Determination of THC-Carbonic Acid in Urine by NCI

Keywords forensics THC-A Cannabis Marinol Dronabinol metabolite derivatization negative chemical ionization 

Because the THC metabolite THC-COOH is excreted in urine in conjugatedform, a hydrolysis step is necessary to receive the free THC-COOH before extraction. Hydrolysis is done for a 1 mL urine sample with 100 iL 12 N KOH solution after addition of 20 pL ofthe internal standard, equivalent to 100 ng of deuterated THC-COOH. Hydrolysis is achieved by heating the mixture in a crimp-cap septum 

The hydrolysed sample is transferred quantitatively on an Extrelut NTl extraction column. After waiting for 5 min, the sample is first extracted with 4 mL isooctane and then with additional 2 mL isooctane. The combined extracts are collected in glass vials (volume 10 mL) with a conical bottom, evaporated to dryness and concentrated in the conical part of the vial. 

The extracted THC-COOH is transferred completely into Nil glass vials. As derivatization agents 50 pL pentafluoropropanol and 80 pL heptafluorobutyric acid anhydride are added and the reaction mixture is heated to 65 C for 20 min in the crimp-closed vial. The remaining derivatization reagents are then completely evaporated and the remainder is carefully transferred to conical autosampler microvials (ND 8, 1.1 mL) using a microliter syringe and toluene as solvent, so that an end volume of 200 pL is achieved for the injection of 1 pL aliquots of the derivatized extract. 

A single quadrupole DSQ II GC-MS system equipped with a CTC PAL liquid autosampler was used. The GC-MS measurements have been done in the electron ionization (LI) mode for method development and the NCI mode for routine quantitation. 

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