Identification and differentiation of resinous cannabisand textile cannabis:combined use of HPLC and high-resolution GLC

Sections

ABSTRACT
Introduction
Experimental part
Method Chromatographic analyses
Results and comments
Conclusion

Details

Author: D. DEBRUYNE, M. MOULIN, M.-C. BIGOT, R. CAMSONNE
Pages: 49 to 58
Creation Date: 1981/01/01

Identification and differentiation of resinous cannabisand textile cannabis:combined use of HPLC and high-resolution GLC

D. DEBRUYNE
M. MOULIN
M.-C. BIGOT
R. CAMSONNE
Pharmacology Laboratory, Caen Regional and University Hospital Centre, Caen, France

ABSTRACT

The combined use of high-pressure liquid chromatography (HPLC) and high-resolution gas-liquid chromatography (GLC) afforded a means of isolating three substances in samples of cannabis, of determining their retention times in HPLC and in GLC with and without derivation, and of identifying them, by comparison with the data in the literature, with the three major constituents of cannabis: cannabidiol (CBD), Δ-9-etrahydrocannabinol (Δ-9-THC) and cannabinol (CBN), without using either control substances or a mass spectrometer. Furthermore, calculation of the peak area ratios for the different sample varieties can serve as a criterion for the differentiation of resinous cannabis and textile cannabis.

Introduction

Numerous methods have been described for identifying the constituents of cannabis from the plant or from biological liquids: thin-layer chromatography [ 1] - [ 7] , gas-liquid chromatography (GLC) with or without derivation [ 1] , [ 7] - [ 16] , high-pressure liquid c41hromatography (HPLC) [ 3] , [ 18] - [ 22] and radioimmunology [ 21] , [ 23] , [ 24] . All these techniques used independently of each other require one or more pure constituents of cannabis [ 4] , [ 7] , [ 8] , [ 19] , [ 21] , [ 23] as reference substances or specific detection by mass spectrometry [ 6] , [ 9] , [ 11] , [ 13] , [ 15] , [ 16] , [ 20] , [ 21] . These conditions are rarely met m a conventional laboratory and we thought that it would be interesting to develop a means of identifying the major constituents of cannabis by making simultaneous use of the excellent results obtained with two chromatographic techniques: HPLC and high-resolution GLC. Application of this method to several samples of resinous and textile cannabis enabled us to propose a criterion for the differentiation of these two categories.

Experimental part

Materials

The five samples of resinous cannabis were each of different origin and their geographical source unknown. One of the two samples of textile cannabis came from an industrial culture, the other from a laboratory culture from hemp seed.

The Model 3000 [ 1] gas chromatography unit was equipped with a flame ionization detector [ 1] and a solid injector. [ 1] The column was a 12-m high-performance capillary column. [ 2] The thickness of the SE 30 phase was 0.4 μ.

The unit for high-pressure liquid chromatography [ 3] consisted of a Model 6000 A pump [ 3] and a Model 440 ultraviolet detector [ 3] with a fixed wave length of 254 mm.

The 3.9 mm x 30 cm column [ 4] was filled with μ porasil particles measuring 10 μ.

Method Chromatographic analyses

The samples were shaken for five minutes with methanol [ 5] at a ratio of 10 mg of powder per ml of solvent. After centrifugation the methanol phase was removed and used: l

In GLC: 1 μl was injected directly;

In GLC with derivation: 1 ml was evaporated under a current of air. The dry residue was dissolved with 100 μl of BSTFA containing 1 per cent of TMC S [ 6] and left to stand for one hour at 60°C. The sililation agent was evaporated. The dry residue was dissolved in 1 ml of ethyl acetate [ 5] and 1 μl was injected.

In HPLC: 1ml was evaporated and the dry residue dissolved in 100 μl of heptane. [ 5] The amount injected was 10 μ

The chromatographic conditions were as follows:

l In GLC: Injector and detector temperatures, 270°C; column temperature, 240°C; gas vector pressure, 0.3 bar; flow rate of additional gas, 40 ml/min; flow rate of hydrogen, 25 ml/min; flow rate of air, 350 ml/min.

_________

1 Girdel, Suresnes, France.

2No. 2101 - 201 L.K.B., Bromma, Sweden.

3Waters Associates S.A., Paris, France.

4 Ref. P/N 27477 S/N Waters Associates, S.A.

5 Very pure quality, Merck, Darmstadt, Federal Republic of Germany.

6 Bis(trimethylsilyl)-trifluoroacetamide with 1 per cent trimethylchlorosilane, Pierce, Rockford, Illinois, United States of America.

In HPLC: Composition of the mobile phase 10 per cent CHCL 3, 5 90 per cent heptane, 5 flow rate 2 ml/min.

Coupling of HPLC and GLC

The fractions corresponding to the chromatogram peaks in HPLC were collected after injection of 50 μl of heptanoic solution I00 times more concentrated than that used for the analysis. This operation was repeated five times. The homologous fractions were combined and evaporated to dryness and then dissolved in 100 μl of methanol in order to be injected in GLC. The three fractions, each corresponding to one of the three main peaks of the chromatogram in GLC, were again evaporated to dryness, sililated under the same conditions as previously and reinjected in GLC.

Results and comments

In a sample of resinous cannabis we identified in GLC: (a) without prior derivation, three main peaks with retention times of 12.4, 15.2 and 17.8 rain (see figure 1a); (b) after derivation, five main peaks with retention times of 8.7, 9.9, 10.6, 13 and 14.3 rain (see figure 1b); in HPLC, seven main peaks with retention times of 4.5, 5.4, 5.9, 6.6, 7.6, 9.5 and 12 min (see figure 1c).

Figure 1 - Chromatogram of a sample of resinous cannabis in GLC without derivation (a), with derivation (b) and in HPLC (c)

Full size image: 26 kB, Figure 1 - Chromatogram of a sample of resinous cannabis in GLC without derivation (a), with derivation (b) and in HPLC (c)

Figure 2 - Chromatogram of the three fractions isolated in HPLC, each containing one of the major constituents of cannabis in GLC before (a) and after (b) derivation

Full size image: 44 kB, Figure 2 - Chromatogram of the three fractions isolated in HPLC, each containing one of the major constituents of cannabis in GLC before (a) and after (b) derivation

Figure 3 - Chromatogram of a sample of textile cannabis in GLC before derivation (a), after derivation (b) and in HPLC (c)

Full size image: 26 kB, Figure 3 - Chromatogram of a sample of textile cannabis in GLC before derivation (a), after derivation (b) and in HPLC (c)

Table 1

Result of the analysis

Technique

Column

Conditions

tr1 (min)

tr2 (min)

tr3 (min)

tr3-tr1/tr2-tr1

GLC
CapillarySE 30 28 m
240 0.3 bar 12.4 15.2 17.8 1.9
GLC with derivation
CapillarySE 30 28 m
240 0.3 bar 8.7 10.6 13 2.25
HPLC
μporasil
10% CHCl 3-90% heptane
  5.4 6.6 7.6 1.8

Note: tr1, retention time of peak 1; tr2, retention time of peak 2; tr3, retention time of peak 3.

Note

:tr 1 , retention time of peak 1: tr 2, retention time of peak 2: tr 3 , retention time of peak 3.

Table 2

Review of the literature

Technique

Author

Column

Conditions

tr CBD (min)

tr Δ-9-THC(min)

tr CBN (min)

tr CBN-trCBD/tr Δ-9-THC-tr CBD

 
 
ZEEUW
SE 30
5%
230
20ml/min
16.8 21.5 25.9 1.9
 
PARIS
OV 1
3%
207
60 ml/min
7 9.2 11 1.8
GLC
STROMBERG
OV 17
3%
100 300 23.5 27 30.5 2
 
MECHOULAM
OV 17
2%
235
30 ml/min
5.7 7.9 10.2 2
 
FETTERMAN
OV 17
2%
210
30 ml/min
14 20.5 26 1.85
 
VREE
OV 1
3%
190
30 ml/min
27.5 39.5 50 1.9
GLC with derivation
ZEEUW
SE 30
5%
230
20 ml/min
12 15.7 20.5 2.3
 
PARIS
OV 1
3%
207
60 ml/min
5.2 6.8 8.8 2.25
 
HARVEY
SE 30
3%
 
30ml/min
22.7 23.5 24.3 2
HPLC
GARRETT
μporasil
 
20% CHCI 3 -80% heptane
  5.2 6 6.7 1.9
 
KANTER
Spherisorb
 
3% CHCI 297% (3% CH 3OH-97% hexane)
  2.8 3.4 4 2

Note: tr CBD,retention time of cannabidiol; tr Δ-9-THC, retention time of Δ-9-tetrehydrocannabinol; tr CBN, retention time of cannabinol.

Note: tr CBD, retention time of cannabidiol; tr @-9-THC, retention time of @-9-tetrahydrocannabinol: tr CBN. retention time or cannabinol.

Table 3

Relative propositions of the main constituents of cannabis in various samples

 

Resinous cannabis

Textile cannabis

Technique

Ratio of peak areas

Sample 1

Sample 2

Sample 3

Sample 4

Sample 5

Sample 1

Sample 2

CPG
Δ-9-THC/CBD
1.09 1.04 0.29 0.76 0.27 0.02 0.03
 
CBN/CBD
0.40 0.93 1.36 1.2 1.21 0.01
Below 0.01
CPG with derivation
Δ-9-THC
0.82 1.52 0.05 1.06 0.08 0.02 0.02
CBN/CBD
  0.77 2.55 1.88 3.53 1.6
Below 0.01
Below 0.01
CPL
Δ-9-THC
0.90 0.25 0.03 0.27 0.06 0.02 0.01
CBN/CPD
 
Above 5
10
Above 5
Above 7
Above 10
0.13 0.08

Among the seven fractions collected in HPLC and corresponding to the seven main peaks, three fractions contained for the most part one of the three main substances identified in GLC (see figure 2a). The fractions 1, 2 and 3 were isolated and derived, we were able to determine which ones corresponded to the five main peaks identified in GLC after derivation (see figure 2b).

Three substances were isolated in this way and their retention times were determined in GLC, in GLC after derivation and in HPLC. By comparison with data from the literature, [ 7] substance 1 could be identified as cannabidiol (CBD), substance 2 as @-9-tetrahydrocannabinol ( @-9-THC) and substance 3 as cannabinol (CBN) (see tables 1 and 2).

In the samples of textile cannabis (see figure 3), three peaks (GLC and HPLC) corresponding to the major constituents of cannabis were likewise identified but in very different proportions from those observed in the case of resinous cannabis.

Since the conditions of analysis were the same for all samples, an estimate of the content of @-9-tetrahydrocannabinol, the principal active substance [ 25] , and of CBN was carried out in relation to CBD, the only constituent present in large amounts in the samples.

Calculation of the peak area ratios

@-9-THC/CBD and CBN/CBD

in the three techniques under consideration (see table 3) indicated that these two ratios were low for the textile cannabis samples. For the resinous cannabis samples, at least one of these two ratios

@-9-THC/CBD and CBN/CBD

was high (generally greater than 1), thus indicating the presence of considerable amounts of at least two of the major constituents of cannabis. It should be noted that the small proportion of @-9-THC in some samples (3 and 5) is not inconsistent with their resinous character in so far as it is associated with a large proportion of cannabinol, a degradation product [ 26] of @-9-THC.

Conclusion

This method, which combines two chromatographic techniques (GLC and HPLC), affords a means of identifying and differentiating cannabis samples by establishing the presence of the major constituents of cannabis and determining their relative proportions.

7 In order to be able to compare these data which were heterogeneous because of the diverse conditions of chromatography (column phase, temperature, flow rate), we calculated the ratios of the distances between the peaks.

References

001

M. Paris and D. Demesy, "Nouvelles méthod d'identification et d'évaluation de l'activité du Cannabis" Plantes médicinales et phytothérapie , vol. 5, No. 1 (1971), pp. 28 - 38.

002

J.A. Vinson, D. D. Patel and A. H. Patel, "Detection of tetrahydrocannabinol in blood and serum using a fluorescent derivative and thin-layer chromatography'', Analytical Chemistry , vol. 49, No. 1 (1977), pp. 163 - 165.

003

S.L. Kanter, L. E. Hollister and K. O. Loeffier, "Marihuana metabolites in the urine of man: VIII - Identification and quantitation ol 9-tetrahydrocannabinol by thin-layer chromatography and high-pressure liquid chromatography'', Journal of Chromatography , vol. 150, 1978, pp. 233 - 237.

004

R. A. De Zeeuw, Th. M. Malingre and F. W. H. M. Merkus, "l-Tetrahydrocannabinolic acid, an important component in the evaluation of Cannabis products", Journal of Pharmacy and Pharmacology , vol. 24, No. 1 (1972), pp. 1 - 6.

005

S.N. Tewari and J. D. Sharma, "Separation and identification of cannabinoids from Cannabis indica L. by thin-layer chromatography", Pharmazie, vol. 34, No. I (1979), p. 54.

006

W.W. Just, N. Filipovic and G. Werner, "Detection of 9-tetrahydrocannabinol in saliva of men by means of thin-layer chromatography and mass spectrometry, Journal of Chromatography , vol. 94, 1974, pp. 189 - 194.

007

K. D. Parker and others, "Preliminary report on the separation and quantitative determination of cannabis constituents, present in plant material and when added to urine, by thin-layer and gas-chromatography", Bulletin on Narcotics , vol. 20, No. 4 (1968), pp. 9 - 14.

008

P.S. Fetterman and others, "Mississippi-grown Cannabis sativa L.: preliminary observation on chemical definition of phenotype and variations in tetrahydrocannabinol content versus age, sex and plant part", Journal of Pharmaceutical Sciences , vol. 60, No. 8 (1971), pp. 1246 - 1249.

009

C. E. Turner and others, "Constituents of Cannabis sativa L.: VII - use of silyl derivatives in routine analysis", Journal of Pharmaceutical Sciences , vol. 63, No. 2 (1974), pp. 1872 - 1876.

010

C. W. Waller, "Chemistry of marihuana", Pharmacological Reviews , vol. 23, No. 4 (1971), pp. 265 - 271.

011

D. 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.

012

Y. Gaoni and R. Mechoulam, "The isolation and structure of 1-tetrahydrocannabinol and other neutral cannabinoids from hashish", Journal of the American Chemical Society , vol. 93, No. I (1971), pp. 217 - 224.

013

T. B. Vree and others, Identification in hashish of tetrahydrocannabinol, cannabidiol and cannabinol analogues with a methyl side-chain", Journal of Pharmacy and Pharmacology , vol. 24, No. 1 (1972), pp. 7 - 12.

014

N. K. McCallum and E. R. Cairns, "A simple gas chromatographic method lot routine 1-tetrahydrocannabinol analysis of blood and brain", Journal of Analytical Toxicology, vol. 2, 1978, pp. 89 - 93.

015

J. Rosenfeld, "The simultaneous determination of 9-tetrahydrocannabinol and 11-hydroxy-9-tetrahydrocannabinol in plasma", Analytical Letters, vol. 10, No. 12 (1977), pp. 917 - 930.

016

T. B. Vree and others, "Gas chromatography of Cannabis constituents and their synthetic derivatives", Journal of Chromatography, vol. 74, 1972, pp. 209 - 224.

017

L. E. Stromberg, "Minor components of cannabis resin. I. - Their separation by gas chromatography, thermal stability, and protolytic properties", Journal of Chromatography, vol. 63, 1971, pp. 391 - 396.

018

P. J. Twitchett, P. L. Williams and A. C. Moffat, "Photochemical detection in high-performance liquid chromatography and its application to cannabinoid analysis", Journal of Chromatography, vol. 149, 1978, pp. 683 - 691.

019

E. R. Garrett and C. A. Hunt, "Separation and analysis of 9-tetrahydrocan-nabinol in biological fluids by high-pressure liquid chromatography and GLC", Journal of Pharmaceutical Sciences, vol. 66, No. 1 (1977), pp. 20 - 26.

020

J. L. Valentine and others, "High-pressure liquid chromatographic-mass spectrometric determination of 9-tetrahydrocannabinol in human plasma following marijuana smoking", Journal of Pharmaceutical Sciences, vol. 66, No. 9 (1977), pp. 1263 - 1266.

021

P. L. Williams, A. C. Moffat and L. J. King, "Combined high-pressure liquid chromatography and radioimmunoassay method for the quantitation of 9-tetrahydrocannabinol and some of its metabolites in human plasma", Journal of Chromatography, vol. 155, 1978, pp. 273 - 283.

022

S.L. Kanter, M. R. Musumeci and L. E. Hollister, "Quantitative determination of 9-tetrahydrocannabinol and 9-tetrahydrocannabinolic acid in marihuana by high-pressure liquid chromatography", Journal of Chromatography, vol. 171, 1979, pp. 504 - 508.

023

R. Rodgers and others, "Homogeneous enzyme immunoassay for cannabinoids in urine", Clinical Chemistry, vol. 24, No. 1 (1978), pp. 95 - 100.

024

J. D. Teale and others, "Radioimmunoassay of cannabinoids in blood and urine", The Lancet, vol. 2, No. 7880 (1974), pp. 553 - 555.

025

J. L. Neumeyer and R. A. Shagoury, "Chemistry and pharmacology of marihuana", Journal of Pharmaceutical Sciences, vol. 60, No. 10 (1971), pp. 1433 - 1457.

026

J. Levine, "Origin of Cannabinol", Journal of the American Chemical Society, vol. 66, 1944, p. 1868.