Forensic toxicology of drug abuse. Status report - Analytical Chemistry

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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 regulations for analytical practice do not exist. State-of-the-art, problems, and anticipated future developments are treated HE NATIONAL PALAVER concernTing 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 narcotic addicts (6),this expenditure represents almost $1000 per addict. Government estimates indicate that there are at least 2000 rehabilitation programs nationwide. Some are officially sponsored; some are private agencies, and between them they adhere to almost every possible theory of treatment, operational management, and functional requirements. An astonishing 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 outnumber the addicts by a considerable 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 manifest problem t o a point where it will no longer be s dominating and political issue. Under the Drug Abuse Office and Treatment Act of 1972, the President’s SAODAP will be abolished effective June 30, 1973. 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 magnitude requiring millions of urine analyses per year), the task of investigating, controlling, curbing, and finally preventing drug abuse in the civilian population has reached into almost every part of the medical and scientific community 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 ramifications of drug abuse in the United States are legion, and as a drugoriented society, with a four-billiondollar retail trade in patent medicines and where allowing your natural metabolism to take life as it comes is passQ,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

ABUSE: A Status ReNort 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 hIethadone” 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. It 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 t o 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. It 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 scientiJic 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. It 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 Propoxy phene Amphetamine Methamphetamine Barbiturates Amobarbital Pentobarbital Secobarbit a1 Phenobarbit a1 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 (&IO), 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

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

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Report for Analytical Chemists

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drug metabolism and excretion rates, concentration ranges following acute and chronic therapcutic dosage, and those concentrations anticipated in the drug abuser. No realistic sensitivity limits can be set without regard to sample type and size. Current analytical methods and the need for uncomplicated patient management 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 bc detected in blood samples from living subjects by current routine procedures. This is generally true for the organic bases as a group. By use of urine samples of greater than 10 ml in volume and obtained less than 48 hr after ingestion, the following are reasonable sensitivity limits: 0.5 pg/ml of urine for morphine (total, Le., free drug plus bound, conjugated metabolite) and barbiturates. 2.0 pg/ml for cocainc and propoxyphene; amphetamine and methamphetamine. A thorough understanding of the human metabolism of these drugs (11-17, 28) is essential to meeting the above sensitivity limits and making a correct interpretation of the analytical results. Much 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. To be intensely concerned over detection limits for cocaine in urine, before fully understanding whether the drug is excreted in humans unchanged or as benzoyl ecgonine and ecgonine metabolites (64) is a typical cart-before-the-horse attitude which does much to damage the credibility of clinicians and drug abuse program personnel requesting laboratory service. Without a knowledge of naturally occurring interfering substances, a clear analytical appreciation of other therapeutic agents likely to be present, such as phenothiazines and antimalarial drugs, and a certain ability to recognize urine metabolic profiles for propoxyphene (It?), methadone (19), and cocaine (and nicotine), the analyst will be unable to serve the often bewildered but demanding physician. The quality of the analytical

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results required will vary for each laboratory operation and will be conditioned by the intended use of the results. For example, quantitative analysis and unequivocal qualitative specificity, often mandatory in forensic work where criminal guilt is at issue, are not necessary in surveillance and preemployment screening programs supporting private clinics and industrial companies. Similarly, programs monitoring methadone clinic pstients and analysis of samples required for emergency medical diagnosis of overdosed patients in the emergency room rarely require quantitative work. Each has its own peculiar requirement, and the ultimate choice of analytical method and equipment should be decided within this framework . Confirmation that 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 “yes” or “no” answer that is without any false positive results, whereas extraction and multiple identification techniques are essential in the more exacting forensic context. The use of gas chromatography-mass spectrometry is the best tool to provide a specific answer, but it would be folly to waste its power to screen thousands of samples per week when it may be anticipated that less than 10% of the samples is likely to yield a positive result. What is really needed is an inexpensive simple, mechanized or automated technique whereby those samples which are negative (as defined by sensitivity limits and drugs encompassed by the 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 continual demand for production, often within a 24-hr (‘turnaroundtime.” Cost efectiveness in the laboratory usually means getting the most from the least-the greatest output for the smallest cost-but what of quality? Is it really wise to insist on a gold-plated answer when the

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copper penny product is equally effective? The deluge of funds recently available in support of methadone and other programs has unfortunately tended to push such basic considerations into the shadows of a scientific world already noted for its aloofness from industrial management techniques. The fact is, with the advent of largescale drug abuse detection programs often requiring analyses in excess of half a million samples a year, such mundane 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-cxchange resin extraction of the urine at controlled pH (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 by oncolumn derivative chemistry, is the most common method employed for identification (29-34). The nonspecific nature of fluorometry and immunology, in addition to potential cross-reaction interferences in the latter, must be appreciated if an over-reliance on results is to be avoided. The advantages of these procedures are speed, sensitivity, and their ability to indicate negative samples. I n mechanized form both are capable of analyzing more than 60 samples/hr to a sensitivity limit below 0.5 pg/ml of urine for morphine. The free-radical antibody method, FRAT (65, 6 6 ) , has the advantage of detecting free and conjugated morphine directly, without the need for prior hydrolysis. Greater than 90% of morphine is excreted conjugated with glucuronic acid (11,35) and as the sulfate. Conversely, it is an expensive method at more than $1.00 per test and uses a unique antibody reagent available 22A

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 than five cents per test and require only common laboratory chemicals in addition spectrophotofluorometer. to a 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 the production of morphine fluorophores (36-38, 22), one using potassium ferroferricyanide to generate fluorescent pseudomorphine, and the other depending upon sulfuric acid oxidation. The products of the latter are not known precisely, but the resulting multiple fluorescence peaks seen in the total emission spectrum enhance the qualitative significance of the test. There are no reported commonly occurring interfering substances. The antibody reagent used in the FRAT test is not specific for morphine. It will react with any phenanthrene, notably codeine, and dextromethorphan which occurs in nonprescription Romilar cough syrup. Although not as sensitive to the 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 many as 13% false positive tests may be anticipated (39, 40) in high-volume routine screening. Ascorbic acid interferes, but this is simply overcome by adding a dichromate oxidation step to the test procedure. These limitations in no way diminish the usefulness of the technique as a reliable screening method for opiate narcotics, and with the promise of further reagents for phenethylamine derivatives, methadone, barbiturates and cocaine metabolites, this approach could become the screening method of choice, only requiring gas chromatography or gc/ms confirmation to complete the analysis. However, reliable antibody reagents suitable for routine use are currently not available for these drug groups.


Similarly, the addition of a hydrolysis module to the mechanized Autoanalyzer (Technicon Instruments Corp., Tarrytown, N.Y.) fluorescence device and channels to detect more drugs of abuse than just morphine would greatly increase the utility of the instrument and justify the capital cost to many more laboratories. Recent studies have shown that without hydrolysis, at least 20% and perhaps greater than 90% of routinely submitted urine samples containing morphine would be reported negative by thin-layer chromatography (40, 41, 42). The choice of hydrolysis method is important. The 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 but maintains the integrity of the drugs and the urine, permitting simple extraction and clean residues. A 2: 1 enzyme mixture of p-glucuronidase and aryl sulfatase at a concentration of 1000 Fishmann units/ml of urine a t pH 4-5, incub a t d for 12 hr at 45"C, will release better than 90% of the bound morphine (43). The need for hydrolysis is mandatory 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 to the organic chemist (21, 44, 47, 48). High-pressure liquid chromatography offers perhaps the newest rapidly developing method and adds separation and detection parameters in a continuous system (68, 69). It will also include many polar, highly water-soluble metabolites 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 pH value (44-48) and absorption of the drugs on a column of amberlite XAD-2 (21) nonionic-exchange resin followed by solvent elution. Cation exchange paper has been proposed but has found only limited use, probably because of its lack of adequate sensitivity (44,55).

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The XAD-2 resin technique has the advantages of avoiding emulsions which can plague liquid-liquid methods and extracting some of the polar drug metabolites. Preprepared columns are available commercially (Brinkman Instruments, Inc., “Drug Sheen,” Westbury, N.Y.), and they provide the added nicety of a phaseseparating interface at the end of the column which substantially avoids the problem of eluting unwanted absorbed water. It also overcomes the operational disadvantages of washing and purifying the resin before use and the necessity to assemble and pack the columns. The columns cannot be reused indefinitely, and tests must be made to determine their life span. The loss of activity can be precipitous. There are a variety of organic solvents and solvent mixtures used to elute the drugs from the column. Ethylene dichloride-ethyl acetate is advised for the commercially prepared columns, but ethyl acetate followed by methanol is most frequently used and is efficient. Its disadvantage is that 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

(W.

The efficiency of extraction of the drugs is dependent upon the pH of the urine as it passes through the resin column, and unfortunately, the optimal pH of 8.5-9.5 for morphine and other amphoteric bases is not ideal for phenethylamines such as amphetamine (pH 7-9) or the barbiturates (pH 5-7). Either an unhappy compromise pH must be

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selected, or appropriate pH adjusjustments 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 75580% recovery of the drugs, including the weakly acidic bar47). biturates (48, Similar pH considerations are implicit in solvent extraction procedures. Although there is seemingly an infinite list of possibilities to the imaginative chemist, a 4 : l mixture of chloroform: isopropanol is simple and highly efficient, particularly in extracting morphine. It 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 (88). The amphoteric nature of morphine demands critical pH control of the urine. Using approximately equal volumes of urine and solvent, the urine must be at pH 8.5 + 0.1 if a single extraction is to suffice. At these conditions 90-95Y0 of the morphine will he extracted (49, 49). Immediately beyond the pH tolerance of +0.1, the recovery will be drastically reduced, to 40-50% morphine extraction at pH 8.3 and pH 8.8. pH 8.5 is not ideal for extracting all common drugs of ahuse as it is for morphine, hut it is satisfactory for all of the bases aud, surprisingly, the weaMy acidic barbiturates to

Figures 1-3. Rolling devices used in the extraction of free drugs from urine. Efficient extraction is complete in 10 m i n without emulsion formation. The device shown a t left was designed and con. structed by D. M. Taylor, Laboratory of Criminalistics, San Jose, Calif.

sensitivity limits previously d i 5 cussed, when a 10-20-ml urine sample is used. The more acidic phenobarbital is extracted to only approximately 30% hut can nevertheless he easily detected at 1.0 pg/ml in the 20-ml urine sample. This extraction condition is of considerable importance because it permits a single-step procedure. The problem of emulsion formation between urine and solvent is a vexing one but 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 the free drugs is complete in 10 min without any emulsions. Solvent evaporation to concentrate solutions or generate residues for chromatography must he done under a stream of dry air or nitrogen and without heat if the integrity of labile free-hase drugs such as amphetamine and meperidine is to he 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, but rather in separation, and as a device for rapidly and certainly indicating those samples which are negative. It 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. It is of crucial importance that the quality of the answer is understood. Silica gel G or H as a 250-p layer on 20 X 20-em glass plates, either commercially or laboratory prepared, are universally used. De veloping solvent systems are legion, hut those of Davidow (50) (ethyl acetate-methanol-ammonium hydroxide; 85: 10: 5) and Cochin and Daly (51) (benzene, 50; dioxane, 40; ethanol, 5; ammonium hydroxide, 5) are the most common. Both systems provide excellent separation of the hasic drugs in less than 1 hr hut equally share the disadvantage that the barbiturates cannot be isolated from the bases on the same plate. This problem must of necessity he overcome if the analyst wishes to detect all of the drugs on one plate, particularly as the spray reagents used for the bases interfere with those for the barbiturates. If not, an aliquot of the residue may he applied to a separate plate and developed in a solvent system appropriate for the barbiturates, e.g., chloroform:acetone, 90: 10 (5.2). In the analytical scheme described below, the solvent system isolates the harhiturates at Rf values greater than 90. It does not separate the individual barbiturate drugs, but that is unneeessary inasmuch BS a discriminating confirmation analysis will be carried out in the event of a tlc positive. Locating spray systems are for the most part uniform amongst laboratories. Thry are adequate to detect the drugs in question, but great caution must be exercised in assigning sensitivity and specificity to the reactions. All of the spray


reagents are general and react with organic groups; for example, ninhydrin used to detect amphetamine reacts with all primary amines, including p-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 the procedure below, although more sensitive reagents than ninhydrin are now being used in a few laboratories. A lightly applied spray of 0.4% ninhydrin in pH 10 phosphate buffer followed by 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 to chlorine vapor followed by a 1.0% benzidine in chloroform spray will develop primary amines as green spots (54). These reactions are sensitive to approximately 5 pg of amphetamine extracted from urine and represent a sensitivity factor 4-8 times greater than the ninhydrin reaction viewed under long-wave ultraviolet light. Morphine detection can also be improved. By using 0.1% solution of potassium ferroferricyanide in pH 8.5, 0.1M phosphate buffer as spray reagent, and viewing the resulting bright yellow-green fluorescent spots under short-wave (250 nm) ultraviolet light, less than 1 pg of the drug can be seen. Recognition of tlc patterns associated with commonly occurring metabolites, such as those from methadone (sometimes appearing without the parent drug), chloroquin, a frequently encountered antimalarial drug, phenothiazines, and nicotine, is mandatory to a correct evaluation of the plate. Frequently prescribed therapeutic agents which may appear at the same Rf value as a drug of abuse can cause confusion. An example is oxymetazoline, a nasal decongestant, which is located close to morphine and, like the opiates, stains purple with iodoplatinate reagent and gives a positive silver reduction reaction. Like tlc, gas chromatography ie empirical by nature and cannot be classified with techniques such as mass spectrometry and infrared spectrophotometry which relate to molecular structure and are truly 26A

specific. The value of gc in this context is its sensitivity and ability to resolve drug components in mixtures of biological artifacts. Oncolumn derivative chemistry can add significantly to its potential as an identification tool; but, again, the precise quality of the analytical result must be appreciated before any clinical interpretation is made. During the past 12 years gas chromatography has become fundamental to most laboratories and is now well recognized as a method ideal for confirming thin-layer chromatography findings in drug abuse analysis. There is a wealth of published reference relative retention data (29, 32, 48, 56). The gc system of Finkle et al. (29) is particularly suited to resolving drug mixtures and utilizes two columns, 2 ft and 6 ft, respectively, packed with 2.5% SE 30 and operated isothermally at selected temperatures. O W 7 and QF-1 are liquid phases which are also well proven and provide adequate chromatography. The former is especially good for free-base morphine, one of the most difficult drugs to chromatograph satisfact orily . Experience in gc practice and analytical toxicology is required to evaluate gas chromatograms of urine residues. Previously mentioned naturally occurring urine extractives and spurious peaks given by dihexyl and butyl phthalate plasticizers solvent extracted from plastic laboratory equipment can be misleading and perplexing to an unsuspecting analyst. The gas chromatograph can be used as a tool for performing microorganic chemistry by reacting functional groups to form derivatives. Acylation of primary and secondary amines (amphetamine and methamphetamine) by use of acetic anhydride, halogenation of unsaturated barbiturates (secobarbital) 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 the case of propoxyphene, and silylation by use of a variety of reagents (58,’ 59) has wide app reciation.


In lieu of a single, specific identification technique, cumulative analytical 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 procedure to indicate which extracts are negative and provide the first suggestion of identity in the positive samples, and finally an independent parameter such as derivative gc to indicate probable identity, will satisfy most demands. However, the artisan’s talent and conscientious experience are still indispensable factors in chromatographic methods, and a frank acceptance of this fact will avoid dangerous and costly errors. Gas chromatography-mass spectrometry offers the best available answer to the need for unequivocal identification of drugs and their metabolites extracted from biological samples. It is direct; the mass spectrometer acts as a specific gc detector, is extremely sensitivecapable 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, SI),and data will undoubtedly continue to grow as the cost of instrumentation falls, and mass spectrometry finds wider acceptance 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 subject. 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 n-ork is undertaken. There are numerous published schemes which serve well for preliminary testing. The simplest, most reliable and efficient methods for processing large numbers of drugs involve microcrystalline tests (66, 63). The direct


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

tubes from the water bath and transfer the residues to 5-ml tubes by sequential washing with 2 X 1.0-ml aliquots of methanol-chloroform 1 : l . Use a vortex mixer to ensure solution of the residue. Evaporate the solvent to dryness as before and use the residue for thin-layer chromatography.

RECOMMENDED ANALYTICAL PROCEDURE 1. Preliminary Tests for Morphine

3. Thin-Layer Chromatography

(a) Immunological free-radical assay technique, or (b) Technicon Autoanalyzer fluorescence method after hydrolysis, or (c) Dal 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 pH 4.5-5.0 with 4 ml of sodium acetate buffer. Add 2 ml of enzyme mixture (20,000 FU as described) and incubate for approximately 1 2 hr at 55°C. (This hydrolysis step may be omitted if the preliminary trst for morphine LTas negative.) After enzymatic hydrolysis, adjust pH to S.5 + 0.1 with .joy0 W/V sodium hydroxide [This can be accomplished without using a p H meter. Two drops of mixed indicator, cresol red (pH 8.2) and thymal blue (pH 8.8) will cause a dark blue color at pH S.5.1and add 20 ml 4:1 chloroform-isopropanol solvent. Cap the 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 0.025111 phosphate buffer p H 7.0. Shake vigorously, allow phases to separate, and aspirate off top buffer layer to waste. Transfer half of the solvent phase to another glass tube and evaporate, without heat under a stream of air, to low bulk (approximately 50 p1) for gas chromatography analysis. The 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 evaporate the solvent to dryness under a stream of dry air. Remove the 28 A

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

Solvent Systems

I 70 15 8 0.5

Ethyl acetate Cyclohexane Methanol Water Ammonium hydroxide

2

Each solvent system must thoroughly mixed before use.

I1 80

..

15 3

.. be

Spray Reagents Mercuric sulfate: Mix 5 grams of mercuric oxide in 100 ml of water. Slonly add 20 ml of concentrated sulfuric to dissolve the oxide, then make to 230 ml with water. Xnhydrin: 0.4% W/V in pH 10 phosphate buffer. Sulfuric acid : 0.1N. Potassium iodoplatinate (a) 6 grams of potassium iodide in 100 ml of methanol (b) 3 ml 10% W/V platinum chloride in 100 ml of water Nix equal parts of (a) and (b) just prior to use. Procedure Allow solvent I to migrate approximately half way up the plate. Remove the plate and allow it to air dry. Place plate in solvent I1 and allow migration to the top of the plate. Air dry. Location of Drugs Mask off all but the top 1 in. of the plate. Spray exposed portion x i t h mercuric sulfate. Barbiturates will appear as white spots on a gray background. Remove the mask and spray the plate in the following sequence: (a) Sinhydrin followed by phenylacetaldehyde. Ampheta-

ANALYTICAL CHEMISTRY, VOL. 44,

NO. 9, AUGUST

1972

mine will appear as a gold spot enhanced under uv light. (b) Sulfuric acid: observe brilliant blue fluorescence of quinine under uv light. (c) Iodoplatinate. The 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 gc/nis analysis. 4. Gas Chromatography-Mass Spectrometry

According to Reference 29, aliquots analyzed on appropriate gc system-A, B, D, or E. A 5-ft X 1/8-in. i.d. glass column packed with 3Yc OV-17 on supelcoport and operated at 250°C is particularly suitable for the detection of submicrogram amounts of morphine free base. On-column derivativization as previously discussed can be implemented at this stage. Better, however, that the gc peaks of interest be transferred to the mass spectrometer for final identification. Almost all of the published reference data and drugs analysis applications have resulted from the Finnigan Model 3000-003 gc/ms peak identifier by use of an all-glass jet separator as an interface (60). The equipment is simple to use and provides a mass spectrum as the gc peak is eluted at its retention time. The foregoing procedure is easily capable of supporting a n-ork load of 100 samples per hr and has proved capable of operating with consistent efficiency up 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. Drug concentrations in urine samples taken randomly can rarely be interpreted t o yield any meaning with respect to behavior or clinical condition. The only sure interpretation is that the presence of the drug dem-

At $3150, the new HP 33738 resembles two or three other good quality, low cost GC integrators. The difference is performance: it i s . , .

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The only low-cost integrator with area threshold control as a standard feature. The 33738 eliminates false prints due to noise or unwanted small peaks.

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The 33738 includes a quiet built-in printer at the $3150 price. For a little more, you can get total area capability, recorder output with superimposed event marks, and a choice of four interchangeable CIRCLE 100 ON READER SERVICE CARD

output boards for automatic systems. For complete information, call your nearest HP sales office or write for literature. Hewlett-Packard, Route 41, Avondale, Pa. 19311. In Europe: P.O. Box 85, CH-1217 Meyrin 2, Geneva, Switzerland. In Japan: YHP, 1-59-1, Yoyogi, Shibuya-Ku, Tokyo, 151. 43206

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onstrates that subject did in fact ingest the material. The dearth of reliable information correlating dosage, time, and clinical condition with blood and urine drug and metabolite concentrations in humans attests that this is an area of almost total neglect. Whereas urine concentrations are often so extraordinarily high (especially for amphetamine and related phenethylamines which may reach concentrations up to 330 pg per ml) that it is obvious that the drug \vas not ingested under therapeutic conditions, it would be foolhardy to predict the behavior of the subject at a specified time with the urine concentration as the 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 that the drug was also in the circulatory system and consequently exerting some physiological effect on the subject during the period of time between voiding and sample. Quality control and proficiency testing by means of blind samples containing drugs at realistic sensitivity limits is mandatory. The scheme should be designed to test all aspects of the operation, from sample receipt and identification to the clerical preparation of the laboratory report. Required analytical performance must be defined and rigorously applied. False positive results cannot be tolerated. The ability to meet performance standards in greater than 93% of the samples should be routine. Any laboratory operation continuously processing in excess of 10,000 samples each week will inevitably make some mistakes. Even if the analytical method is ideal, mistakes will occur a t the data processing and clerical stage. Lessons can be Iearned, and advice should be solicited from large hospital clinical chemistry laboratories where similar problems have been faced and solved in recent years. The quality control program should begin with the urine sample. The addition of boric acid at 10 mg/ml of urine will preserve the sample from bacterial action and maintain the urine in a weakly acid condition, thus preventing degradation of the drugs. The program must include urine 30A

samples containing drugs at known concentrations approximating required sensitivity limits. The sample should be entered into the analytical system on a completely blind basis. Human urine containing excreted morphine should be included to test hydrolysis efficiency, and control drug mixtures must be applied 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. T H E 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 matter which must be decided by the physicians and administrators of treatment programs, district attorneys, probation officers, and legislators in consultation with analytical toxicologists. If purposeful guidelines were issued through the President’s Special Action office, it would be of inestimable help. There is an overwhelming need for trained toxicologists. There are only 145 members of the American Academy of Forensic Sciences toxicology section and but a handful of accredited training programs in the nation. Carefully conceived programs in which academic and vocational training is allied between schools staffed by experienced toxicologists and existing forensic toxicology laboratories, to assist analytical and clinical chemists enter this exacting but challenging field, are a first-order priority. Drug abuse analysis is no job for the unskilled technician who carries much of the bench-work burden today. Technical advances will undoubtedly 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 the day of direct testing. Analyzing more conveniently obtained samples such as saliva, fingerprick blood, and breath is in prospect, and the results may offer a better base for predicting the drug user’s clinical condition. Immunological techniques will


certainly advance in the near future, as experience is gained in operating laboratories. A highly innovative variation has already been reported (67) and reputedly is more sensitive than free-radical detection. It is the EMIT (Enzyme 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 that currently being developed at Jet Propulsion Laboratory under the SASA Space Technology Applications program, offers perhaps the most sophisticated approach to toxicology analysis ever conceived, but 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 the challenge of a difficult job, in the knowledge that the professional chemist can assist in the Sation’s major public health problem and help improve the quality of life for thousands of American citizens. ACKNOWLEDGMENT

The author thanks Loren Price and Dale Gray of United Medical Laboratories, Portland, Ore., for their technical assistance and practical suggestions which added substantially to this work. Similarly, the experimental Tvork performed by the staff of the Laboratory of Criminalistics, San Jose, Calif., is acknowledged and appreciated. REFERENCES (1) The White House Fact Sheet, Special Action Office for Drug Abuse, hlarch 21, 1972. (2) Medical World Sews, p 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 AIethadone, Federal Register, April 6, 1972, 37 F.R. 6940. ( 5 ) julius Axelrod et al., Ann. S.Y . Acad. Sci., 66 (3),435 (1957). (6) L. A. Dal Cortivo and J. R. Broich, Anal. Chem., 38, 1959 (1966).

(7) I. C. Nigam and J. L. Holmes, J . Pharm. Sei.,58, 506 (1969). (8) S. J. Mule, J. Chmmatog., 55, 255 (1971). ( 9 j H. Stone and H. M. Stevens, J . Forensic Sci. Soe., 9,31 (1969). (10) E. J. Woodhouse, Insurance Institute for Highway Safety, Washington, D.C., March 1972. (11) E. Leong Way and T. K. Adler, Bull. W . H.0.. 27,359 (1962). (12) J. Grove and P. A. Toseland, Clin. Chem. Aela. 29. 213 (1970). (13) M. S. Moss'and J. V.'Jackson, Ezcelpla Med. Found. Int. Congr. Ser., 80, 104 (19631. . . (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, J . B i d . Chem., 214, 753 (1958). (17) J. Axelrod, Fed. Proe:, 13,332 (1954). (18) R. E. McMahon e t al., Tozicol. Appl. Pharmacol., 19,427 (July 1971). (19) A. H. Beckett e t al., J . Pharm. Pharmaeol.. 20. 754 (19681.

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,

(21) N. Weissman e t al., Clin. Chcm., 17, 875 (1971). (22) L. Dal Cortivo, E. Kallet, and W. Matusiak, Proe. of Committee on Problems of Drug Dependency, Nat. Aead. Sci., Nat. Res. Comm. 6397, 1970. (23) A. E. Takemori, Bwehem. Pha-col., 17, 1627 (1968). (24) S. J. Mule and P. L. Hushin, Anal. Chem., 43,708 (1971). (25) G. Nadeau and G. Sobolenski, Can. J . Bwehem. Phvsiol.. . . 36. 625 (1958). (26) S. Spector, J . Pharmacol. i z p . Ther., 178, 253 (1971). (27) Medical World News, pp 15-16, July Ifi., lQ71. ~. (28) T . K. Adler et al., J . Pharmaeol. Ezp. Ther., 114,251 (1955). (29) B. S. Finkle e t al., J . Chromalogr. Sci., 9, 393 (1971). (30) N. C. Jain, Diss. Abstr., 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. Pismo, Anal. Biochem., 3,337 (1969). (34) M. J. Barrett, Clin. Chem. Newslett., 3. 1 f1971L (35) J. M. Fujimato and E. L. Way, J. Pharmacol. Ezp. Ther., 121.340 (1957). (36) H. Kupferberg e t al., ibid., 145, 247 (1964). (37) A. E. Takemori, Biochem. P h a m col.., 17., 1627 (1968). . , (38) D. J. Blackmore et %I., Clin. Chem., 17, 896 (1971). (39) B. ,Hamman, Proe. A m ? . Aead. Forenszc Set., unpublished (1972). (40) B. S. Finkle and L. Price, unpublished work, United Medical Lahore tones, Portland, Ore., 1972. (41) J. Wallace and J. T. Payte, Curr. Ther. Res., Clin. Ezp., 13, 412 (June (1971). (42) T., J. Butler, Southern Memorial Hospital, Las Vega, Nev., private communication. 1972. I~

(43) B. S. Finkle, unpublished work, I.ahoratory of Criminalistics, S m Jose, (hlif., 1971. (44) V. P. Dole e t al., J. A m r . Med. >Iss., 198,349 (1966). (45,L..~~~~-.~.. K. K. Kaistha and J. H. Jaffe, J . CrbT"mr"y.r.,

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ha

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(46) S. J. Mule. ibid.., 39.. 302 (1969) (47) B. Dwidow e t al., Amer J . Clin. Palhol., 50, 714 (1968). (48) S. J. Mule, Anal. Chem., 36, 1907

Report for Analytical Chemists in a Drug Abuse Screening Program. K. K. Kaistha and J. H. Jaffe, J. Chromalogr., 60, 83 (1971). An Improved Method for Rali d LargeScale Thin Layeyer Chromeitographie of Abuse. Urine T " D_^:^L Screening- 1 forT Drugs n e. U I U L G U t.b ai., 4 . wrumatogr., 60, 95 (1971). 11.

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(49) D. Reed, Orange County Cor0"el'S Laboratory, Santa Ana, Calif., 1)rivate communication, 1971. (50) B. Davidow 'et al., A m ? . J . Clin. Paliwl., 50, 714 (1968). (51) J. Cochin and J. W. Daly, Ez,perienl a . 18. 294 (19621. , , (52) I . Sunshine, Arner. J . Clin. 1'aalhol., 40, 576 (1963). (53) R. Bath, Cuyahoga County Coroner's Office Laboratory, Cleveland, Ohio, private communication, 1972. (54) R. Porter, Proc. Calif.Ass. Tozicol., unpublished ( M a y 1972). (65) P. Dole e t ;I., N.Y. State 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. Neursletl., 3, l(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, Ill.. 1970. (60) B. S. Finkle e t al., J . Chromatogr. Sci., 10,312 (1972). (61) N. C. LEWe t al., Clin. Chem. Acta, 32,221 (1971). (62) C. C. Fulton, "Mpdern 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 . Arner. Med. Ass., in press (1972). (66) R. Leute, E. Ullman, A. Goldstein, and L. A. Herzenberg, Nature, New Bwl., 236,93 (1972). (67) K. E. Ruhenstein, R. S. Schneider, and E. Ullman, Bwchem. Bwphys. Res. Commun., 47,846 (1972). (68) H. Felton, J . Chromalogr. Sei., 7, 13 (1969). (69) 0. N. Hinsvark, W. Zazulak, and A. I. Cohen, ibid., 10, 379 (1972).

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BIBLIOGRAPHY

The following additional references are important: Drug Ahuse Screening Programs: Detection Procedures, Development Costs, StreeMample Analyses, and Field Tests. K. K. Kaistha, J . PhaTm. 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. Xaistha and I. H. Jaffe, J . Phann. Sci., 61, 679 (1972). Extraction Techniques for Narcotics, Barbiturates and C.N.S. Stimulants

Bryan S. Finkle is a forensic toxicoloyist at the Laboratory of Criminali s t i c ~ ,Department of District Attorney, Santa Clara County in S u n 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 1963-66,first as a research associate in toxicology at Cuyahoga County Coroner's O&e 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 i s 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 &motes time to drug abuse organizations in Santa Clara County. For the past 16 years, he has been closely associated with problems of alcohol and drugs. H i s main professional interests are in the study of operations management in toxicology, instrumental, automated analytical methods, yc/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 i s coauthor of a "Manual of Toxicology Methods."

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

31 A

Forensic toxicology of drug abuse. Status report - Analytical Chemistry

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