Dept. of Biochemistry, St. Boniface General Hospital, Winnipeg, Manitoba, R2H 2A6, USA

The urine was hydrolysed with E. coli b-glucuronidase, alkaninized to pH 9 and extracted with methylene dichloride/trifluoroethanol (92:8). The dried extract was subjected to sequential derivatization. First, the keto-opioids were converted to their respective oximes with the addition of 2% methoxyamine HCl in pyridine. The reaction occurs at room temperature and is complete within 15 minutes. Next, propionic anhydride was added to the pyridine. The available hydroxyl groups at O₃ and O₆ are converted to propionyl esters. The reaction occurs at 56°C and is complete within 15 minutes. The excess derivitizing reagents were evaporated. The residue was purified by liquid extraction from 100 µL of 15% ammonium hydroxide into 1 mL of hexane/chloroform (3:1). The organic phase is dried, reconstituted in 50 µL of ethyl acetate and 1 µL analysed by GC-MS in full scan, m/z 50 to 550 at 1 scan/s.

Good separation was obtained between all opioids on a 15 m x 0.25 mm DB-1 column (J&W Scientific); a unique mass spectrum is obtained for each. Using naltrexone as the internal standard for the keto-opioids and nalorphine for the others, the day-to-day CVs for hydrocodone, hydromorphone, oxycodone, oxymorphone, codeine and morphine are 7.6, 8.7, 10.9, 8.3, 8.6 and 6.7% at 300 ng/mL and 9.1, 6.0, 10.2, 9.0, 8.3, and 7.9% at 1500 ng/mL. CVs for 6-MAM are 12.0% at both 30 and 150 ng/mL. The method is linear from 25 to 2,000 ng/mL.