Bryan S. Finkle Center for Human Toxicology 3 8 Skaggs Hall University of Utah Salt Lake City. vtah 841 12
Forensic Toxicology in the 1980s The Role of the Analytical Chemist
If it is accepted that the broadest possible definition of toxicology is thi study of the adverse effects of chemical suhshices on living systems, and that forensic toxicology applies the results and opinions from such studies in a medicelegal context, then it is an immediate reminder that toxicology is neither new nor limited to man. From ancient Greece through the Middle Ages the understanding and application of poisons were developed through knowledge of their chemistry, and indeed, the chemical extraction and identification of a poison in man were usually sufficient evidence of ita culprit role in times when elements such as arsenic or mercury, plant alkaloids such as atropine, morphine, and ergotamine, and glycosides such as digitalis were regarded as unnatural constituents of man’s normal hiochemical makeup. Today, arsenic and mercury are almost environmentally ubiquitous and are accepted as part of man’s normal body burden, and analogs of digitalis are among the most widely used drugs for the treatment of cardiac illness. Their very presence alone is, then, hardly evidence of toxicity, and may, in fact, represent just the opposite. The earliest toxicologists were necessarily chemist., and following the examples of Mathieu Orfila (1839, first extracted arsenic from human organs), Jean Sevais Stas (1850, developed methods for the extraction of alkaloids in postmortem tieaues and the quantitation of metals in human organs), F.J. Otto (1856, modified Stas’s method to include nonalkaloidal substances), and Wilhelm Autenrieth OW3-27W/82/0351-433A$O~.W/O 0 1982 Amerlcan Chamlcal Sociehl
I
(1905, wrote the first practical book on “The Detection of Poisons and Powerful Drugs”), they developed analytical techniques by which they could literally isolate and identify postmortem the offending poison for all to see. This trend, of the chemist becoming the toxicologist-pathologist and applying analytical skills to the postmortem investigation of suspicious death, continued well into the 20th century. Organic and analytical chemists bad become fascinated with the difficult problems involved in the extraction and identification of poisons from biological specimens, and the rise of the pharmaceutical industry gave impetus to their task. . Since the 19308, a generation of analytical toxicologists has h e n almost ANALYTICAL
exclusively concerned with the extraction, identification, and ultimately the quantitation of poisons in autopsy specimens. With this historical background it is difficult to see how any qualified toxicologist can deny the fundamental essential of his trade, that he be an exquisitely skilled analytical chemist. In a historical parallel, pbarmacologists have inquisitively pursued an understanding of the mechanisms of drug action and the adverse effects of drugs. Many of these biomedical scientists specialized in the study of toxicities and earned the title toxicologist. It is interesting that it was a forensic problem that eventually brought the chemist-toxicologist and (continued on page 440 A )
CHEMISTRY, VOL. 54, NO. 3. MARCH 1982
433A
Wide choice of injectors, each oatimized for a specific injection technique.
Pneumatics sufficient for any type of analysis, with up to four ionization detector gas controls.
-
Wide choice of modular detectors -hot wire, differential flame ionization, nitrogen-phosphorous,electron capture, photoionization.
----
Four oven ports accommodate extra valves or injectors.
L
The new PerkinlElrner SIGMA 2000 is today's most advanced GC, Tomorrow's too. Easy and intelligent A 9-inch interactive CRT helps you set up and monitor the system. A simple keyboard makes changes on-the-fly easy and fast. The memory can store nine methods while running a tenth. What's more, the CRT and keyboard can be used to set up four satellite Perkin-Elmer SIGMA 2100 GCs (right), each with its own Each module is specially keyed, microprocessor. And each of these so the SIGMA 2000 recognizes it can store nine methods while and identifies its parametersrunning a tenth. You can even such as temperature maximum, for transfer methods between instruexamole. No more cumbersome, ments to increase efficiency and time-consuming "reconfigurations" save time. when you change applications.
It's modular and expandablewith computer power to spare
I
Our SIGMA 2000 GC is modular to a degree you've never seen before. It's easy to reconfigure. Its open-end design makes it readily upgradeable for future needs. Its microprocessor control with our built-in software lets you add as many as four satellite GCs set UD from.one simple keyboard. Instantly adaptable Injectors. detectors-even the va1ves-m in modules YOU simply plug into the oven ports (right).
I
434A
ANALYTICAL CHEMISTRY,
VOL.
54. NO. 3, MARCH 1982
-
Innovative column oven permts up to tour-column operation
1 I
"Smart" CRT helps the operator by prompting. guiding, tracking changes. Simple, easy-to-use keyboard,
/ with prompting by the CRT
Powerful microprocessor stores up to nine methods while a tenth is running.
All this makes the SIGMA 2ooo today's advanced GCand an ideal example of Computer Aided Chemistry, the Perkin-Elmer concept that integrates instruments, computers, and software to revolutionize productivity.
Complete chromatography The SIGMA 2000 aives YOU a full selection of easilyher6hangeable SIGMA 2100 Gas Chromatograph components and a variety of Add computer power accessories. And we back everyFor higher levels of sophistication, thing with Perkin-Elmer service, add our Model 3600 Data Station including training courses, applicaand Chromatographics 2 software tions support, and publications. package to get data reduction, Get all the details on the SIGMA graphic display and manipulation 2000-the gas chromatograph of chromatograms, and floppy-disk that's like the one youu design for storage. Or control the whole yourself. Return the coupon today network with a large computer or contact your Perkin-Elmer represystem such as our LIMSi2000. sentative or one of the offices shown here
Perkin-Elmer Corp.. Analytical Instruments. Main Ave (MS-lZ), Norwalk, CT 06856 U.S.A Tel: (203) 762-1000. Telex 965-954 Bodenseewerk Perkin-Elmer 8 Co.. GmbH. Postfach 1120, 7770 Ueberlingen. Federal Republic of Germany. Tei: (07551) 811 Perkin-Elmer Ltd., post Office Lane, Beaconsfield, Bucks HW IQA, England. Tel: Beaconsfield(049 46) 6161
,
r-------1 Perkin-Elmer Corp. Main Avenue (MS-12), Norwalk, CT 06856 U.S.A.
I Please send information on the new SIGMA 2000 and 2100 GCs. Name Institution Street my State/ Province
Zip
I I I I I I I I
Country
I IL Phone -( - - -) - - - - J DM-266~0382-CPN
PERKIN-ELMER ResponsiveTechnology Circle 180 for literature.
Circle 181 for sales call. ANALYTICAL CHEMISTRY,
VOL.
54,
NO. 3. MARCH
1982
-
435A
Judge our newest M by how much it tells you.
At last, there's an atomic absorption spectrophotometer that gives you all the informationyou need for an analysis. Automatically, Instantaneously.And you actually become part of the system, through its remarkable information center. The Perkin-Elmer Model 3030 AA spectrophotometer. Its big 12-inch screen is your window into the analysis, Perkin-Elmersoftware, unmatched in scope and power, is your guide. Exclusive "soft" keys. The "soft" keys on the simplified keyboard are a Perkin-Elmer exclusive in AA instrumentation.Their functions change to fit the analysis, so when you need a key, it's there. Moreover, you're guided and prompted through the routine. You're given the options. With the operating system and "cookbook" data on a floppy disk, you enjoy complete flexibility for upgrading (made easy by an extra key). Another disk stores user-defined analytical methods.
The Model 3030 is a system that grows with your needs. And in AA, it's also the i g p e r ultimate expression of Chemlstry Computer Aided Chemistry, the Perkin-Elmerconcept that integrates instruments,computers, and software to revolutionize productivity.
0
Routine operation. With your first keystroke,complete analytical conditions appear. Now you can program the system, as shown on the page at left. The bar graphon the left screen above facilitates lamp and burner alignment. The result of this flame analysis appears on the right screen. Super software. The high-resolutionflame graphics display software is most helpful. In fact, all our graphics software is the most comprehensive anywhere -flame, HGA, calibration curve. Both corrected atomic absorption signal and background signal are displayed together. Of course, you can get hard-copy printouts of everything you see on the screen.
The Model 3030 is page-oriented and delivers information step-by-step. YOU can instantly call up the parameters for a given mode, Mode pages are accessed with a single keystroke. Widen your horizons. The Model 3030 comes complete with a 4-lamp turret. Attractive options include a 2-way RS-232C interface and a quickchange mount for speedy changsover from flame to graphite furnace.
Find out more. For details, contact your Perkin-Elmer representative or one of the offices below. Perkin-Elmer Corp.,Analytical Instruments, Main Ave. (MS-12),Norwalk,CT 06856 U S A Tel: (203) 762-1 000 Telex: 965-954 Bodenseewerk Perkin-Elmer & Co., GmbH, Postfach 2230 7770 Ueberlingen, Federal Republic of Germany. Tel: (07551) 81 1 Perkin-Elmer Ltd.,Post Office Lane, Beaconsfield, Bucks HP9 1QA, England. Tel; (049 46) 6161
PERKIN-ELMER Responsive Technology Circle 178 For Literature. Circle 179 For Sales Call.
New Perkin-Elmer SERIES 4 LC offers 4-solvent flexibility at 2msolvent prices. The new firkin-Elmer SERIES 4 liquid chromatography system brings you 4solvent flexibility for less than you'd pay for many 2- or 3-solvent systems. It accommodates the full array of Perkin-Elmer LC detectors and accessories. The state-of-the-artdesign makes it easy to accept future develoDmenls.
438A
It's also designed for simplicity of operation. The fully-promptingcontrol system communicates in common terms. All analysis parameters are displayed in commonterms. No codes. No conversion tables. In fact,the SERIES 4 isa striking example of Computer Aided Chemistry,the Perkin-Elmerconcept that integrates instruments,computers and software to revolutionize productivity.
ANALYTICAL CHEMISTRY, VOL. 54, NO. 3, MARCH 1982
More data-handling flexibility The SERIES 4 lets you store, chain and develop methods.The standard video display shows you boththe selected method and the actual status of the analysis. Or substitute the optional Chromatographics2 Intelligent Terminal to get high-powered data processing and store supplemental programs and data on disk.
Liquid Chromatography
make your work easier Its pump has no complex controls.All components and connections are easily accessible. Solvent degassing and pressurization are standard. The optionaltotal-contact oven delivers SuDerior column temDerature control And the optional ISS-100 IntelligentSampling System (see inset) provides unmatchedversatility, programmabilityand performance More of everything you need The IS-100 Intelligent Perkin-Elmer also gives you a wide Sampling Systemisa highly selection of high-speed and convenflexible, fully-automatedliquid tional columns of unexcelled perforsampling system S p e C i f M l y mance.And you can choose from the designed for modern liquid broadest variety of LC detectors availchromatography able: high-speed and conventional UVNisible,fluorescence,polarimetric, electrochemical,conductimetric, refractive index,and more. More of the support you want With the SERIES 4, Perkin-Elmer stays out in front in bringing you the most
,.
diversified line in all liquid chromatography Everythingyou need for successful liquid chromatography is here, including applicationssuppofl and literature,user and sewice training courses,and worldwide service backup. And the SERIES 4 is under warranty for a full 12 months. Let us help you solve your LC needs. Just contact your Perkin-Elmer representative or one of the offices below Perkin-Elmer Corp.. Analyfica Instruments. Main Ave. (MS-12).Norwalk,CT 06856 U.S.A. Tel: (203) 762-1000.Telex 965-954. Bodenseewerk PerkinElmer&Co.,GmbH, FmUach 1120,7770 Ueberlingen,Federal RepublicolGermany. Tel: (07551) 811. Perkin-Elmer Ltd., Post mice Lane, Beaconsfield, Bucks HF9 1 0 4 England. Tel: Beaconsfleld(O4946) 6161.
.
PERKIN-ELMER ResponsiveTechnology Circle 175 for literature.
Circle 176 for sales call. ANALYTICAL CHEMISTRY, VOL. 54, NO. 3. MARCH 1982
439,.
pharmacologist-toxicologist together. It was clear that they were both needed if reasonable legislation was to be promulgated concerning the problem of alcohol, the drinking driver, and highway safety. The chemist possessed the expertise to accurately and precisely assay ethanol in biological samples such as blood and urine, and to invent and test devices for the determination of alcohol in breath. The pharmacologist, however, best understood the mechanisms of action, the kinetics and metabolism by which ethanol disturhed the body biochemistry and physiology. The chemists came from industry and the world of pharmaceuticals, and from government laboratories such as Customs and Excise, where they analyzed and assayed contraband, including narcotics; the pharmacologists came from biomedicine and pharmacological research laboratories. This 20th-century partnership, sometimes easy and sometimes strained, has matured to a point where, under the pressure of social needs (for example, the problems of drug misuse and abuse, and chronic toxicity from passive exposure to environmental poisons or from lifelong use of therapeutic agents) it has melded, so that today there is at least a cadre of toxicologists who are wholly educated and professionally experienced as both analytical chemists and pharmacologists and who devote their intellect solely to the problems of toxicology. These prohlems are often those especially difficult matters that must he adjudicated through litigation in the courts. These professionals are too few, and there is little sign that the current limited university programs will add to their number significantly in the near future. For instance, the toxicology section of the American Academy of Forensic Sciences has only 298 members (November 1981) and only a fraction of these are eraduate analvtical chemists. The asnects of both analvtical chemistr; and pharmacoloiy have in recent times become dauntingly more complex than a simple extrapolation of historical precedent might have anticipated. The macabre, beautiful art of homicidal poisoning may have hecome unfashionable in the face of the crude, violent handgun, hut accidental and suicidal poisoning as well as unintended chronic toxicity have grown at an alarming rate during the past 20 years. In the same period, the chemist has henefited most from extraordinary technological developments in scientific instrumentation that now permit the isolation of drugs and poisons and their biotransformation produds and allow for their absolute identification even when only infinitesimal amounts I
440A
are present. Toxicological understanding of the mechanisms of action of these substances has not henefited similarly; and the ability to understand the meaning of the analytical data in terms of toxicity is by contrast very crude. The disparity between the remarkable analytical skills and the intellectual capacity to interpret the data is a professional burden that all forensic toxicologists are seeking to lighten.
Current Capabliities and Limitations Most forensic toxicology lahoratories are associated with state or local government services. These are usually either medical examiner-coroner offices or state attorney general or county attorney departments. Some are even directly associated with police departments, sheriffs offices, or departments of public safety. Historical development is again responsible for this unfortunate situation. The scientist-opinion witness should not he an advocate for anything but his own scientific opinions properly based upon facts in the case and should, therefore, be a “friend of the court,” but is all too often seen and treated as accusatorial and in league with the prosecutor. The advent of public defender systems and aggressive legal investigation in pursuit of civil litigation has hegun to change the public perception of the forensic scientist’s role. In contrast, it must be clearly recognized that forensic toxicologists cannot be, and are not, isolated professionals; they are part of a team that at the very least involves an investigator, a forensic pathologist, and certainly at least one attorney. It can be stated flatly that virtually no case can be resolved on the basis of toxicology data alone-that is, without the integration of the circumstantial evidence, the medico-legal autopsy findings, and an understanding of the exact legal question p d in the case. Ideally, toxicol-
ANALYTICAL CHEMISTRY, VOL. 54, NO. 3, MARCH 1982
ogists and their laboratories should be either associated with a medical examiner’s or coroner’s office, the local jus. tice department (hut entirely divorced from any direct connection with law enforcement), or in a university chemistry department or medical center. Forensic toxicologists should he administratively placed within the bureaucracy so that no direct association exists with either side of the adversary system. Most of the forensic toxicology service laboratories in the U S . today are inadequately supported and without the minimal complement of staff. A forensic toxicology laboratory investigating 30004000 cases per year in support of a medical examiner, a drinking and drugged driver program, and drug abuse enforcement, as well as perhaps jail and probation surveillance work, cannot fulfill its duties without a chief toxicologist fully qualified and with several years’ experience and at least four or five technical laboratory staff members, including graduate-level analytical chemists. Quite apart from the usual chemicals, glassware, and laboratory bric-a-brac essential for any analytical work, there is a need for scientific instruments that are costly and sophisticated. These include spectrophotometers, scintillation and gamma counters, gas chromatographs and high-pressure liquid chromatographs, mass spectrometers, and the inevitable minicomputers, microprocessors, and calculators for statistical analysis and control. To equip such a laboratory may cost between $500 000 and $1 million. The sensitivity and specificity required in contemporary analytical toxicology, quite apart from the taxing legal demands made by the courts for unimpeachable factual evidence, make these instruments essential and have rendered classic steam distillations, most test-tube color reactions, and volumetric chemistry inadequate and obsolete. In addition, forensic toxicology laboratories that have teaching and research responsibilities should be able to accept visiting scientists from service laboratories and postdoctoral candidates who have graduated in chemistry, pharmacology, or biochemistry and who wish to be trained to hoard certification level in forensic toxicology. It is often said that forensic-analytical toxicology is one of the easiest things to do badly and one of the most difficult things to do well. Unfortunately, when compromise is made the negative dividend can he guaranteed to be vastly disproportionate to the small cuts or equipment denials made in good faith by a budget-conscious bureaucrat. The Drug Enforcement
.
Sensitivity and Selectivity.. The Foundations of Fluorescence.
The NEW LS.5 Fluorescence Spectrophotometer shown with the popular Perhin Elmer Data Station will increase laboratory efficiency and productivity Perhin Elmer also provides software to make your application faster and more accurate
.
The complete product line lor research and routine analysis A complete line of fluorescence spectrophotometers, sample handling accessories and data handling accessories, along with service back-up and applications support. is available from one source, Perkin-Elmer. Our product line includes instruments that Provide the benchmark in o p tical performance and micro. processor innovation. Can be interfaced to the Perkin-Elmer Data Station to enhance productivity. Can be used as a detector for liquid chromatography or thin layer chromatography.
Computer Aided Chemistry and Laboratory Automation Add the PerkiwElmer Data Station to your fluorescence system. Supported software enables you to control the instrument from the Data Station console and acquire, disDlav. . ~. file and retrieve swctra: even accumulate spectra'to average transients and correct excitation and emission spectra. You can even Write your own programs in BASIC for custom applications. An optional printer is available to provide hard copy reports formatted to suite your needs. Ask for this free booklet describing
Wide Variety of Sample Handling Accessories Accessories to aid in your analyses include: Polarization, Phosphorescence, Ultramicro flowcells (for LC detection), TLClSlab Gel Scanning, solid sampling and many more.
...
To Receive More Information call Toll Free 80,,323-7,55 (in 3,28874770) or write perki&lmer Gorp, Analytical Iw struments, 2ooo Yr;k Road, Oak Brook, IL 6052,, to receive literature about our Fluorescence Spectrophotometers.
PERKIN-ELMER ResponsiveTechnology Circle 171 for literature. Circle 172 for sales call. ANALYTICAL CHEMISTRY, VOL. 54. NO. 3, MARCH 1982
441 A
4
with Nicolet's GC-IR.
We have optimized our FT-IR systems to serveashlshtyspedtlc
md intelligent detectors for today's ~ p h b t b t e high-mlud tion CaplusrY gas chromatograph. This combination of state-ofhe-art IR with state-of-the-art GC zives rapid solutions to complex zhemical problems. The Nicolet K - I R system is easy to use while xoviding fully automated analysis xpabilities. Our newly designed GC-IR interace has been optimally designed to iccommodate the small elution volimes characteristic of capillary GC. iystem versatility allows for the use )f flexible fused srlica WCOT columns IS well as packed columns, SCOT colimns, and WCOT columns. A makeip gas option IS included for use with WCOT columns to maintain their hracteristic high resolution E&.ient l i n k i n of the GC column with h e infrareglight pipe provides zero lead volume, thus maintaining GC esolution. The versatility of the GC-
in real time. The exampre presented here is an automated analysis of a corn-
ance peaks during elution. In photo, the spectral data are mmpu
1650
-
800
,030
.oao
,020
-
1215
1185
,030
-
,080 -
tbe low nanogram
,030 -
.om
-.020 -
the entire analysis can be fully auto-
,055
2980
I
-
2920
-
1700
,005
sfMvf@, wttb rouMn
.030 ,005
1 .
-
.005 1760
--
-
,055
,055
an Automated GCtion of sample to n of compounds,
be seen eluting approximately five seconds after acetone
so accomm unattended over
-.020
These instrument features, combined with an integrated GC-IR software package, provide truly exceptional analytical performance coupled with rapid and convenient experimental automation.
scans. Methylene chloride can
-
-
I
I 1Hf TIBLE COITIIIS Pll
1 2
17 GC P h FIRST
CfYlER
n
tinf
LIS1
241 278
23V
241
I75
la1
298 115 144
281 3Ok
1 4
317
5
311
6
15v
7 8
179
356 377
3VO
188
I4
486 519
58k 63)
I7
I205 OL 5LCON05 INTO RUN
3IV
151 361 111 3V2
15 I6
RffRfYCI
k83
491
511 581
541
627
L12
587
f R PEIK 10 B f CUIYGE9 k UALUES mf 8nou11 EIIIER ufu UILUES
514
e reconstructed cbra the Nicolet F l I R
This table shows the file numbers ana file widths of the spectra correspond-
-__
WRYENUMBERS
mpb: Hewlett-Pack& mn: 30 meter x 0.3 mm fus
don: 5 microliters
C. The system automatically a recoasauaion of the chro gram after the run is compl window vs. retention time, or a GramSchmidt reconstruction. Here, the reconstructed chromatogram overlaps the chromatogram obtained with the FID. Note that the IR time resolution is identical to that of the chromatogram, even though the peak intensi-
SADILER SN3111 POSSIBLE HITS
' Y R I G H I 1981
1599
METHANE. DIBROMO-.
2151
DIRENZOTHIOPHEHE
wtile, allowing you to cuncentr'ite on vour chrorn,itogrdph\ while n e take care ot the \pectr,il inedwreiiiei and interpretdtion
278 I E656 HSJ 4889 THIOXANTHENE 286 I666 BSJ
52 BENZENESULFORYL CHLORID 289 USGR 123 EIHANE. 1.2-DIBROMO-. 291 E2E 5167 FLUORENE 146
292 2229
TOLUENE. A.A-DICHLORO-. 6Y6R
ETHANE. I-BROMO-2-CHLOR
dwide sales?servrce,
uppllrcal
C i R W 153 ON READER SERVICE CARD
Agency has established a national network of laboratories that employs and trains forensic chemists; during the past 10years the federal Law Enforcement Assistance Administration (LEAA) has done much to support the physical improvement of existing laboratory facilities; and the National Institute on Justice has recognized the need to support research in this applied science. Much of this valuable effort has gone for naught, however, because there has been so little support for training new forensic toxicologists or for encouraging mature chemists and biomedical scientists to enter this challenging field. In consequence, mass spectrometers and other equipment bought with federal largess, although vitally needed, have not been used to their full potential. Training and research programs in support of forensic toxicology nationwide are desperately needed, and until improvement occurs, current knowledge will be only slowly assimilated and applied, new knowledge and techniques will be neglected, and the courts will be inadequately served. Almost all toxicological investigations proceed in two stages. The first involves the analytical chemistry, which provides the data upon which the second depends, namely, interpretation of that data within a particular case context. There are five sequential steps to be considered in the first stage of analytical toxicology: the selection and acquisition of appropriate biological specimens for analysis, extraction of any drugs or poisons and their metabolites that may be present in the specimens, separation of the extract into its individual constituents, identification of the separated constituents, and finally, the quantitation of any drugs and poisons and their metabolites. The analysis always proceeds in this order, although some steps may be omitted (e.g., extraction, in direct immunological testing) or partially integrated (e.g., separation-identification by gas chromatographylmasa spectrometry). Each step is dependent upon the preceding steps, and defined controls are necessary for each step if the final result is to have acceptable quality assurance that can withstand the rigors of legal examination. What is a forensic toxicology case? It is a n y situation involving one or more individuals, living or deceased, in which it is suspected that drugs, poisons, or other xenobiotics may have played a role which wholly or in part accounts for the case circumstances into which there is legal inquiry. The forensic toxicologist begins by trying to state clear questions that address the case problem to be answered. Without this clear statement of pur444A
pose, the toxicologist will be involved in little more than a fishing expedition or data-gathering exercise that is unlikely to prove rewarding or relevant to the legal inquiry. It is, therefore, extremely important that lawyers and also pathologists discuss the case in sufficient detail with the toxicologist to allow these auestions to be formulated.
Perhaps the most common cases are those involving postmortem investigation, in which the cause and manner of death are at issue. Certainly there are routine analyses that are carried out in almost every case; for example, the quantitative determination of alcohol is a requirement in every case no matter what the cirumstances or who the victim. On the other hand, the brief facts of medical history and possible therapeutic use of drugs would immediately signal the need for particular analyses. If the deceased victim was an epileptic, for example, it would be very important to determine if commonly prescribed anticonvulsant drugs were in the blood at effective concentrations or not, at the time of death. In another case this class of drugs may be of no significance. The scope of a given analysis is first defined by the circumstances prevailing in the case, the known history of the victim, and on the basis of local experience. The local experience must also include what is normal in the population. Miners may carry a heavy burden of metals, coast dwellers eating shellfish may have elevated arsenic levels, farmers and those in rural areas may be exposed to particular pesticides, and all without any overt, acute toxicity.
The Technical Principles of Analytical Toxicology Not all drugs and poisons can be detected in all biological specimens at any particular time and, therefore, the choice and availability of samples for analysis are crucial. Obviously, speci-
ANALYTICAL CHEMISTRY, VOL. 54, NO. 3. MARCH 1982
mens from living individuals are limited and generally only blood or plasma and urine are available. Occasionally gastric contents, cerebrospinal fluid, feces, and saliva may be available and useful. Although in many special circumstances unusual autopsy specimens may be required when a particular poison is suspected (such as fat in the pesticide cases and spleen in cyanide poisoning), there are five specimens that should be taken in all cases: blood from a major vessel and blood from a peripheral vessel (the latter for alcohol determination), gastric contents (total), liver, hile (total), and urine (total). Often, lung, brain, and heart tissues are useful, and in the absence of gastric contents or urine, small intestine contents and kidney tissue are valuable substitutes, respectively. As a general approach, the peripheral blood sample will be used for the analysis of ethanol, methanol, acetone, isopropanol, and volatile hydrocarbons, usually by gas chromatography without prior extraction. Some other direct testing of blood and urine specimens for major groups of drugs such as opiate narcotics, barbiturates, and amphetamine and its derivatives can be accomplished by either enzyme or radio immunoassay methods. These methods are rapid, very sensitive, and their principal value lies in the quick determination of a negative resultthat is, the absence of particular drug groups in the specimen tested. Positive findings must always be confirmed by a different independent method. Blood, urine, gastric contents, or tissue homogenates can be extracted with organic solvents so that drugs, poisons, and metabolites are isolated from the biological matrix according to their chemical character-strong and weak acids, strong and weak bases, neutral compounds, and some amphoteric substances. Chromatography in its various forms is the technique used almost universally to separate extracts into their individual constituents. Although imaginative development of chromatographic systems has permitted their use for partial identification, it must be emphasized that chromatography is first and foremost a separation technique. Secondarily, it is very important in the quantitation of the separated drugs, and only lastly should it be considered a method of identification. In general, thin-layer chromatography (TLC) with a wide variety of developing reagents is used to separate extracts of urine and gastric contents, and gas liquid chromatography (GLC) and highperformance liquid chromatography (HPLC) are used with a variety of detectors for blood and tissue extracts.
Each of these chromatographic techniques has its own advantages and limitations. For example, GLC by definition can only separate compounds that can be volatilized and carried through the separation column as a gas, but with selective detectors and long capillary columns it can be supremely sensitive and can provide remarkable discrimination hetween closely related compounds. In contrast, HPLC will separate nonvolatile compounds and polar molecules that are very difficult to separate any other way. By judicious use of these different forms of chromatography, analytical toxicologists can today separate very small quantities (in the nanogram to picogram range) of almost every drug or poison of interest. Information leading to the identity of the isolated compounds is gathered sequentially as the analysis proceeds through extraction and separation, hut final unequivocal identification lies with either a composite of data concerning the chemical character of the compound so that all other reasonable possihilities are eliminated, or by use of mass spectrometry. Electron impact (EI) and chemical ionization (CI) mass spectrometry can provide the most sensitive and specific identification. By comparing the unknown spectra with reference standards or libraries of reference mass spectra of compounds pertinent to toxicology, an identification is usually made. The mass spectrometer has the additional very important advantage %at it can he engineered in conjunction with GLC, and even HPLC, so that the chromatographically separated compounds can he transferred directly into the instrument source for immediate analysis. Most mass spectrometers used by analytical toxicologists today are GLC-quadrupole MS instruments with positive- and negative-ion CI and selected multiple-ion mode capabilities. The ultimate quality of the results hinges critically on every stage of the analysis, beginning with the type and condition of the biological specimens available. It would he simply inappropriate to analyze gastric contents for a drug that was known to be used intravenously. Many drugs concentrate in particular organs or may he excreted rapidly into the urine, which raises this specimen to a level of first importance. Toxicological analyses may be compromised because of the condition of the specimens, perhaps from fire deaths, exhumations, and bodies that have putrefied and degraded through exposure. The sedsitivity limit of each method must be known and stated hy the analyst so that a report that indicates that no drugs were detected in the sample
has meaning with respect to some minimum detectable value. Generally, in forensic toxicology, sensitivity limits should at least be within the concentration range known to be therapeutically effective for a drug, and within the known range of body hurden values for other chemicals, except for those that are known poisons at any value, but these are very few. The
accuracy and precision of quantitative methods should be determined and documented in every laboratory. Standard deviations or coefficients of variat a reasonable toxicoation of 5 1 0 9 ~ logical concentration are acceptable for most chromatographic procedures, even though some methods can achieve much more precision than this. The toxicologists themselves should, by virtue of their academic training and professional experience, be credible to the courts. Ideally, the toxicologist should have had the training and experience that has led to hoard certification in forensic toxicology through written examination and inspection of credentials. This certification should be current and based upon periodic reexamination and continuing education activities. To reach this stage of professional maturity, chief forensic toxicologists should he drawn from the ranks of doctoral (Ph.D.) scientists who have graduated from a university with organic chemistry (analytical) and a biomedical science, preferably pharmacology. They should then have experienced at least one year of postdoctoral training in an existing forensic toxicology laboratory and made some contribution to the scientific literature during that time. During this period, or at its end, they should be eligible for acceptance into the American Academy of Forensic Sciences, Toxicology Section, and then he prepared to undergo board esamination. There are other important matters that hear on the quality of toxicology case reports. Development of state-
of-the-art quantitative analytical methods is needed so that routine methods used in the laboratory can he evaluated fully with respect to their limitations. At present, quantitative mass spectrometry techniques offer the best approach for reference methods; several mch procedures do exist and have been published but many more are needed desperately. Acceptable protocols to determine accuracy are not in general practice. With few exceptions, fundamental wet-chemistry methods based in stoichiometry are not available for toxicological analyses. The best alternative is to have the reference solution analyzed by at least two completely different methods, including a reference procedure if it exists, and hy more than one analyst in different laboratories. This is a lahorious approach but it does not have to he done often. Proficiency testing involves externally prepared, simulated case specimens containing drugs or poisons in known concentrations, to he sent to forensic laboratories for analysis. These analyses permit toxicologists to evaluate their own analytical results against those of other participants. The data can be u&d as a laboratory management tool to improve performance or most importantly, it can he used to determine very clearly areas in which education or research and development are required. This last aspect, of course, again leads to the need for reference laboratory centers, preferably in universities, at which workshops, seminars, and other educational activities can be made available to practicing toxicologists. Forensic toxicology proficiency testing lags far behind its clinical medicine counterpart. Although recently supported by the National Institute on Justice, it was withdrawn after just one year because government funds were terminated. In summary, although the analytical methods available are marvelous in contrast with practice just a generation ago, programs to ensure creditable technical quality in forensic toxicology laboratories are urgently needed. Above all, an integrated effort to educate and train professional chemists to hecome toxicologists who can truly fulfill the needs of the courts should he given the highest priority.
Analytical Advances and Critical issues The most rapidly developing area in analytical toxicology is immunology, in which antibodies have been raised against a particular drug or drug class or poisonous chemical so that they can be used as test reagents for their complementary compound. These antigen-antibody reactions depend upon either an enzyme, or a radioactive or
ANALYTICAL CHEMISTRY, VOL. 54, NO. 3. MARCH 1982
445A
fluorescent marker for their detection. They are rapid, direct testing methods with exquisite sensitivity, hut completely controlling the selectivity of the antibody for the suhstance(s) for which it was designed is not yet achievable. Development of these immunoassays is very costly and involves more than a little “scientific art.” Ultimate selectivity-in other words, unique specificity-is not necessarily always the most desirable goal. Through new techniques of genetic engineering, monoclonal antibodies unique for a single drug or chemical have heen developed in research laboratories. What is really needed are antibody reagents tagged for detection that have predesigned, known reaction characteristics. When this is possible, antihodies with broad cross-reactivity could be made for screening purposes, multiple antibodies could he prepared as mixed reagents so that different classes of drugs could he detected in one test, and monoclonal antibodies could still he used to detect single species. Among the analytical methods that are developing to the benefit of toxicologists are improved mass spectrometry (e.g., MS/MS and fast-atom homhardment), and of course, microprocessors and computer data systems. Technologically,’the computers and microprocessors are already here, but their application in the toxicology lahoratory needs to he evaluated further. Particularly, their role as easy suhstitutes for the human brain needs to he carefully considered if it is the human who must present and defend the lahoratory results in a court of law. Without doubt, the role of mass spectrometers will grow, especially in the area of reference quantitative methods. It could he convincingly argued that the greatest power and value of GC/MS lies in its ability to perform quantitative analyses a t sensitivity levels generally beyond any other technique, and with great accuracy and precision. To use the instrument in this way, however, requires considerable experience and technical skill, and many toxicologists do not currently take advantage of this capability. Some headway has heen made in the past two or three years through the encouragement and support of the National Institute on Drug Abuse (NIDA) Research Technology Branch, and under its auspices a monograph of quantitative GC/MS methods for drugs of abuse has been published. A lack of available, appropriate internal standards for GC/MS assays has also inhibited methods development. Ideally, carbon-13 or deuterium-laheled analogs of the drugs and metabolites to he assayed should he used as internal standards, although
they are not absolutely necessary in all cases. The synthesis of these stable isotopes requires considerable expertise hy organic chemists and is very expensive. In consequence, there is a considerable exchange mart in these substances, and they are jealously guarded when acquired. The need for a store of these compounds of known chemistry and purity is essential to the future of analytical toxicology. Particular drugs of abuse and suhstances that are not otherwise available from commercial sources have heen synthesized under contract for NIDA and small quantities are available to biomedical researchers and toxicologists by special request. More needs to be done, however, on a much broader base if quantitative GC/MS techniques are to be used to develop reference methods for forensic toxicology
1
There is really only one critical issue ultimately, and that is: Can mechanisms of toxicity uniquely characteristic of particular drugs and poisons he demonstrated in humans? If there is cause and effect, can the analytical toxicologist, alone or with colleagues from allied professions, especially pathology, provide proof sufficient for legal adjudication? I t is the mission and the professional responsibility of forensic toxicologists to do so whenever possible, but unfortunately there are relatively few classes of drugs and chemicals for which clearly defined mechanisms of action and toxicity are known. There are few for which lesions can he demonstrated chemically or pathologically in hiological specimens for living individuals or even postmortem. It is the critical issue and challenge to the analytical toxicologist to become knowledgeable about the known biochemistry and physiology of his or her science and to understand more about the fundamentals of pathology that results from chemical insult.
4 4 6 A * ANALYTICAL CHEMISTRY, VOL. 54. NO. 3, MARCH 1982
The problems of defining and demonstrating toxicity strike at the heart of the relationship between laboratory analytical data and actual toxic events, in a science in which the technical capacity to produce data bas outstripped our ability to understand its forensic significance or interpret its toxicological message. A redress of this imbalance is urgently needed, hut it seems unlikely that forensic toxicologists will undertake it unless appropriate research support is forthcoming for those who are relatively free of the daily case travail and are able to devote themselves to research. Forensic toxicologists who have primary appointments in universities and who have a mandate to conduct research of this type are a small minority. One unfortunate outcome of this situation has heen the premature promulgation of law and regulations before adequate scientific data and knowledge are available to support their enforcement. For example, there are many statutes that state that it is illegal to drive an automobile on the highway while under the influence of a drug. These laws are weakly enforced, in part because being under the influence of a drug to such an extent that normal driving ability is impaired almost defies definition on the hasis of current scientific knowledge. It is known that millions of drivers take drugs legally under the supervision of a physican for illness hut they are not over-represented in highway accidents. How then is toxicity and legal infringement to be defined in these circumstances? Analytical, behavioral, and epidemiological research data should he the foundation of reasonable law and should precede enforcement. Virtually no research (except a few ad hoc studies) is in progress on this problem, hut it is critical, and until it is addressed, the courts will continue to look askance a t toxicologists when these cases come before them for adjudication. There are several areas in forensic toxicology that need immediate study and critical review so that synopses of current knowledge can he passed on to practicing professionals. For example, adverse drug interactions are constantly reported in the medical literature but many are anecdotal cases and not substantiated hy appropriate laboratory studies. Those documented by pharmacologists rarely include analytical data, and those for which the mechanism is known are too few relative to the case reports. This knowledge needs to he distilled into a concise and useful form for forensic toxicologists. The workplace is no exception to the use of drugs, and it is also the place where occupational toxicities
cmr. If thene lead (0 rcrinun medical
prohlemn. dinahility or wen death. then the forensic t i i x i r n l i ~ i ncan t almost lr axrured of a part in the legal proceedings t hat are rertain to ensue. 'I'hrse matters have grown in recent years to the point where they ninsume a significant part of thecivil niurt's activities. Often huge sums of money are involved in the way of compensation and thr iqiiniims id fnrensic torinilcwists are. therefore. of critiral nm. rem t o workers' compenuatiiin Inmrdn. trailr unions. insuronre nimpnnies. emplt,yers. ani1 the virtimsor their rrlatives. It hnu h m e abundantly clear lhal thr tcixinilagint. in paanernhipwith
pathdogirts end hinmedicaladleagues. plays an incrrnsingly important ride as an innhudsmanof puhlic health iunrw and that this new dimension of profennional reqn~nsiliilityis of almost frighteninn prolwrtiim. The slnrm that i s rightly felt in thr profen. siim can only tr a~~uaged and tirinighl into pernpective by inrreasing thr knciwledge hare from whirh twxicnlagical npiniiins are rmdernl. This meann morr wicntisw. pnrtirulnrly those skillnl in analytical rhrmiztry and hii~mrdicinc.must Ir nttrncted into thr field. .Irrat a few arras of immediatr n 4 have I ~ mrntionnl. n hit they are critical. they ranniit Ir awided. and thry mu..( receivr apprnpriate attentiiin unin.
7 d Bryan Finblr is dinctnr n l t h r Crnt r r /nr Human Tnxiciilngy at t h r ( I n i c m i t y ii/ 1 'tah Hralth Srirnrrn Crnt r r . aunnriatr pru/rmur II/ hiachrmiral pharmarnlogy and tnxirnlogy at 1 'tah i ('n//rReii/ I'harmary. and orrixtant prii/wsnr o/ pathology at t h r u n i c r n i t y i (bllrgr o/ M r d i r i n r . Edu r a t r d in h g l a n d as an analytical chrmirt. Finklr latrr rarnrd a dnrtnra1 drgrrr in mrdiral p h a r m a c i i l ~ ~at~ y thr 1 'niwrsity o/ 1 ?ah. His prii/rxrinnal intrrrsta inrludr rrrrarrh into t h r pmhlrmr ri/alriihiil and drugs. lahriratnry iiprratinnrr in toxrre/nRy. autematrd analytrral inrtrumrntaIron. mom rprrtriimrtry ax a toxiroIiigiral t w l . and impriicrd h n r r /or i n t r r p r r t a t i m o/ analytiral tiixirnlo#y data.
KC,*: RPTLG A true C18 reversed phase TLC plate. Better resolution. Faster. Higher capacity. Fully silanized. Close correlation to c18 HPLC columns. In inexpensive microslides and standard TLC plate formats. KC,,: high efficiency, true reversed phase precoaled glass TLC plates. C~sgroupsSi-0-Si-C bonded to a special silica gel. Minimum residual silanols. Fast developing: 2 min cm. Rf deviation only 0 . 5 O b . High capacity allows prep TLC (milligrams applied) on the same plate. KCl.: available with or without fluorescent indicator in microslides. 1" x 3"(particularly useful for solvent system development. and HPLC correlations) 5 x 20 cm. 10 x 10 cm and 20 x 20 cm formals. Whatman quality. Immediate delivery. KCl.:
For high efficiency RPTLC.
Write for free samples. To order call Toll Free: 800-631-7290 (in N.J. call COlleCl201-773-5800), Whatman Chernlcal Separation Inc. 9 Bridewell Place. Clifton. New Jersey 07014
