Quantification of acetaminophen (paracetamol) in human plasma and urine by stable isotope-dilution GC–MS and GC–MS/MS as pentafluorobenzyl ether derivative

We report on the quantitative determination of acetaminophen (paracetamol; NAPAP-d0) in human plasma and urine by GC–MS and GC–MS/MS in the electron-capture negative-ion chemical ionization (ECNICI) mode after derivatization with pentafluorobenzyl (PFB) bromide (PFB-Br). Commercially available tetradeuterated acetaminophen (NAPAP-d4) was used as the internal standard. NAPAP-d0 and NAPAP-d4 were extracted from 100-μL aliquots of plasma and urine with 300 μL ethyl acetate (EA) by vortexing (60 s). After centrifugation the EA phase was collected, the solvent was removed under a stream of nitrogen gas, and the residue was reconstituted in acetonitrile (MeCN, 100 μL). PFB-Br (10 μL, 30 vol% in MeCN) and N,N-diisopropylethylamine (10 μL) were added and the mixture was incubated for 60 min at 30 °C. Then, solvents and reagents were removed under nitrogen and the residue was taken up with 1000 μL of toluene, from which 1-μL aliquots were injected in the splitless mode. GC–MS quantification was performed by selected-ion monitoring ions due to [M−PFB]− and [M−PFB−H]−, m/z 150 and m/z 149 for NAPAP-d0 and m/z 154 and m/z 153 for NAPAP-d4, respectively. GC–MS/MS quantification was performed by selected-reaction monitoring the transition m/z 150 → m/z 107 and m/z 149 → m/z 134 for NAPAP-d0 and m/z 154 → m/z 111 and m/z 153 → m/z 138 for NAPAP-d4. The method was validated for human plasma (range, 0–130 μM NAPAP-d0) and urine (range, 0–1300 μM NAPAP-d0). Accuracy (recovery, %) ranged between 89 and 119%, and imprecision (RSD, %) was below 19% in these matrices and ranges. A close correlation (r > 0.999) was found between the concentrations measured by GC–MS and GC–MS/MS. By this method, acetaminophen can be reliably quantified in small plasma and urine sample volumes (e.g., 10 μL). The analytical performance of the method makes it especially useful in pediatrics.