GC-MS and GC-MS/MS measurement of ibuprofen in 10-μL aliquots of human plasma and mice serum using [α-methylo-2H3]ibuprofen after ethyl acetate extraction and pentafluorobenzyl bromide derivatization: Discovery of a collision energy-dependent H/D is

- Quantitative GC-MS and GC-MS/MS methods for ibuprofen were developed.
- Ibuprofen and the internal standard d3-ibuprofen were solvent extracted.
- Ibuprofen and d3-ibuprofen were derivatized with pentafluorobenzyl (PFB) bromide.
- ECNICI of the PFB esters of Ibuprofen and d3-ibuprofen yields their carboxylates.
- In GC-MS/MS collision induces a remarkable H/D isotope effect.

GC-MS and GC-MS/MS methods were developed and validated for the quantitative determination of ibuprofen (d0-ibuprofen), a non-steroidal anti-inflammatory drug (NSAID), in human plasma using α-methyl-2H3-4-(isobutyl)phenylacetic acid (d3-ibuprofen) as internal standard. Plasma (10 μL) was diluted with acetate buffer (80 μL, 1 M, pH 4.9) and d0- and d3-ibuprofen were extracted with ethyl acetate (2 × 500 μL). After solvent evaporation d0- and d3-ibuprofen were derivatized in anhydrous acetonitrile by using pentafluorobenzyl (PFB) bromide and N,N-diisopropylethylamine as the base catalyst. Under electron-capture negative-ion chemical ionization (ECNICI), the PFB esters of d0- and d3-ibuprofen readily ionize to form their carboxylate anions [M-PFB]− at m/z 205 and m/z 208, respectively. Collision-induced dissociation (CID) of m/z 205 and m/z 208 resulted in the formation of the anions at m/z 161 and m/z 164, respectively, due to neutral loss of CO2 (44 Da). A collision energy-dependent H/D isotope effect was observed, which involves abstraction/elimination of H− from d0-ibuprofen and D− from d3-ibuprofen and is minimum at a CE value of 5 eV. Quantitative GC-MS determination was performed by selected-ion monitoring of m/z 205 and m/z 208. Quantitative GC-MS/MS determination was performed by selected-reaction monitoring of the mass transitions m/z 205 to m/z 161 for d0-ibuprofen and m/z 208 to m/z 164 for d3-ibuprofen. In a therapeutically relevant concentration range (0-1000 μM) d0-ibuprofen added to human plasma was determined with accuracy (recovery, %) and imprecision (relative standard deviation, %) ranging between 93.7 and 110%, and between 0.8 and 4.9%, respectively. GC-MS (y) and GC-MS/MS (x) yielded almost identical results (y = 4.00 + 0.988x, r2 = 0.9991). In incubation mixtures of arachidonic acid (10 μM), d3-ibuprofen (10 μM) or d0-ibuprofen (10 μM) with ovine cyclooxygenase (COX) isoforms 1 and 2, the concentration of d3-ibuprofen and d0-ibuprofen did not change upon incubation at 37 °C up to 60 min. The trough pharmacokinetics of an inhaled arginine-containing ibuprofen preparation in mice was studied after once-daily treatment (0.0, 0.07, 0.4 and 2.5 mg/kg body weight) for three days. A linear relationship between ibuprofen concentration in serum (10 μL) and administered dose 24 h after the last drug administration was observed.