ABSTRACT
Introduction
Experimental part
Findings
Discussion
Conclusion
Author: D. DEBRUYNE., F. ALBESSARD, M. C. BIGOT, M. MOULIN
Pages: 109 to 121
Creation Date: 1994/01/01
The development of chromatography technology, with the increasing availability of easier-to-use mass spectrometers combined with gas chromatography (GC), the use of diode-array or programmable variable-wavelength ultraviolet absorption detectors in conjunction with high-performance liquid chromatography (HPLC), and the availability of scanners capable of reading thin-layer chromatography (TLC) plates in the ultraviolet and visible regions, has made for easier, quicker and more positive identification of cannabis samples that standard analytical laboratories are occasionally required to undertake in the effort to combat drug addiction. At laboratories that do not possess the technique of GC combined with mass spectrometry, which provides an irrefutable identification, the following procedure involving HPLC or TLC techniques may be used: identification of the chromatographic peaks corresponding to each of the three main cannabis constituents - cannabidiol (CBD), Δ - 9 - tetrahydrocannabinol (Δ - 9 - THC) and cannabinol (CBN) - by comparison with published data in conjunction with a specific absorption spectrum for each of those constituents obtained between 200 and 300 nm. The collection of the fractions corresponding to the three major cannabinoids at the HPLC system outlet and the cross-checking of their identity in the GC process with flame ionization detection can further corroborate the identification and minimize possible errors due to interference.
Between 1968 and 1987, references appeared in the literature to numerous methods for identifying cannabis constituents through the use of simple techniques involving TLC on silica gel plates with visual detection by colour reaction [ [ 1] -6], as well as more elaborate techniques involving overpressured layer chromatography [ 7] and HPLC using normal or reversed phases and detection by absorption at different wavelengths [ [ 8] -13] or electrochemical means [ 14] , and also more complex techniques combining capillary or packed-column GC with mass spectrometry [ [ 15] - 19].
The subsequent appearance on the market of diode-array or programmable variable -wavelength ultraviolet absorption detectors that can produce the absorption spectrum of a detected component, and of scanners capable of reading TLC plates in the ultraviolet and visible regions, together with the increased availability of mass spectrometers combined with gas chromatography, which are simpler for non-specialists to use, means that analytical laboratories working in cooperation with the judicial authorities in drug addiction cases can now identify cannabis samples easily, quickly and positively.
Since the new technologies were available and the authors were in possession of 15 hashish samples from various sources with significant differences in the relative proportions of the three major cannabis constituents, that is, Δ-9-tetrahydrocannabinol (Δ-9-THC), cannabidiol (CBD) and cannabinol (CBN), it was considered useful to compare their chromatographic profiles and correlate the findings using three advanced and highly efficient chromatographic techniques (GC, HPLC and TLC) in order to determine the best strategy or strategies for reliable identification of cannabis samples, depending on the equipment resources of the individual laboratory.
The analyses were carried out on 15 samples of hashish taken from resins of different and unknown geographical origins. Various dried organs of cannabis plants cultivated under laboratory conditions from hemp seed were also studied.
The HPLC apparatus consists of a Rheodyne 7125 injector, a Merck 6000 A pump, an LDC SM 4000 programmable variable-wavelength ultraviolet detector and a Merck D 2000 integrator.
The GC apparatus consists of a Carlo Erba Fractovap 2900 chromatograph equipped with a Ros injector and a flame ionization detector, and a Varian Star 3400 chromatograph equipped with a programmable capillary septum injector (SPI injector) and coupled to a Varian Saturn II ion-trap mass spectrometer.
The TLC apparatus comprises a Camag II scanner connected to a Merck D 2500 integrator.
The extracts are obtained by ultrasound mixing (for 15 minutes) of each of the samples, in the ratio of 10 mg of substance to 1 ml of solvent in a mixture consisting of 90 per cent hexane and 10 per cent chloroform, which corresponds to the mobile phase used in the HPLC process. The extracts are ultracentrifuged for 15 minutes at 10,000 revolutions per minute.
Waters μ- Porasil column (15 cm x 4.6 mm); the mobile phase is made up of 90 per cent hexane and 10 per cent chloroform; the flow rate is 2 ml per minute and detection is effected at 220 nm. Three main peaks are identified, and the three fractions that correspond respectively to those three peaks are collected at the HPLC system outlet. The fractions are concentrated and reinjected into the HPLC equipment to check their purity. In order to determine the exact identity of each of the cannabis constituents, a full spectrum is produced during the chromatography in the ultraviolet and visible regions from 200 to 300 nm.
GC:One microlitre of supernatant is injected into a capillary column (25 m x 0.32 mm) coated with methyl silicone having a phase thickness of 0.25 μ. The oven temperature is 240° C and the carrier gas (N2) pressure 0.5 bar. Three main peaks are identified by means of flame ionization detection, and those peaks are matched to the three peaks obtained in the HPLC process through separate injection of each of the three fractions recovered from the HPLC system.
By coupling the same column to the mass spectrometer, a mass spectrum is obtained for each of the three main cannabis constituents, which can thus be positively identified.
TLC: Five microlitres of supernatant is spotted on a Merck 60 F 254 silica gel plate. Once the mobile phase - consisting of a mixture of hexane, chloroform and dioxane (by volume, respectively, 89, 8.75 and 2.25 per cent) - has migrated to a height of 11 cm, the plate is scanned at 220 nm. Each of the three peaks of the main peak area is identified by comparison with each of the three fractions that have been recovered from the HPLC process, identified in the GC and mass spectrometry process and separately deposited on the plate. Each of the spots is scanned from 200 to 300 nm in increments of 2 nm, with the absorption spectrum of the substance in question reconstituted in this way.
The chromatograms obtained from the GC, HPLC and TLC processes are grouped together in figure I. The retention times (t R) of the three main cannabis constituents - cannabidiol (peak marked 1), Δ-9-tetrahydrocannabinol (peak marked 2) and cannabinol (peak marked 3) - are 3.95, 4.6 and 5.15 minutes, respectively, for the GC process, and 4, 4.9 and 5.7 minutes, respectively, for the HPLC process. The retardation factor (R f)values (the distance from the deposit to the tip of the peak divided by the distance from the deposit to the solvent migration front) observed in the TLC process are 0.30 for CBD, 0.27 for Δ-9-THC, and 0.23 for CBN.
The mass spectra of the three cannabis constituents are characteristic (figure II): a mass peak at 314 and a base peak at 232 for CBD; a mass peak at 314 but a base peak at 300 for Δ-9-THC; and a low-intensity mass peak at 310 and a base peak at 296 for CBN.
The absorption spectra of those three constituents obtained during the HPLC process and from the scanning of the TLC plate are comparable (figures III and IV): intense absorption from 200 to 230 nm, minimum absorption between 250 and 260 nm, and a slight increase between 270 and 280 um for CBD; intense absorption from 200 to 230 nm, minimum absorption at around 250 nm, and a slightly more pronounced increase between 270 and 280 nm for A-9-THC; and less intense relative absorption between 200 and 230 nm, minimum absorption between 240 and 250 nm and maximum absorption at 280 nm for CBN.
The correlation between the areas located below, the curves formed by the chromatographic peaks obtained in the GC, HPLC and TLC processes (those areas being proportional to the absolute quantities of CBD, Δ-9-THC and CBN), and also the ratios of those areas (which are proportional to the relative percentage of the constituents CBD/Δ-9-THC and CBN/Δ-9-THC) were calculated for 10 hashish or cannabis resin samples containing substantial quantities of each of the three main constituents (see table). These correlations are excellent between HPLC and TLC, and less good, but nevertheless significant, between GC and TLC.
|
Consituents |
TLC/HPLC |
HPLC/GC |
GC/TLC |
|---|---|---|---|
|
Cannabidiol
|
r = 0.991
|
r = 0.949
|
r = 0.962
|
|
p < 0.01
|
p < 0.01
|
p < 0.01
|
|
|
Δ-9-tetrahydrocannabinol
|
r = 0.994
|
r = 0.839
|
r = 0.696
|
|
p < 0.01
|
p < 0.01
|
p < 0.02
|
|
|
Cannabinol
|
r = 0.858
|
r = 0.896
|
r= 0.730
|
|
p < 0.01
|
p < 0.01
|
p < 0.02
|
|
|
Cannabidiol
|
r = 0.992
|
r = 0.989
|
r = 0.915
|
|
Δ-9-tetrabydrocannabinol
|
p < 0.01
|
p < 0.01
|
p < 0.01
|
|
Cannabinol
|
r = 0.990
|
r = 0.926
|
r = 0.986
|
|
Δ-9-tetrahydrocannabinol
|
p < 0.01
|
p < 0.01
|
p < 0.01
|
|
Note. r correlation ratio.
|
|||
|
p probability.
|
|||
Mass spectrometry combined with GC allows positive identification of CBD, A-9-THC and CBN. The relative retention times (the t R of Δ-9-THC being considered equal to 1) of CBD and CBN - 0.86 and 1.12, respectively - are consistent with values cited in the literature: 0.87 and 1.11 [ 11] , 0.69 and 1.31 [ 17] , 0.69 and 1.03 [ 20] ; and 0.87 and 1.22 [ 21] . Identification by comparison with published data can thus be envisaged as a first step [ 12] . Also, the relative retention times observed in the HPLC process - 0.82 and 1.16 - are comparable with those obtained by Kanter and others [ 10] using a standard column: 0.79 and 1.28. The fairly characteristic absorption spectra of each of the major cannabis constituents enable the identification to be subsequently confirmed.
The use of the TLC technique, while requiring less sophisticated apparatus, is a more delicate operation. Separation is difficult, and the resolution, although sufficient for visualization by means of colour test reagents, is barely adequate for reading by a scanner. The phase -composition must be extremely precise. Any slight excess of chloroform moves the Δ-9-THC close to the CBD, whereas any slight excess of dioxane moves it close to the CBN. However, variable wavelength readings of the spots make it possible to reconstitute the characteristic absorption spectra of the three major cannabis constituents and thus to confirm the nature of each of the three cannabinoids.
The analyses carried out on the samples in the authors' possession made it possible to verify some of the data that should not be overlooked during cannabis sample identification and led to the following conclusions:
The psychotropic and behavioural effects of cannabis are essentially associated with Δ-9-THC [ [ 22] ];
It is the relative percentages of the three major constituents - Δ-9-THC, CBD and CBN - that distinguish fibre hemp from resin hemp [ [ 12] , [ 23] ];
CBD or CBN may be absent even from fresh samples [ [ 5] , [ 24] ]. The presence of the three main constituents is not constant, and only the presence of Δ-/9-THC in substantial quantities qualifies a sample of hemp as a drug;
Samples taken from one and the same block of cannabis resin display fairly comparable chromatographic profiles, indicating the fairly good homogeneity of that form of preparation;
Samples taken from different blocks of resin,even if originating from the same batch seized by the customs authorities, may produce very different profiles. It is thus not possible to determine the precise origin of a sample solely on the basis of the relative proportions of Δ-9-THC, CBD and CBN. Only a knowledge of the minor constituents will make it possible to ascertain the provenance of different samples of marijuana, hashish or other forms of cannabis [ [ 16] , [ 17] , [ 25] ];
-9-THCconcentration decreases over time in resin and hashish samples [ [ 26] , [ 27] ]. Accordingly, it will be difficult to distinguish between a fresh fibre hemp sample and an old resin hemp sample;
-9-THCconcentration is at its highest in the female inflorescences, less in the male inflorescences, lower in the leaves, and virtually nil in the stems and seeds [ [ 26] ].
There is no doubt that the combination of gas chromatography and mass spectrometry guarantees positive identification of cannabis samples. However, for laboratories lacking that technology, it would appear that the separation of the major cannabis constituents by means of TLC or, better still, HPLC in conjunction with the absorption spectra of those components, or else a combination of two chromatographic techniques (gas or liquid), can guarantee reliable analysis findings and a virtually irrefutable identification. In most cases the use of those chromatography techniques in combination make it possible to overcome the problem of errors due to interference, which are often unavoidable when a single, simple method with comparison of retention times is used.
H. Aramaki and others, "Forensic chemical study on Marihuana 1. A detection method of the principal constituents by thin-layer and gas chromatography", Chemical and Pharmacological Bulletin, vol. 16, No. 5 (1968), pp. 822-826.
002K. D. Parker and others, "Rapport préliminaire sur la separation et la determination quantitative par la chromatographic sur couches minces et la chromatographic en phase gazeuse des constituents cannabiniques présents dans la plante ou ajoutés A l'urine", Bulletin des stupéfiants, vol. 20, No. 4 (1968), pp. 7-12.
003R. A. de Zeeuw, Th. M. Malingre and F.W.H.M. Merkus, " Δ-1-Tetrahydro- cannabinolic acid, an important component in the evaluation of cannabis products", Journal of Pharmacy and Pharmacology, vol. 24, No. 1 (1972), pp. 1-6.
004M. R. Paris and R. R. Paris, "Importance de la chromatographic pour I'étude des constituents du cannabis sativa L", Bulletin de la Société chimique de France, vol. 1, 1973, pp. 118-122.
005C. E. Turner and K. Hadley, "Constituents of Cannabis sativa L. 11: Absence of cannabidiol in an African variant", Journal of Pharmaceutical Sciences, vol. 62, No. 2 (1973), pp. 251-255.
006S. N.Tewari and J. D. Sharma, "Separation and identification of cannabinoids from Cannabis indica L. by thin-layer chromatography', Pharmazie,vol. 34, No. 1 (1979), pp. 54-55.
007P. Oroszlan and others, "Separation, quantitation and isolation of cannabinoids from Cannabis sativa by overpressure layer chromatography", Journal of Chromatography, vol. 388, No. 1 (1987), pp. 217-224.
008P. B. Baker and others, 'Determination of the distribution of cannabinoids in cannabis resin using high-performance liquid chromatography", Journal of Analytical Toxicology, vol. 4, 1980, pp. 145-152.
009R. N. Smith and C. G. Vaughan, "High-pressure liquid chromatography of cannabis: quantitative analysis of acidic and neutral cannabinoids", Journal of Chromatography, vol. 129, 1976, pp. 347-354.
010S. L. Kanter, M. R. Musumeci and L. E. Hollister, "Quantitative determination of Δ-9-tetrahydrocannabinol and Δ-/9-tetrahydro- cannabinolic acid in marihuana by high-pressure liquid chromatography", Journal of Chromatography, vol. 171, 1979, pp. 504-508.
011D. J. Harvey, "Chemistry, metabolism and pharmacokinetics of the cannabinoids", in Marihuana in Science and Medicine, G. G. Nahas and others, eds. (New York, Raven Press, 1984), pp. 37-107.
012D. Debruyne and others, "Identification et différenciation des chanvres résineux et textile: utilisation conjuguée de HPLC et GLC haute resolution", Bulletin des stupéfiants, vol. 33, No. 2 (1981), pp. 45-54.
013I.Barni Comparini and F. Centini, "Packed-column chromatography, high-resolution gas chromatography and high-pressure liquid chromatography in comparison for the analysis of cannabis constitu ent", Forensic Science International, vol. 21, No. 2 (1983), pp. 129-137.
014I. Nakahara, "Studies on confirmation of cannabis use. I. Determination of the cannabinoid contents in marijuana cigarette, tar, and ash using high-performance liquid chromatography with electrochemical detection", Journal of Analytical Toxicology, vol. 9, No. 3 (1985), pp. 121-124.
015T. B. Vree and others, "Gas chromatography of cannabis constituents and their synthetic derivatives", Journal of Chromatography, vol. 74, 1972, pp. 209-224.
016M. Novotny and others, "Analysis of marijuana samples from different origins by high resolution gas-liquid chromatography for forensic application", Analytical Chemistry, vol. 48, No. 1 (1976), pp. 24-29.
017L. Stromberg, "Minor components of cannabis resin. VI. Mass spectrometric data and gas chromatographic retention times of components eluted after cannabinol", Journal of Chromatography, vol. 121, 1976, pp. 313-322.
018D. J. Harvey, "Comparison of fourteen substituted silyl derivatives for the characterization of alcohols, steroids and cannabinoids by combined gas-liquid chromatography and mass spectrometry", Journal of Chromatography, vol. 147, 1978, pp. 291-298.
019H. Grote and G. Spiteller, "Neue cannabinoide. II", Journal of Chromatography, vol. 154, 1978, pp. 13-23.
020M. Novotny and others, "Fractionation and capillary gas chromatographic-mass spectrometry characterization of the neutral components in marijuana and tobacco smoke condensates", Journal of Chromatography, vol. 238, 1982, pp. 141-150.
021T. B. Vree and others, "Identification in hashish of tetrahydro-cannabinol, cannabidiol and cannabinol analogues with a methyl side - chain", Journal of Pharmacy and Pharmacology, vol. 24, No. 1 (1972), pp. 7-12.
022G. G. Nahas, "Toxicology and Pharmacology", in Marihuana in Science and Medicine, G.G. Nahas and others, eds. (New York, Raven Press, 1984), pp. 109-246.
023J. Bruneton, Eléments de phytochimie et de pharmacognosie (Paris, Lavoisier - collection technique et documentation, 1987.
024P. S. Fetterman and others, "Mississippi-grown Cannabis sativa L.: Preliminary observation on chemical definition of phenotype and variations in tetra-hydrocannabinol content versus age, sex, and plant part", Journal of Pharmaceutical Sciences, vol. 61, No. 8 (1971), pp. 1246-1249.
025R. Brenneisen and M. A. Elsohly, "Chromatographic profiles of cannabis of different origins: Part I", Journal of Forensic Sciences, vol. 33, No. 6 (1988), pp. 1385-1404.
026J. W. Fairbairn, J. A. Liebmann and M. G. Ronan, "The stability of cannabis and its preparations in storage", Journal of Pharmacy and Pharmacology, vol. 28, 1976, pp. 1-7.
027D. J. Harvey, "Stability of cannabinoids in dried samples of cannabis dating from around 1896-1905", Journal of Ethnopharmacology, vol. 28, 1990, pp. 117-128.
