3.6 Isolation of D9-THC-a from hemp and analytical aspects concerning the determination of D9-THC in cannabis products,
by Dussy, Hamberg, Luginbiihl, Schwerzmann, and Briellmann,
from Institute of Legal Medicine
as published in Forensic Science International on line18-Aug-2004
Wow, lookee what Dr Fischedick shared with me, after being kind enough to listen to my enigma. Still working on the puzzlement, but getting closer and had some major insights reading this study done in Switzerland by the Institute of Legal Medicine.
Take a look at their extraction method at 2.2 and column chromatography purification procedure at 2.3 for lots of new ideas, and decarboxylation optimization results at 3.3, which with the Figure 8 graph, puts a whole new slant on thangs.
Hee, hee, hee, enjoy and hats off to Dr Fischedick for the link! Copies of the study at:
Here are some high points:
The extraction was done according to the procedure described by Lehmann and Brenneisen [4].
In brief, 100 g pulverized plant material with a total D9-THC content of 5% was extracted with 500 mL petrol ether acidified with acetic acid (0.5 mL CH3COOH in 500 mL PE).
The filtrated extract was re-extracted three times with 400 mL of an aqueous solution of NaOH and Na2SO3 (2% each). These combined extracts were acidified with ca. 500 mL of glacial 5 % sulfuric acid until pH reached 3, and immediately extracted with three times 400 mL TBME.
These combined organic extracts were dried with Na2SO4, filtrated and concentrated in a rotary evaporator at 25–30 8C by aid of cryostatic cooling of the vapors. The concentrate was dried overnight at vacuum conditions, yielding 1.71 g brown amorphous material.
***
2.3. Purification of the crude extract by column chromatography.
One hundred and seventy-seven milligrams of the crude extract was chromatographed on a 30 mm _ 400 mm conventional column filled with 100 g silica 60 (0.063–0.2 mm).
The elution solvent consisted of a mixture of hexane (650 mL), toluene (215 mL), Acetone (135 mL) and acetic acid (20 drops), which were passed through the column at
1.5 mL/min. The fractions, sized 20 mL and in the critical moments of the chromatography 10 mL, were controlled by thin layer chromatography (TLC).
Elution of D9-THCA-A began at 280 mL. The fractions from 290 mL until 510 mL
were collected and concentrated in a rotary evaporator at 25–30 8C. The concentrate was dried overnight at vacuum conditions to yield 99 mg pale yellow amorphous material.
The purity of this material was assigned to 96% D9-THCA-A by 1H-NMR, the main impurity being the cannabinol analogue of D9-THCA-A. All NMR analyses were measured at the Institute of Organic Chemistry (Basel, Switzerland) on a Bruker
DRX500. The chemical shifts were assigned by several two dimensional NMR techniques (HMBC, HMQC, COSY, DEPT 135 and NOE).
The NMRdata are listed in the annexe.
****
3.3. Optimization of the decarboxylation temperature for the determination of the total D9-THC content by HPLC As D9-THCA-A is commercially available only since this
year, most laboratories measuring the total D9-THC content by HPLC (a method without thermal stress) needed to convert D9-THCA-A prior to analysis.
Fig. 8 shows that optimum is reached at about 150 8C with about 70% yield. At higher temperature, D9-THC is oxidized to form cannabinol.
As the sum of D9-THCA-A, D9-THC and cannabinol does not reach 100%, it is assumed that polymeric material is formed also.
If the time of exposure to temperature is changed, temperature needs to be adjusted to maximal conversion. With the chosen time, the maximal conversion is in the same range as it is in the injector of the GC system.
Furthermore, in between 140 and 160 8C, there is no significant temperature dependence observed such that an exact temperature adjustment is not of importance in that
range.
The significance that I see in graph Fig 8, is that the previous assumption has been that at 70% decarboxylation that the rate of conversion of the remaining 30% THC-a tp THC, was at a lower rate that the conversion of THC to CBN, but this graph shows THC-a to THC is maxed out at 70% and the decline in total THC is due to conversion to CBN.
