title: 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 isotope effect and pharmacokinetic application to inhaled ibuprofen-arginine in mice.
abstract: 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 (10μL) was diluted with acetate buffer (80μL, 1M, pH 4.9) and d0- and d3-ibuprofen were extracted with ethyl acetate (2×500μL). 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 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 d0-ibuprofen and m/z 208 to m/z 164 for d3-ibuprofen. In a therapeutically relevant concentration range (0-1000μM) 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 (10μM), d3-ibuprofen (10μM) or d0-ibuprofen (10μM) 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 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.
authors.affiliation: Centre of Pharmacology and Toxicology, Hannover Medical School, Hannover, Germany. Electronic address: tsikas.dimitros@mh-hannover.de.
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Tsikas
authors.affiliation: Centre of Pharmacology and Toxicology, Hannover Medical School, Hannover, Germany.
authors.forename: Arslan Arinc
authors.initials: AA
authors.lastname: Kayacelebi
authors.affiliation: Centre of Pharmacology and Toxicology, Hannover Medical School, Hannover, Germany.
authors.forename: Erik
authors.initials: E
authors.lastname: Hanff
authors.affiliation: Institute of Clinical Pharmacology, Hannover Medical School, Hannover, Germany.
authors.forename: Anja
authors.initials: A
authors.lastname: Mitschke
authors.affiliation: Institute of Occupational Medicine, Hannover Medical School, Hannover, Germany.
authors.forename: Bibiana
authors.initials: B
authors.lastname: Beckmann
authors.affiliation: Inhalics Pharmaceuticals GmbH, Vacallo, Switzerland.
authors.forename: Hanns-Christian
authors.initials: HC
authors.lastname: Tillmann
authors.affiliation: Institute of Clinical Pharmacology, Hannover Medical School, Hannover, Germany.
authors.forename: Frank-Mathias
authors.initials: FM
authors.lastname: Gutzki
authors.affiliation: Fraunhofer Institute for Toxicology and Experimental Medicine, Hannover, Germany.
authors.forename: Meike
authors.initials: M
authors.lastname: Müller
authors.affiliation: Inhalics Pharmaceuticals GmbH, Vacallo, Switzerland.
authors.forename: Corrado
authors.initials: C
authors.lastname: Bernasconi
mesh_terms: D000085:Acetates; D000818:Animals; D003903:Deuterium / Q000097:blood / Q000737:chemistry / Q000378:metabolism; D005260:Female; D005464:Fluorobenzenes; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006801:Humans; D007052:Ibuprofen / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D057230:Limit of Detection; D016014:Linear Models; D059625:Liquid-Liquid Extraction / Q000379:methods*; D051379:Mice; D008807:Mice, Inbred BALB C; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*; D005466:Fluorocarbons
chemical_list: D000085:Acetates; D003903:Deuterium; D005464:Fluorobenzenes; D007052:Ibuprofen; C025063:pentafluorobenzyl bromide; D005466:Fluorocarbons; C007650:ethyl acetate
keywords: Collision-induced dissociation; Derivatization; Electron-capture negative-ion chemical ionization; H/D isotope effects; Quantification; Solvent extraction