ R2­ PA is 
I 

5. Micro-Sample Modification: When a suitable mechanical diluter is 

available, exactly 50.0 ul of well-mixed specimen are diluted with exactly 

50.0 ul of the acetonitrile internal standard solution, with fully quantitative technique, and the mixture is delivered into a single dry, clean numbered septum vial which contains 0.1 gram of crystalline sodium chloride. Alternately, larger identical volumes of the specimen and of the internal standard solution are premixed with fully quantitative technique, and approximately 

100 pl of the resultant mixture is employed as the sample for analysis. Calibrators are treated identically. 

6. Ultra-Micro Sample Modification: Using a suitable mechanical diluter, 

exactly 20.0 pl of well-mixed specimen are diluted with exactly 80.0 u1 of a 

modified 0.09375% v/v acetonitrile internal standard solution (containing 

0.9375 ml of acetonitrile per liter of aqueous solution) and delivered directly into a single, dry, clean, numbered septum vial which contains approximately 0.1 gram of crystalline sodium chloride. Calibrators are treated 

identically,. Appropriate change in, electrometer range and/or attenuation setting may be needed to achieve suitably large recorder response. 

3.3 MANUAL METHOD 

3.3.1 Materials: Apparatus, Reagents, Supplies 

Apparatus 

1.­ Gas Chromatograph with Flame Ionization Detector (Model 1860-3, Varian 

Instruments Div., Palo Alto, Calif. 94303). 

2.­ Potentiometric Strip-Chart Recorder, 250 mm,(Model 194, Honeywell, Inc., 

Ft. Washington, Pa. 19034); equipped with Mechanical Trace Integrator (Model 252-A, Disc Instruments, Inc., Santa Ana, Calif. 

92705) and with Automatic Printing Integrator (Model 610, Disc 

Instruments, Inc., Santa Ana, Calif 92705). 
3.­ Hydrogen Generator, Elhygen (Model ER-150, Milton Roy Co., St. Petersburg, Fla. 33733) 

4.­ Constant-Temperature Water Bath, 1000 Watts, Temp. Regulation ±0.02°C 

(Lauda Model K-4, Brinkmann Instruments, Inc., Westbury, N.Y. 11590) 

5.­ Automatic Diluter (Cat. No. 2112, York Instruments Corp., Berkeley, 

Calif. 94710) 

6.­ Gas-Tight Syringe, 1 ml., with Chaney Adaptor (Model 1001LLCH, Hamilton 

Co., Reno, Nev. 89510 or Plastipak Disposable Tuberculin Syringe, 

Cat. No. 5602, Becton-Dickinson, Rutherford, N.J. 07070 with 

Chaney Adapter, Model 14725, Hamilton Co., Reno, Nev. 89510); 

and Disposable Hypodermic Needle, 25 gauge 1.5 inch (38 mm) 

(Cat. No. 5028, Becton-Dickinson, Rutherford, N.J. 07070) 

7.­ Culture Incubator (Model 1A6825E, Thermolyne Corp., Dubuque, Ia. 52001). 

Reagents 

1.­ Calibration Reference Materials (Calibrators) 

a.­ Ethanol, 0.10, 0.20, 0.40% w/v (1.00, 2.00, 4.00 g/Liter) 

Aqueous Solutions 

b.­ Mixed Calibrator, "Low" (Acetone 1.00 g/Liter; Ethanol 2.00 g/ 

Liter; Isopropanol 1.00 g/Liter; Methanol 1.00 g/Liter Aqueous 

Solution) 

c.­ Mixed Calibrator, "High" (Acetone 1.00 g/Liter; Ethanol, 4.00 g/ 

Liter; Isopropanol 1.00 g/Liter; Methanol 1.00 g/Liter Aqueous 

Solution) 

2.­ Internal Standard Solution

Acetonitrile, A.R., Nanograde, 0.15% v/v

(1.50 ml. per Liter Aqueous Solution) 

3.­ Sodium Chloride, A.R., Granular. 
Supplies 

Glass Septum Vials, Screw-capped, Erlenmeyer, 10 ml or 25 ml­

(Products No. 13310 and 13320, Pierce Chemical Co., Rockford, Ill. 

61105); or Cylindrical Glass Septum Vials, Screw-capped, 14 ml. 

(Product No. 13043, Pierce Chemical Co., Rockford, 111. 61105) 

with polymer septa; or Hypo-Vials, 15 ml. (Stock No. 12911, Pierce 

Chemical Co., Rockford, Ill. 61105) with elastomer septum stoppers 

and aluminum seals. 

3.3.2 Summary of Operations 

The liquid sample (20 microliters to 1 ml) is placed into a glass septum 

vial, together with sufficient sodium chloride to assure saturation of the 

liquid (and after dilution in fixed proportion with the internal standard 

solution when that procedural modification is used). Reference samples are 

treated identically, and all sealed vials are inserted into a thermostated 

water bath. After a 45-minute equilibration period, a fixed quantity of 

the headspace vapor of each septum vial is sequentially sampled with a gastight syringe and injected into the preset gas chromatograph. The analysis 

proceeds for a fixed time interval, and the response of the flame ionization 

detector is recorded, as a function of time, on a potentiometric stripchart recorder. 

3.3.3 Instrument and Analysis Conditions 

The following are typical instrument and analysis conditions which have been 

found satisfactory in the author's laboratory. Numerous modifications of 

these conditions are also acceptable, or occasionally preferable in special 

circumstances. 

Column: Carbowax 1500 (0.4%) on 60/80 mesh Carbopack A (51, 53) 

1.8 M x 3.2 mm O.D. (6 ft x 1/8 in) stainless steel column 
1 

1 7 

Carrier Gas: Helium ; inlet pressure 55 psig 

rotameter 85-90% of maximum scale 

flow rate . 45 ml/min. 

Temperatures: Column Oven 80°C 

Injection Port 175°C 

Detector 225°C 

FID: Hydrogen; inlet pressure 20 psig 

flow rate 45 ml/min. 

Air: inlet pressure 15 psig 

flow rate 330 ml/min. 

Temperature 

Program: Isothermal 

Electrometer: Range 10-11 amps/mv 

Attenuation x 32 

Recorder: Input Range 1 my 

Chart Speed 5 mm/minute. 

3.3.4 Procedure - Direct Analysis 

1. A well-mixed aliquot of the blood specimen approximately I ml in 

volume is delivered into a clean, dry numbered septum vial which contains 

gram of crystalline sodium chloride. The vials are closed with a polymeric 

stopper or septum and a screw cap or crimped aluminum cap, as appropriate, 

after which the contents are thoroughly mixed by horizontal rotation. 

2. Ethanol or mixed-volatiles reference solutions(normally the "Low" 

and "High" mixed calibrators) are similarly treated, and all sealed vials 

are inserted into the preheated water bath, at 38±0.05°C, being immersed to 

just below the closures, and allowed to equilibrate for at least 45 minutes. 

Sufficient calibrators are equilibrated to allow their analysis at the beginning, middle, and end of a run. 

17Nitrogen can also be employed as the carrier gas, at slightly greater sensitivity 
1 

3. After the minimum equilibration period, a fixed, identical volume 

of the headspace vapor is withdrawn, using the gas-tight syringe adjusted 

to fixed volume (usually 250 microliters) with the Chaney Adaptor, the syringe.having been preheated to 38°C in the incubator or similar device. Repeated rapid "pumping" of the syringe is employed to obtain a fully-representative aliquot of the headspace vapor, which is then immediately injected into the gas chromatograph. Immediately following the headspace sample withdrawal, each septum vial is vented with a 25 gauge needle to reestablish atmospheric pressure. The syringe and needle are thoroughly purged by rapid, 

repetitive intake and expulsion of room air, and the syringe is returned to 

the incubator until its next use. 

4. The analysis is allowed to proceed for approximately 5 minutes (or 

3 minutes if ethanol is the only compound of concern in the sample). 

5. The same gas-tight syringe, with volume calibration intact, is used 

for all unknown and reference samples. Each specimen is analyzed by this 

procedure in at least independent duplicate, as is each calibrator, after 

allowing each septum vial to re-equilibrate for at least 20 minutes after 

the prior headspace sample withdrawal. 

6. The analysis record is appropriately identified (we routinely employ a date/time stamp imprint also naming the laboratory) and a stamp with 

the chief particulars of the analytical conditions (Figure 4) is affixed. 

7. Results are computed as follows: 

a. By Peak Height Measurements 

PHunknown BAC unknown x Conc. EtOH, calibrator PHcalibrator 

where PH = Peak Height of the Ethanol Detector Response Recording. 

b. By Peak Area Measurements 

PAunknown BAC x Conc• unknown EtOH, calibrator PAcalibrator 

where PA = Peak Area. of the Ethanol Detector Response Recording. 

- 45 ­
4 

8. Ethanol and other volatile substances in the sample, if present, are 

identified by their relative retention time(s) in comparison with those of 

contemporaneous reference solutions. 

9. Micro-Sample Modification: If indicated because of restricted speciment volume, the above procedure is modified as follows, without other changes 

in preparation or analysis of samples or computation of results: A wellmixed 100 ul aliquot of the specimen is delivered into a single clean, dry 

numbered septum vial which contains 0.1 gram of crystalline sodium chloride. 

Calibrators are treated identically. 

10. Ultra-Micro Sample Modification: An aliquot of exactly 20.0 ul of 

well-mixed specimen is diluted with fully quantitative technique (using pipettes calibrated "to contain") with 0.90% w/v aqueous sodium chloride to a 

total volume of 100 ul and that diluted sample is then delivered into a single 

clean, dry. numbered septum vial which contains 0.1 gram of crystalline sodium 

chloride. Appropriate change in electrometer range or attenuation settings 

may be needed to achieve suitably large recorder response. Calibrators are 

treated identically. 

3.3.5 Procedure - Internal Standard Analysis 

1. A well-mixed aliquot of the blood specimen is mixed with an identical volume of the acetonitrile internal standard solution, using fully 

quantitative technique (including pipettes calibrated "to contain" when an 

automatic diluter is not used). Normally, the respective volumes are both 

500 ul. The sample measurement and internal standard solution measurement 

and mixing are carried out with an automatic diluter (appropriately preset 

with calibration verified by weighing of sampled and dispensed quantities), 

and the sample-internal standard mixture is delivered directly into one 

clean, dry numbered septum vial containing 1 gram of crystalline sodium 

chloride. 

2. Ethanol or mixed volatiles reference solutions (usually the "Low" 

and "High" mixed Calibrators) are treated identically. 
3. The remainder of the procedure for analysis is identical with 

"that described in Section 3.3.4 above. It may be desirable to employ 

an attenuation setting 0.5x that normally used with the Direct 

Analysis procedure, in order to achieve suitably large recorder responses 

to the detector signal. 

4. Results are computed as follows: 

a. By Peak Height Measurements 

R1 

x BACunknown = R2 Conc.EtOH, calibrator 

where PH =­ Peak Height of the Indicated Detector Response 

Recording (e.g., PHis = Peak Height of the Acetonitrile Internal Standard Response) 

R =­ PHEtOH 

1 for the unk nown samp l e-i n terna l stand ard PH is mixture 

=­ PHEtOH R2 for the ca libra tor-i n terna l std . m i x tu re. 

PHis 

b. By Peak Area Measurements 

R1 

BACunknown R2 x Conc.EtOH, calibrator 

where PA =­ Peak Area of the Indicated Detector Response Recording (e.g.; PAis = Peak Area of the Acetonitrile Internal Standard Response) 

PAEtOH R = for the unknown sample-internal standard 1 

PAis mixture 

PAEtOH R = 2 for the calibrator-internal std . mixture . PA is 
. S. Micro-Sample Modification: When a suitable mechanical diluter is 

available, exactly 50.0 pl of well-mixed specimen are diluted with exactly 

50.0 pl of the acetonitrile internal standard solution, with fully quantitative technique, and the mixture is delivered directly into a single dry, 

clean numbered septum vial which contains 0.1 gram of crystalline sodium 

chloride. Alternately, larger identical volumes of the specimen and of the 

internal standard solution are premixed with fully quantitative technique, 

and approximately 100 pl of the resultant mixture is employed as the sample 

for analysis. Calibrators are treated identicaZZy. 

6. Ultra-Micro Sample Modification: Using a suitable mechanical diluter, exactly 20.0 p1 of well-mixed specimen are diluted with exactly 80.0 

pl of a modified 0.09375% v/v acetonitrile internal standard solution (containing 0.9375 ml of acetonitrile per liter of aqueous solution) and delivered directly into a clean, dry numbered septum vial which contains 

0.1 grams of crystalline sodium chloride. Calibrators are treated identicaZZy. 

Appropriate change in electrometer range and/or attenuation setting may be 

needed to achieve suitably large recorder response. 
3.4 PROCEDURAL NOTES AND COMMENTS 

Innumerable modifications of certain, details of the above procedures are 

feasible with respect to techniques, instrument operating parameters, data 

treatment, etc. Some of the more significant factors are discussed below. 

Gas Chro»xztographic CoZwmcs. Many column packings have been used successfully 

for alcohol analysis in biological specimens, and several of these have proven 

useful in the author's laboratory. Table 1 reflects, in part, this laboratory's 

experience with several commonly-employed GC column packings with respect to 

absolute ethanol retention time under the stated conditions, using a manual 

gas chromatograph as the comparison device. It should be recognized that 

other significant factors may also vary, such as capability or ease of separation of certain sample components of interest (e.g., acetaldehyde, formaldehyde, or methanol). 

The column specified in the procedural section, 0.4% Carbowax 1500 on 60/80 

mesh Carbopack A, was introduced about 1974 but is no longer commercially 

available. A replacement, Carbopack C, has been marketed for Carbopack A 

as the support material, and the liquid phase loading is O.Sx that previously 

used on Carbopack A (53). Among the voluminous literature on column packings 

are two reports which specifically deal with graphitized carbon and open-pore 

polyurethane as column packings for the gas chromatographic analysis of ethanol 

in blood (54-55). In general, increasing the column oven temperature decreases 

absolute retention times for ethanol and related compounds of interest, shortening total elapsed time per analysis; but column temperature increases also 

reduce the efficiency of separation of closely adjacent sample components, 

sometimes to the point of inadequate resolution. In practice, therefore, 

the optimum conditions, for a given column packing, of column geometry, oven 

temperature, and carrier gas pressure and flow rate represent compromises to 

attain the currently most important goal, whether speed of analysis or best 

resolution. 
TABLE 1. 

GAS CHROMATOGRAPHIC COLUMN CHARACTERISTICS FOR ETHANOL 

UNDER THE STATED GC CONDITIONS (52) 

Column Packing 

Column 

Temperature, °C 

Absolute Retention Time 

for Ethanol (min_L_ 

1 0.5% Carbowax K-600 + 3.8%

Hallcomid 18 on Teflon 

6 HC, 40/60 mesh

100 0.24

2 0.5% Carbowax K-600 + 3.8%

Hallcomid 18 on Teflon 

6 HC, 40/60 mesh

50 1.80

3 0.4% Carbowax 1500 on Carbopack A, 60/80 mesh

80 1.32

4 Porapak Q, 80/100 mesh 155 1.60

5 Chromosorb 102, 100/120 mesh 155 2.16

6 15% Carbowax 1500 on Celite 

525, 80/100 mesh

80 2.58

7 Porapak S, 80/100 mesh 155 2.51

8 30% Carbowax 20M on Chromosor 

W, AW, 60/80 mesh

130 2.12

*1.8M x 6.4mm o.d. column, rather than the 1.8M x 3.2mm size used for

the other supports
I 

The several column packings listed above also differ significantly in the 

absolute retention times for volatile compounds other that ethanol, and consequently in their ability to achieve satisfactory and practical separations 

of the components of interest in biological liquids. Table 2 lists the retention times relative to ethanol of several commonly encountered volatile 

substances on a Porapak S column and a Carbowax 1500 on Carbopack A column. 

TABLE 2, 

TYPICAL RELATIVE RETENTION TIMES OF VOLATILE SUBSTANCES 

ON PORAPAK S AND CARBOWAX 1500 ON CARBOPACK A COLUMNS 

Relative Retention Timea 

Compound Porapak S Carbowax 1500 

Acetone 1.44 1.35 

Acetonitrile 1.23 0.87 

Ethano^ 1.00 1.00 

Isopropanol 1.80 1.98 

Methanol 0.48 0.52 

aRelative to the retention time of ethanol 

Machata (47) has reported absolute retention times for various volatile 

compounds on three commonly employed columns, as shown in Table 3. 

It is evident that greatly increased certainty of identification of ethanol 

and other volatile substances from their retention times can be obtained by 

the simultaneous or sequential GC analysis of headspace vapors on two different columns, and that procedure is recommended. 
TABLE 3. 

TYPICAL ABSOLUTE RETENTION TIMES OF VARIOUS SUBSTANCES OBSERVED ON 

THREE COLUMNS (47) 

Carbowax 1500 Hallcomid 18 Porapak 

Column Temperature, °C 90 90 175 

Compound Absolute Retention Time, Minutes 

Acetaldehyde 1.1 1.7 1.5 

Acetone 1.5 2.1 3.2 

Chloroform 2.8 2.4 7.2 

Diethyl Ether 0.8 1.3 4.2 

Ethanol 2.6 4.0 2.1 

Isopropanol 2.5 4.8 3.4 

Methanol 2.1 2.6 1.1 

Methyl Ethyl Ketone 2.1 4.2 6.9 

n-Butanol 7.2 19.3 10.2 

n-Propanol 4.2 8.7 4.5 

Paraldehyde 4.6 12.7 4.3 

tert. Butanol 2.2 5.4 5.4 
Gas Chromatographic Instrument Parameters. The several GC instrument temperatures specified in the procedural section have proven suitable in the 

author's laboratory; others are feasible. Since the headspace samples always contain water vapor in addition to any volatile substances present, 

the entire analysis train inclusive of the sample inlet must be maintained 

at a sufficiently high temperature to prevent condensation of any sample 

component. Analysis time per sample is partly determined by the foregoing 

column considerations and partly by the desired information. When ethanol 

or other volatiles eluting earlier (e.g., acetaldehyde) are the only components of interest, chromatography can be terminated after the complete ethanol 

peak has been recorded. Temperature programming can be employed to accelerate elution of any later-eluting sample components, or tined injections of 

succeeding samples can be made while the preceding sample is being chromatographed. Acetonitrile has the advantage, as an internal standard in this 

system, of eluting before ethanol and thus not prolonging the analysis. 

It is usually desirable to record (or otherwise measure) GC detector response 

at 75-80% of maximum full-scale response for the highest expected sample component concentration, where feasible. The interaction of sample quantity 

chromatographed, electrometer sensitivity setting (e.g., range and attenuation), and recorder full-scale span is suitably adjusted toward this goal, 

with due attention to short-term baseline stability and long-term baseline 

drift. In the automated method, length of injection time governs the headspace sample quantity chromatographed; in the manual procedure it is determined by the headspace volume transferred to the gas chromatograph. For 

large specimen numbers, fully automatic result calculation and reporting accessories for the automated gas chromatograph are available (46, 56). 

Equilibration Considerations. Isothermal equilibration is a complex phenomenon which is affected by such factors as temperature, time, volume, and geometry of the equilibration vessel, and presence and corcentration of inorganic 

salts in the liquid phase. In general, the headspace volume should be kept 

as small as is compatible with the sample volume required by the sensitivity 

- 53 ­
of the gas chromatograph employed and by the number of replicate headspace 

vapor samplings contemplated; 10-25 ml are appropriate volumes for the reccommended scheme. While the cylindrical equilibration vessel form is dictated by the turntable requirements of the F-40 instrument, larger interface 

areas accelerate equilibration, and the Erlenmeyer flask shape is thus more 

efficient. Close temperature regulation is required and the equilibration 

vessel must be immersed in the water bath to cap level; spring retainers or 18 

weighting with suitable washers or lead doughnuts are used as required. 

The vapor pressure of volatile compounds increases as a function of the temperature, but the vapor pressure curves of ethanol and many other volatile 

compounds potentially useful as internal standards are not parallel over the 

temperature range of interest. Hence, calibration factors and curves established at one temperature cannot be used at other temperatures. While the 

concentration of ethanol in headspace vapor can be increased by equilibration at higher temperatures, other factors (such as the greater differential 

between equilibration and room temperatures) may offset any advantage gained 

through condensation effects, etc. Hence, the manual procedure, which involves manual headspace transfer, employs a compromise equilibration temperature of 38°C. In theory, equilibration could be accomplished at room 

temperature if the latter were sufficiently stable over time. In practice, 

the requisite identity of equilibration temperatures for unknowns and reference specimens can only be assured by use of high-precision thermostating. 

Time to reach complete equilibrium of volatile components of the sample between the liquid and gaseous/vapor phases in the equilibration vessel is a 

key factor which is often disregarded. Incomplete equilibration will yield 

variable results, and failure to achieve identical equilibration of reference 

and unknown samples (e.g., because of analysis of insufficiently-equilibrated 

reference samples at the beginning of a run) will yield erroneous results. 

Figures 5 and 6 illustrate this phenomenon. 

Instruments for Research and Industry, Cheltenham, Pa. 19012. 

- 54 ­

18 
 *

200 o ACETONITRILE 1 1.0

150 0.75

ETHANOL/ACETONITRILE o 0

100 0.50

1-^p ETHANOL /

0.25

0 0 0 10 20 30 40 50 60

TIME, min.

FIGURE 5. TIME COURSE OF GC DETECTOR RESPONSES TO ETHANOL AND ACETONITRILE AND THEIR RATIO, SHOWING TIME REQUIRED FOR EQUILIBRATION OF HEADSPACE VAPOR AT 65°C. DATA POINTS SHOWN ARE

SINGLE OBSERVATIONS (100 MG/DL AQUEOUS ETHANOL SAMPLE IN

THE PRESENCE OF SODIUM CHLORIDE)

200 1.0

Iy

ACETONITRILE

9150 0.75

100 0.50

ETHANOL/ACETONITRILE

50 c ETHANOL 0.25

0 10 20 30 40 50 60 0 * TIME, min.

FIGURE 6. TIME COURSE OF GC DETECTOR RESPONSES TO ETHANOL AND ACETONITRILE, AND THEIR RATIO, SHOWING TIME REQUIRED FOR EQUILIBRATION OF HEADSPACE VAPOR AT 38°C. DATA POINTS SHOWN ARE

SINGLE OBSERVATIONS. (100 MG/DL AQUEOUS ETHANOL SAMPLE IN

THE PRESENCE OF SODIUM CHLORIDE)

*

-55-
Addition of inorganic salts decreases the vapor pressure of water and increases that of organic substances with proportionate effects upon their 

relative partial pressures in the headspace gas. Saturation of the liquid 

phase with sodium chloride yields an approximate 3-fold increase in alcohol 

concentration of the vapor phase compared with the same system in the absence of added sodium chloride; e.g., at 40°C, the GC peak height ratio 

for ethanol in the headspace gas above NaCl-saturated liquid vs. that 

above untreated-liquid specimens is 3.2. Machata (47) has reported the 

following "vapor pressure increases" for ethanol at 60°C by addition of various salts to an aqueous ethanol solution (the respective salt concentrations not being specified): 

Anhydrous Salt Added Vapor Pressure Increase 

Ammonium chloride 2 x 

Ammonium sulfate 5 x 

Potassium carbonate 8 x 

Sodium chloride 3 x 

Sodium citrate 5 x 

Unionized dissolved solids in the specimen exert much smaller effects on 

the relative fugacity of volatile compounds; e.g., addition of 1 gram of 

sucrose to 1 ml of a 100 mg/dl aqueous ethanol specimen increases the gas 

chromatographic ethanol peak height at 35°C to 1.3x that yielded by the untreated specimen. The observations are in accord with theories regarding 

decreased mutual solubility of solvents or complete or partial removal of 

one solvent in such systems via preferential solvation processes. 

In the manual method, equilibration should preferably occur at atmospheric 

pressure. Partial loss of a headspace sample can occur during its transfer 

with a syringe whose needle is open to the atmosphere when equilibration 
has been carried out above atmospheric pressure, e.g., at elevated temperature in an unvented vessel; or dilution of a headspace sample obtained below atmospheric pressure can occur from air influx. Atmospheric pressure 

is, therefore, established by hypodermic needle puncture through the equilibration vessel septum stopper prior to headspace sampling, and again thereafter if sampling is to be repeated. 

Sample Measurement. In the manual method direct procedure, measurement of 

the liquid specimens placed into equilibration vessels need only be approximate. In both the manual and automated methods, the internal standard 

technique requires exact, reproducible measurement of unknown or reference 

specimen and internal standard reagent quantities. Further, in the automated method direct procedure, the headspace vapor quantity actually transferred pneumatically into the chromatograph is significantly affected by 

the total liquid volume in the septum vial. Figure 7 illustrates the relation between total liquid volume in the 24 ml septum vial and the peak 

height of the GC ethanol trace for a 200 mg/dl aqueous ethanol specimen 

with added sodium chloride in the usual (1 g NaCl/l ml sample) proportions. The experimental curve corresponds to a'power equation (y=axb), 

but apparent best-fit regression of the data is with the parabolic or quad2 

ratic curve equation y = 0.400 + 0.748x - 0.201x (r = 0.93). 

Since the sample is not diluted in the direct procedure, wash-out ("to 

contain") pipettes cannot be employed for delivery of the sample. Whole 

blood can be measured with Ostwald-Folin pipettes calibrated "to deliver," 

allowing drainage to proceed slowly enough to avoid blood film residues. If19 

necessary, treatment of pipettes with a suitable silicone coating compound 

will reduce wettability of pipette walls and avoid adhesion of blood. When 

the volume of analyses is not large enough to warrant use of automated mechanical pipetting or pipetting-diluting machines, manually-operated repetitive 

19 SILICLAD water soluble silicone concentrate (Cat. No. 1950, Clay Adams Div., 

Becton-Dickinson & Co., Parsippany, N. J. 07054) has been found suitable. 

- 57 ­
1.0 

K 

w

05 

0L I 

0 I 2 3 4 

VIAL CONTENTS, ml. 

FIGURE 7.­ EFFECT OF SAMPLE VOLUME IN 24-ML SEPTUM VIAL ON DETECTOR RESPONSE 

TO ETHANOL WITH MULTIFRACT F-40 AUTOMATED GAS CHROMATOGRAPH. 

EACH DATA POINT IS MEAN OF FOUR OBSERVATIONS. DASHED CURVE 

REPRESENTS BEST-FIT PARABOLIC REGRESSION EQUATION Y = 0.400 + 

0.748X - 0.201X2 (R=0.93) (200 MG/DL AQUEOUS ETHANOL SAMPLE 

IN PRESENCE OF SODIUM CHLORIDE) 

20 

pipetters or pipetter-diluters are convenient; the syringe-based types 

have proven suitable for the dilutions involving internal standard reagent, 

while hand-held automatic pipets of the Eppendorf type with disposable 

polypropylene tips have proven suitable for measurement and delivery of 

blood and other liquid samples. 

Mini-Repipet Diluter (Cat. No. Mini-80501, Labindustries, Berkeley, CA 94710). 

- 58 ­

20 
i 

The high concentration of sodium chloride, incidentally, aids in hemolysis 

of the whole blood sample, a desirable effect since the ethanol concentration of the sample is thus equalized. Mixing an aqueous internal standard 

with the whole blood specimen, of course, further effectively promotes rupture of the walls of the cellular components. 

Headspace Sample Measurement and Transfer. In these procedures, a headspace 

vapor aliquot is chromatographed; hence, measurement of that aliquot is critical. In the automated method, measurement and introduction of the headspace 

vapor sample occurs automatically through suitably heated connections; but 

in the manual method manipulation by the analyst is required. Although many 

devices and schemes can be used for measuring and transferring the headspace 

vapor aliquot, we have found use of a fixed-volume syringe to be the most 

convenient suitable procedure. The plastic tuberculin-pattern syringe is 

readily heated to a temperature slightly above that of the septum vial and 

retains the heat adequately during the brief headspace aspiration-and-transfer operation, when handheld. A specialized gas-tight glass syringe with 

heating mantle can be similarly employed. Reproducible-volume sampling is 

assured by means of a Chaney adaptor or similar volume-limiting device. 

The entire headspace aliquot removed by syringe is then immediately injected 

into the gas chromatograph. 

Alternately, a heated gas-sample valve, maintained at a fixed temperature 

slightly above that of the septum vial,-can be used to measure and inject 

the headspace vapor aliquot, after a suitably larger roughly-measured volume 

of headspace gas has been transferred from the septum vial to the gas-sample 

loop. Identity of volumes of unknown and reference sample headspace vapors 

is assured by using the same syringe, after repeated air-purging between 

samples. Absence of carry-over or sample-memory effects can be documented, 

if necessary, by chromatographing such a blank sample. 

When an internal standard is used, the ratio of GC detector signals for ethanol and the internal standard (e.g., acetonitrile) is identical for any 

- 59 ­
appropriate sample volume, and hence minor sample losses through leaks, etc., 

do not affect the results. In the direct procedures, however, the chromatographed sample quantity is a critical variable, and steps are required to exclude unrecognized losses of the headspace vapor aliquot. The simplest 

routine procedure toward that end is that employed here; namely, independent 

replicate (i.e., at least duplicate) sampling of the headspace, from two or 

more separately but identically equilibrated specimen aliquots, in order to 

avoid the effects of rapidly repeated withdrawals of headspace aliquots upon the analysis results. Coincidence of these replicate results within the 

precision limits for the procedure then excludes unrecognized sample losses 

or alterations. If the volume of analyses to be accomplished militates against 

the replicate analysis practice, internal standard use becomes the mandatory 

alternative. When time permits, replicate sampling from a single vial suffices. 

Of the various volatile compounds proposed as candidate internal standards, 

many have limitations of one sort or another. Acetonitrile was found in 

this laboratory to be acceptable on general practical grounds and to yield 

the same GC detector response ratio (e.g., peak heights) for ethanol/internal 

standard in both aqueous reference solutions of ethanol and biological-fluids 

of the same ethanol concentration when both are saturated with sodium chloride. 

(This limitation does not apply to use of internal standards as biological 

fluid diluents for direct liquid sample injection in gas chromatography.) 

Quantitation of GC Detector Response. Many of the typical detector-response 

quantitation problems encountered in gas chromatography of injected biological liquid samples are obviated by use of the equilibrated headspace technique. Asymmetry and tailing of peaks, significant baseline offset or drift, 

and other problems resulting from flash-vaporization effects or column changes 

induced by liquid samples are absent. As illustrated in Figure 8, under 

routine operating conditions the headspace analysis method described herein 

yields highly symmetrical component peaks for all substances of interest and 

good return to baseline. Under these conditions, quantitation of detector 
1 

1 

' I 

1 4 3 2 If • 

FIGURE 8.­ TYPICAL HEADSPACE CHROMATOGRAM OBTAINED WITH MULTIFRACT F-40 

AUTOMATED GAS CHROMATOGRAPH (DIRECT PROCEDURE), UNDER CONDITIONS DESCRIBED IN TEXT. (HITACHI PERKIN-ELMER MODEL 159 RECORDER WAS USED.) SAMPLE COMPONENTS SHOWN ARE: 1 = METHANOL, 

2 = ETHANOL, 3 = ACETONE, 4 = ISOPROPANOL 

response can be carried out equally satisfactorily by measurement of peak 

height or peak area. In the internal standard mode, the peak height (and 

area) of the internal standard should ideally be identical to that of the 

peak of interest, ethanol; in practice, similarity is, of course, all that 

can be attained when the ethanol concentrations of unknown specimens vary. 

The peak height for the internal standard should therefore be adjusted to 

match that of the most commonly occurring unknown value according to prior 

experience (i.e., the modal concentration). 

- 61 ­
The various methods of recording, measuring, and computing GC detector response as a function of sample mass all have characteristic inherent limitations as well as advantages. For the gas chromatographic system described 

herein, the basic detector signal output is a d.c. voltage as a function of 

time. For a narrow Gaussian peak, the peak height at maximum is directly 

proportional to the total peak area; therefore the d.c. voltage as a 

function of time (i.e., my x min) is also proportional to the peak area and 

hence to the mass of analyte present in the carrier gas at the relevant peak 

time. The several common result computation methods all utilize the two key 

variables, d.c. voltage and time, of the relevant chromatographic peak, 

either directly or indirectly. Direct quantitation from the potentiometric 

strip-chart recorder tracing (whether by manual peak height measurements 

or manual peak area estimates or by accessory mechanical integrator reflecting recorder response over time) necessarily entails the detector signal degradation and variability attributable to the signal conditioning and display by a separate device, especially one of limited quantitative discriminating capacity such as a recorder with 200-250 mm (or possible lesser) chart 

width. Ideally, therefore, an electronic quantitation device of precision 

and accuracy at least equal to the detector signal output should be employed, 

such as a suitable electronic integrator capable of accepting the electrometer output signal directly. 

The printing (Disc) integrator employed in this laboratory is an accessory 

for the (Disc) mechanical integrator, itself an accessory to a potentiometric 

strip-chart recorder. It has proven satisfactory for the application described 

and has yielded results equivalent to those obtainable with more sophisticated 21 

direct digital voltage measurements, direct electronic integrators, or the 

fully automatic data processing system. 

The Multifract F-40 instrument has a direct electrometer output signal for 

integrators (100, 250, or 1000 my f.s.d.) . 

21 
For large-scale forensic alcohol analysis, use of an electronic integrator 

directly coupled to the electrometer output signal and preferably equipped 

with hard-copy reporting (e.g., by means of a digital printer or teletypewriter) is desirable and recommended because of the substantial savings in 

personnel time and the probable reduction in errors in computing and transcribing results which can be achieved. 

When instrumental conditions are optimal, quantitation of detector response 

by either peak height or peak area measurements is acceptable and appropriate. 

It may, however, be more conservative to base quantitation of ethanol in the 

headspace sample on the peak areas ratio for ethanol and an appropriate internal standard. 

Several common quantitation methods employing either peak height or peak area 

measurements assume direct proportionality between the peak height or peak 

area and the concentration of the analyte. Over a narrow concentration range 

this is normally a tenable assumption when gas chromatgraphic analysis conditions are optimized. It is, however, good practice to use at least three 

reference samples of different concentrations over the full range of interest 

to assure that adequate linearity of detector response is present. 

Specificity Considerations. In routine gas chromatographic analysis, putative identification of the unknown is universally based on comparison of 

its retention time with that of contemporaneously chromatographed reference 

compounds. In the absence of sophisticated instrumentation with inherently 

high specificity for qualitative identification of unknowns (e.g., by combined gas chromatography-mass spectrometry (57) or mass fragmentometry (58)) 

several simple expedients can significantly increase the certainty of identification of ethanol and other sample components based on their relative 

retention time. The first, and simplest, involves employing two or more 

different GC columns, so selected that the expected analyte (e.g., ethanol) 

elutes at substantially different absolute times after injection and prefer­

- 63 ­
ably in altered sequence relative to a reference compound such as the internal standard. (Cf. Tables 1-3) Coincidence of these significant variables 

for the unknown and for a contemporaneous authentic ethanol-internal standard reference, with two or more different columns, normally increases the 

confidence in the identification to the extent required. 

The alternative approach of Blume et al. (59), a recent variant of a classic 

analytic technique, is to demonstrate disappearance of the putative ethanol 

peak of a gas chromatogram following treatment of the unknown specimen with 

alcohol dehydrogenase. Essentially complete disappearance of the supposed 

ethanol peak is the requisite identity-confirmation criterion, since alcohol 

dehydrogenase also partially oxidizes higher alcohols (e.g., isopropanol, 

n-propanol, and n-butanol) under certain conditions. 
3.5 ANALYTICAL AND PERFORMANCE CHARACTERISTICS OF THE METHOD 

The following information illustrates some key performance characteristics 

of the above method for gas chromatographic analysis for ethanol of headspace 

above biological liquids. These data are not intended to constitute a full 

method evaluation; rather, they are typical of a much larger body of data 

obtained in the author's laboratory in the process of developing, modifying, 

and validating the basic method and its procedural subsets presented herein. 

This presentation is limited to several of the major characteristics of the 

method, including accuracy, precision, sensitivity, specificity, and to typi­ 22 

cal examples of its performance in the analysis for alcohol of reference 

specimens, of proficiency survey specimens, and of routine case specimens 

submitted for alcohol analysis in connection with traffic law enforcement. 

Equilibration Time. Time to achieve presumably complete equilibration of 

the volatile components of aqueous reference specimens, as reflected by 

peak heights of the relevant gas chromatographic responses, is illustrated 

in Figure 5 for the 65°C equilibration temperature employed in the automated 

analysis and in Figure 6 for the 38°C equilibration temperature employed in 

the manual method, for a nominal sample ethanol concentration of 100 mg/dl. 

It is evident that at least 35 minutes are required to reach steady state; 

hence 45 minute pre-analysis equilibration times are specified in the procedural sections above, to afford an appropriate safety margin. The peak 

heights ratio of ethanol and acetonitrile internal standard becomes asymptotic (or nearly so) more rapidly than the ethanol response per se. How22 

In order better to illustrate the characteristics of the method and to 

facilitate statistical comparisons, the ethanol concentrations in this 

section are generally reported to three significant figures. However, 

it deserves reemphasis that the results of alcohol analyses of biological specimens, when intended for traffic law enforcement or similar for. 

ensic applications, should be reported in percent w/v to two decimal 

places, truncated; i.e., to two significant figures only. 

- 65 ­
ever, since the time course of the alcohol and the internal standard responses are not entirely parallel, sampling of the headspace vapor should 

be avoided until complete equilibration has occurred. These effects are 

also concentration related, and the illustrated time courses may not be 

applicable to specimens with substantially different compositions. 

Chromatographic Separations and Specificity. A typical gas chromatogram 

for the automatic headspace analysis method, internal standard mode, is 

shown in Figure-9. Certain specificity considerations, such as order of 

elution of various volatile substances, absolute and relative retention 

times, are given in Tables 1 to 3 for several commonly employed columns. 

k 

2 4 6 

WiM 

FIGURE 9.­ TYPICAL HEADSPACE CHROMATOGRAM OBTAINED WITH MULTIFRACT F-40 

AUTOMATED GAS CHROMATOGRAPH (INTERNAL STANDARD PROCEDURE), 

UNDER CONDITIONS DESCRIBED IN TEXT. SAMPLE COMPONENTS SHOWN 

ARE: 1 = METHANOL, 2 = ACETONITRILE, 3 = ETHANOL, 4 = ACETONE, 

5 = ISOPROPANOL; MARKER "10" IS SAMPLE IDENTITY INDICATION. 

CORRESPONDING INTEGRATOR VALUES ALSO SHOWN. 

- 66 ­
 *

When alcohol is the only analyte of immediate concern, the chromatography

time can be reduced to 3 min. or less per sample since the internal standard,

acetonitrile, elutes before ethanol. Alterations in the gas chromatographic

instrument parameters (e.g., column packing and temperature), can, of

course, substantially alter the chromatogram.

Figure 10 is a_chromatogram of a blood specimen from a human subject intoxicated with isopropanol, followed by the chromatogram of the "Low" mixed

Calibrator; analysis was performed by the manual headspace analysis method,

direct (non-internal standard) mode. By coincidence, the acetone peak

height and peak areas of unknown reference samples are virtually identical;

the concentration of blood acetone (100 mg/dl) and of blood isopropanol

(62 mg/dl) are typical of moderate isopropanol intoxication after substantial * 

metabolic conversion of the latter to the former has occurred.

FIGURE 10. HEADSPACE CHROMATOGRAMS OBTAINED WITH MANUAL METHOD (DIRECT PROCEDURE), UNDER CONDITIONS DESCRIBED IN TEXT. LEFT = HUMAN

BLOOD FROM SUBJECT INTOXICATED WITH ISOPROPANOL, RIGHT = CONTEMPORANEOUS MIXED "LOW" CALIBRATOR RUN. SAMPLE COMPONENTS SHOWN

ARE: 1 = METHANOL, 2 = ETHANOL, 3 = ACETONE, 4 = ISOPROPANOL

- 67 -
Calibration Curves and Linearity. Our typical calibration curves for the

automated headspace analysis method, in both the direct and internal standard modes, are illustrated in Figure 11. The curves all demonstrate high

200 500

150 400

300

100

R 200

50 K 100

0

0 100 200 300 400

0

0 100 200 300 400

ETHANOL, mg/di. ETHANOL, mg/dI.

(A) (B)

0 100 200 300 400 0 100 200 300 400

ETHANOL, mg/dI. ETHANOL, mg/d1.

(C) (D)

FIGURE 11. TYPICAL CALIBRATION CURVES FOR ETHANOL WITH AUTOMATED GAS

CHROMATOGRAPHIC HEADSPACE METHOD, UNDER CONDITIONS DESCRIBED

IN TEXT. EACH DATA POINT IS MEAN OF FIVE OBSERVATIONS.

A. PEAK HEIGHT, DIRECT PROCEDURE

B. PEAK AREA, DIRECT PROCEDURE

C. RATIO OF PEAK HEIGHTS, INTERNAL STANDARD PROCEDURE

D. RATIO OF PEAK AREAS, INTERNAL STANDARD PROCEDURE

- 68 -
reproducibility and linearity of calibration of the respective analysis

modes and means of quantitating the ethanol content of the reference specimens. Hence, each of these four quantitation schemes is potentially acceptable and satisfactory for determination of ethanol in liquid specimens.

Linearity of response over the immediate range of interest in any given analysis run is, of course, an important consideration. Typical direct experimental verification of such linearity over the immediate range of interest

for ethanol concentrations in blood is illustrated in Figure 12.

.

BAC, mg/dL

I'l1111llIfo

FIGURE 12. LINEARITY OF DETECTOR RESPONSE TO EHTANOL WITH AUTOMATED GAS

CHROMATOGRAPHIC HEADSPACE METHOD (DIRECT PROCEDURE). EACH

ETHANOL PEAK IS PRECEDED BY A SAMPLE IDENTITY MARKER

-69-

 *

 *

 *

 *

 *

 *

 *

 *

 *
It can be estimated from the gas chromatographic instrument parameters and 

the typical calibration results that the automated method described, conservatively, has a functional sensitivity of 10 ng of ethanol per ml of 

specimen and a functional absolute detection limit for ethanol of approximately 1 nanogram. 

Precision. Reproducibility of the automated headspace analysis method is 

shown in Tables 4 and 5, respectively, for the direct and internal standard 

modes. 

TABLE 4.

PRECISION OF AUTOMATED GC HEADSPACE ANALYSIS FOR ETHANOL WITH MULTIFRACT F-40

INSTRUMENT IN REPLICATEa DETERMINATIONS BY DIRECT PROCEDURE

at 50 mg./dl. Mean = 50.0 mg./dl. S.D. = 0.27 mg./dl. C.V. = 0.55% 

100 mg./dl. 100.0 mg./dl. 1.05 mg./dl. 1.05% 

150 mg./dl. 150.0 mg./dl. 0.95 mg./dl. 0.63% 

200 mg./dl. 200.0 mg./dl. 1.77 mg./dl. 0.89% 

a 

(N=29, 28, 27, 24) 

TABLE 5. 

PRECISION OF AUTOMATED GC HEADSPACE ANALYSIS FOR ETHANOL WITH MULTIFRACT F-40 

INSTRUMENT IN REPLICATEa DETERMINATIONS BY INTERNAL STANDARD PROCEDURE 

at 100 mg./dl. Mean = 100.0 mg./dl. S.D. = 0.62 mg./dl. C.V. = 0.62% 

200 mg./dl. 200.0 mg./dl. 0.55 mg./dl. 0.28% 

300 mg./dl. 300.0 mg./dl. 1.65 mg./dl. 0.55% 

400 mg./dl. 400.0 mg./dl. 2.01 mg./dl. 0.50% 

a 

(N=14, 15, 15, 15) 

Figures 13 and 14 depict the typical precision of replicate analyses for 

alcohol with the automated method in the direct and internal standard modes, 

respectively. 

- 70 ­
 *

--L-I

1

1

I I

1^- ^- t I ` I

I

 * 

FIGURE 13. CHROMATOGRAM ILLUSTRATING TYPICAL PRECISION OF REPLICATE ANALYSES

FOR ETHANOL IN BLOOD WITH AUTOMATED GAS CHROMATOGRAPHIC HEADSPACE

METHOD (DIRECT PROCEDURE), UNDER CONDITIONS DESCRIBED IN TEXT.

SIXTEEN CONSECUTIVE ANALYSES ARE SHOWN (SAMPLE VOLUME = 100

MICROLITERS; BAC = 200 MG/DL). EACH ETHANOL PEAK IS PRECEDED

BY A DOTTED SAMPLE IDENTITY MARKER

-- -

7 77-7

*

I ^ b

 *

LA uFIGURE 14. CHROMATOGRAM ILLUSTRATING TYPICAL PRECISION OF REPLICATE

ANALYSES FOR ETHANOL IN BLOOD WITH AUTOMATED GAS CHROMATOGRAPHIC HEADSPACE METHOD (INTERNAL STANDARD PROCEDURE),

UNDER CONDITIONS DESCRIBED IN TEXT. TEN CONSECUTIVE ANALYSES ARE SHOWN (SAMPLE VOLUME = z ML, BAC = 167 MG/DL), EACH

ETHANOL PEAK IS PRECEDED BY A NUMBERED SAMPLE IDENTITY MARKER

- 71 -
The precision illustrated for the automated method is substantially better 

than can be achieved with manual methods, even with careful manipulations. 

Typical precision for the manual headspace method (direct non-internal standard mode) using peak area measurements, at a specimen concentration of 100 

mg /dl is: Standard deviation of replicate analyses = ±2.80 rig /dl ; coefficient of variation = 2.8%. 

Accuracy. The term "accuracy" is generally understood, in scientific disciplines, to mean concordance or exact agreement between an experimentally 

determined result and the true value of the item under consideration. Although the "true" value for the alcohol content of any unknown or reference 

specimen is, of course, unknown, several experimental approaches can yield 

adequate information regarding the accuracy of.a candidate method for alcohol 

analysis. Findings for two of these are documented here. 

Table 6 reflects the results of independent routine analyses for alcohol on 

a series of whole blood specimens submitted for forensic blood-alcohol analysis by the automated headspace analysis method in the direct non-internal 

standard mode, employed independently by two geographically separted laboratories. The agreement of the results is remarkably good, especially when 

it is considered that individual calibrations were carried out independently 

in the two laboratories and that all of the specimens were stored at 4°C 

for intervals up to 60 days between separate analyses. The mean difference 

between the independent BAC results, 0.0012% w/v, is 0.53% of the mean BAC 

of the 25 specimens (0.226% w/v), and the high correlation coefficient 

(r=0.998) confirms the excellent agreement of these independently obtained 

results. Statistical correlation analysis of these results reveals a 

negligibly slight bias between the two series of results, reflecting the 

independent calibrations. 

When a large number of laboratories carry out independent analyses upon aliquots of the same specimen pool, using several different analytical methods, 

the mean, mode, and median of the combined results closely reflect the "true" 

value, provided suitable precautions have been taken against specimen alter­

-72­
TABLE 6.

RESULTS OF INDEPENDENT ROUTINE ANALYSES FOR ETHANOL ON IDENTICAL BLOOD SPECIMENS,

IN TWO SEPARATE LABORATORIES, BY AUTOMATED GC HEADSPACE ANALYSIS (DIRECT PROCEDURE)

BAC Found, % w/va Difference B b 

Specimen Laboratory A Laboratory B % w/v 

1 0.16 0.16 0 

2 0.33 0.33 0 

0.18 0.17 0.01 

4 0.17 0.17 0 

5 0.25 0.25 0 

6 0.40 0.40 0 

7 0.17 0.16 0.01 

8 0.10 0.09 0.01 

9 0.27 0.27 0 

10 0.17 0.18 -0.01 

11 0.37 0.36 0.01 

12 0.12 0.12 0 

13 0.19 0.19 0 

14 0.32 0.32 0 

15 0.37 0.37 0 

16 0.04 0.05 -0.01 

17 0.12 0.13 -0.01 

18 0.27 0.27 0 

19 0.22 0.22 0 

20 0.19 0.19 0 

21 0.24 0.24 0 

22 0.31 0.30 0.01 

23 0.26 0.25 0.01 

24 0.15 0.15 0 

25 0.29 0.29 0 

Mean 0.0012 

S.D. ±0.0060 

aAnalysis in Laboratory A preceded those in Laboratory 

B by intervals of up to 60 days, with intervening specimen 

storage at 4°C. Both laboratories employed the automated GC headspace analysis method. 

bCorrelation Coefficient: r = 0.998 for Laboratory A Results 

vs. Laboratory B Results 

- 73 ­
ation during aliquoting and against decomposition in transit. These conditions exist in "double-blind" proficiency surveys of alcohol analysis in 

aqueous and biological specimens. Table 7 illustrates performance of the 

automated method (direct non-internal standard mode) in a monthly state 

alcohol-testing proficiency survey, vis-'-vis the participant median results 

for 31 laboratories and the mean results for the 3 reference laboratories. 

Very high correlation was found for the automated method results with the 

reference laboratory mean results as well as with the participant laboratory 

median results. The "blood" specimens in this survey are whole blood hemolysates with significant concentrations of fluoride. 

The same high degree of correlation is shown in Table 8 between the automated 

method (direct, non-internal standard mode) results and the mean values of 

the results of all 85 participant laboratories. The differences probably relate, at least in part, to alterations of the whole blood specimen pool during aliquoting as revealed by differences in the respective hematocrit values 

in the preparing and this laboratory, and to the substantially different 

receipt dates for these specimens in different laboratories. Some bias resulting from different calibrations is also probable. 

These comparisons adequately establish the accuracy of the method described 23 

herein for alcohol analysis. 

23 

"...Analytical procedure(s) for determining alcohol in blood should meet the 

following performance requirements: a. The accuracy and sensitivity of the 

procedure should be such as consistently to attain results within ±0.01% w/v 

of the known value over the range of 0 to 0.30% w/v in analyses of appropriate reference materials of known ethyl alcohol concentration. b. The precision of the procedure should be such as consistently to attain a standard 

deviation not greater than 0.003% w/v in replicate analyses. c. The blank 

values yielded by the procedure in analyses of alcohol-free blood specimens 

consistently should be not greater than 0.01% w/v. d. The specificity of 

the procedure should be adequate and appropriate for. the analysis of biological specimens for the determination of the blood alcohol concentration in 

traffic law enforcement and highway crash investigations." (71) 

- 74 ­
TABLE 7.

TYPICAL PERFORMANCE OF THE AUTOMATED GC HEADSPACE ANALYSIS (DIRECT PROCEDURE)

IN "DOUBLE-BLIND" ANALYSES FOR ETHANOL IN A STATE PROFICIENCY SURVEYa

Reported Ethanol Concentrations, mg/dld 

Ref. Labs. Mean Participant Labs. 

Specimenb Nature F-40 Result (N=3) Median (N=31) 

1 U 196 209 201 

2 A 323 325 320 

3 B 80 82 83 

4 A 232 233 232 

5 B 157 159 162 

6 U 281 283 286 

7 U 197 196 198 

8 A 325 326 320 

9 B 155 162 160 

10 A 320 324 312 

11 B 295 298 286 

12 U 236 239 239 

13 U 160 160 162 

14 A 72 71 73 

15 B 296 300 290 

16 A 323 320 319 

17 B 0 2 0 

18 U 240 249 240 

19 B 250 256 250 

20 U 158 167 162 

21 B 325 336 316 

22 U 201 206 204 

23 U 86 90 90 

24 A 296 297 293 

25 B 254 259 252 

26 A 157 161 159 

aWisconsin State Laboratory of Hygiene Alcohol Testing Evaluation Program (60)

bConsecutive paired monthly specimens (e.g., 1 + 2, 3 + 4, etc.)

cB = Blood; U = Urine; A = Aqueous

dCorrelation Coefficients: r=0.999 for Reference Labs. Mean vs. F-40 Result; 

r=0.978 for Participant Labs. Median vs. F-40 Result 

- 75 ­
TABLE 8. 

TYPICAL PERFORMANCE OF THE AUTOMATED GC HEADSPACE ANALYSIS (DIRECT PROCEDURE)

IN "DOUBLE-BLIND" ANALYSES FOR ETHANOL IN A NATIONAL PROFICIENCY SURVEY"

Reported Ethanol Concentrations, mg/dld 

Specimenb Nature F-40 Result Participant Labs. Mean (N=85) 

1 B 127 123

2 B 76 76

3 A 191 185

4 A 48 49

5 B 216 225

6 B 165 167

7 A 76 76

8 A 178 179

9 B 193 194

10 B 99 99

11 A 196 198

12 A 96 99

13 B 213 216

14 B 121 125

15 A 82 83

16 A 148 149

17 B 91 94

18 B 145 149

19 B 92 97

20 B 195 193

21 B 268 272

22 B 53 61

23 B 155 164

24 B 85 91

25 B 214 229

26 B 110 114

27 B 68 71

28 B 158 161

aU. S. Department of Transportation, Transportation Systems Center 

Blood-Alcohol Proficiency Testing Program (61) 

bConsecutive Sets of 4 quadrimonthly specimens (e.g., 1-4, 5-8, etc.) 

CB = Blood; A = Aqueous 

dCorrelation Coefficient: r = 0.998 for Participant Labs. Mean vs. 

F-40 Result - 76 -
4. QUALITY ASSURANCE: SELECTED ASPECTS 

In modern laboratory practice, quality assurance is generally understood 

to encompass those plans and activities which are intended to provide certainty that the laboratory operations are proper and suitable to the assigned tasks, and that the reported laboratory results are correct, reliable, 

and valid. It is evident that quality assurance is a very large subject, 

only a few selected aspects of which can be considered here. 

4.1 REFERENCE MATERIALS AND THEIR VALIDATION 

Gas chromatographic methods are basically comparison procedures in which 

qualitative and quantitative results are obtained by comparison of the instrument response generated by an unknown sample with those generated by 

samples representing reference materials or calibrators, commonly (but improperly) referred to as "standards." The selection, validation and use 

of reference materials and calibrators is thus critically important to the 

quality and validity of the gas chromatographic analysis data. 

Reference Materials. Pending the development and marketing by the National 

Bureau of Standards of an ethanol SRM (=Standard Reference Material) to 

which working ethanol reference specimens can be traced, alternate means of 

validating the latter must be employed. 

Ethanol reference specimens (often referred to as "calibrators") can be purchased from commercial sources or prepared locally from reagent-grade ethanol. 

Typical aqueous calibrators understood by the author to be available commercially at the time of this writing (June 1976) are: 
Alcohol Stock Solutions (Aqueous) 

Cat. No. 8988/0001 Ampoules, 0.5 mg in 1 ml 

Cat. No. 8991/0001 Ampoules, 1.0 mg in 1 ml 

Cat. No. 9003/0001 Ampoules, 2.0 mg in 1 ml 

Cat. No. 9008/0001 Ampoules, 4.0 mg in 1 ml 

Cat. No. 8985.0001 Large Series (0.8-1.9 mg in 1 ml 

in Steps of 0.1 and 2.0-3.0 mg in 

1 ml in Steps of 0.2) 

Manufacturer: E. Merck, Darmstadt, Germany (E. M. Laboratories, Inc., 500 Executive Blvd., Elmsford, 

N. Y. 10523) 

Ethanol Standard Solution, 0.08% w/v 

Product No. 330-20 

Manufacturer: Sigma Chemical Co., P. 0. Box 14508, 

St. Louis, Mo. 63178. 

Ethanol Calibrators are readily prepared from reagent-grade ethanol, commercially available at 190 Proof ( -95% v/v) or 200 Proof (- 100% v/v). 

Typical sources understood by the author to be available at the time of 

this writing (June 1976) are24. 

Pure Anhydrous Ethyl Alcohol 

Manufacturer: Commercial Solvents Corporation 

245 Park Ave., New York, N.Y. 10017 

Absolute Pure Ethyl Alcohol 

Manufacturer: U. S. Industrial Chemicals Co., 99 Park 

Ave., New York, N.Y. 10016. 

24 

Purchase and use of "pure" (i.e., undenatured) ethyl alcohol is subject to 

Federal regulations (26 CRF), whether tax-paid or tax-free use is involved. 

Details of these regulations are available from the Bureau of Alcohol, 

Tobacco and Firearms, U. S. Department of the Treasury. 

- 78 ­

E 
1 

Specifications for reagent-grade Ethyl Alcohol (190 Proof) and Ethyl Alcohol, 

Absolute are given in the AMERICAN CHEMICAL SOCIETY SPECIFICATIONS for reagent 

chemicals (62). Since anhydrous ethanol is highly hygroscopic, it should be 

obtained, for this use, in the smallest unit size available. Absolute ethanol 

in original, tightly sealed commercial containers is generally water-free 

prior to opening. If necessary, traces of water can be removed with calcium 

oxide or molecular sieve 3A. Because it is much less hygroscopic, 190-Proof 

ethanol is much easier to use as a stock reagent, but does require the additional step of determining its exact initial ethanol concentration by suitable quantitative analysis. Dilute ethanol solutions, such as those prepared 

as calibrators, are stable when properly stored and protected from chemical 

and microbial contamination. Stored in screw-capped borosilicate glass containers at 0-5°C, such solutions are stable for at least one year; in individual 5-10 ml portions in flame-sealed borosilicate glass ampoules, they 

appear to be stable indefinitely (63, 64). 

Aqueous calibrators of desired alcohol concentrations are readily prepared 

by critically careful dilution of appropriate stock reference ethanol solutions, with fully quantitative technique. Stock reference ethanol solutions, 

in turn, are best prepared by weighing the required quantity of absolute ethanol into a conveniently-sized, tared flask; diluting with approximately 50 

ml of reagent-grade water and quantitatively transferring the mixture to a 

liter volumetric flask; and finally, diluting to volume with reagent-grade 

water, with due attention to the flask calibration temperature, normally 20°C 

for Grade A volumetric ware. Alternately (and less preferably), the desired 

calibrator concentration is established by volumetric measurement of the required ethanol quantity, calculated from the desired mass of anhydrous ethanol and its density at the existing laboratory temperature (e.g., 0.78522 

g/cm. 3 at 25°C), obtained from various handbooks. 

Aqueous calibrators yield results identical to those yielded by calibrators 

based on biological liquids such as whole blood or whole blood hemolysates 
in the methods detailed here. That is not necessarily true for other methods of analysis; in fact, it is generally not true for many alcohol analysis 

methods. On purely theoretical grounds, calibrators should be identical to 

unknown specimens in terms of biological matrix composition or properties 

and should cover the entire range of alcohol concentrations of interest. 

Further, at least three calibrators of different alcohol content should be 

used in any given calibration to provide appropriate information about the 

linearity of calibration in the specific instance. In practice, complete 

identity of composition and other relevant properties for calibrators and 

unknowns is often both unachievable or unnecessary (as in the methods 

described here). Surrogate calibrators are often employed effectively; 

these are specimens which possess the critical properties of the unknowns 

(e.g., viscosity, protein content, nonhomogeneity) to an extent sufficient to duplicate or simulate the behavior of the unknowns in the various 

steps of the analysis. Considerable sophistication is required to make 

and implement appropriate choices of surrogate materials, and specialized 

references on this topic should be consulted. Human whole blood, for example, differs substantially from that of some animal species in respect 

to erythrocyte fragility (and hence extent of hemolysis of samples), concentration and nature of plasma proteins, cellular composition, sedimentation 

rate, etc. Whole blood of animal origin, especially that obtained incident 

to slaughter, is usually not a suitable substitute for preparation of calibrators or control specimens which are to duplicate the properties of human blood. In turn, human whole blood obtained in the form of outdated 

blood-bank materials is not identical in properties or composition to 

freshly shed whole human blood, often being substantially diluted with 

liquid anticoagulants and of greatly different carbohydrate and/or electrolytes composition. Additional safety aspects are also relevant such as the 

need to guard against hepatitis transmitted by contact with human blood or 

blood components. For these and other cogent reasons, calibrators and control specimens should be prepared with commercially available products of 

human origin which include proper safeguards, if possible. 
Validation of Reference Specimens and Calibrators. The exact ethanol concentration of stock reference solutions and dilute calibrators, prepared as 

described above, should be validated by independent analysis. The usual 

progression is to employ three linked sets of reference materials: 1) A 

Primary standard such as the NBS potassium dichromate SRM, of known and 

validated chemical characteristics and established quantitative chemical 

reaction relationship to ethanol; 2) a Secondary Standard such as an a-. 

queous solution of ethanol whose concentration has been validly established 

by quantitative analysis using or traceable to a Primary Standard (e.g., by 

oxidimetry with NBS potassium dichromate) and which serves as a stock solution; 3) a Tertiary Standard, such as a ready-to-use, dilute aqueous ethanol 

solution prepared by dilution from the Secondary Standard, to be employed 

directly as a calibrator or for the preparation of other reference samples 

or of control specimens of known alcohol content. 

A variety of chemical and physical methods can be used for the establishment and/or verification of the ethanol concentration of reference materials and calibrators. Suitable physical methods of analysis include pycnometry and refractometry, the experimental results being converted to the 

corresponding ethanol concentration from handbook tables (e.g., 65, 66). 

Osmometry has been advocated by Redetzki (63) for analysis and control of 

aqueous alcohol reference solutions; only osmometers operating on the 

freezing-point depression principle are suitable for this application. The 

author has found this technique useful for the rapid, simple pre-use checking of reference solutions to exclude gross dilution errors or unsuspected 

deterioration. 

Chemical analysis by oxidimetry using a potassium dichromate reagent is a 

suitable method forinitial and periodic verification of the alcohol content of stock and dilute alcohol reference solutions and calibrators, since 
the results are traceable directly to a true Primary Standard ;2 5 it is 

the method of choice recommended here. The reaction, for the oxidation of 

ethanol utilizing potassium dichromate in the presence of sulfuric acid, is 

2Cr2O7 + 3C2H5OH + 16H+ ---• 4Cr+++ + 3CH3000H + 11H20­

Under suitably moderate oxidation conditions (acid concentration below 18N, 

etc.) the reaction does not proceed beyond acetic acid. It follows that 

under such conditions 4.257 mg of K2Cr2O7 are equivalent to 1.000 mg of 

C2H5OH. Most commonly, a measured aliquot of the ethanol sample is reacted 

with excess standard dichromate solution and the unreacted dichromate determined spectrophotometrically or titrimetrically. The Dubowski spectrophotometric microdetermination method with final measurement at 350 nanometers 

(67) is well-suited to this oxidimetric analysis, since it requires only a 

single stable reagent and is rapid, simple, and sensitive. When an aqueous 

alcohol reference solution is to be analyzed', the distillation step of the 

procedure can be omitted, and the reaction is carried out directly on a 1:5 

dilution of the aqueous specimen. Oxidimetry with potassium dichromate is 

also the procedure for determination of the ethanol concentration of standard 

stock solutions stipulated in the DOT Performance Standard for Calibrating 

Units for Breath Alcohol Testers (68). 

Enzymatic oxidation of ethanol in the presence of NAD oxidoreductase, EC 

1.1.1.1 (=alcohol dehydrogenase) can also be used to quantitate ethanol in 

reference solutions and calibrators, the reaction being: 

+ ADH + 

C2H5OH + NAD CH3CHO + NADH + H% 

The ultraviolet spectrophotometric measurement method based on this reaction 

Potassium dichromate, oxidimetric, SRM 136c, Office of Standard Reference 

Materials, Rm. 8311, Chemistry Building, National Bureau of Standards, 

Washington, D. C. 20234. 

- 82 ­

25 
is very sensitive and rapid; it can also be simplified by eliminating the 

primary separation step (e.g., distillation) when analyzing suitably-diluted 

aqueous alcohol solutions. Under ideal analysis conditions, the alcohol 

concentration of aqueous samples can be determined solely from the difference 

between the measured absorbances at 340 nanometers of the unknown and blank 

samples and the established molar absorptivity of NADH; in practice, an 

ethanol calibrator is always included in the analysis series (69, 70). 

Whatever scheme is employed to establish the actual concentration of ethanol 

in stock and dilute reference solutions and calibrators, it should be emphasized that an experimental confirmation of the ethanol concentration independent of the preparation procedure should be made initially (after preparation) 

and at appropriate intervals thereafter if the materials are stored. 

4.2 PERFORMANCE MONITORING 

Both internal and external performance monitoring are accepted quality assurance practices. Of the many variants, those considered here are use 

of within-run and between-run controls, use of internally-prepared "unknown" specimens, and participation in internal and external performance 

monitoring (often referred to as "proficiency testing"). 

Use of Within-run and Between-run Controls. Individual aliquots of a suitably large biological specimen pool, so treated and stored as to maintain 

stability of composition, are included in each series of analyses. The 26 

results should lie within acceptable limits of the previously-estab26"Procedures for blood analysis should include the following controls in 

conjunction with each batch of samples analyzed: ...b. Analysis of a suitable reference or control blood sample of known alcohol content within the 

range 0.10 to 0.30% w/v; the result of which analysis must coincide with 

the known blood alcohol value of the reference specimen within ±0.01% w/v 

if validity is to be assigned to the results for the batch analyzed." (72) 

- 83 ­
lished concentration. To avoid contamination of the specimen pool by repeated entry to withdraw aliquots, as well as the necessity to thaw or bring 

it to room temperature on each occasion of use, it is good practice to divide the homogeneous or well-mixed specimen pool into single-use aliquots 

(e.g., 1 ml portions) to be stored preferably in flame-sealed ampoules. 

Whole-blood alcohol control specimens are commercially available. At the 

time of this writing (June 1976), the following such item was understood 

.by the author to be marketed: 

Product No. 2930-12, LederEth ® Whole Blood Alcohol Control 

Manufacturer: Lederle Diagnostics, American Cyanamid Co., 

. Pearl River, N. Y. 10965. 

These materials consist of human whole blood containing ethanol (typically 

at a BAC of 0.15% w/v) and are furnished with analysis results applicable 

to the specific lot of control material. Commercial or laboratory-prepared 

controls (i.e., surrogate specimens identical (if possible) or appropriately 

similar to the unknown specimens in overall composition and expected alcohol content) should be included in every analysis series ("run"). Only if 

the experimental result obtained for the control specimen in that run coincides acceptably with the established target value should the results for 

unknowns in that analysis series be cleared for release. Conversely, 

should the control specimen result not be acceptable, search for the cause 

of the discrepancy should be instituted and any indicated remedial measures 

completed before analysis of unknown specimens is resumed. It should be 

emphasized that the control specimen(s) are to be included in each run in 

addition to and apart from the calibrators and should not be aliquots of 

any calibrator used in that run. 

- 84
Internal and External Pro_ficiencz, Testing. Performance evaluation with survey or proficiency testing specimens has become an indispensable part of laboratory quality assurance practice and of laboratory regulatory programs. 

Internal and external proficiency testing have some similarities and several 

significant differences, especially in objective. Internal proficiency testing is a useful and objective but limited technique for detecting and documenting presence or absence of acceptable laboratory performance (in the tested 

area) according to predetermined criteria and perhaps pinpointing one or 

more commonly encountered sources of error or indicating gradual changes in 

laboratory performance. In this respect, it serves in part as a control 

function. 

External proficiency testing has the advantage of a larger data base from 

(often many) participating laboratories and, usually, from a separate group 27 

or "reference" or "referee" laboratories. It thus permits relative assessment of the laboratory's performance in comparison with a peer group and often compared with different an4lytical methodologies which may not be in use 

locally. Since the external proficiency test specimens are identified as 

such upon receipt, whether delivered by common carrier or hand-carried by inspectors, they clearly often receive special treatment not accorded routinely 

submitted specimens. To that extent, the results of participation in external proficiency testing or surveys constitute a measure of best laboratory 

capability rather than an indication of standard or routine performance. 

This limitation also exists to some degree in internal proficiency testing 

but can be more easily overcome, in part at least, by use of split-specimens, 

masked submissions by supervisors, and similar techniques. External proficiency testing periodically documents elements of laboratory performance, 

As commonly understood, "reference" laboratories determine the true identity 

or composition of a specimen, or characterize it; "referee" laboratories establish the level of acceptable performance for participant laboratories, 

usually for quantitative or semiquantitative results.(74) 

- 85 ­

27 
and continued satisfactory performance according to predetermined criteria 

thus established is often a requirement for laboratory licensure in the 

tested category of laboratory activity. The techniques of proficiency 

testing and its multiple aspects are a highly specialized field and the 

applicable literature should be consulted. (73-78). 

Use of internal proficiency testing is recommended as a good management 

practice for surveillance of laboratory performance. If "blind" specimen 

handling is possible (i.e., use of proficiency test specimens sufficiently 

similar to those routinely analyzed and submitted like the latter with respect to containers, laboratory request forms, putative identification, use 

of the mails, etc.) it adds an element of credibility, but analysts quickly 

become adept in identifying such "blind" submissions unless extraordinary 

care is taken in their camouflage. 

External proficiency testing in the health laboratory field is widely carried out by Federal agencies, units of many state governments, and professional organizations in the private sector. Although occasional probes and 

periodic surveys of laboratory performance in analyses of aqueous or biological specimens for alcohol have been carried out by several organizations 

for varying intervals in the past (e.g., by the American Academy of Forensic 

Sciences Toxicology Section, California Association of Criminalists, Center 

for Disease Control), the sole current national program of proficiency testing in alcohol analysis known to the writer is the Blood-Alcohol Proficiency 

Testing Program of the National Highway Traffic Safety Administration, U. S. 

Department of Transportation (operated by Transportation Systems Center, U. S. 

Department of Transportation, Cambridge, Mass. 02142). Many states, however, 

operate individual statewide proficiency testing programs in alcohol analysis. 

4.3 OTHER QUALITY ASSURANCE PRACTICES 

There is at present no universally recognized equivalent, in forensic or 

health laboratory practice, to the overall generally-accepted operational 

- 86 ­
standards encompassed under the term "GMP" (Good Manufacturing Practices) 

in, for example, the pharmaceutical industry. In their absence, each 

laboratory must strive individually to identify and implement operational 

practices which the consensus of informed practitioners would identify 

as "Good Laboratory Practices," and to eliminate any local practices 

inconsistent therewith. 

Certain desirable practices are obvious, and many are common to all forensic 

laboratories (and often health laboratories as well). These include the full, 

contemporaneous, unique labeling and identification of every incoming specimen and its associated items (laboratory request forms, shipping containers, 

etc.) and of every laboratory-generated hard-copy data form. Raw data 

should be entered into permanent bound data log books if handwritten or 

assembled into permanent data log books if computer-terminal generated, etc. 

There is no substitute for dating (and timing when indicated, as when multiple analytical runs are made in a given day) and identifying with the name 

of the writer all record entries. The usual bookkeeping rules should be 

followed; all entries are made in permanent reproducing ink and are 

thereafter never obliterated or eradicated; errors or deletions are treated 

by AeR-eb 4 e^at$Ag-thFedgh- #p pg-a€-the-}te^fs^-eeReeined, associated 

ibidem with any corrections or superseding information. Liberal use should 

be made of time stamps, numbering machines, etc. In addition to raw data 

compilations associated with each specimen analyzed, all computations entering into the final reported result should be shown in full in the permanent 

record log, including any unique factors (special dilutions, individual 

sample quantities other than routinely used, etc.). Data files should be 

maintained for an adequately long period to meet all legal and operational 

requirements; when necessary, they can be microfilmed. 

A separate permanent, complete, chronological file should be maintained for 

methods. This includes complete documentation of all procedural steps for 

a given analysis, and literature reference(s), reagent preparation directions, 
sample calculation, instruction for reporting of results, instrumental parameter listings, etc. The initial method description should bear the date of 

preparation and full name of the preparer, and all subsequent changes should 

consist of dated, signed additions (perhaps on differently colored paper 

stock), but never of physical deletions. Separate bench reminder cards can 

be used to summarize salient steps in the procedure for time/point-of-use 

availability. 

Reports of examination of blood and other biological liquids for alcohol 

should be complete, correct, and fully self-contained. Hence, they should 

always include the following data: 

a. Laboratory case identification number 

b. Complete subject and specimen identification (Cf. Section 1.3) 

c. Unequivocal statement of the examination(s) or analyses performed, 

together with brief identification of the analysis method employed 

d. Unequivocal report of the findings, always inclusive of concentration 

units employed 

e. Interpretation of the findings, if required 

f. Significant date/time information relating to specimen collection, 

specimen receipt in the laboratory, analysis, and reporting 

g. Legible name, title, signature of the reporting analyst(s) and 

official(s) 

h. Authentication device(s). 

The last item deserves further comment. In this era of efficient, ubiquitous 

copying machines, it seems obvious that original authentic reports should be 

readily distinguishable from unofficial copies and forgeries. Report forms 

can be printed on security paper which is uniquely identifiable and reveals 

attempted alterations; versions are available which resist copying by xerographic methods. It is, additionally, recommended good practice to authenticate the completed report and any authorized copies with an embossed, ser­
rated, or penetrating seal or legend, such as those used for commercial 

checkwriters, passport photograph attachment, bank cancellation of checks, 

etc. The embossed authentication used for reports originating from the 

writer's laboratory is illustrated in Figure 15. Distribution of official 

copies should be shown on the original of the report. 

i o I 

FIGURE 15.­ EMBOSSED AUTHENTICATION FOR TOXICOLOGY LABORATORY REPORTS, 

EMPLOYED IN AUTHOR'S LABORATORY 

It has a salutary effect on the credibility of the laboratory and of its 

reports, as well as on the conscientiousness of laboratory personnel in recording information and data, if the laboratory observes a universal "open 

book" policy. By this is meant that original raw data entries, chromatograms, 

calculations, proficiency testing information, etc., are available for inspection in the laboratory, at reasonable times, by any appropriately interested 

party (e.g., attorneys representing a tested subject) without further legal 

process. The advantages of such a policy far outweigh any potential disadvantages, and it should be pursued whenever it is statutorily permissible. 

- 89 ­
5. INTERPRETATION OF RESULTS 

In forensic and clinical laboratory practice, proper interpretation of results depends in substantial measure on the use to be made of those laboratory results. Further, many jurisdictions have statute law governing the 

interpretation of the results of alcohol analyses in relation to the alleged 

offense of operating a (motor) vehicle under the influence of alcohol, which 

remains among the most common applications of breath-alcohol and blood-alcohol analyses. Virtually all jurisdictions also have applicable case law relating to the interpretation of traffic law-related alcohol analyses. The 

material which follows is not intended to supersede or replace such applicable statute or case law, but rather to supplement them. 

An understanding of the somewhat unique position of ethanol in the broad 

spectrum of interpretative toxicology may be useful at this point. With 

most other drugs (e.g., the barbiturates), the usual practice is to define 

expectable therapeutic (where applicable), toxic, and lethal concentrations 

in a specified medium (e.g., blood plasma). In this context, therapeutic 

concentration is generally considered that accompanying effective therapeutic action of the drug and/or its metabolites in humans, and toxic concentration is often regarded as that associated with serious toxic symptoms 

in humans (79). With alcohol, interpretation of concentrations found in 

whole blood (for example, and limited to that medium for the moment to simplify the discussion) is typically required for the following purposes: 

1) To establish whether or not alcohol is a significant factor in 

a patient's clinical condition, or whether a patient is intoxicated 

2) To establish whether or not the alcohol-related element of a 

motor vehicle or other traffic offense is present. 
Thus, in the former, the BAC becomes an element in decisions concerning diagnosis, prognosis, or treatment, while in the latter it becomes an element in 

the investigation and prosecution of an alleged traffic offense. It is easier to consider these two applications separately since they involve significant differences. In general, however, and in contrast to the usual practice 

with respect to most other drugs subject to abuse, four special features 

apply: (1) There is no accepted therapeutic concentration in the usual sense; 

(2) the demonstrable effects of alcohol are of substantial legal (and clinical) significance at other than the toxic and lethal concentrations in blood, 

and hence lesser concentrations are critically important; (3) in many instances existence of a BAC less than, or equal to or greater than a statutoryspecified value is per se the conclusive decisive criterion (or more commonly is a presumptive evidential factor); and (4) unequivocal demonstration 

of the presence of alcohol is the sole decisive criterion in certain special 

situations. 

5.1 BLOOD-ALCOHOL CONCENTRATIONS 

While artificial in a sense, it is convenient to consider interpretation of 

BACs separately with respect to clinical and law enforcement applications. 

Figure 16 is a schematic flow-chart illustrative of the interpretation of 

BACs (or other alcohol findings) in typical civil and criminal justice proceedings. 

Clinical Applications. Disregarding its social-and-recreational-drug use 

aspects, the acute effects (if any) of alcohol intake on a person can only 

be properly evaluated in the light of the total individual situation and of 

the entirety of his or her environment at the time concerned. Most often, 

the acute effects of alcohol of interest are those related to alcoholic intoxication, usually understood to mean the demonstrable, significantly deleterious acute effects of alcohol attributable to its direct pharmacological 

actions. 

- 91 ­
Question of Alcoholic 

Influence/Intoxication 

Raised; or AlcoholRelated Arrest

I

Procurement of 

Blood Specimen 

Blood Alcohol Analysis 

Alcohol Present Alcohol Absent 

BAC = X BAC = 0 

BAC Per Se Interpretation, Dismissal of Charges, 

Controlling of BAS Required' or Disregard of their 

Alcohol Elements 

Dismissal of Charges, 

or Conviction, Guilty V'171 1Co 1111 

// Co clusi n: 

Plea, etc. Alcoholic^Influence///// e

Alcoholic Influence

/Absent ////Present

at Material Time ^I at Material Time

Dismissal of Charges, Conviction, Plea, etc. 

FIGURE 16.­ SCHEMATIC FLOWCHART ILLUSTRATING ROLE OF INTERPRETATION OF 

BLOOD ALCOHOL FINDINGS IN TYPICAL CRIMINAL AND CIVIL JUSTICE 

PROCEEDINGS (INTERPRETATION FUNCTIONS CROSSHATCHED) 

- 92 ­
-Against the background that these acute effects of alcohol can range continuously from subclinical to lethal, it can be said that (acute) alcoholic 

intoxication constitutes 

1) a state of unfitness, attributable to the acute effects of 

alcohol upon the human body and mind and their several processes and functions, safely to undertake the task(s) attempted; 

or 

2) a state of significant danger to the life of the individual 

concerned, resulting from the acute effects of alcohol upon 

the human body and mind and their several processes and 

functions. 

The second situation is analagous to any life-threatening drug overdose. 

The continuum of the acute effects of alcohol upon the human body and mind 

and their function and processes is reflected in Table 9, developed by 

Dubowski (80). It should be noted that there is deliberate overlap between the designated stages of alcoholic influence and cross-over between 28 

these stages and the adjacent clinical signs and symptoms, as well as between both of these categories and the BAC ranges shown. While the tabulation is, in the author's experience, typical, exceptions to the categorized 

information are encountered. 

Space does not permit further consideration herein of the nature and extent 

of individual fluctuations in consumption tolerance and constitutional tolerance to alcohol. It is also recognized that interpretations analagous to 

those required for clinical applications are frequently called for in connec•­

In common clinical usage, signs denote objective indications or manifestations of the existence of health, disorder, or disease; symptoms refer to 

subjective functional evidence or manifestations of a patient's condition. 

The former are thus observable, but the latter require reporting by the 

subject. 

- 93 ­

28 
TABLE 9. STAGES OF ACUTE ALCOHOLIC INFLUENCE/INTOXICATION (80) 

BLOOD-ALCOHOL 

CONCENTRATION, oW/V 

STAGE OF 

ALCOHOLIC INFLUENCE 

CLINICAL SIGNS/SYMPTOMS 

No apparent influence

0.01-0.05 Sobriety Behavior nearly normal by ordinary observation

Slight changes detectable by special tests

Mild euphoria, sociability, talkativeness 

0.03-0.12 Euphoria

Increased self-confidence; decreased inhibitions

Diminution of attention, judgment, and control

Loss of efficiency in finer performance tests

Emotional instability; decreased inhibitions 

Loss of critical judgment 

0.09-0.25 Excitement

Impairment of memory and comprehension 

Decreased sensitory response; increased reaction time 

Some muscular incoordination 

Disorientation, mental confusion; dizziness

Exaggerated emotional states (fear, anger, grief, etc.

Disturbance of sensation (diplopia, etc.) and of perception of color,

0.18-0.30 Confusion form, motion, dimensions 

Decreased pain sense 

Impaired balance; muscular incoordination; staggering gait, slurred 

speech 

Apathy; general inertia, approaching paralysis 

Markedly decreased response to stimuli 

0.27_0.40 Stupor Marked muscular incoordination; inability to stand or walk 

Vomiting; incontinence of urine and feces 

Impaired consciousness; sleep or stupor 

Complete unconsciousness; coma; anesthesia 

Depressed or abolished reflexes 

0.35-0.50 Coma

Subnormal temperature

Incontinence of urine and feces

Embarrassment of circulation and respiration

Possible death

0.45' Death

Death from respiratory paralysis 
tion with judicial and quasi-judicial proceedings. Common problems in these 

.categories involve questions regarding the existence and extent of alcoholic 

intoxication as a partial or complete defense against workmen's compensation 

claims or as an issue in other types of civil litigation; questions regarding the defendant's capacity to form intent for (or execute) an alleged criminal act; and_questions concerning a subject's ability adequately to understand his rights in an investigative or custodial situation and his or her 

ability intelligently, knowingly, and understandingly to waive Mirandaderived rights. 

Applications to Traffic Law Enforcement. These fall into two principal Z 

categories: 

1) To establish whether or not the alcohol element of the alleged 

offense is present in a jurisdiction which prohibits operation 

or actual physical control of a (motor) vehicle by a person 

whose BAC is equal to or greater than a specified value 

2) To establish whether or not the alcoholic influence or alcoholic 

impairment element of the alleged offense is present in a jurisdiction which prohibits operation or actual physical control of 

a (motor) vehicle by a person while under the influence of 

alcohol (etc.) or while such person's ability to operate a 

(motor) vehicle is impaired by the presence of alcohol in the 

blood. 

29 

UVC §11-902: "Driving while under influence of alcohol or drugs. (a) A 

person shall not drive or be in actual physical control of any vehicle 

while: 1. There is 0.10 percent or more by weight of alcohol in his blood; 

2. Under the influence of alcohol... 4. Under the combined influence of 

alcohol and any drug to a degree which renders him incapable of safely 

driving..." (81) 
In the first instance, the reported BAC result becomes a controlling element 

in the adjudication of this so-called per se" offense which makes driving illegal at or in excess of a stipulated blood-alcohol concentration. In the second instance, the statutory provisions merely create a factual, rebuttable 

presumption of being under the influence of alcohol, based upon the same evidence (i.e., BAC 2 a specified value). Suitably expert interpretation is 

therefore required in the latter situation on the issue of whether the BAC 

in evidence is indicative of existence of alcoholic intoxication, influence, 

or impairment at the material time, to the requisite standard of proof ­

customarily, in criminal litigation, beyond a reasonable doubt. In reaching 

that opinion, the expert witness may, depending upon the jurisdiction involved, need to be cognizant of applicable case law defining such relevant 

terms as "intoxicated," "under the influence of alcohol (or intoxicating 

liquor)," "impaired," etc. There is massive case law on this subject (82); 

the following classical examples show the diversity of these interpretations: 

a.­ Deskin v. State (Oklahoma, 1951): "Not drunk is he who from 

the floor can arise and drink once more, but is he drunk who 

prostrate lies and can neither drink nor rise." (83) 

b.­ State v. Stout (Iowa, 1956): "...if by reason of the use of 

alcoholic liquors.. .he has lost control in any manner or to 

any extent of his reason or faculties, or the control of motion 

of his person or body, then he is in an intoxicated condition 

within the meaning of the law..." (84) 

c.­ Steffani v. State (Ariz., 1935): "The expression 'under the 

influence of intoxicating liquor' covers not only all the well 

known and easily recognized conditions and degrees of intoxication, but any abnormal mental or physical condition which is 

the result of indulging in any degree in intoxicating liquors, 

and which tends to deprive him of that clearness of intellect 

and control of himself which he would otherwise possess. If 

the ability of the driver of an automobile has been lessened in 

the slightest degree by the use of intoxicating liquors, then 

- 96 ­
the driver is deemed to be under the influence of intoxicating liquor..." (85) 

Other jurisdictions have appellate decisions which promulgate as the standard for establishment of alcoholic influence or intoxication that the subject lack to "an appreciable degree the ability to function properly..." or 

that he be "so affected by the ingestion of... alcohol as to impair his 

ability to drive in an appreciable degree..." (86). It is altogether esoteric 

and treacherous territory, and the interpretation of alcohol concentrations 

in blood (or in other specimens) in relation to alleged traffic law violations or other offenses is distinctly no task for a tyro, but requires an 

experienced, seasoned toxicologist with directly applicable first-hand experience in this field. 

There is a further complication. In traffic law enforcement, the time of 

blood sampling is commonly not the material time of evidential interest; 

hence, retrograde extrapolation of the BAC found to an earlier time 

(e.g., that of a vehicular crash or an alleged traffic law violation) is 

not infrequently requested. Unfortunately, that material time is virtually 

30 never identified statutorily as the time of collection of the specimen. 

This is a most complex matter involving biological and other factors, which 

has been covered in detail elsewhere (3, 4, 87). For the present purpose 

it suffices to note that various assumptions of uncertain validity would 

30 

What,never? Well, hardly ever: Illinois Vehicle Code 1972: "§11-501. Persons under the influence of intoxicating liquor or narcotic drugs... c) evidence of the amount of alcohol in the person's blood at the time of the act 

alleged as shown by a chemical analysis of his breath, blood, urine, saliva 

or other bodily substances, is admissible... and the result of any such analysis shall give rise to the following presumptions: 1. If there was at the 

time of such analysis 0.05 percent or less by weight of alcohol in the person's blood, it shall be presumed ...2. If there was at the time of such analysis in excess of 0.05 percent but less than 0.10 percent by weight of alcohol in the person's blood, such fact shall not give rise to any presumption ...3. If there was at the time of such analysis 0.10 percent or more by 

weight of alcohol in the person's blood, it shall be presumed..." (emphasis 

added) (It seems probable that the legislature intended to equate "time of 

analysis" with "time of test" and "time of specimen collection."), 

- 97 ­
generally be required for such an exercise in a given real-world case; hence, 

in the view of the writer, such speculative retrograde extrapolation of the 

BAC found is unwarranted and should not be undertaken. Recent case law supports the view that courts are becoming alerted to these problems and will 

not require inappropriate speculative retrograde extrapolation. To quote 

the Idaho Supreme Court in State v. Sutliff (1976): 

"...The primary question for resolution by this Court is whether the 

results of a properly administered blood alcohol test must be related 

back to the time of the alcohol related offense as a foundational prerequisite to admissability... 

...we hold that this statute does not require extrapolation back but 

establishes that the percentage of blood alcohol as shown by chemical 

analysis relates back to the time of the alleged offense for purposes 

of applying the statutory presumption. This holding is in accord with 

those of other jurisdictions who have considered the question... 

A contrary result could defeat the statute entirely since an extrapolation, particularly to a period prior to the defendant's 'peak' period, 

would often be based solely on the defendant's own testimony as to the 

amount of alcohol ingested, the period of time over which it was ingested and the time of the last consumption of alcohol..." (88) (Emphasis 

added.) 

Similar considerations apply to the attempt to state unequivocally what 

dose of alcohol or quantity of alcoholic beverage(s) was ingested by a subject, solely on the basis of the SAC and such general factors as the subject's body weight, sex, and the supposed time of alcohol intake. 

A synopsis of information bearing on the interpretation of BACs for legal 

purposes has been published (89). 
5.2 ALCOHOL CONCENTRATIONS IN OTHER SPECIMEN MATERIALS 

In autopsy toxicology, studies of alcohol distribution in various body liquids and tissues may be warranted in special circumstances, e.g., to estimate the state of alcohol distribution in the subject at the time of death, 

or for statistical purposes. The uses and limitations for alcohol analysis 

of specimens in living subjects other than blood and breath have been covered previously (Section 2.1.1). It is clear from that discussion that 

interpretation of alcohol findings in most other specimens is fraught 

with pitfalls for most purposes. 

In the absence of blood, such findings can establish presence or absence of 

alcohol in the body, which (infrequently) may be useful to rebut, say, alleged lifelong abstention from alcohol. Brief consideration of other specimens follows and documents the hazards of estimating BACs from other materials. 

Cerebrospinal Fluid. This has not been extensively utilized for alcohol determination in recent years. At alcohol distribution equilibrium (which is 

always an uncertain factor in routine cases), a mean alcohol quotient for 

human lumbar CSF/blood of 1.14 in 46 samples was reported by Harger et al. 

(90). Gettler and Freireich reported a mean CSF/blood quotient of 1.08 

for 15 subjects (91). Other tabulations give a mean ethanol content value 

for CSF of 1.27 relative to that in blood (92). It is relevant that alcohol 

in CSF lags behind that in blood during uptake as well as during elimination. 

The reported data indicate that fluctuation in experimentally determined alcohol ratios for CSF/blood are so aside as to make attempts to estimate the 

coexisting BAC from CSF-alcohol analysis futile. 

Saliva. Alcohol apparently reaches saliva by passive diffusion and should, 

therefore, partition between it and plasma according to their relative water 

content. The weighted average quotient of saliva to venous blood-alcohol 

was reported by Coldwell and Smith (93) as 1.12 for 244 sample pairs; Friede­

-99­
ma-n et al. (43) had earlier reported a mean saliva/venous blood quotient of 

1.10. With renewed interest in saliva as a practical medium for drug determinations of clinical relevance and improved routine collection techniques 

for secreted parotid saliva now available, it is likely that the prediction 

of BACs from saliva-alcohol analysis can be improved to the point of practical utility by modern standards. 

Urine. The voluminous literature on the relationship of alcohol concentrations of alcohol concentrations in blood and urine (5) has been referred to 

above. It supports virtually any ratio anyone would wish to use for the 

estimation of BACs from urine-alcohol values, a practice to be deprecated 

for the reasons stated above (Section 2.1.1). Under special, experimentallycontrolled conditions the relationship may be reasonably consistent (e.g., 

Coldwell and Smith (93) found a urine/venous blood alcohol quotient of 

1.24 at the half-time period of urine accumulation in the bladder); however, 

in routine applications to case material that consistency is notably absent. 

There is no assurance that any of the many suggested formulae will yield 

BAC estimates from urine-alcohol values sufficiently valid for forensic 

or clinical use in a given instance. 

To quote Kaye and Cardona (94): "Our data clearly confirm what other investigators have claimed: that the relationship (ratio-range) between the 

concentrations of alcohol in urine and in blood may vary widely. This 

renders it unreliable to use an average conversion factor in medicolegal 

cases." 

Vitreous Humor. Comparative studies of alcohol and other drugs in vitreous 

humor and blood are of relatively recent origin. For Sturner and Coumbis' 

series of paired blood and vitreous humor specimens (19), a mean vitreous 

humor/venous blood alcohol ratio of 1.10:1 (s.d. ±0.74:1) can be calculated 

for 38 specimen pairs. For the series of 30 paired vitreous humor and heart/ 
great vessel blood specimens of Leahy et al. (20) a mean quotient of 1.07 

±0.09 can be calculated. Felby and Olsen (21) postulated that the relative 

alcohol concentrations in vitreous humor and blood, at diffusion equilibrium, 

should conform to their respective water content and proposed the theoretical 

quotient 1.27 for vitreous humor/blood alcohol. However, their data for 27 

pairs of specimens yields a mean quotient of 1.38 ±0.17. In Coe and Sherman's 

174 cases a vitreous humor/blood alcohol quotient of 1.12 was indicated (22). 

Neumann and Pribilla's series of 36 paired vitreous and serum specimens (23) 

yields a mean vitreous/serum alcohol quotient of 1.04 ±0.26. Norheim (25) 

analyzed the distribution of alcohol between vitreous humor and blood in 

73 cases and found a mean quotient of 1.19 with a range of 0.90-1.89. 

Finally, Scott et al. (26) reported 38 cases in which blood obtained prior to 

embalming was compared for alcohol content with vitreous humor obtained after 

embalming (and with pre-embalming vitreous humor in 9 of these cases). The 

data yielded a mean post-embalming vitreous humor/blood alcohol quotient of 

1.03 ±0.33 for the 27 pairs in which alcohol was found in both specimens, 

and a mean pre-embalming vitreous humor/blood alcohol quotient of 0.90 ±0.16 

for 8 such pairs. 

The use of vitreous humor has been increasing as a postmortem specimen in 

situations in which blood is unavailable, and it is a useful material in that 

circumstance. As with other relatively isolated body components, the vitreous 

humor lags behind blood in alcohol uptake and elimination. Hence, although 

congruence between the alcohol content of vitreous humor and blood at alcoholdistribution equilibration has been experimentally demonstrated in animal 

models, it often cannot be established with the requisite certainty for vitreous humor - blood alcohol conversions whether a real-world subject was in 

the postabsorptive state. In the circumstances, only guarded interpretations 

can validly be made, such as ruling out significant body alcohol presence at 

the time of death when the vitreous humor is alcohol-free. 
5.3 DECOMPOSED SPECIMENS 

This is a large, tedious, and controversial subject which continues to generate literature. Because it has been convincingly demonstrated that neoformation of ethanol can take place in body fluids and tissues during decomposition under both aerobic and anaerobic conditions, interpretation of 

the ethanol content demonstrated in such specimens is a treacherous, hazardour, and generally unrewarding task. It complicates matters that decomposition and alcohol neoformation can occur in postmortem specimens both before 

and after their removal from the body; but specimens from living subjects 

are also subject to decomposition after procurement when specimen preservation and/or storage conditions are inappropriate or improper. The simplest 

response is to forgo alcohol analysis on decomposed specimens. 

Excynination of Decomposed Specimens. The interpretation of findings in decomposed specimens (if their analysis is undertaken at all) involves three 

separate but related tasks: 

1)­ Chemically valid establishment of whether ethyl alcohol is 

conclusively present, and if so, in what concentration 

2) Chemically valid establishment of the absence, or presence 

and concentration, of certain other compounds of interest 

in relation to assessing the presence and extent of putrefaction and the likelihood of alcohol neoformation 

3) Assessment of the significance of these findings. 

The common initial step is to identify and document whether consequential decomposition (especially to the point of putrefaction) has occurred in the 

specimen under consideration. In the event extensive putrefaction has occurred, the indicia are usually obvious (using blood as an exemplar): The distinctive odor representing inorganic sulfur compounds and mercaptans, abnormal color reflecting the changes in hemoglobins, complete hemolysis, abnormal a 

- 102 ­
physical state of the specimen with respect to viscosity and liquefaction, 

etc. Less extreme putrefaction is more subtle to detect and more difficult 

to document. Gas chromatographic evidence of the presence of methane and 

other decompositional products is usually present; Osterhaus (11, 16), for 

example, demonstrated presence of acetone, isopropanol, n-propanol, n-amyl 

alcohol, and tert-butanol in trace quantities in some (but not all) decomposed blood specimens after long-term storage. 

It is, of course, necessary to establish conclusively that a volatile substance present in a decomposed specimen and tentatively identified as ethanol on the basis of its GC retention time is, in fact ethanol, and this 

matter was covered previously. Conclusive identification of ethanol does 

not per se exclude the possibility of neoformation. Some authors have 

suggested elaborate schemes for the attempted differentiation between neoformed alcohol resulting from putrefactive processes and that initially 

present in the specimen. Smith et al. (96), for example, routinely culture 

postportem specimens derived in connection with aircraft accident investigations. They assess the ability of contaminating microorganisms to produce alcohol by 24-hour incubation in a brain-heart infusion-medium containing 2% glucose, subtracting the alcohol value thus found from the total 

in the original specimen. When such subtraction leaves an alcohol remainder 

equal to or greater than 0.05% they consider "it is highly probable that 

alcohol has been ingested by the subject," although they have documented 

occasional instances of positive, alcohol-producing bacterial or fungal 

cultures yielding alcohol values corresponding to 0.067 and 0.075%. (Presumably, blood is the specimen material to which these findings pertain.) 

Others have reported substantially higher blood alcohol concentrations 

attributed to putrefactive neoformation. 

Assessment of the Significance of Findings. As already noted, the literature pertaining to decomposed specimens, postmortem changes of drug concentrations, and postmortem generation of alcohol is substantial and still increasing. Two recent summaries are pertinent (97, 98). 
Perhaps the most conservative approach to interpretation of the results of 

alcohol analyses on specimens from decomposed cadavers is the 1963 position 

of Dubowski: "No valid conclusions are routinely possible from alcohol determinations performed on blood specimens obtained from decomposed bodies or 

those subjected to prolonged submersion" (7). The same view can be held 

with respect to the significance of findings on specimens which have decomposed subsequent to their procurement from living or dead subjects. For 

the reasons discussed, the intervening widescale use of gas chromatography 

and the consequent increased specificity of alcohol analyses in biological 

specimens does not add a proportionately greater element of validity to the 

interpretation of the findings. It is evident that both alcohol loss and neoformation can occur in postmortem specimens before and after their removal 

from the body but that only the latter can occur in specimens from living 

subjects. The resulting combinations and permutations of conditions and 

effects can yield various final results which are capable of ready differentiation, the principal ones being: 

a.­ A specimen can become alcohol-free if its initial concentration 

was low 

b.­ A moderate initial alcohol concentration can be reduced to an 

insignificant one 

c.­ An initially alcohol-free specimen can gain a low or moderate 

alcohol concentration 

d.­ An initially high alcohol concentration can be reduced to a 

moderate concentration 

e.­ An initially moderate alcohol concentration can be increased 

to a high one 

f.­ The initial alcohol concentration can be accidentally reproduced 

by offsetting losses and neoformation. 
I 

} 

If three time factors (before/after/before and after removal from body) and 

three directional factors (alcohol increase/decrease/no change) are considered, there are nine possible combinations for postmortem specimens. Rarely, 

if ever, will the ancillary information be available to decide which of these 

possibilities led to the observed result. If any assessment of the results 

is to be made, it should probably be limited to the opinion that complete 

absence of alcohol negates, with high probability, the initial existence of 

a highly elevated BAC and that a very high BAC (in or near the lethal concentration range) is unlikely to be attributable only to neoformation. 
APPENDIX - REPORT OF INVENTIONS 

After a diligent review of the work performed under this contract, no 

discovery, improvement, or invention deemed patentable was 

found. Details are given for a highly reliable and practical method of 

analysis of alcohol. This information should assist forensic scientists in performing valid and reliable analysis for alcohol in biological liquids and interpreting the results of such analysis for traffic 

law enforcement purposes. 

r 

- 107/108 ­
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1.­ Dubowski, K. M., "Quality Control in Toxicology: The Necessity," in 

PROFICIENCY TESTING - TOXICOLOGY CRITIQUE October 1972, Atlanta, Center 

for Disease Control, U. S. Dept. of Health, Education, and Welfare, 1972. 

2.­ IMPROVEMENT AND STANDARDIZATION OF BLOOD ALCOHOL ANALYSIS (Results of the Meeting at DOT Headquarters, Washington, D.C., May 10, 1973). 

3.­ Mason, M. F., and Dubowski, K. M., "Alcohol, Traffic, and Chemical Testing in the United States: A Resume and Some Remaining Problems," Clin. 

Chem. 20: 126-140 (1974). 

4.­ Mason, M. F., and Dubowski, K. M., "Breath-Alcohol Analysis: Uses, Methods, 

and Some Forensic Problems - Review and Opinion," J. Forensic Sci. 21: 9­

41 (1976). 

5.­ "Selected References on Analysis of Alcohol in Urine for Traffic Law Enforcement Purposes and on Relationship of Alcohol Concentrations in Blood 

and Urine," {Appendix B to "Recommendation of the Ad Hoc Committee on 

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Safety Council, April 29 & 30, 1968, Florissant, Colorado"} in TESTING

THE DRINKING DRIVER, TRAFFIC SAFETY MONOGRAPH No. 2, ed. by M. Impellizzeri,

Chicago, National Safety Council, 1970, pp. E-12 to E-14.

6.­ Ibid., p. E-2. 

7.­ Dubowski, K. M., "Postmortem Analysis of Blood for Alcohol," J. Amer. 

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8.­ Gifford, H., and Turkel, H. W., "Diffusion of Alcohol through Stomach 

Wall after Death," J. Amer. Med. Assoc. 161: 866-868 (1956). 

9.­ Turkel, H. W., and Gifford, H., "Erroneous Blood Alcohol Findings at 

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164: 1077-1079 (1957). 
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Deut. Z. gerichtl. Med. 47: 619-624 (1958) (in German). 

11.­ Osterhaus, E., and Johannsmeier, K., "Postmortem Alcohol Formation Caused by 

Putrification," Deut. Z. gerichtl. Med. 57:281-284 (1964) (in German). 

12.­ Plueckhahn, V. D., and Ballard, B., "Diffusion of Stomach Alcohol and 

Heart Blood Concentration at Autopsy," J. Forensic Sci. 12: 463-470 (1967). 

13.­ Plueckhahn, V. D., "Alcohol Levels in Autopsy Heart Blood," J. Forensic 

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14.­ Plueckhahn, V. D., "The Evaluation of Autopsy Blood Alcohol Levels," 

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15.­ Christopoulos, G., Kirch, E. R., and Gearien, J. E., "Determination of 

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455-472 (1973). 

16.­ Joachim, H., Wuermeling, H. B., and Wust, U., "The Postmortem Formation of 

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17.­ Blackmore, D. J., "The Bacterial Production of Ethyl Alcohol," J. Forensic 

Sci. 8: 73-78 (1968). 

18.­ Moritz, A. R., and Jetter, W. W., "Antemortem and Postmortem Diffusion of 

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19.­ Sturner, W. Q., and Coumbis, R. J., "The Quantitation of Ethyl Alcohol in 

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349-351 (1966). 

20.­ Leahy, M. S., Farber, E. R., and Meadows, T. R., "Quantitation of Ethyl 

Alcohol in the Postmortem Vitreous Humor, " J. Forensic Sci. 13: 498­

502 (1968). 

21.­ Felby, S., and Olsen, J., "Comparative Studies of Postmortem Ethyl Alcohol 

in Vitreous Humor, Blood, and Muscle," J. Forensic Sci. 14: 93-101 (1969). 
r 22.­ Coe, J. F., and Sherman, R. E., "Comparative Study of Postmortem Vitreous 

Humor and Blood Alcohol," J. Forensic Sci. 15: 185-190 (1970). 

23.­ Neumann, R., and Prioilla, 0., "On the Correlation of the Alcohol Content 

in Vitreous Body Fluid in the Blood Serum and Urine of Corpses," 

Blutalkohol 7: 463-469 (1970) (in German). 

24.­ Audrickly, I., and Pribilla, 0., "Comparative Studies of the Alcohol 

Concentration in the Blood, the Vitreous Body Fluid, the Synovial 

Fluid, and the Urine," Blutalkohol 8: 116-121 (1971) (in German). 

25.­ Norheim, G., "Postmortem Alcohol in Vitreous Humor," Blutalkohol 9: 

187-191 (1972). 

26.­ Scott, W., Root, I., and Sanborn, B., "The Use of Vitreous Humor for 

Determination of Ethyl Alcohol in Previously Embalmed Bodies," J. Forensic Sci. 19: 913-916 (1974). 

27.­ Breithaupt vs. Abram, 352.U.S 432, 77 S Ct 408, 412 U. S. Supreme Court 

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28.­ Schmerber vs. California,' 384 U. S. 757, 771-72 U. S. Supreme Court (1966). 

29.­ Ibid.,384 U.S. 757, 760 (1966). 

30.­ Strub, C. G., and Frederick, L. G., THE PRINCIPLES AND PRACTICE OF EMBALMING, 4th ed., Dallas, Texas, Lawrence G. Frederick, Publisher, 1967. 

31.­ THOMAS REGISTER OF AMERICAN MANUFACTURERS AND THOMAS REGISTER CATALOG 

FILE 1975, New York, Thomas Publishing Co., 1975. 

32.­ Weatherford vs. State, Okla. Court of Criminal Appeals, No. M-75-686, 

May 10, 1976; 549 P. 2d 1221. 

33.­ Dubowski, K. M., "Some Practical Laboratory Aspects of Forensic Alcohol 

Determinations," Proc. Iowa Acad. Sci. 63: 364-390 (1956). 

34.­ Glendening, B. L., and Waugh, T. C., "The Stability of Ordinary Blood 

Alcohol Samples Held Various Periods of Time Under Different Conditions," 

J. Forensic Sci. 10: 192-200 (1965). 

- 111 ­
35....­ Plueckhahn, V. D., "The Significance of Alcohol and Sugar Determinations 

in Autopsy Blood," Med. J. Australia 1: 46-51 (1970). 

36.­ Blume, P., and Lakatua, D. J., "The Effect of Microbial Contamination of 

the Blood Sample on the Determination of Ethanol Levels in Serum," Am. 

J. Clin. Path. 60: 700-702 (1973). 

37.­ Bradford, L. W., "Preservation of Blood Samples Containing Alcohol," 

J. Forensic Sci. 11: 214-216 (1966). 

38.­ Brown, G. A., Neylan, D., Reynolds, W. J., and Smalldon, K. W., "The 

Stability of Ethanol in Stored Blood. Part I. Important Variables and 

Interpretation of Results," Anal. Chim. Acta 66: 271-283 (1973). 

39.­ Smalldon, K. W., and Brown, G. A., "The Stability of Ethanol in Stored 

Blood. Part II. The Mechanism of Ethanol Oxidation." Anal. Chim. Acta 

66: 285-290 (1973). 

40.­ Smalldon, K. W., "Ethanol Oxidation by Human Erythrocytes," Nature 245: 

266-267 (1973). 

41.­ Marbach, E. P., et al., "Sodium Iodoacetate as an Antiglycolytic Agent 

in Blood Samples," Clin. Chem. 21: 1810-1812 (1975). 

42.­ Fabre, R., and Kahane, E., "Determination of Alcohol in Saliva," Ann. 

Med. leg. 17: 1010 (1937) (in French). 

43.­ Friedemann, T. E., Motel, W. G., and Necheles, H., "The Excretion of 

Ingested Ethyl Alcohol in Saliva," J. Lab. & Clin. Med. 23: 1007-1014 

(1938). 

44.­ Nanikawa, R., and Kotoku, S., "A Routine Method for the Determination of 

Ethanol in Biological Materials Using Gas Chromatography,". Japan J. 

Studies on Alcohol 4: 27-33 (1969). 

45.­ Machata, G., "On the Gas Chromotographic Determination of the Blood-Alcohol 

Level," Blutalkohol 4: 252-260 (1967) (in German). 

46.­ Machata, G., "On the Gas Chromotographic Determination of the Blood-Alcohol 

Level, Part II," Blutalkohol 7: 345-348 (1970) (in German). 

- 112 ­
47.­ Machata, G., "Determination of Alcohol in Blood by Gas Chromatographic 

Head Space Analysis," Clin. Chem. Newsletter (Perkin-Elmer Corp.) 4: 

29-32 (1972). 

48­ Glasstone, S., TEXTBOOK OF PHYSICAL CHEMISTRY, 2nd ed., New York, D. 

Van Nostrand Co., Inc., 1946, p. 697. 

49.­ Ibid., p. 699. 

50.­ Harger, R. N., Raney, B. B., Bridwell, E. G., and Kitchel, M. F., "the 

Partition Ratio of Alcohol between Air and Water, Urine and Blood; 

Estimation and Identification of Alcohol in these Liquids from Analysis 

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51.­ CARBOPACK (GRAPHITIZED CARBON BLACK), Bulletin 738, Bellefonte, Pa., 

Supelco, Inc., 1974. 

52.­ Dubowski, K.M., "Ethanol, Type C Procedure," in METHODOLOGY FOR ANALYTICAL TOXICOLOGY, ed. by I. Sunshine, Cleveland, CRC Press, Inc., 1975, 

pp. 149-154.

53.­ CARBOPACK GRAPHITIZED CARBON BLACK, Bulletin 738A, Bellefonte, Pa., 

Supelco, Inc., 1976. 

54.­ Moffat, A. C., and Isaacs, M. D. J., GRAPHITIZED CARBON AS A GC COLUMN 

PACKING FOR BLOOD-ALCOHOL ANALYSES, CRE Report No. 142. Aldermaston, 

Berks. (U.K.), Home Office Central Research Establishment, 1974. 

55.­ Moffat, A. C., and Walker, G. W., OPEN PORE POLYURETHANE G.C COLUMNS 

FOR THE ANALYSIS OF ETHANOL IN BLOOD, CRE Report No. 175, Aldermaston, 

Berks. (U.K.), Home Office Central Research Establishment, 1975. 

56.­ Goke, G., "Fully Automatic Determination of the Blood-Alcohol Level With 

a Gas-Chromatography Data System," Blutalkohol 10: 281-299 (1973) (in German). 

57.­ Bonnichsen, R., Hadfjall, B., and Ryhage, R., "Determination of Ethyl 

Alcohol by Computerized Mass Chromatography," Z. Rechtsmedizin 71: 

134-138 (1972). 

58.­ Pereira, W. E., et al., "The Determination of Ethanol in Blood and Urine 

by Mass Fragmentography," Clin. Chim. Acta 51: 109-112 (1974). 

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59.­ Blume, P., Berchild, K. M., and Cawley, L. P., "A Gas Chromatographic 

Analysis of Ethanol with Identity Confirmation," Analyt. Biochem. 54: 

429-433 (1973). 

60.­ Laessig, R. H., et al., "Performance Evaluation of Wisconsin Laboratories 

Performing Implied Consent Alcohol Determinations," Health Lab. Sci. 12: 

82-90 (1975). 

61.­ Flores, A. L._, "Blood-Alcohol Proficiency Test Program," U.S. Dept. of 

Transportation, National Highway Traffic Safety Administration, 

Washington, D.C., Jan. 1975, HS 801 332. 

62.­ REAGENT CHEMICALS: AMERICAN CHEMICAL SOCIETY SPECIFICATIONS, 4th ed., 

ed. by American Chemical Society Publications, Washington, D.C., American 

Chemical Society, 1968, pp. 228-231. 

63.­ Redetzki, H. M., "Use of the Osmometer for Preparation, Analysis and 

Control of Standard Solutions of Alcohol." Quart. J. Studies on Alcohol 

34: 206-208 (1973). 

64.­ Pella, P. A., and Diamondstone, B. I., "Stability of Aqueous Ethanol 

Solutions Stored in Glass Ampoules," J. Forensic Sci. 20: 532-538 (1975). 

65.­ "Percentages by Volume at 15.56°C (60°F) of Ethyl Alcohol Corresponding 

to Apparent Specific Gravity at Various Temperatures," in OFFICIAL METHODS OF ANALYSIS OF THE ASSOCIATION OF OFFICIAL ANALYTICAL CHEMISTS, 11th 

ed., ed. by W. Horwitz et al., Washington, Assn. of Official Analytical 

Chemists, 1970, pp. 889-907. 

66.­ "Alcohol Table for Calculating Percentages of Alcohol by Volume at 

15.56°C (60°F) in Mixtures of Ethyl Alcohol and Water from the Zeiss 

Immersion Refractometer Readings and Refractive Indices at 17.5-25°C," 

in op.cit. (65), pp. 908-913. 

67.­ Dubowski, K. M., "Methods for the Determination of Ethyl Alcohol in the 

Blood and Urine - Dubowski Method," in ALCOHOL AND THE IMPAIRED DRIVER. 

A MANUAL ON THE MEDICOLEGAL ASPECTS OF CHEMICAL TESTS FOR INTOXICATION, 

Chicago, National Safety Council, 1970 (Reprinted 1976), pp. 78-81. 

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Federal Register 40: 36167-36171 (Aug. 19, 1975). 

69.­ Bonnichsen, R., "Ethanol - Determination with Alcohol Dehydrogenase and 

DPN," in METHODS OF ENZYMATIC ANALYSIS, ed. by H.-U. Bergmeyer, New York, 

Academic Press, Inc., 1965, pp. 285-289. 

70.­ Dubowski, K. M., "Methods for the Determination of Ethyl Alcohol in the 

Blood and Urine - Enzymatic Procedure," in op.cit. (67), pp. 87-91. 

71.­ "Highway Safety Program Manual No. 8: Alcohol in Relation to Highway 

Safety," U. S. Dept. of Transportation, National Highway Traffic Safety 

Administration, Washington, D.C., March 1975, p. IV-4. 

72.­ Ibid. p. IV-3­

73.­ A GUIDE FOR ESTABLISHING AND INTRASTATE PROFICIENCY TESTING PROGRAM, 

Atlanta, Communicable Disease Center, U.S. Dept. of Health, Education 

and Welfare, August 1967. 

74.­ PROFICIENCY TESTING: GUIDE TO PROFICIENCY TESTING, Atlanta, Center 

for Disease Control,U.S. Dept. of Health, Education and Welfare, 

December 1972. 

75.­ Finkel, P.W., and Miller, T.R., "A Proficiency Test Assessment of 

Clinical Laboratory Capability in the Unites States," U.S. Dept. 

of Commerce, National Bureau of Standards, Washington, D.C., May 

1973, NBSIR 73-163. 

76.­ Finkel, P.W., and Miller, T.R., "A Proficiency Test Assessment of 

Clinical Laboratory Capability in the United States - Appendix 

Volume," U.S. Dept. of Commerce, National Bureau of Standards, 

Washington, D.C., May 1973, MBSIR 73-162 Appendix. 

77.­ Finkel, P.W., and Miller, T.R., "Clinical Laboratory Performance 

Analysis Using Proficiency Test Statistics," U.S. Dept. of Commerce, 

National Bureau of Standards, Washington, D.C., Dec. 1973, NBSIR 

73-197. 

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78.­ PROCEEDINGS OF SECOND NATIONAL CONFERENCE ON PROFICIENCY TESTING SPONSORED BY NATIONAL COUNCIL ON HEALTH LABORATORY SERVICES, Washington, 

D. C., October 6-7, 1975, ed. by M. Davis, Bethesda, Md., Information 

Services, Inc., 1975. 

79.­ Winek, C. L., "Drug and Chemical Blood Levels," in TOXICOLOGY ANNUAL 

1974, ed. by C. L. Winek and S. P. Shanor, New York, Marcel Dekker, 

Inc., 1975, pp. 203-228. 

80.­ Dubowski, K. M., "Measurement of Ethyl Alcohol in Breath," in LABORATORY DIAGNOSIS OF DISEASES CAUSED BY TOXIC AGENTS, ed. by F. W. Sunderman and F. W. Sunderman, Jr., St. Louis, Warren H. Green, Publisher, 

1970, pp. 316-342. 

81.­ UNIFORM VEHICLE CODE AND MODEL TRAFFIC ORDINANCE, SUPPLEMENT II, 1976, 

Washington, National Committee on Uniform Traffic Laws and Ordinances, 

1976, p. 41. 

82.­ Donigan, R. L.: CHEMICAL TESTS AND THE LAW, 2nd ed., Evanston, the 

Traffic Institute, Northwestern University, 1966; see also 1976 POCKET 

SUPPLEMENT TO CHEMICAL TESTS AND THE LAW. 

83.­ Deskin v. State, 94 Okla. Cr. 107; 230 P. 2d 939 (1951). 

84.­ State v. Short, 247 Iowa 453; 74 N.W. 2d 208, 210 (1956). 

85.­ Steffani v. State, 45 Ariz. 210; 42 P 2d 615-618 (1935). 

86.­ Donigan, R. L., op.cit, (32), pp. 8-10. 

87.­ Dubowski, K. M., "Human Pharmacokinetics of Ethanol-Further Studies," 

Clin. Chem. 22: 1199 (1976). 

88.­ State v. Sutliff, Idaho Sup. Ct., 547 P. 2d 1128 (1976). 

89.­ Forster, B., and Joachim, H.: BLUTALKOHOL UND STRAFTAT. NACHWEIS UND 

BEGUTACHTUNG FUR ARZTE UND JURISTEN ("Blood-Alcohol Level and Criminal 

Offense: Evidence and Opinions for Physicians and Lawyers") Stuttgart, 

West Germany, Georg Thieme Verlag, 1975 (in German). 

90.­ Harger, R. N., Hulpieu, H. R., and Lamb, E. B., "The Speed with which 

Various Parts of the Body Reach Equilibrium in the Storage of Ethyl 

Alcohol," J. Biol. Chem. 120: 684-704 (1937). 

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91. Gettler, A. 0., and Freireich, A. W., "Determination of Alcoholic Intoxication During Life by Spinal Fluid Analysis," J. Biol. Chem. 

92: 199-209 (1931). 

92.­ Kalant, H., "Absorption, Diffusion, Distribution of Ethanol: Effect 

on Biological Membranes," in THE BIOLOGY OF ALCOHOLISM. VOL. 1: 

BIOCHEMISTRY, ed. by B. Kissin and H. Begleiter, New York, Plenum 

Press, 1971, p. 17. 

93.­ Coldwell, B. B., and Smith, H. W., "Alcohol Levels in Body Fluids 

after Ingestion of Distilled Spirits," Can. J. Biochem. Physiol. 

36: 43-52 (1959). 

94.­ Kaye, S., and Cardona, E., "Errors of Converting a Urine Alcohol 

Value into a Blood Alcohol Level," Am. J. Clin. Path. 52: 577-584 (1969). 

95.­ Osterhaus, E., "Concluding Results of Gas-Chromatographic Studies on 

Postmortem New Formation of Methanol, Acetone, and n-Amyl Alcohol," 

Blutalkohol 10: 305-309 (1973) (in German). 

96.­ Smith, P. W., Lacefield, D. J., and Crane, C. R., "Toxicological Findings in Aircraft Accident Investigation," Aerospace Med. 41: 760-762 (1970). 

97.­ Parker, J. M., "Postmortem Drug Level Changes," in TOXICOLOGY ANNUAL 1974, 

ed. by C. Winek and S. P. Shanor, New York, Marcel Dekker, Inc., 1975, 

pp. 151-165. 

98.­ Collom, W. D., "Postmortem Synthesis of Alcohol," in TOXICOLOGY ANNUAL 

1974, ed. by C. Winek and S. P. Shanor, New York, Marcel Dekker, Inc., 

1975, pp. 269-274. 

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