GC-MS and GC-MS/MS measurement of ibuprofen in 10-μL aliquots of human plasma and mice serum using [α-methylo- 2 H 3 ]ibuprofen after ethyl acetate extraction and pentafluorobenzyl bromide derivatization: Discovery of a collision energy-dependent H/D isotope effect and pharmacokinetic application to inhaled ibuprofen-arginine in mice.

GC-MS and GC-MS/MS methods were developed and validated for the quantitative determination of ibuprofen (d ₀ -ibuprofen), a non-steroidal anti-inflammatory drug (NSAID), in human plasma using α-methyl- ² H ₃ -4-(isobutyl)phenylacetic acid (d ₃ -ibuprofen) as internal standard. Plasma (10μL) was diluted with acetate buffer (80μL, 1M, pH 4.9) and d ₀ - and d ₃ -ibuprofen were extracted with ethyl acetate (2×500μL). After solvent evaporation d ₀ - and d ₃ -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 d ₀ - and d ₃ -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 CO ₂ (44 Da). A collision energy-dependent H/D isotope effect was observed, which involves abstraction/elimination of H ^- from d ₀ -ibuprofen and D ^- from d ₃ -ibuprofen and is minimum at a CE value of 5eV. 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 d ₀ -ibuprofen and m/z 208 to m/z 164 for d ₃ -ibuprofen. In a therapeutically relevant concentration range (0-1000μM) d ₀ -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, r ² =0.9991). In incubation mixtures of arachidonic acid (10μM), d ₃ -ibuprofen (10μM) or d ₀ -ibuprofen (10μM) with ovine cyclooxygenase (COX) isoforms 1 and 2, the concentration of d ₃ -ibuprofen and d ₀ -ibuprofen did not change upon incubation at 37°C up to 60min. 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 2.5mg/kg body weight) for three days. A linear relationship between ibuprofen concentration in serum (10μL) and administered dose 24h after the last drug administration was observed.
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