Quantitative determination of phenacetin and its metabolite acetaminophen by GLC-chemical ionization mass spectrometry

A quantitative GLC-mass spectrometric procedure was developed for the determination of phenacetin and its O-desethyl metabolite, acetaminophen, in human plasma. The assay utilizes selective ion detection to monitor, in a GLC effluent, the MH+ molecular ions of both phenacetin and the methyl derivative of acetaminophen, p-acetanisidine, generated by isobutane chemical ionization. Deuterated analogs of phenacetin and acetaminophen, phenacetin-d3 and acetaminophen-d3, respectively, are added to the plasma before extraction to serve as internal standards. To determine phenacetin and unconjugated acetaminophen, 1.0 ml of plasma is extracted with 5 ml of benzene-dichloroethane (7:3). The extraction solvent is removed, and the residue is methylated with diazomethane. Th solution is again evaporated to dryness, and the residue is reconstituted in ethyl acetate. A portion of this solution is then analyzed by GLC-mass spectrometry, with the mass spectrometer set to monitor m/e 166 (p-acetanisidine), 169 (p-acetanisidine-d3), 180 (phenacetin), and 183 (phenacetin-d3). To determine total acetaminophen, 0.1 ml of plasma is treated with a mixture of beta-glucuronidase and sulfatase, extracted with ethyl acetate, methylated, and analyzed by GLC-mass spectrometry. The procedure has a sensitivity limit of 1 ng of phenacetin/ml and 0.1 mug of acetaminophen/ml. The curves relating the amount of phenacetin and acetaminophen added versus the amount of phenacetin and acetaminophen found for 12 known phenacetin concentrations over the 9.9-246.6-ng/ml range and for 16 known acetaminophen concentrations over the 0.52-13.10-mug/ml range are straight lines with intercepts of nearly zero and with slopes of unity. Analyses of six separate plasma samples, each containing 25 ng of phenacetin/ml and 1.31 mug of acetaminophen/ml, had a precision of +/- ng/ml for phenacetin and +/- 0.08 mug/ml for acetaminophen.