forensic toxicology of drug bryan - ACS Publications

FORENSIC TOXICOLOGY OF DRUG BRYAN S. FINKLE, Laboratory of Criminalistics, Department of District Heavy responsibilities accrue to chemists and toxicologists who provide analyses of blood and urine samples for drugs and narcotics and interpret results to physicians, nurses, probation officers, police, and the courts. The mission of laboratories so involved is ill-defined, and regu lations for analytical practice do not exist. State-of-the-art, prob lems, and anticipated future de velopments are treated ΤΉΕ

NATIONAL PALAVER COIlCem-

ing drug dependency and abuse in the United States has led to an unparalleled release of the federal purse strings. The Federal Government will pour over $600 million in fiscal year 1973 (1) into a multiplicity of drug abuse programs across the country. Even at the dubiously high official figure of 600,000 nar cotic addicts {2), this expenditure represents almost $1000 per addict. Government estimates indicate that there are at least 2000 re habilitation programs nationwide. Some are officially sponsored; some are private agencies, and between them they adhere to almost every possible theory of treatment, op erational management, and func tional requirements. An astonish ing bureaucracy has mushroomed around the drug abuser and addict. There are some 173 federal offices charged with some aspect of the drug problem, and to them must be added a forbidding number of state, county, and city organizations. The bureaucrats may now out number the addicts by a consid erable margin. Despite the laudable efforts of the President's Special Action Office for Drug Abuse Prevention (SAODAP), under the direction of Dr.

Jerome Jaffe, to guide and cajole the national melee of activity, there is considerable confusion of purpose behind the bureaucratic structure. No one is sure if the job is to get at the causes of drug abuse and remove them by patient effort or to simply reduce the mani fest problem to a point where it will no longer be a- dominating and political issue. Under the Drug Abuse Office and Treatment Act of 1972, the President's SAODAP will be abolished effective June 30,1975. Discounting the current efforts by the Department of Defense to rationally address the drug abuse problem in the Armed Forces (the triservices' screening and prevention programs are of a stunning magni tude requiring millions of urine analyses per year), the task of in vestigating, controlling, curbing, and finally preventing drug abuse in the civilian population has reached into almost every part of the medical and scientific com munity and is the concern of every citizen—from children in grade school, to whom "dropping Reds" (illicit secobarbital capsules) is just part of life's tapestry, through the adult often sustained by a pot pourri of tranquilizers and drugs to adjust a gamut of body functions, to the aged with minor, chronic ills controlled by self-medication at the local drug store. The social and scientific ramifica tions of drug abuse in the United States are legion, and as a drugoriented society, with a four-billiondollar retail trade in patent medi cines and where allowing your natural metabolism to take life as it comes is passé, the naive readiness with which people reach for a tube or bottle has only served to defy a clear definition of the real problems the scientist must address if a fruitful and realistic outcome is to be achieved. The result of this uncertainty and confused state is that although

REPORT FOR ANALYTICAL CHEMISTS

ABUSE: A Status Report Attorney, County of Santa Clara, 875 N. San Pedro Street, San Jose, Cailf. 95110 there are many federally funded research programs into the nature of drug abuse, the major effort is now in detecting the drug abuser and in the control of legally proscribed drugs through law enforcement (3). The national methadone treatment program for heroin addicts is the one exception, but to date, even it has no common operational guidelines and standards of practice. "Proposed Special Requirements for Use of Methadone" has been published by the Federal Food and Drug Administration, Division of HEW (4); and in cooperation with the National Institute of Mental Health and SAODAP, the FDA is undertaking an intensive inspection of existing programs to ascertain that federal requirements are being met. The latest requirements become effective in July 1972. I t is a laudable activity but, unfortunately, does not include regulations for analytical practice in the laboratories. No segment of the effort reflects the confusion of purpose more poignantly than the laboratories where toxicologists and chemists are charged with the responsibility to analyze blood and urine samples for drugs and narcotics and to interpret the results to physicians, nurses, probation officers, police, and the courts. The analyst is in the invidious position of attempting to provide a support service to a service user who often seems incapable of defining the problem and, therefore, cannot pose an intelligent question to the laboratory staff beyond, "Are there any drugs in this sample?" or, "Is this tablet a narcotic?" A toxicologist cannot design an analysis, much less provide a useful professional opinion to a physician, lawyer, or social worker in a context of such broad, general inquiries without a rationale and explicit purpose to each analysis. The plethora of laboratories involved in drug abuse analysis is not a realistic guide to the need for

their service. I t is unfortunate that many laboratories serve only to fulfill a contract requirement, to help convince a government agency to grant money, or to assist the entrepreneur to garner his share of the dollar bonanza now synonymous with drug abuse programs. It is hardly surprising in this situation that the mission of even the best laboratories is often woefully ill-defined, and the standards of analytical practice are lamentably below par. LABORATORY RESOURCES

Consideration of available laboratory resources is a prerequisite to any analytical scheme and dictates the fundamentals and methodology upon which the analytical result will stand or fall. Laboratory resources may be thought of simplistically as dollars, staff, space, and equipment, and providing these items are in adequate balance, scientific capability will be born as a natural consequence. This superficial approach has bred a glut of laboratory operations which are overwhelmed by demands and choked in a morass of analytical results, attempting to answer redundant questions. It is the inevitable penalty of ignoring a clear definition of the service to be rendered and a detailed understanding of both operational and scientific constraints involved. The following are principal requirements which must be considered before an analytical scheme can be designed: Name of drugs to be detected Sensitivity limits Sample type and size Quality of results required Intended use of the analytical results Cost effectiveness, including work load and required "turnaround time" Available laboratory equipment and trained personnel

Drugs to be detected should not mean any and every drug available to a would-be user. Five years of experience has taught that the "drug scene" is dynamic and local with respect to both time and place. Focus should be on those drugs which are in vogue locally. I t will be a surprisingly small number —less than 10 (excepting alcohol) will account for over 75% of all positive analyses, and less than 20 will cover practically 100% of those drugs likely to be encountered. Laboratory capability to detect and identify the following drugs and their metabolites will permit practical work and useful results : Morphine (heroin metabolite) Codeine Methadone Cocaine Propoxyphene Amphetamine Methamphetamine Barbiturates Amobarbital Pentobarbital Secobarbital Phénobarbital and "cutting agents," quinine and procaine. Although there is intense current research activity, there are no means at present to prove LSD or marijuana use by analysis of body fluids. There are useful procedures applicable to controlled clinical and pharmacological studies, by use of fluorometry and mass spectrometry {5-10), but they are unsuitable for samples taken randomly from suspected users in which dosage, time interval, and conditions of ingestion are unknown. Sensitivity limits are often whatever is imposed by the physician in charge (frequently a psychiatrist), government agency or contracting officer to the limit of the toxicologist's forebearance; and seldom determined on the basis of available analytical capability, knowledge of

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Report

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drug metabolism and excretion rates, concentration ranges following acute and chronic therapeutic dosage, and those concentrations anticipated in t h e drug abuser. No realistic sensi tivity limits can be set without re gard to sample type and size. Cur rent analytical methods and the need for uncomplicated patient man agement dictate urine, in excess of 10 ml, as the sample of choice. I n any event, with the exception of the barbiturates, choice of sample is a moot point because most of the common drugs of abuse cannot be detected in blood samples from living subjects b y current routine procedures. This is generally t r u e for t h e organic bases as a group. By use of urine samples of greater t h a n 10 ml in volume and obtained less t h a n 48 hr after ingestion, the following are reasonable sensitivity limits: 0.5 jug/ml of urine for mor phine (total, i.e., free drug plus bound, conjugated metabolite) and barbiturates. 2.0 μg/ml for cocaine and propoxyphene; amphetamine and methamphetamine. A thorough understanding of the h u m a n metabolism of these drugs (11-17, 28) is essential to meeting the above sensitivity limits and making a correct interpretation of t h e analytical results. M u c h work needs t o be done in this area, and an appreciation of drug metabolites must be conveyed to administrators and those responsible for drug abuse program regulations. T o be in tensely concerned over detection limits for cocaine in urine, before fully understanding whether the drug is excreted in humans un changed or as benzoyl ecgonine and ecgonine metabolites (64) is a typical cart-before-the-horse atti t u d e which does much to damage t h e credibility of clinicians and drug abuse program personnel requesting laboratory service. Without a knowledge of naturally occurring interfering substances, a clear ana lytical appreciation of other thera peutic agents likely to be present, such as phenothiazines and anti malarial drugs, and a certain ability to recognize urine metabolic profiles for propoxyphene (18), methadone (19), and cocaine (and nicotine), t h e analyst will be unable t o serve the often bewildered b u t demanding physician. T h e quality of the analytical

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Chemists

ANALYTICAL CHEMISTRY, VOL. 4 4 , NO. 9, AUGUST 1 9 7 2

results required will vary for each laboratory operation and will be conditioned by the intended use of the results. For example, quan titative analysis and unequivocal qualitative specificity, often man datory in forensic work where crim inal guilt is a t issue, are not neces sary in surveillance and preemployment screening programs support ing private clinics and industrial companies. Similarly, programs monitoring methadone clinic pa tients and analysis of samples required for emergency medical diagnosis of overdosed patients in the emergency room rarely require quantitative work. E a c h has its own peculiar requirement, and the ultimate choice of analytical method and equipment should be decided within this framework. Confirmation t h a t a patient is taking methadone as prescribed and not reverting to heroin use may be achieved by a simple, nonspecific chromatography system providing an adequate " y e s " or " n o " answer t h a t is without any false positive results, whereas extraction and mul tiple identification techniques are essential in t h e more exacting foren sic context. T h e use of gas chrom a t o g r a p h y - m a s s spectrometry is the best tool to provide a specific answer, b u t it would be folly to waste its power to screen thousands of samples per week when it m a y be anticipated t h a t less t h a n 1 0 % of the samples is likely to yield a positive result. W h a t is really needed is an inexpensive simple, mecha nized or automated technique whereby those samples which are negative (as defined by sensitivity limits and drugs encompassed by t h e analysis) can be rapidly and certainly identified. I n this way, time gained can be applied against the difficult problems of accurate assay and drug identification in the samples indicated as positive. Such naive efficiency considerations should be the foundation of any laboratory faced with a potentially overwhelming work load and a con tinual demand for production, often within a 24-hr " t u r n a r o u n d t i m e . " Cost effectiveness in the laboratory usually means getting t h e most from t h e least—the greatest o u t p u t for t h e smallest cost—but what of quality? Is it really wise to insist on a gold-plated answer when t h e

Report for Analytical Chemists

copper penny product is equally effective? T h e deluge of funds recently available in support of methadone and other programs has unfortunately tended t o push such basic considerations into t h e shadows of a scientific world already noted for its aloofness from industrial management techniques. T h e fact is, with t h e advent of largescale drug abuse detection programs often requiring analyses in excess of half a million samples a year, such m u n d a n e aspects as money, efficiency, production, and effectiveness have arrived on the scientist's laboratory doorstep with a vengeance. ANALYTICAL METHODS

A national survey of laboratory practice would reveal a symphony of variations on only three preliminary screening themes and one or two qualitative identification techniques. Liquid-liquid or nonionic-exchange resin extraction of t h e urine at controlled p H {21) followed by thin-layer chromatography, in addition to either a fluorescence method {22-25) or a free-radical immunological procedure for the detection of morphine {26-27, 65, 66), is common to almost all laboratories. Similarly gas-liquid chromatography, often supplemented b y oncolumn derivative chemistry, is t h e most common method employed for identification {29-84). T h e nonspecific n a t u r e of fluorometry and immunology, in addition to potential cross-reaction interferences in t h e latter, must be appreciated if an over-reliance on results is t o be avoided. T h e advantages of these procedures are speed, sensitivity, and their ability to indicate negative samples. I n mechanized form b o t h are capable of analyzing more t h a n 60 samples/hr t o a sensitivity limit below 0.5 /xg/ml of urine for morphine. T h e free-radical antibody method, F R A T {65, 66), has t h e a d v a n t a g e of detecting free and conjugated morphine directly, without t h e need for prior hydrolysis. Greater t h a n 9 0 % of morphine is excreted conjugated with glucuronic acid {11,35) and as t h e sulfate. Conversely, it is an expensive method at more t h a n $1.00 per test and uses a unique antibody reagent available

from only one manufacturer (Syva Corp., Palo Alto, Calif.), and an electron spin resonance instrument is required to detect the displaced free radical. By contrast, fluorescence procedures cost less t h a n five cents per test and require only common laboratory chemicals in addition to a spectrophotofluorometer. There is at least one instrument available with a specially designed sample head for multiple urine analysis (Farrand Optical Co., Inc., Valhalla, N . Y . ) . There are two well-demonstrated methods for t h e production of morphine fluorophores {86-38, 22), one using potassium ferroferricyanide to generate fluorescent pseudomorphine, and the other depending upon sulfuric acid oxidation. T h e products of t h e latter are not known precisely, b u t t h e resulting multiple fluorescence peaks seen in t h e total emission spectrum enhance t h e qualitative significance of t h e test. There are no reported commonly occurring interfering substances. T h e antibody reagent used in t h e F R A T test is not specific for morphine. I t will react with any phenanthrene, notably codeine, and dextromethorphan which occurs in nonprescription Romilar cough syrup. Although not as sensitive to t h e reagent as the phenanthrenes, a positive test can be obtained from other drugs in urine, e.g., diphenoxylate (in Lomotil), phenothiazines (particularly thioridazine), and high concentrations of methadone and propoxyphene. As m a n y as 1 3 % false positive tests may be anticipated {89, 40) in high-volume routine screening. Ascorbic acid interferes, b u t this is simply overcome b y adding a dichromate oxidation step to the test procedure. These limitations in no way diminish the usefulness of t h e technique as a reliable screening method for opiate narcotics, and with t h e promise of further reagents for phenethylamine derivatives, methadone, barbiturates and cocaine metabolites, this approach could become t h e screening method of choice, only requiring gas chromatography or g c / m s confirmation t o complete the analysis. However, reliable antibody reagents suitable for routine use are currently n o t available for these drug groups.

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Similarly, t h e addition of a hydrolysis module to the mechanized Autoanalyzer (Technicon I n s t r u m e n t s Corp., Tarrytown, N.Y.) fluorescence device and channels t o detect more drugs of abuse t h a n just morphine would greatly increase t h e utility of t h e instrument and justify t h e capital cost to m a n y more laboratories. Recent studies h a v e shown t h a t without hydrolysis, a t least 2 0 % and perhaps greater t h a n 9 0 % of routinely submitted urine samples containing morphine would be reported negative by thin-layer chromatography {40, 41, 42). The choice of hydrolysis method is imp o r t a n t . T h e most efficient, by use of strong hydrochloric acid, also degrades other drugs of interest, e.g., methadone, propoxyphene, cocaine. Enzymatic hydrolysis is less efficient and costs about ten cents per sample b u t maintains the integrity of the drugs and the urine, permitting simple extraction and clean residues. A 2 : 1 enzyme mixture of 0-glucuronidase and aryl sulfatase at a concentration of 1000 Fishmann u n i t s / m l of urine at p H 4 - 5 , incubated for 12 hr at 45°C, will release better t h a n 9 0 % of the bound morphine {43). T h e need for hydrolysis is m a n d a t o r y if chromatography procedures are to be used and realistic sensitivity limits met for morphine. There are as many different, possible methods for extracting drugs from urine as there are techniques known t o t h e organic chemist {21, 44, 47, 48). High-pressure liquid chromatography offers perhaps t h e newest rapidly developing method and adds separation and detection parameters in a continuous system {68, 69). I t will also include m a n y polar, highly water-soluble m e t a b olites difficult to include in a simple, conventional extraction scheme. A review of existing laboratory methods reveals only two extraction techniques: solvent extraction of the urine at a specific p H value {44~48) and absorption of the drugs on a column of amberlite X A D - 2 {21) nonionic-exchange resin followed by solvent elution. Cation exchange paper has been proposed b u t has found only limited use, probably because of its lack of adeq u a t e sensitivity {44, 55).

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ANALYTICAL CHEMISTRY, VOL. 44, NO. 9, AUGUST 1972

T h e X A D - 2 resin technique has t h e advantages of avoiding emulsions which can plague liquid-liquid methods a n d extracting some of t h e polar drug metabolites. Preprepared columns are available commercially (Brinkman I n s t r u m e n t s , Inc., " D r u g Skreen," Westbury, N . Y . ) , and they provide t h e added nicety of a phase-separating interface at t h e end of t h e column which substantially avoids t h e problem of eluting unwanted absorbed water. I t also overcomes t h e operational disadvantages of washing and purifying t h e resin before use a n d t h e necessity t o assemble a n d pack t h e columns. T h e columns cannot b e reused indefinitely, a n d tests m u s t be m a d e t o determine their life span. T h e loss of activity can be precipitous. T h e r e are a variety of organic solvents and solvent mixtures used t o elute t h e drugs from t h e column. E t h y l e n e dichloride-ethyl acetate is advised for t h e commercially prepared columns, b u t ethyl acetate followed by methanol is most frequently used a n d is efficient. I t s disadvantage is t h a t methanol removes water and in common with all other eluting systems, also removes absorbed urine pigments resulting in intolerably dirty residues. Clean-up steps are necessary before acceptable gas or thin-layer chromatography can be achieved {21). T h e efficiency of extraction of t h e drugs is dependent upon t h e p H of the urine as it passes t h r o u g h t h e resin column, and unfortunately, the optimal p H of 8.5-9.5 for morphine and other amphoteric bases is not ideal for phenethylamines such as amphetamine (pH 7-9) or t h e barbiturates (pH 5-7). Either an u n h a p p y compromise p H must be


selected, or appropriate p H adjustments made, and then sequential passes made through the column. This is a tedious and operationally unsatisfactory procedure. pH values in the range 6-9 will permit 7 5 - 8 0 % recovery of the drugs, including the weakly acidic barbiturates (43, 47). Similar p H considerations are implicit in solvent extraction procedures. Although there is seemingly an infinite list of possibilities to the imaginative chemist, a 4 : 1 mixture of chloroform : isopropanol is simple and highly efficient, particularly in extracting morphine. I t has become the solvent of choice although other mixtures may be better in specialized methods, e.g., 5 : 4 : 1 chloroform : carbon tetrachloride : isobutanol used in the Autoanalyzer (38). T h e amphoteric nature of morphine demands critical p H control of the urine. Using approximately equal volumes of urine and solvent, the urine must be at p H 8.5 ± 0.1 if a single extraction is to suffice. At these conditions 9 0 - 9 5 % of the morphine will be extracted (48, 49)· Immediately beyond the p H tolerance of ± 0 . 1 , the recovery will be drastically reduced, to 4 0 - 5 0 % morphine extraction at p H 8.3 and p H 8.8. p H 8.5 is not ideal for extracting all common drugs of abuse as it is for morphine, b u t it is satisfactory for all of the bases and, surprisingly, the weakly acidic barbiturates t o

Figures 1-3. Rolling devices used in the extraction of free drugs from urine. Efficient extraction is complete in 10 min w i t h o u t emulsion f o r m a t i o n . The device shown at left was designed a n d constructed by D. M. Taylor, Laboratory of Criminalistics, San Jose, Calif.

sensitivity limits previously discussed, when a 10-20-ml urine sample is used. T h e more acidic phénobarbital is extracted to only approximately 3 0 % b u t can nevertheless be easily detected at 1.0 jug/ml in the 20-ml urine sample. This extraction condition is of considerable importance because it permits a single-step procedure. T h e problem of emulsion formation between urine and solvent is a vexing one b u t can be overcome by using screw-capped tubes or bottles and a rolling device, such as those shown in Figures 1-3. An efficient extraction of t h e free drugs is complete in 10 min without any emulsions. Solvent evaporation to concent r a t e solutions or generate residues for chromatography must be done under a stream of dry air or nitrogen and without heat if the. integrity of labile free-base drugs such as amphetamine and meperidine is to be maintained. Converting the drugs to their stable base salts by addition of acid to the solvent before evaporation is a simple and expedient alternative. Notwithstanding carefully designed solvent and locating spray systems, the forte of thin-layer chromatography does not lie in identification, b u t rather in separation, and as a device for rapidly and certainly indicating those samples which are negative. I t also lends itself to processing large numbers of samples simultaneously and, as such, has become an indispensable part of drug abuse analysis. Although operationally tempting, multiple solvent systems, color reactions, and correlated Rf values are not adequate for unequivocal identification. They are, however,

often properly used in laboratories supporting programs which do not require more exacting answers. I t is of crucial importance t h a t the quality of the answer is understood. Silica gel G or H as a 250-μ layer on 20 X 20-cm glass plates, either commercially or laboratory pre pared, are universally used. De veloping solvent systems are legion, b u t those of Davidow (50) (ethyl acctate-methanol-ammonium hy droxide; 85:10:5) and Cochin and Daly (51) (benzene, 50; dioxane, 40; ethanol, 5; ammonium hy droxide, 5) are t h e most common. Both systems provide excellent sep aration of t h e basic drugs in less t h a n 1 hr b u t equally share the disadvantage t h a t the barbiturates cannot be isolated from the bases on the same plate. This problem must of necessity be overcome if the analyst wishes to detect all of t h e drugs on one plate, particularly as t h e spray reagents used for the bases interfere with those for the barbiturates. If not, an aliquot of the residue may be applied t o a separate plate and developed in a solvent system appropriate for the barbiturates, e.g., chloroform : ace tone, 90:10 (52). I n the analytical scheme de scribed below, the solvent system isolates the barbiturates at Rf values greater t h a n 90. I t does not separate the individual bar biturate drugs, b u t t h a t is unneces sary inasmuch as a discriminating confirmation analysis will be carried out in the event of a tic positive. Locating spray systems are for the most part uniform amongst laboratories. They are adequate to detect the drugs in question, b u t great caution must be exercised in assigning sensitivity and specificity to the reactions. All of the spray

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reagents are general and react with organic groups; for example, ninhydrin used t o detect amphetamine reacts with all primary amines, including /3-phenethylamine which occurs as an artifact in urine, and potassium iodoplatinate will locate any substance containing a tertiary nitrogen. A typical spray sequence is given in t h e procedure below, although more sensitive reagents t h a n ninhydrin are now being used in a few laboratories. A lightly applied spray of 0 . 4 % ninhydrin in p H 10 phosphate buffer followed b y 0 . 5 % phenylacetaldehyde in ethanol will detect amphetamine as a slowly developing gold spot which appears bright orange under ultraviolet light (53, 43). Similarly, exposure t o chlorine vapor followed by a 1.0% benzidine in chloroform spray will develop primary amines as green spots (54). These reactions are sensitive t o approximately 5 μg of amphetamine extracted from urine and represent a sensitivity factor 4 - 8 times greater t h a n t h e ninhydrin reaction viewed under long-wave ultraviolet light. Morphine detection can also be improved. B y using 0 . 1 % solution of potassium ferroferricyanide in p H 8.5, 0.1M phosphate buffer as spray reagent, a n d viewing t h e resulting bright yellow-green fluo rescent spots under short-wave (250 nm) ultraviolet light, less t h a n 1 μg of t h e drug can be seen. Recognition of tic p a t t e r n s as sociated with commonly occurring metabolites, such as those from methadone (sometimes appearing without t h e parent drug), chloroquin, a frequently encountered anti malarial drug, phenothiazines, and nicotine, is mandatory t o a correct evaluation of t h e plate. Fre quently prescribed therapeutic agents which m a y appear a t t h e same Rf value as a drug of abuse can cause confusion. An example is oxymetazoline, a nasal decon gestant, which is located close t o morphine and, like t h e opiates, stains purple with iodoplatinate reagent and gives a positive silver reduction reaction. Like tic, gas chromatography is empirical b y n a t u r e and cannot be classified with techniques such as mass spectrometry and infrared spectrophotometry which relate t o molecular structure and are truly

specific. T h e value of gc in this context is its sensitivity and ability to resolve drug components in mixtures of biological artifacts. Oncolumn derivative chemistry can a d d significantly t o its potential as an identification tool; b u t , again, t h e precise quality of t h e analytical result must be appreciated before any clinical interpretation is made. During t h e past 12 years gas chro matography h a s become funda mental t o most laboratories a n d is now well recognized as a method ideal for confirming thin-layer chro matography findings in drug abuse analysis. There is a wealth of published reference relative retention d a t a (29, 32, 48, 56). T h e gc system of Finkle et al. (29) is particularly suited t o resolving drug mixtures and utilizes two columns, 2 ft and 6 ft, respectively, packed with 2 . 5 % SE 30 and operated isothermally a t selected temperatures. OV17 and QF-1 are liquid phases which are also well proven and provide ade quate chromatography. T h e former is especially good for free-base morphine, one of t h e most difficult drugs t o chromatograph satis factorily. Experience in gc practice and analytical toxicology is required t o evaluate gas chromatograms of urine residues. Previously mentioned nat urally occurring urine extractives and spurious peaks given b y dihexyl and butyl phthalate plasticizers solvent extracted from plastic lab oratory equipment can be mislead ing and perplexing t o an unsus pecting analyst. T h e gas chromatograph can be used as a tool for performing microorganic chemistry by reacting func tional groups t o form derivatives. Acylation of primary and secondary amines (amphetamine and methamphetamine) b y use of acetic anhydride, halogenation of u n saturated barbiturates (seco barbital) by use of chlorine, and methylation of other barbiturates and opiates by use of trimethyl anilinium hydroxide (57) are simple reactions which can be performed on-column. Acid hydrolysis is also useful, producing unique products in t h e case of propoxyphene, a n d silylation b y use of a variety of reagents (58,' 59) has wide app reciation.


In lieu of a single, specific identi fication technique, cumulative ana lytical data are acceptable in most programs. An extraction method designed to isolate particular groups of drugs according to their chemical nature, followed by a thin-layer chromatography screening proce dure to indicate which extracts are negative and provide the first suggestion of identity in the posi tive samples, and finally an inde pendent parameter such as deriva tive gc to indicate probable identity, will satisfy most demands. How ever, the artisan's talent and con scientious experience are still in dispensable factors in chromato graphic methods, and a frank ac ceptance of this fact will avoid dangerous and costly errors. Gas chromatography-mass spec trometry offers the best available answer to the need for unequivocal identification of drugs and their metabolites extracted from bio logical samples. It is direct; the mass spectrometer acts as a specific gc detector, is extremely sensitive— capable of identifying nanogram amounts of material—and extremely fast. These qualities place the technique well ahead of infrared spectrophotometry which is tedious and insensitive by comparison. Reference mass spectra of drugs are now published (60, 61), and data will undoubtedly continue to grow as the cost of instrumentation falls, and mass spectrometry finds wider ac ceptance and use in toxicology laboratories. Gc/ms is the only applicable technique which will provide certain identification and avoid false positive reports so socially damning and damaging to the life of the subj ect. All of the analytical methods discussed lend themselves to the identification of solid dosage drugs, tablets, capsules, and powders. However, it is appropriate that screening or presumptive tests are made to determine whether the drug is a legally defined narcotic or scheduled substance (3) before sophisticated confirmatory work is undertaken. There are numerous published schemes which serve well for preliminary testing. The sim plest, most reliable and efficient methods for processing large num bers of drugs involve microcrystalline tests (62, 63). The direct


chemical reactions on microscope slides and observation of consequent crystal growth and form require training and experience before re liable interpretation can be made, b u t the method is operationally ideal for rapid testing of selected drugs of abuse.

1.

RECOMMENDED ANALYTICAL PROCEDURE Preliminary Tests for Morphine

(a) Immunological free-radical assay technique, or (b) Technicon Autoanalyzer fluo rescence method after hy drolysis, or (c) D a l Cortivo spectrofluorescence method (22) 2. Solvent Extraction

(Can be carried out on batches of 100 samples in 10 X 10 racks.) Transfer 20 ml of each urine sample to a 50-ml glass, screw-capped tube and adjust to p H 4.5-5.0 with 4 ml of sodium acetate buffer. Add 2 ml of enzyme mixture (20,000 F U as described) and incubate for approximately 12 hr at 55° C. (This hydrolysis step m a y bo omitted if the preliminary test for morphine was negative.) After enzymatic hydrolysis, ad just p H t o 8.5 ± 0.1 with 5 0 % W / V sodium hydroxide [This can be accomplished without using a p H meter. T w o drops of mixed indicator, cresol red (pH 8.2) and t h y m a l blue (pH 8.8) will cause a dark blue color at p H 8.5. ] and add 20 ml 4 : 1 chloroform-isopropanol solvent. Cap t h e tubes and extract in a roller extractor (Figures 1-3) for 10 min. Allow the phases to separate and aspirate off the top urine phase to waste. Add 5 ml of 0.025M phosphate buffer p H 7.0. Shake vigorously, allow phases to separate, and as pirate off top buffer layer t o waste. Transfer half of t h e solvent phase to another glass t u b e and evaporate, without heat under a stream of air, to low bulk (approximately 50 μΐ) for gas chromatography analysis. T h e solvent must not go to dryness. Add approximately 0.1 ml 0.2N hydrochloric acid to the remaining solvent phase. Transfer the tubes to a waterbath at 60°C and evapo r a t e the solvent to dryness under a stream of dry air. R e m o v e the

tubes from the water b a t h and transfer the residues to 5-ml tubes by sequential washing with 2 X 1.0-ml aliquots of methanol-chloroform 1:1. Use a vortex mixer to ensure solution of the residue. E v a p o r a t e t h e solvent to dryness as before and use the residue for thin-layer chromatography. 3. Thin-Layer Chromatography

Plates : Commercially prepared glass plates coated with silica gel G without fluorescent indicator.

Solvent Systems Ethyl acetate Cyclohexane Methanol Water Ammonium hydroxide

I 70 15 8 0.5

II 80 15 5

2

E a c h solvent system must thoroughly mixed before use.

be

Spray Reagents Mercuric sulfate: Mix 5 grams of mercuric oxide in 100 ml of water. Slowly add 20 ml of concentrated sulfuric to dissolve the oxide, then make to 250 ml with water. Ninhydrin : 0.4% W / V in p H 10 phosphate buffer. Sulfuric acid: 0.1N. Potassium iodoplatinate (a) 6 grams of potassium iodide in 100 ml of methanol (b) 3 ml 1 0 % W / V platinum chloride in 100 ml of water Mix equal p a r t s of (a) and (b) just prior to use. Procedure Allow solvent I to migrate ap proximately half way u p t h e plate. R e m o v e the plate and allow it to air dry. Place plate in solvent I I and allow migration to the top of t h e plate. Air dry. Location of Drugs Mask off all b u t the top 1 in. of the plate. Spray exposed portion with mercuric sulfate. Barbiturates will appear as white spots on a gray background. R e m o v e the mask and spray the plate in t h e following sequence: (a) Ninhydrin followed b y phenylacetaldehyde. Ampheta


mine will appear as a gold spot enhanced under uv light. (b) Sulfuric acid: observe bril liant blue fluorescence of quinine under u v light. (c) Iodoplatinate. T h e opiate narcotics, methadone and its metabolite, and other basic drugs of abuse will appear as blue-purple spots. Samples which are negative at this point in the procedure need not be pursued further. Samples which react positively are subject to gc and g c / m s analysis. 4. Gas Chromatography—Mass Spectrometry

According to Reference 29, ali quots analyzed on appropriate gc system—A, B , D , or E . A 5-ft X 1/8-in. i.d. glass column packed with 3 % OV-17 on supelcoport and operated at 250°C is particularly suitable for the detec tion of submicrogram amounts of morphine free base. On-column derivativization as previously discussed can be imple mented at this stage. Better, how ever, t h a t the gc peaks of interest be transferred to t h e mass spectrom eter for final identification. Almost all of t h e published reference d a t a and drugs analysis applications have resulted from the Finnigan Model 3000-003 g c / m s peak identifier by use of an all-glass jet separator as an interface (60). T h e equipment is simple to use and provides a mass spectrum as the gc peak is eluted at its retention time. T h e foregoing procedure is easily capable of supporting a work load of 100 samples per hr and has proved capable of operating with consistent efficiency u p to 12,000 samples per six-day work week (two 10-hr shifts per day) for a year, constrained by a demanding 24-hr "turnaround time." Quantitative analysis is rarely necessary as a routine and should be discouraged unless a practical use of the results can be demonstrated. I n most instances, the additional exacting laboratory work is not justified. D r u g concentrations in urine samples taken randomly can rarely be interpreted to yield any meaning with respect to behavior or clinical condition. T h e only sure interpretation is t h a t the presence of the drug dem-


onstrates t h a t subject did in fact ingest the material. T h e dearth of reliable information correlating dos age, time, and clinical condition with blood and urine drug and metabolite concentrations in humans attests t h a t this is an area of almost total neglect. Whereas urine concentra tions are often so extraordinarily high (especially for amphetamine and related phenethylamines which may reach concentrations u p to 350 μg per ml) t h a t it is obvious t h a t t h e drug was not ingested under thera peutic conditions, it would be fool hardy to predict the behavior of the subject at a specified time with t h e urine concentration as t h e sole basis. If the subject under surveillance voided completely and sometime later gave a urine sample for analysis which proved to contain a drug, then it would be reasonable to state t h a t t h e drug was also in the circulatory system and consequently exerting some physiological effect on the sub ject during the period of time be tween voiding and sample. Quality control and proficiency testing by means of blind samples containing drugs at realistic sen sitivity limits is mandatory. T h e scheme should be designed to test all aspects of the operation, from sam ple receipt and identification to the clerical preparation of the laboratory report. Required analytical per formance must be defined and rig orously applied. False positive results cannot be tolerated. The ability to meet per formance standards in greater t h a n 9 5 % of the samples should be rou tine. Any laboratory operation continuously processing in excess of 10,000 samples each week will in evitably make some mistakes. E v e n if the analytical method is ideal, mistakes will occur at t h e d a t a processing and clerical stage. Lessons can be learned, and advice should be solicited from large hospi tal clinical chemistry laboratories where similar problems have been faced and solved in recent years. T h e quality control program should begin with the urine sample. T h e addition of boric acid at 10 m g / m l of urine will preserve the sample from bacterial action and maintain the urine in a weakly acid condition, thus preventing degradation of the drugs. T h e program must include urine

samples containing drugs at known concentrations approximating re quired sensitivity limits. T h e sam ple should be entered into the ana lytical system on a completely blind basis. H u m a n urine containing ex creted morphine should be included to test hydrolysis efficiency, and control drug mixtures must be ap plied to each thin-layer plate and gc system as references. These are simple considerations but will pay high dividends in confidence and credibility for the toxicologist and requesting agency alike. THE FUTURE

If the toxicologist is to provide a useful service, the future of drug abuse analysis must lie above all in a clearer definition of the need and requirements. This is an urgent m a t t e r which must be decided by t h e physicians and administrators of t r e a t m e n t programs, district at torneys, probation officers, and leg islators in consultation with ana lytical toxicologists. If purposeful guidelines were issued through the President's Special Action office, it would be of inestimable help.

certainly advance in the near fu ture, as experience is gained in operating laboratories. A highly innovative variation has already been reported (67) and reputedly is more sensitive t h a n free-radical detection. I t is the E M I T (En zyme Multiplicity Immunological Technique). Following antibodydrug reaction, displaced lysozyme enzyme is detected spectrophotometrically, as it acts to clear a turbid solution of bacteria. Automated and computer-assisted equipment, such as t h a t currently being developed at J e t Propulsion Laboratory under the NASA Space Technology Applications program, offers perhaps the most sophisti cated approach to toxicology analy sis ever conceived, b u t this is still a long-term prospect. Meanwhile, harassed analysts working in overloaded laboratories faced with high-volume urine testing must steadfastly resist the pressure demanding "instant results," shun mediocrity as the enemy, and with dignity born of social conscience and scientific integrity face t h e challenge of a difficult job, in the knowledge t h a t the professional chemist can assist in the Nation's major public health problem and help improve the quality of life for thousands of American citizens.

There is an overwhelming need for trained toxicologists. There are only 148 members of the American Academy of Forensic Sciences toxi cology section and b u t a handful of accredited training programs in the nation. Carefully conceived pro grams in which academic and voca tional training is allied between schools staffed by experienced tox icologists and existing forensic tox icology laboratories, to assist ana lytical and clinical chemists enter this exacting b u t challenging field, are a first-order priority. D r u g abuse analysis is no job for the unskilled technician who carries much of the bench-work burden today.

T h e author thanks Loren Price and Dale Gray of United Medical Laboratories, Portland, Ore., for their technical assistance and practi cal suggestions which added sub stantially to this work. Similarly, t h e experimental work performed b y the staff of the Laboratory of Crim inalistics, San Jose, Calif., is ac knowledged and appreciated.

Technical advances will un doubtedly continue to dominate and will lead toward simpler, faster, and more reliable analysis. Gc/ms hardware is already in development, which will minimize the necessity for extracting drugs from urine, and heralds t h e day of direct testing. Analyzing more conveniently ob tained samples such as saliva, fingerprick blood, and breath is in pros pect, and the results may offer a better base for predicting the drug user's clinical condition. Immunological techniques will

REFERENCES (1) The White House Fact Sheet, Special Action Office for Drug Abuse, March 21, 1972. (2) Medical World News, ρ 20, February 18, 1972. (3) Code of Federal Regulations, 21 Food and Drugs, January 1, 1972, published by the Office of the Federal Register, General Services Administration. (4) Proposed Special Requirements for Use of Methadone, Federal Register, April 6, 1972, 37 F.R. 6940. (5) Julius Axelrod et al., Ann. Ν. Υ. Acad. Sci., 66 (3), 435 (1957). (6) L. A. Dal Cortivo and J. R, Broich, Anal. Chem., 38, 1959 (1966).


ACKNOWLEDGMENT


(7) I. C. Nigam and J. L. Holmes, J. Pharm. Sci., 58, 506 (1969). (8) S. J. Mule, J. Chromatogr., 55, 255 (1971). (9) H. M . Stone and H. M. Stevens, / . Forensic Sci. Soc, 9, 31 (1969). (10) E. J. Woodhouse, Insurance Insti tute for Highway Safety, Washington, D.C., March 1972. (11) E. Leong W a y and T. K. Adler, Bull. W. H. 0., 27, 359 (1962). (12) J. Grove and P. A. Toseland, Clin. Chem. Acta, 29, 213 (1970). (13) M. S. Moss and J. V. Jackson, Excerpta Med. Found. Int. Congr. Ser., 80, 104 (1963). (14) J. J. Kaman and E. J. Van Loon, Clin. Chem., 12, 789 (1966). (15) A. H. Beckett and M. Rowland, J. Pharm. Pharmacol., 17, 628 (1965). (16) J. Axelrod, / . Biol. Chem., 214, 753 (1958). (17) J. Axelrod, Fed. Proc., 13, 332 (1954). (18) R. E. McMahon et al., Toxicol. Appl. Pharmacol., 19, 427 (July 1971). (19) A. H. Beckett et al., J. Pharm. Phar macol., 20, 754 (1968). (20) E. G. C. Clarke, "Isolation and Identification of Drugs," Pharmaceuti cal Press, London, England, 1969. (21) N. Weissman et al., Clin. Chem., 17, 875 (1971). (22) L. Dal Cortivo, Ε. Kallet, and W. Matusiak, Proc. of Committee on Problems of Drug Dependency, Nat. Acad. Sci., Nat. Res. Comm. 6397, 1970. (23) A. E. Takemori, Biochem. Pharmacol., 17, 1627 (1968). (24) S. J. Mule and P. L. Hushin, Anal. Chem., 43,708(1971). (25) G. Nadeau and G. Sobolenski, Can. J. Biochem. Physiol., 36, 625 (1958). (26) S. Spector, J. Pharmacol. Exp. Ther., 178, 253 (1971). (27) Medical World News, pp 15-16, July 16, 1971. (28) Τ. Κ. Adler et al., J. Pharmacol. Exp. Ther., 114, 251 (1955). (29) B . S. Finkle et al., J. Chromatogr. Sci., 9, 393 (1971). (30) N. C. Jain, Diss. Abslr., 27, 3, 710B (September 1966). (31) N. Ikekawa, Anal. Biochem., 28, 156 (1969). (32) L. Kazyak and E . C. Knoblack, Anal. Chem., 35, 1448 (1963). (33) H. M. Fales and J. J. Pisano, Anal. Biochem., 3 , 337 (1969). (34) M. J. Barrett, Clin. Chem. Newslelt., 3, 1 (1971). (35) J. M. Fujimoto and E. L. Way, J. Pharmacol. Exp. Ther., 121. 340 (1957). (36) H. Kupferberg et al., ibid., 145, 247 (1964). (37) A. E. Takemori, Biochem. Pharma col., 17, 1627 (1968). (38) D . J. Blackmore et al., Clin. Chem., 17,896(1971). (39) Β. Hamman, Proc. Amer. Acad. Forensic Sci., unpublished (1972). (40) B. S. Finkle and L. Price, unpub lished work, United Medical Labora tories, Portland, Ore., 1972. (41) J. Wallace and J. T. Pay te, Curr. Ther. Res., Clin. Exp., 13, 412 (June (1971). (42) T. J. Butler, Southern Memorial Hospital, Las Vegas, Nev., private communication, 1972.

(43) B. S. Finkle, unpublished work, Laboratory of Criminalistics, San Jose, Calif., 1971. (44) V. P. Dole et al., J. Amer. Med. Ass., 198, 349 (1966). (45) K. K. Kaistha and J. H. Jafîe, J. Chromatogr., 60, 83 (1971). (46) S. J. Mule, ibid., 39, 302 (1969). (47) B. Davidow et al., Amer. J. Clin. Pathol., 50, 714 (1968). (48) S. J. Mule, Anal. Chem., 36, 1907 (1964). (49) D . Reed, Orange County Coroner's Laboratory, Santa Ana, Calif., private communication, 1971. (50) B. Davidow et al., Amer. J. Clin. Pathol., 50, 714 (1968). (51) J. Cochin and J. W. Daly, Experientia, 18, 294 (1962). (52) I. Sunshine, Amer. J. Clin. Pathol., 40, 576 (1963). (53) R. Bath, Cuyahoga County Coroner's Office Laboratory, Cleveland, Ohio, private communication, 1972. (54) R. Porter, Proc. Calif. Ass. Toxicol., unpublished (May 1972). (55) V. P. Dole et al., N. Y. Stale J. Med., 72 (February 15, 1972). (56) L. Kazyak and R. J. Permisohn, J. Forensic Sci., 15, 346 (1970). (57) M. J. Barrett, Clin. Chem. Newslett., 3, 1 (1971). (58) G. E. Martin and J. S. Swinehart, Anal. Chem., 38, 345 (1966). (59) A. E . Pierce, "Handbook of Silylation," Pierce Chemical Co., Rockford, 111., 1970. ^60) B. S. Finkle et al., / . Chromatogr. Sci., 10, 312 (1972). '61) N. C. Law et al., Clin. Chem. Acta, 32,221 (1971). ;62) C. C. Fulton, "Modern Microcrystal Test for Drugs," Wiley-Interscience, New York, N.Y., 1969. '63) C. L. Hider, J. Forensic Sci., 11 (4) (October 1971). '64) F . Fish and W. C. C. Wilson, J. Pharm. Pharmacol., 21, 1355 (1969). 65) R. Leute, E. Ullman, and A. Goldstein, J. Amer. Med. Ass., in press (1972). 66) R. Leute, E . Ullman, A. Goldstein, and L. A. Herzenberg, Nature, New Biol. 236, 93 (1972). 67) K. E. Rubenstein, R. S. Schneider, and E. Ullman, Biochem. Biophys. Res. Commun., 47, 846 (1972). 68) H. Felton, J. Chromatogr. Sci., 7, 13 (1969). 69) O. N. Hinsvark, W. Zazulak, and A. I. Cohen, ibid., 10, 379 (1972). BIBLIOGRAPHY

A comprehensive bibliography for drugs of abuse analysis is published in / . Chromatogr. Sci., 10, M a y 1972. The following additional references are important: Drug Abuse Screening Programs: Detection Procedures, Development Costs, Street-Sample Analyses, and Field Tests. K. K. Kaistha, J. Pharm. Sci., 61, 685 (1972). T. L. C. Techniques for Identification of Narcotics, Barbiturates and C.N.S. Stimulants in a Drug Abuse Screening Program. K. K. Kaistha and I. H. Jaffe, J. Pharm. Sci., 61, 679 (1972). Extraction Techniques for Narcotics, Barbiturates and C.N.S. Stimulants

in a Drug Abuse Screening Program. K. K. Kaistha and J. H. Jaffe, / . Chromatogr., 60, 83 (1971). An Improved Method for Rapid LargeScale Thin Layer Chromatographic Urine Screening for Drugs of Abuse. J. R. Broich et al., / . Chromatogr. 60, 95 (1971).

B r y a n S. Finkle is a forensic toxicologist at the Laboratory of Criminalistics, Department of District Attorney, Santa Clara County in San Jose, Calif. He was born and educated in England and spent a 10-year period in forensic science at Scotland Yard, specializing in toxicology. An 18-month leave of absence was spent in the U.S. in 1963-65, first as a research associate in toxicology at Cuyahoga County Coroner's Office and Western Reserve University School of Medicine, and then as a criminalist specializing in toxicology at the Santa Clara Laboratory. He joined the latter permanently in 1966 and began a lectureship in forensic toxicology at the University of California School of Criminology at Berkeley in 1971. Mr. Finkle is a consultant for the Jet Propulsion Laboratory in Pasadena and consultant director of toxicology to United Medical Laboratories in Portland, Ore. He is a Fellow of the American Academy of Forensic Sciences and a member of several state, national, and international organizations of criminalists and toxicologists. He also devotes time to drug abuse organizations in Santa Clara County. For the past 15 years, he has been closely associated •with problems of alcohol and drugs. His main professional interests are in the study of operations management in toxicology, instrumental, automated analytical methods, gc/ms as a tool in toxicology, and studies and experiments to provide a data base for interpretation of analytical toxicology results. He has made many contributions to the technical literature and is coauthor of a "Manual of Toxicology Methods."

ANALYTICAL CHEMISTRY, VOL. 44, NO. 9, AUGUST 1972 • 31 A

forensic toxicology of drug bryan - ACS Publications

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