review of analytical methods for identification and quantification of cannabis products

8/13/2019 Review of Analytical Methods for Identification and Quantification of Cannabis Products

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REGULATOR Y TOXICO LOGY AND PHARMACOLOGY6,348-358 ( 1986)

Review of Analytical Methods for Identification andQuantification of Cannabis Products

L.VOLLNER'

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 con-cerning cannabis (hashish, marijuana) constituents have been reviewed with the aim of summa-rizing the status of analytical detection and quantitation. Detailed protocols of standard analyti-

cal methods are compared in order to recommend uniform methods for field and forensic sam-ples and also to provide guidance to less experienced analysts in countries where cannabis sativaoccurs (T. Maylon. In Big Deal: The Politics ofthe IIIicit Drug Business, the Cannabis Commod-itykfarket, pp. 63-107. Guernsey, London.).

Because of its importance, there has been an increasing number of investigations of the chemi-cal, botanical, pharmacological, clinical and sociological aspects of the marijuana (hashish)problem. Since analytical techniques have improved substantially during the last 10 years, manypapers have been published containing a variety of methods for detection and quantificationof cannabis constituents. Since the analytical situation is becoming increasingly confused andbecause many of the journals are unavailable in less developed countries, the aim of this paperis to give an overview of existing analytical techniques and to attempt to distinguish practicaland 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 ofthe drug are the cannabinoids, which can be regarded as monoterpenoids coupled

To whom correspondence should be addressed.

348

0273-2300186 3.00Cqyi&t 8 1986 by cademic PITS, nc.All tiBllts of cepmdlK tion in any form -cd.


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ANALYSIS OF CANNABIS PRODUCTS 349

with olivetol(5n-pentylresorcinol). Cannabinol(2,3) (CBN), A9-tetrahydrocannabi-no1 (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 R, = COOH, CBNARz=Cs,H,,CBV CBVA

C2s

&-THC

The cannabinoid acids are psychotropically inactive and unstable. Under the in-fluence of light and temperature, decarboxylation to neutral cannabinoids occursrapidly. A new class of cannabinoids, whichc n be regarded as derived from divarinol(5-n-propylresorcinol) rather than olivetol, was discovered in 1969 (5) and the seriesof compounds (6, 7) e.g., cannabivarin (CBV). Meanwhile, about 60 cannabinoidcompounds have been reported and the properties summarized in several review arti-cles and books (S- 13).

A9-THC was found to be the active principle of the drug. The nomenclature of theposition of double bound in the terpene ring is not uniform. Some authors prefer themonoterpene numbering (e.g., A-THC) based on pcymene, but them jority usethe dibenzopyran numbering, which is also used in this paper. In some texts, bothclassifications 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 theplant. In warm climates the samples are rich in A9-THC, in cool climates, such as inEurope, lower concentrations of A9-THC but higher levels of CBD can be found.


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350 VOLLNER, BIENIEK, AND KORTE

TABLE 1

PHYSICAL-CI~E~~ICALPR~PERTIESOF d9-THC, CBN,AND CBD

A9-THC CBN CBD

MWMelting pointSolubility

WaterEthanolHexaneChloroform

314 310Liquid 76-7X

Insoluble Insoluble+ ++ ++ +

31466-67C

Insoluble+++

The physical-chemical properties of the three main constituents are given in Ta-ble I.

ANALYTICAL ASPECTS OF CANNABINOID CONSTITUENTS

Cannabis can be easily identified by simple field tests. These are usually based oncolor reactions. Two such tests which have been in use for many years are the Beamtest (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 colordifferentiations between the cannabinoids. Effective and reliable detection is given bythis technique combined with separation techniques such as column or thin-layerchromatography (TLC) ( 15, 16); TLC separation of cannabinoids is difficult becauseof their similarities in chemical structure. Thus, TLC cannot be used for precise quan-titative 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. Animproved technique, using a two-dimensional chromatographic system ( 17), was re-cently introduced. which allows separation of all major cannabinoids.

After complete separauon and reaction with Fast Blue B, more exact quantitationmay 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 andvery low detection limits (picogram range) have been reported (19). Both stationaryphases (polar and reversed) and both elution techniques (isocratic and gradient) wereapplied. A recently developed chromatographic technique which employs microborecolumns was also tested for cannabinoid separations (3 1). These columns allowhigher 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) andthe use of capillary columns has been found to improve separations by an order ofmagnitude (5 1, 53, 54). Derivatization of the compounds, usually as trimethylsilyl(TMS) ethers (32,33), improves the separation. Cannabinolic acids must be deriva-


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tized for GLC analysis because the free acids decarboxylate in the heated injectionport of the instrument.

Furthermore, the formation of trimethylsilyl derivatives increases the maximumsensitivity of detection from approximately 50 ng to about 10 ng using flame ioniza-tion detection (FID). A few picograms ( lo- g) can be detected by analysis of chloro-acetyl or heptafluorobutyl derivatives using electron-capture detection (ECD). Ofcourse, GC/MS provides the most sensitive and specific assay system for cannabi-noids. Quantities at the femtogram ( lo-l5 g) level have been detected using TMSderivatives (49).

Spectroscopic techniques, such as nuclear magnetic resonance (NMR) and massspectroscopy (57), have facilitated an understanding of cannabinoid structure ratherthan 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 coextractedcompounds, such as lipids, many metabolites, and other cannabinoids which cross-react with the antibody. In many cases, clean-up is needed prior to the assay tech-nique, e.g., chromatography on Sephadex (59). Combination of HPLC and RIA im-proves the effectivity of this assay (60). Detection limits of 100 pg/ml have beenachieved using this technique. Because metabolism of A9-THC is rapid, metabolitedetection is important for forensic reasons. GC/MS is the method of choice for rapidqualitative and quantitative analysis. Even complex mixtures can be handled directlyusing capillary columns.

In the following sections, recommended methods for the identification and analysisof cannabis products are given in detail. To avoid confusion, three different systemsare 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, theenvironment, maturity, sex, part of the harvested plant, and the time which haselapsed between harvesting and chemical analysis, as well as the conditions of storageof the plant. On storage of samples of cannabis products, THC content graduallydecreases 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 canna-bis or cannabis resin.

The plant material is green or brown, often compressed into blocks or slabs. Theresin is yellow-brown, red-brown, or dark-brown, depending on its origin, oftenmixed with sand and compressed into blocks or slabs. Other samples are powdery.

Prior to extraction, samples should be homogenized by mechanical crushing orgrinding. One gram of the-sample is then sufficient for extraction with, e.g., 20 ml ofpetroleum ether, ethanol, or chloroform. After shaking or treatment in an ultrasonicbath followed by centrifugation, aliquots of the extract can be used either for furthertreatment (silylation, decarboxylation, etc.) or for direct analysis (TLC, HPLC, GC).


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PRESUMPTIVE TEST (FIELD TESTS) OF CANNABIS

1. East Blue B Salt Test (15)

Reagent. Solid Fast blue B salt (di-O-anisidinetetrazolium chloride). Dilute Fastblue 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 ofcannabis oil into the center of the paper; add two drops of petroleum ether allowingthem to penetrate to the lower filter paper; allow lower paper to dry; add a very smallamount 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 tech-nique such as column or thin-layer chromatography, even these plants can be elimi-nated. In a recent study (6 I) only cannabis gave a positive result out of 527 plantstested.

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 reagentA; shake with 1 ml of chloroform for 1 min; add 1 ml of reagent B; shake the testtube 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 anequal volume of reagent B; if a color develops within some minutes, shake the mix-ture 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 addi-tive which fluoresces at 254 nm (GF 254).


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Layer thickness. 0.25 mm. (Plates should be stored in dry conditions-over bluesilica 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 ofsamples 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 overcomeedge effect.)

Length of run. Optimum is 10 cm, because this figure allows easy calculation of Rrvalues (Method 1 below). However, if Rfvalues are not required, a simple approachis to allow the solvent to develop to the top of the TLC plate. In such circumstancesthe plates are arranged so that the maximum development does not exceed 10 cm(Method 2 below).

Method 1. For 20X 20-cm plates a line is drawn 11 cm from the spotting endwhich 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 withthe IO-cm sides vertical; by allowing the solvent to flow to the top of the plate 9 cmdevelopment 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 devel-opment 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 thespotting point. The spreading of the solution should be restricted as much as possi-ble, otherwise diffise spots will be produced during development. The ideal size forthe application area is no more than 2 mm in diameter. To achieve this it may benecessary to apply solutions in aliquots rather than by a single discharge of the spot-ting equipment. The aliquots may be dried by cold air between discharges. If hotair 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 belined with adsorbent paper to assist saturation. The lid should be tight fitting to mini-mize solvent loss through evaporation. The glass may be ground and/or a smear ofpetroleum jelly may be applied to the rim.

The developing solvent. If a mixture, it should be made as accurately as possible bycareful use of measuring cylinders. If the same solvent systems are used daily, it maybe convenient to obtain each component via an automatic dispenser. Mixing may bedone 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 berenewed 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/n-hexane/diethylamine, 70:25:5 v/v.


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2. Two-Dimensional Development (I 7)

First development.n-Heptane/dichloromethane/butan-2-one, 83:5: 12 v/v. Whenthe 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. Reversed Phase 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.Reference solutions. Use available methanolic solutions of THC, CBN, CBD in

concentrations 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 tolueneand shake for 1 hr. Filter and wash the residue a few times with a small amount ofthe same solvent. Transfer the filtrate into a 25-ml volumetric flask and make up thetotal volume to 25 ml using toluene.

Cannabis plant. Shake 1 O g of pulverized cannabis plant with 20 ml of toluene for1 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 to25 ml.

HIGH PERFORMANCE LIQUID CHROMATOGRAPHYOF CANNABIS (30)

1. Zsocratic Technique

Operating conditions.Column: 250mm 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: uvat 230 nm.

Sample preparation. Weigh 5- 10 mg cannabis oil, 50- 100 mg resin, or 150-200mg 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 Iiltra-tion, aliquots can be used for injection or for decarboxylation. For decarboxylation100 ~1 should be evaporated and kept for 5 min at 200-2 10C. After cooling, thematerial should be resolved 100 ~1 of the mobile phase.

Injection volume. 2-3~1.


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TABLE 2

HIGHPERFORMANCELIQUIDCHROMATOGRAPHYOFCANNABIS

RI values

Isocratic Gradient Microbore

CBD 6.0 1.8 19.0CBDA 1.1 9.1CBN 10.8 12.0 28.5THC 13.4 13.7 35.0THCA 16.1 15.6CBC 43.5

2. Gradient Technique

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

3. Microbore Column Technique (31)

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

Operating conditions.Column: 250mm 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 anal-ysis 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 silicone.Carrier gas:nitrogen at 45 ml/min.


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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: 25m 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 pro-

gramme: 1. Start at 50C; 2. Increase at 5C per minute to 170C after 1 minute; 3.Increase at 2C per minute to 250C; 4. End of programme.

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 concentra-tions of THC (or other cannabinoids). The first GC/MS method for quantitation ofTHC (in blood) was reported in 1973 (59). When MS is combined with the capillaryGLC technique (see previous section) high resolution and very low detection levelscan be achieved. Because of the relatively high cost of the instrumentation, only alimited number of laboratories are equipped with a GC/MS.

Using the electron-impact ionization (EI), the TMS derivative of THC gives thefollowing 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 successfullyapplied to the detection and measurement of a wide range of drugs including cannabi-noids. For simple and rapid tests in blood and urine, RIA is the most promisingmethod currently available; if used in combination with HPLC techniques, interfer-

ence reactions with the antibody can effectively be avoided.Since the production of antibodies takes several months and since animals such assheep and rabbits are needed, the technique may be limited in its availability. For thisreason no recommended procedures are described here. For interested investigators,references are cited above.


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2. Nuclear Magnetic Resonance and Mass Spectroscopy

Both techniques require extremely highly purified compounds, which limits therapid application for tests. The use of a GC/MS combination was described pre-viously.

3. Ultraviolet Spectrometry (64)

Conventional uv spectrometry has not been used for quantitative analysis of can-nabis constituents because of lack of specific features in the uv spectra of the structur-ally 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 derivativeuv spectrometry was used in a few cases for qualitative and quantitative analysis ofsome illicit drugs. For the measurement, a derivative spectrum transformer is needed.The method enables the analyst to detect THC in mixtures with CBN, more complexmixtures have not yet been tested. At this time, the method seems not to be suitablefor 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.

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review of analytical methods for identification and quantification of cannabis products

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