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

Sections

Introduction
Experimental

Details

Author: Kenneth D. PARKER, James A WRIGHT, Anita F. HALPERN, Charles H. HINE
Pages: 10 to 14
Creation Date: 1968/01/01

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 *

Kenneth D. PARKER
James A WRIGHT
Anita F. HALPERN
Charles H. HINE
The Hine Laboratories, Inc., 1099 Folsom Street, San Francisco, California, USA 94103

Introduction

We have summarized methodology of solvent extraction, thin-layer (TLC) and gas chromatography (GLC) techniques appropriate for the separation and quantitative determination of cannabis plant constituents. The cannabis plant constituents studied include delta-l-tetrahydrocannabinol (Δ-1-THC), delta-6-tetrahydrocannabinol (Δ-6-THC), delta-l-tetrahydrocan-nabinol acetate (Δ-1-THC-acetate), Δ-6-THC-acetate, cannabinol and cannabidiolic acid (1). These micro methods were developed to help in solving the following problems:

  1. To provide a rigorous proof of chemical identity and reliable quantitative determination of various individual constituents of cannabis plant materials.

  2. To form the basis for the development of urinalysis methods for the detection of cannabis use and abuse;

  3. To study biotransformation of cannabis plant constituents in man;

  4. To study chemical alterations which occur during the ageing of the plant material and the smoking process.

EQUIPMENT AND REAGENTS

Gas chromatograph: Aerograph "Hy-Fi" model A-600-C equipped with a hydrogen flame ionization detector and a detector base heater accessory. Range expansion: 1-1000.

Column: 3 % SE-30 on Chromasorb W-H.P., 100/-120 mesh (Johns-Manville) in 1/8 in o.d. 6 ft. length stainless steel. HCl-BSA washed borosilicate glass injector insert (n: 2950 HETP: 0.0020).

This article summarizes portions of more detailed reports of progress dealing with the Detection of Abuse Drug Usage supported by Contract PH 43-64-931 from the National Institutes of Health.

Developing systems: I. n-hexane: 1,4-dioxane (9:1). II. n-hexane:diethyl ether (4:1) -- ambient temp.

Thin-layer plates: 0.3 mm silica gel G prepared according to Stahl (2).

Experimental

  1. Selection of solvent and pH-giving optimal partition between water and organic phase

PROCEDURE

Two water solutions of Δ-l-THC containing 10 μg/ml were prepared and extracted (from acid, basic and neutral solution) using petroleum ether or chloroform as solvents.

Estimations of the respective partition coefficients of Δ-1-THC were made using the determined extinction coefficient values in the UV range.

RESULTS

These results indicated that the Δ-1-THC was extracted as a neutral compound. The recovery from water with petroleum ether as the extracting solvent was approximately 40 % and with chloroform approximately 75%.

  1. Quantitative determination of cannabis resin constituents present in plant material - Δ-1-THC 'and cannabinol

PROCEDURE

We developed the following procedure which gave satisfactory recovery:

  1. Take to dryness 100 μl of a methanol solution of a petroleum ether extract of plant material represent ing 200 mg of the original cannabis plant material in a 3 ml glass-stoppered centrifuge tube at 50 °C and a vacuum of 10 psi.

  1. Prepare in the same manner, at the same time an authentic 100 μg Δ-1-THC standard.

  2. Add to the residues in both tubes 50 μl of a solution of methanol: acetone (1:1) containing 0.5 μg/μl of tetraphenylethylene.

  3. Inject 2.5-3.0 μl into the gas chromatograph for analysis.

  4. Observe the peak heights of the corresponding component peaks and calculate the peak height ratio relative to the internal standard tetraphenylethylene. Using determined peak height response ratios calculate the quantity of each component.

RESULTS

Using this method the Δ-1-THC content of one sample of marijuana plant material was found to be 0.075 %. The same sample was negative for Δ-6-THC (less than 0.001%) and positive for cannabinol, cannabidiolic acid, olivetol and other unidentified compounds. The latter components were not quantitatively determined from the response ratios obtained because appropriate reference standards have not been made available.

  1. Extraction scheme for determination of Δ-1-THC and cannabinol added to urine

Liquid-liquid extraction of compounds from urine and determination by gas liquid chromatography was accomplished using an internal standard response ratio developed from fortified urines carried through every step of the procedure.

PROCEDURE

  1. Add 2 g of sodium chloride to 50 ml of urine.

  2. Confirm that the pH of the mixture is approximately 5.8 to 6.0 and adjust if necessary.

  3. Extract twice with 15 ml portions of chloroform.

  4. Centrifuge the extraction mixture for 10 minutes at 1500 RPM, if necessary, to break any emulsion which may develop.

  5. Combine and dry the removed chloroform fraction by filtering it through anhydrous sodium sulphate into a 50 ml beaker (25 cc/30 cc removed).

  6. Measure 25 ml of the chloroform fraction and evaporate it to dryness at 70 0C under a vacuum of 10 psi.

  7. Quantitatively wash the residue from the beaker into a 3 ml glass-stoppered centrifuge tube usingmethanol: acetone (1:1) (3 times 0.5 ml) and evaporate to dryness at 70 0C and vacuum at 10 psi.

  8. Dissolve the residue using 50 μl of methanol: acetone (1:1) containing 0.5 μg/μl of tetraphenylethylene.

  9. Inject 2-4 μl of the prepared sample for GLC determination by comparison of response ratios as previously described.

  10. Spot the remainder of the extract on thin-layer plates and chromatograph using solvent system I and II and visualize using appropriate spray reagents such as fast blue B.

  11. Estimate the concentrations by comparing intensities and sizes of corresponding spots appearing in the samples and the fortified urine extract positive controls.

RESULTS

In one set of experiments urine was fortified with various graded quantities of cannabis resin and carried through the procedure described. By comparison of GLC responses obtained with and without the extraction treatment, the following recoveries from urine were determined: Δ-l-THC 90 %, cannabinol 86 %. The detectability of the method for Δ-l-THC was 0.01 ppm using a 50 ml sample of urine.

Figure 1, page 11, illustrates the typical GLC separations obtained for cannabis resin constituents. Table 1 summarizes the GLC retention data for cannabis compounds and corresponding trimethylsilyl (TMS) derivatives formed using N,O-bis-(trimethylsilyl)-acetamide (BSA).

TABLE 1

Retention data obtained for cannabis compounds and corresponding trimethylsilyl derivates

 

Retention values

Compound

Rt a (min)

Rrt

Cannabidiolic acid
3.10 0.31
Δ-6-THC-TMS derivative b
3.20 0.32
Δ-1-THC-TMS derivative
3.40 0.34
Δ-6-THC-acetate
6.70 0.68
Δ-1-THC-acetate
6.95 0.70
Δ-6-THC
8.30 0.84
Δ-1-THC
8.80 0.89
Tetraphenylethylene c
9.90 1.00
Cannabinol
10.70 1.09

a Operating parameters:

SE-30 3 % on Silanized Chromasorb W, 100/120 mesh

Column temp. 240° C

Detector temp. 280°C

Injector temp. 275°C

Inlet pressure 35 psig

Additional column data based on Δ-1-THC:

HETP: 0.002; n: 2950

Sensitivity: 0.01 μg

b TMS = Trimethylsilyl derivatives.

c Internal qualitative and quantitative reference standard used.

Full size image: 12 kB

Fig. 1: Typical GLC separation obtained for cannabis resin constituents using a SE-30 3 % column at a temperature of 240 0C. Cannabidiolic acid (E), Δ-l-THC-acetate (C), Δ-1-THC (A) and cannabinol (D).

  1. Multidimensional chromatography (GLC and TLC) of petroleum ether extracts of cannabis plant material

PURPOSE

The purpose of this experiment was to correlate different qualitative tests on petroleum ether extracts of cannabis plant material using GLC and TLC retention data and colour reactions.

TECHNIQUE

Gas liquid and thin-layer chromatography were used.

EQUIPMENT AND REAGENTS

Gas chromatograph: Varian 600 C as described previously.

Column: 5% SE-30 on W-H.P. 100/20 (Johns-Manville) mesh in 1/8 in. o.d. 5 ft. length stainless steel. HCl-HMDS washed borosilicate glass injector insert.

Oven temperature: 255 0C. Carrier gas: nitrogen-30 psig.

Developing systems: Solvent systems I and II as described previously.

Thin-layer plates: As described previously.

Post column splitter: For GLC fraction collection on TLC plates.

Spray reagents: Fast blue B, MeOH:FeCl 3 and Ghamravy.

  1. Identification of thin-layer fractions of cannabis resin extracts using spray reagents after separation by two TLC solvent systems

PROCEDURE

  1. Spot two plates with two spots each of mixed standards containing 5 μg each of Δ-l-THC, Δ-6-THC, Δ-1-THC acetate and 10 spots each of 5 μl cannabis resin. Solution in methanol represents 10 mg of original plant material.

  2. Develop one plate in system I and one plate in solvent system II.

Fig. 2: TLC chromatogram of cannabis plant resin extract and reference standards using solvent system II.

Full size image: 29 kB, Fig. 2: TLC chromatogram of cannabis plant resin extract and reference standards using solvent system II.
  1. Allow the solvent front to move about 16 cm (about 35 minutes) and then remove the plates from the tank and air dry for 20 minutes.

  2. Block off appropriate columns of the plate with a glass plate and spray with the visualizing reagents - fast blue B, methanol: ferric chloride, Marquis and Ghamravy.

RESULTS

Figure 2 illustrates typical chromatographic results obtained using solvent system II. Table 2 summarizes the results obtained with the spray reagents giving TLC R f values for Δ-l-THC, Δ-6-THC, cannabinol and cannabidiolic acid with solvent systems I and II. Table 3 lists TLC retention values for cannabis compounds obtained using various solventsystems. Table 4 summarizes colours and approximate sensitivities obtained for cannabis compounds on TLC plates after the application of each of 15 different visualizing reagents as a spray.

  1. Simultaneous separation of cannabis fractions by GLC and TLC (I)

PROCEDURE

  1. Constant drive stage is fitted with thin-layer plates. Fraction splitter oriface is adjusted to a height of 1 mm above the plate. Five μl of resin representing 10 mg of cannabis plant material and 5 μg of mixed standard are spotted on plate.

TABLE 2

Summary of TLC Rf values for constituents of cannabis resin and corresponding colour reactions observed with selected spray reagents

 

R f values

 

Δ-l-THC

Δ-6-THC

Cannabinol

Cannabidiolic acid

System I
0.38 0.42 0.30 0.05
System II
0.59 0.67 0.46 0.10
Spray reagent used
   
Colours observed
 
Fast blue B
Violet
Red
Purple
Violet
Methanol: ferric chloride
N.C. a
N.C.
N.C.
Yellow-Brown
Ghamravy
Purple
Purple
Red-Brown
Violet
Marquis
Brown
Rose
Rose
Purple

a N.C. indicates no change.

TABLE 3

TLC retention values for cannabis compounds obtained using various solvent systems

 

Retention value (R f)

Solvent system

Δ-l-THC

Δ-6-THC

Δ-1T-HC-acetate

Cannabinol

CHCl 3: CHCOOH (4:1)
1.00 1.00 1.00
- a
ETOH: NH 4OH: 1, 4, Dioxane: Benzene (1:1:8:10) b
1.00 1.00 1.00
-
Methanol: NH 4OH (200:3)
0.87 0.87 0.85
-
n-hexane: CHCl 3 (8:1)
0.10 0.10 0.10
-
n-hexane: diethylamine (9:1)
0.66 0.66 0.98
-
Cyclohexane: benzene diethylamine (75:15:10)
0.89 0.89 0.89
-
2-ethoxyethanol: diethyl ether (8:1)
0.28 0.25 0.91
-
n-hexane (dimethyl formamide: CHCl 3 impregnation 6:4)
0.25 0.30 0.90
-
Petroleum ether: diethyl ether (9:1)
0.31 0.38 0.62 0.27
n-hexane: 1:4 Dioxane (9:1)
0.38 0.42 0.81 0.30
n-hexane: diethyl ether (4:1)
0.59 0.67 0.87 0.46

a Values not determined because no qualitative reference sample was available at that time.

b (1:5) means parts by volume.

TABLE 4

Summary of results obtained for cannabis compounds with various spray reagents and corresponding sensitivities on TLC plates

 

Compound

 

Spray reagent

Δ-1-THC

Δ-6-THC

Cannabinol

Cannabidiolic acid

Δ-l-THC-acetate

Sensitivity a (μg)

Duquenois
Aqua
Violet
- b
-
Lt. Blue
2
Marquis
Brown
Brown
-
Purple-Brown
White with Brown Centre
1
Frohdes
Grey
Grey
-
-
Grey
5
Aq. FeCl 3
Org-Brn
Org-Brn
Org-Brn
-
Org-Brn
2
MeOH FeCl 3
N.C. c
N.C.
N.C.
Yellow-Brown
N.C.
5
Ghamravy
Purple
-
Red
-
N.C.
1
Tetracyanoethylene
Brown
Brown
Brown
Brown
Brown
5
Pauly's Reagent
Yellow
Yellow
-
-
Yellow (Lt)
1
Diazo benzidine
Red-Org
Red-Org
-
-
Orange
1
Diazo p-nitro aniline
Yel-Org
Yel-Org
-
-
Yel-Org
1
Fast blue B
Red-Viol
Crimson
Purple
Violet
N.C. d Red (H+)
0.2
Scarlet 2G
Yellow
Yellow
-
-
N.C. Yellow (H+)
0.5
Scarlet R
Yellow
Yellow
-
-
Yellow
0.5
Dragendorffs
Orange
Orange
-
-
Orange
2

a Sensitivities for Δ-l-THC, Δ-6-THC and Δ-1-THC-acetate.

bNo observation made.

cN.C. = no change observed.

d(H+) indicates change occurred after TLC Plate was first sprayed with hydrochloric acid to hydrolyze acetate.

  1. Move thin-layer plate to starting position and start drive motor. Inject 5 μl representing 10 mg of resin into gas chromatograph.

  2. As areas of interest appear mark TLC plate at apex of peak on GLC recorder chart.

  3. Develop TLC plate in solvent system of choice.

  4. Air dry and spray with appropriate reagent, i.e. fast blue B.

RESULTS

Figure 3 illustrates GLC separation obtained and Figure 4 shows corresponding GLC effluent collection and separation by TLC with direct application of cannabis plant resin extract for comparison. The TLC solvent system II was used and the developing spray was fast blue B. The GLC column and conditions used for figure 3 and 4 illustrations were different than those previously described.

The THC reference standards were supplied by the National Institutes of Health. This paper was presented at the semi-annual seminar of the California Association of Criminalists, November 4, 1967, in San Jose, California, and The California Toxicology Seminars.

We are indebted to Dr. E. T. Hsi and Mrs. Elizabeth Thomas for technical assistance.

Fig. 3: Corresponding GLC results obtained from cannabis plant resin extract using SE-30 3% column at 255 0 C.

Full size image: 15 kB, Fig. 3: Corresponding GLC results obtained from cannabis plant resin extract using SE-30 3% column at 255 0C.

Fig. 4: TLC chromatogram of cannabis plant resin extract from direct application to the plate and the GLC profile collection developed in solvent system II and sprayed with fast blue B.

Full size image: 16 kB, Fig. 4: TLC chromatogram of cannabis plant resin extract from direct application to the plate and the GLC profile collection developed in solvent system II and sprayed with fast blue B.

References

001

Parker, K. D., Wright, J. A. and Hine, C. H., J. Forensic Sci. Soc . 7, 162-170 (1967).

002

Stahl, E., Schröter, G., Kraft, G. and Renz, R., Pharmazie 11 , 633-638 (1956).