REGULATORY

TOXICOLOGY

AND

PHARMACOLOGY

6,348-358 ( 1986)

Review of Analytical Methods for Identification Quantification of Cannabis Products

L.VOLLNER'

and

Joint FAO/IAEA Division, Wagramerstrasse 5. A-1400 Vienna, Austria

AND
D.BIENIEK

ANDF.KORTE

Gesellschaft fir Strahien und Umweltforschung mbH Mtkchen, ingolstiidter Landstrasse I, 8042 Neuherberg, Federal Republic of Germany

Received June II. 1986

About 100 recently published original papers, reviews, books and other communications concerning cannabis (hashish, marijuana) constituents have been reviewed with the aim of summarizing the status of analytical detection and quantitation. Detailed protocols of standard analytical methods are compared in order to recommend uniform methods for field and forensic samples and also to provide guidance to less experienced analysts in countries where cannabis sativa occurs (T. Maylon. In Big Deal: The Politics ofthe IIIicit Drug Business, the Cannabis Commoditykfarket, pp. 63-107. Guernsey, London.). Because of its importance, there has been an increasing number of investigations of the chemical, botanical, pharmacological, clinical and sociological aspects of the marijuana (hashish) problem. Since analytical techniques have improved substantially during the last 10 years, many papers have been published containing a variety of methods for detection and quantification of cannabis constituents. Since the analytical situation is becoming increasingly confused and because many of the journals are unavailable in less developed countries, the aim of this paper is to give an overview of existing analytical techniques and to attempt to distinguish practical and effective methods from those which are complex or which provide questionable results.
0 1986 Academic Press, Inc.

CANNABINOID

CHEMISTRY

The chemical composition of the plant Cannabis sativa (hashish, marijuana, etc.) has been well studied. The compounds responsible for the pharmacological effects of the drug are the cannabinoids, which can be regarded as monoterpenoids coupled
To whom correspondence should be addressed. 348
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0273-2300186 $3.00 8 1986 byAcademic PITS,Inc.

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ANALYSIS

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349

with olivetol(5n-pentylresorcinol). Cannabinol(2,3) (CBN), A9-tetrahydrocannabino1 (A9-THC), and cannabidiol(4) (CBD) have been identified as the major neutral constituents. The neutral compounds may be accompanied by their respective acids (A), which have a carboxyl group at one of the free positions of the aromatic ring.

R, = H, R2 = Cs, H,,, CBN Rz=Cs,H,,CBV

R, = COOH, CBNA CBVA

C2s

&-THC

The cannabinoid acids are psychotropically inactive and unstable. Under the influence of light and temperature, decarboxylation to neutral cannabinoids occurs rapidly. A new class of cannabinoids, which can be regarded as derived from divarinol (5-n-propylresorcinol) rather than olivetol, was discovered in 1969 (5) and the series of compounds (6, 7) e.g., cannabivarin (CBV). Meanwhile, about 60 cannabinoid compounds have been reported and the properties summarized in several review articles and books (S- 13). A9-THC was found to be the active principle of the drug. The nomenclature of the position of double bound in the terpene ring is not uniform. Some authors prefer the monoterpene numbering (e.g., A-THC) based on pcymene, but the majority use the dibenzopyran numbering, which is also used in this paper. In some texts, both classifications may be used indiscriminately. The third main component of Cannabis sutiva is cannabidiol (CBD).

CBD

The relative ratio of cannabinoids varies greatly, depending on the source of the plant. In warm climates the samples are rich in A9-THC, in cool climates, such as in Europe, lower concentrations of A9-THC but higher levels of CBD can be found.

350

VOLLNER, BIENIEK, AND KORTE TABLE 1

PHYSICAL-CI~E~~ICALPR~PERTIESOF

d9-THC, CBN, AND CBD CBN 310 76-7X Insoluble + + + CBD 314 66-67C Insoluble + + +

A9-THC MW Melting point Solubility Water Ethanol Hexane Chloroform 314 Liquid Insoluble + + +

The physical-chemical ble I.

properties of the three main constituents are given in Ta-

ANALYTICAL

ASPECTS

OF CANNABINOID

CONSTITUENTS

Cannabis can be easily identified by simple field tests. These are usually based on color reactions. Two such tests which have been in use for many years are the Beam test (13) and the Duquenois-Negm test (14). Probably the most specific color test involves reaction of the cannabinoid with an alkaline solution of Fast blue salt B, Merck (15). This method provides high sensitivity (50-ng level) and gives color differentiations between the cannabinoids. Effective and reliable detection is given by this technique combined with separation techniques such as column or thin-layer chromatography (TLC) ( 15, 16); TLC separation of cannabinoids is difficult because of their similarities in chemical structure. Thus, TLC cannot be used for precise quantitative analysis and its use for comparison of samples is also limited. It is, however, valuable as an aid to establishing the geographical origin of cannabis products. An improved technique, using a two-dimensional chromatographic system ( 17), was recently introduced. which allows separation of all major cannabinoids. After complete separauon and reaction with Fast Blue B, more exact quantitation may be achieved by photodensitometric scanning or by spectrophotometry. High-performance liquid chromatography (HPLC) has recently been successfully applied to cannabinoid analysis ( 18-3 1). Most cannabinoids have been separated and very low detection limits (picogram range) have been reported (19). Both stationary phases (polar and reversed) and both elution techniques (isocratic and gradient) were applied. A recently developed chromatographic technique which employs microbore columns was also tested for cannabinoid separations (3 1). These columns allow higher separation effectivity at a high level of sensitivity. Gas-liquid chromatography (GLC or GC) and combined GLC/mass spectrometry (GC/MS) are the most specific and sensitive separation techniques for cannabinoids. A large variety of stationary phases provide excellent separations (32-56, 68) and the use of capillary columns has been found to improve separations by an order of magnitude (5 1, 53, 54). Derivatization of the compounds, usually as trimethylsilyl (TMS) ethers (32,33), improves the separation. Cannabinolic acids must be deriva-

ANALYSIS

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351

tized for GLC analysis because the free acids decarboxylate in the heated injection port of the instrument. Furthermore, the formation of trimethylsilyl derivatives increases the maximum sensitivity of detection from approximately 50 ng to about 10 ng using flame ionization detection (FID). A few picograms ( lo- g) can be detected by analysis of chloroacetyl or heptafluorobutyl derivatives using electron-capture detection (ECD). Of course, GC/MS provides the most sensitive and specific assay system for cannabinoids. Quantities at the femtogram ( lo-l5 g) level have been detected using TMS derivatives (49). Spectroscopic techniques, such as nuclear magnetic resonance (NMR) and mass spectroscopy (57), have facilitated an understanding of cannabinoid structure rather than providing methods of analysis for cannabis products, For quantification of A9-THC in body fluids only radioimmunoassay (57, 58) (RIA) offers high sensitivity and selectivity (besides GC/MS and GLC). However, sensitivity and selectivity is considerably reduced by the interference of coextracted compounds, such as lipids, many metabolites, and other cannabinoids which crossreact with the antibody. In many cases, clean-up is needed prior to the assay technique, e.g., chromatography on Sephadex (59). Combination of HPLC and RIA improves the effectivity of this assay (60). Detection limits of 100 pg/ml have been achieved using this technique. Because metabolism of A9-THC is rapid, metabolite detection is important for forensic reasons. GC/MS is the method of choice for rapid qualitative and quantitative analysis. Even complex mixtures can be handled directly using capillary columns. In the following sections, recommended methods for the identification and analysis of cannabis products are given in detail. To avoid confusion, three different systems are described. The validity of these methods is well established. CANNABIS PRODUCTS AND SAMPLING

The hemp plant, Cannabis sativa L., can be distinguished as fiber, intermediate, and drug types (66). The fiber type is characterized by a high CBD to THC ratio (>5) and the drug type by a much lower ratio (~0.2). The wide variations in the relative amounts of cannabinoids depend on the genetic characteristics of the seedstock, the environment, maturity, sex, part of the harvested plant, and the time which has elapsed between harvesting and chemical analysis, as well as the conditions of storage of the plant. On storage of samples of cannabis products, THC content gradually decreases as a result of oxidation to CBN. Similarly, tetrahydrocannabivarin (THV) gives rise to CBV (67). The two products CBN and CBV are absent from fresh cannabis or cannabis resin. The plant material is green or brown, often compressed into blocks or slabs. The resin is yellow-brown, red-brown, or dark-brown, depending on its origin, often mixed with sand and compressed into blocks or slabs. Other samples are powdery. Prior to extraction, samples should be homogenized by mechanical crushing or grinding. One gram of the-sample is then sufficient for extraction with, e.g., 20 ml of petroleum ether, ethanol, or chloroform. After shaking or treatment in an ultrasonic bath followed by centrifugation, aliquots of the extract can be used either for further treatment (silylation, decarboxylation, etc.) or for direct analysis (TLC, HPLC, GC).

352 PRESUMPTIVE

VOLLNER,

BIENIEK,

AND

KORTE

TEST

(FIELD

TESTS)

OF CANNABIS

1. East Blue B Salt Test (15) Reagent. Solid Fast blue B salt (di-O-anisidinetetrazolium chloride). Dilute Fast blue B salt with anhydrous sodium sulfate ( 1: 100). Solvent. Petroleum ether; 60/8OC boiling range is suitable. Procedure. Fold two filter papers into quarters and open partly to form a funnel; place a small amount of pulverized cannabis plant or resin or a very small drop of cannabis oil into the center of the paper; add two drops of petroleum ether allowing them to penetrate to the lower filter paper; allow lower paper to dry; add a very small amount of reagent to the lower paper; add two drops of water. Results. A mixture of the following colors results: THC = scarlet; CBD = orange; CBN = violet. Remarks. A few other plants such as nutmeg (Myristica fragrants Houtt) also give positive results, but by combining the simple color reaction with a separation technique such as column or thin-layer chromatography, even these plants can be eliminated. In a recent study (6 I) only cannabis gave a positive result out of 527 plants tested. 2. The modified Fast Blue B Salt Test Reagents. A. Solid Fast blue B salt, as above; B. 0.1 NNaOH. Solvent. Chloroform. Procedure. Place a small amount of pulverized cannabis plant or resin material or a very small drop of cannabis oil in a test tube; add a very small amount of reagent A; shake with 1 ml of chloroform for 1 min; add 1 ml of reagent B; shake the test tube for 2 min; allow the test tube to stand for 2 min. Results. Violet color indicates a positive result. 3. Rapid Duqu&ois Test (63)

Reagents. A. Five drops of acetaldehyde and 0.4 g of vanillin in 20 ml of ethanol; B. concentrated hydrochloric acid. Solvent. Chloroform. Procedure. Place a small amount of pulverized cannabis plant or resin material or a very small drop of cannabis oil in a test tube, shake with 2 ml of reagent A; add an equal volume of reagent B; if a color develops within some minutes, shake the mixture with l-2 ml of chloroform. Results. Violet color indicates a positive result. THIN LAYER CHROMATOGRAPHY OF CANNABIS

1. Standard Technique Coating. Activated silica gel G on glass backed plates; the coating contains an additive which fluoresces at 254 nm (GF 254).

ANALYSIS

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PRODUCTS

353

Layer thickness. 0.25 mm. (Plates should be stored in dry conditions-over blue silica gel inside a desiccator. The plates should be protected from chemical vapors. Plates should be activated before use at 110C for a minimum of 30 min.) Size of plate. 20 X 20 cm; 20 X 10 cm; 10 X 5 cm; choice depends on number of samples to be simultaneously developed. Starting point of run = spotting line. 1 cm from bottom of plate. Depth of developing solvent in TLC tank. Not more than 0.5 cm, not less than 0.3 cm. Distance between applications (spotting points). Usually 1 cm, never less than 0.8 cm. (Spots must be positioned at least 1.5 cm from edge of plate to overcome edge effect.) Length of run. Optimum is 10 cm, because this figure allows easy calculation of Rr values (Method 1 below). However, if Rfvalues are not required, a simple approach is to allow the solvent to develop to the top of the TLC plate. In such circumstances the plates are arranged so that the maximum development does not exceed 10 cm (Method 2 below). Method 1. For 20 X 20-cm plates a line is drawn 11 cm from the spotting end which gives 10 cm development for spots applied 1 cm from the bottom. Method 2. Plates of 20 X 10 cm and 10 X 5 cm are placed in the TLC tank with the IO-cm sides vertical; by allowing the solvent to flow to the top of the plate 9 cm development is produced. It is important that the analyst monitor the progress of solvent in both methods; plates must be removed from the TLC tank as soon as the solvent reaches the development line or the top of the TLC plate. Otherwise diffuse spots will result. Size of spot. The solution being applied to the plate spreads outward from the spotting point. The spreading of the solution should be restricted as much as possible, otherwise diffise spots will be produced during development. The ideal size for the application area is no more than 2 mm in diameter. To achieve this it may be necessary to apply solutions in aliquots rather than by a single discharge of the spotting equipment. The aliquots may be dried by cold air between discharges. If hot air is used care must be taken to ensure that no component of the mixture under investigation is thermally labile. The TLC tank and lid. Preferably both should be of clear glass; the tank should be lined with adsorbent paper to assist saturation. The lid should be tight fitting to minimize solvent loss through evaporation. The glass may be ground and/or a smear of petroleum jelly may be applied to the rim. The developing solvent. If a mixture, it should be made as accurately as possible by careful use of measuring cylinders. If the same solvent systems are used daily, it may be convenient to obtain each component via an automatic dispenser. Mixing may be done within the TLC tank. The developing solvent, mixture or single component, should be placed within the TLC tank in sufficient time to allow saturation to be achieved. With paper-lined tanks this should take approximately 5 min. It is important to note that for certain developing systems the solvent must be renewed after each development, or at least after 2 to 3 runs. Developing solvents: A. benzene; B. benzene/n-hexane, 60:40 v/v; C. benzene/nhexane/diethylamine, 70:25:5 v/v.

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VOLLNER,

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AND

KORTE

2. Two-Dimensional Development (I 7) First development. n-Heptane/dichloromethane/butan-2-one, 83:5: 12 v/v. When the solvent front reaches 12 cm, the plates have to be dried at room temperature, rotated 90, and developed using n-hexanelacetone, 86: 14 v/v. Color spray reagent. Dissolve 45 mg of Fast blue B salt in 20 ml of 0.1 N NaOH. (Important. This reagent should always be freshly prepared.) 3. ReversedPhase Technique (62) (RP) Coating. RP- 18, HPTLC-plates, Merck 15037. Solvent system.Acetonitrile/water 90: 10 v/v. Reagent. 0.5 g Fast blue salt B dissolved in 10 ml distilled water and then 90 ml
acetone added. solutions of THC, CBN, CBD in of 0.5 mg/ml. Cannabis oil. Dissolve 0.025 g of hashish oil in 25 ml of toluene. Cannabis resin. Triturate 0.25 g of cannabis resin in a mortar with a small amount of toluene to a paste. Transfer into an Erlenmeyer flask using about 20 ml of toluene and shake for 1 hr. Filter and wash the residue a few times with a small amount of the same solvent. Transfer the filtrate into a 25-ml volumetric flask and make up the total volume to 25 ml using toluene. Cannabis plant. Shake 1.O g of pulverized cannabis plant with 20 ml of toluene for 1 hr. Filter and wash the residue a few times using a small amount of the same solvent. Transfer the filtrate into a 25-ml volumetric flask and make up the total volume to 25 ml. concentrations HIGH PERFORMANCE LIQUID OF CANNABIS CHROMATOGRAPHY (30)

Reference solutions. Use available methanolic

1. Zsocratic Technique Operating conditions. Column: 250 mm X 4.6 mm i.d. Packing material: Spherisorb ODS 5- or 1O-pm diameter. Mobilephase: methanol/water/acetic acid, 75:23.7: 1.3 v/v. Flow rate: 1.8 ml/min. Detection: uv at 230 nm. Sample preparation. Weigh 5- 10 mg cannabis oil, 50- 100 mg resin, or 150-200
mg cannabis plant into a small vial, add 1 ml of methanol/chloroform 9: 1 and 0.8% w/v dioctylphthalate as internal standard. Close the vial tightly and extract for 15 min, using an ultrasonic bath. After centrifugation at 3500 cpm for 5 min and Iiltration, aliquots can be used for injection or for decarboxylation. For decarboxylation 100 ~1 should be evaporated and kept for 5 min at 200-2 10C. After cooling, the material should be resolved 100 ~1 of the mobile phase. Injection volume. 2-3 ~1.

ANALYSIS

OF CANNABIS TABLE 2

PRODUCTS

355

HIGHPERFORMANCELIQUIDCHROMATOGRAPHYOFCANNABIS RI values Isocratic CBD CBDA CBN THC THCA CBC 6.0
1.1

Gradient 1.8 9.1 12.0 13.7 15.6

Microbore 19.0 28.5 35.0 43.5

10.8 13.4 16.1

2. Gradient Technique
development 1. 80% methanol/20% 0.02 Nsulfuric acid; 2.20-min linear gradient; 3. Final composition, 90% methanol/ 10% 0.02 N sulfuric acid. Flow rate: 1.5 ml/min.

Column. As above. Mobile phase. At start of chromatographic

3. Microbore Column Technique (31)

For the microbore HPLC technique a special low-volume detector cell and a special HPLC pump with an extremely low flow rate are needed. The great advantages of this technique are higher separation efficiency and higher sensitivity by using very low volumes of the mobile phase (0. 1- 1 ml) per analysis.

Operating conditions. Column: 250 mm X 1.35 mm. Packing material: CP tm spher C 18. Mobile phase: acetonitrile/H,O 80:20. Flow rate: 0.06 ml/min. Injection volume: 1 ~1. Detector cell volume: 1 ~1. Results.The results are shown in Table 2. Alternative HPLC methods for the analysis of cannabis are given in Refs. (2822). GAS-LIQUID CHROMATOGRAPHY OF CANNABIS

I. Packed Column Technique Operating conditions. Detector: FID (hydrogen 30 ml/mm, air 450 ml/min). Column: length 6 R (or 2 m), i.d. 2 to 4 mm. Packing: 3% OV- 1; OV- 17, i.e., methyl silicone or methylphenyl Carrier gas: nitrogen at 45 ml/min.

silicone.

356

VOLLNER,

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AND KORTE

Operating temperatures: injector temperature 275C; oven temperature 250C;

detector temperature 275C.

Derivatizing agents:BSA, MSTFA. 2. Capillary Column Technique Operating conditions. Detector: HD. Column: OV- 1 crosslinked capillary. Film thickness:0.20 pm. Length: 25 m X 0.32 mm i.d. Carrier gas: nitrogen. Injection technique: split mode (ratio l/60). Flow rate: circa 110 cm/set measured at oven temperature of 150C. Make-up gas: nitrogen at 18 ml/min. Operating temperatures: injector 250C; detector 280C. Temperature
gramme: 1. Start at 50C; 2. Increase at 5C per minute to 170C after 1 minute; Increase at 2C per minute to 250C; 4. End of programme.

pro3.

3. Mass SpectrometricDetection (CC/MS) (63, 64,65)

GC/MS analysis using multiple ion and particularly selected-ion monitoring (SIM) technique, is currently the most reliable method for measuring very low concentrations of THC (or other cannabinoids). The first GC/MS method for quantitation of THC (in blood) was reported in 1973 (59). When MS is combined with the capillary GLC technique (see previous section) high resolution and very low detection levels can be achieved. Because of the relatively high cost of the instrumentation, only a limited number of laboratories are equipped with a GC/MS. Using the electron-impact ionization (EI), the TMS derivative of THC gives the following main ions: M+ = 386 (60%), 371(80%), 343 (20%), 315 (45%), 303 (50%), 73 (TMS, 100%). For sample preparation and chromatographic conditions see GLC techniques. OTHER TECHNIQUES

1. Radioimmunoassay of Cannabinoids (57, 58)

RIA is a sensitive and specific analytical procedure which has been successfully applied to the detection and measurement of a wide range of drugs including cannabinoids. For simple and rapid tests in blood and urine, RIA is the most promising method currently available; if used in combination with HPLC techniques, interference reactions with the antibody can effectively be avoided. Since the production of antibodies takes several months and since animals such as sheep and rabbits are needed, the technique may be limited in its availability. For this reason no recommended procedures are described here. For interested investigators, references are cited above.

ANALYSIS

OF

CANNABIS

PRODUCTS

357

2. Nuclear Magnetic Resonance and Mass Spectroscopy Both techniques require extremely highly purified compounds, which limits the rapid application for tests. The use of a GC/MS combination was described previously. 3. Ultraviolet Spectrometry (64)

Conventional uv spectrometry has not been used for quantitative analysis of cannabis constituents because of lack of specific features in the uv spectra of the structurally related cannabinoids. It has been recently shown that the value of uv and visible spectrometric data can be enhanced by transformation to the second or higher derivative. Second derivative uv spectrometry was used in a few cases for qualitative and quantitative analysis of some illicit drugs. For the measurement, a derivative spectrum transformer is needed. The method enables the analyst to detect THC in mixtures with CBN, more complex mixtures have not yet been tested. At this time, the method seems not to be suitable for rapid testing of cannabinoids. ACKNOWLEDGMENT
The authors wish to acknowledge Dr. K. Szendrci, UN Narcotics Laboratory, Vienna, for his kind sup port in literature, and Dr. J. R. Plimmer, Joint FAO/IAEA Division, for critical comments.

REFERENCES
T. (1985). In Big Deal: The Politics of the Illicit Drug Business, the Cannabis Commodity Market, pp. 63-107. Guernsey, London. 2. WOOD, T. B., SPIVEY, W. T. N., AND EASTER~IELD, T. M. (1899). J. Chem. Sot. 75,20. 3. CAHN,R.S. (1930-1933).J. Chem. Sot. 986(1930),630(1931), 1342(1932), 1400(1933). 4. ADAMS, R., PEASE, D. C., AND CLARK, J. M. (1940). J. Amer. Chem. Sot. 62,2 194. 5. VOLLNER, L., BIENIEK, K. D., AND KORTE, F. (1969). Tetrahedron Z&t. 3,145- 147. 6. GILL, E. W., PA~ON, G. W. D. M., AND PERTWEE, R. G. (1971). J. Chem. Sot. Ser. C 1971,579. 7. MERKUS, F. W. H. M. (1971). Pharm. Weekblad 106,69. 8. MECHOULAM, R. (1970). Science U&1159. 9. KORTE, F., AND BIENIEK, D. (1968). Mater. Med. Nordmark XX/l 1,607-6 12. 10. NAHAS, G. G. (1976). Marihuana: Chemistry, Biochemistry, and Cellular Effects. Springer-Verlag, New York/Heidelberg/Berlin. 11. MECHOULAM, R., AND GAONI, Y. (1967). Recent advances in the chemistry of hashish. Fortschr. Chem. Org. Naturstofle 25,175-2 13. 12. BAKER, P. B., ANDFOWLER, R. (December 1978). Proc. Anal. Div. Chem. Sot.. pp. 347-349. 13. STEININGEN, M. (1970). Pharm. Zeitung50,1939-1943. 14. DE FAUBERT MAUNDER, M. J. (1974). Bull. Narc. 26(4), 19-26. 15. KORTE, F., AND SIEPER, H. (1964). J. Chromatogr. 13,90. 16. PARKER, J. M., AND FISKE, H. L. (1972). J. Assoc. Og Anal. Chem. 55,876-879. 17. FOWLER, R., GILHOOLEY, R. A., AND BAKER, P. B. (1979). J. Chromatogr. 171,509. 18. WHEALS, B. B., AND SMITH, R. N. (1975). J. Chromatogr. 105,396-400. S. R., ABU-SHUMAYS, A., LOEFFLER, K. O., AND FORREST, I. S. (1975). Res. Commun. Chem. 19. Aeon, Pathol. Pharmacol. 10,9. 20. SMITH, R. N. (1975). J. Chromatogr. 115, 101-106. 21. SMITH, R. N., AND VAUGHAN, C. G. (1976). J. Chromatogr. 129,347-354. 1. MALYON,

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43.
44. 45. 46. 47. 48. 49. 50. 5 1. 52. 53. 54. 55. 56.

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