Profiling primaquine metabolites in primary human hepatocytes using UHPLC‐QTOF‐MS with 13C stable isotope labeling

Therapeutic efficiency and hemolytic toxicity of primaquine (PQ), the only drug available for radical cure of relapsing vivax malaria are believed to be mediated by its metabolites. However, identification of these metabolites has remained a major challenge apparently due to low quantities and their reactive nature. Drug candidates labeled with stable isotopes afford convenient tools for tracking drug‐derived metabolites in complex matrices by liquid chromatography‐tandem mass spectrometry (LC‐MS‐MS) and filtering for masses with twin peaks attributable to the label. This study was undertaken to identify metabolites of PQ from an in vitro incubation of a 1:1 w/w mixture of ¹³C₆‐PQ/PQ with primary human hepatocytes. Acquity ultra‐performance LC (UHPLC) was integrated with QTOF‐MS to combine the efficiency of separation with high sensitivity, selectivity of detection and accurate mass determination. UHPLC retention time, twin mass peaks with difference of 6 (originating from ¹³C₆‐PQ/PQ), and MS‐MS fragmentation pattern were used for phenotyping. Besides carboxy‐PQ (cPQ), formed by oxidative deamination of PQ to an aldehyde and subsequent oxidation, several other metabolites were identified: including PQ alcohol, predictably generated by oxidative deamination of PQ to an aldehyde and subsequent reduction, its acetate and the alcohol's glucuronide conjugate. Trace amounts of quinone‐imine metabolites of PQ and cPQ were also detected which may be generated by hydroxylation of the PQ/cPQ quinoline ring at the 5‐position and subsequent oxidation. These findings shed additional light on the human hepatic metabolism of PQ, and the method can be applied for identification of reactive PQ metabolites generated in vivo in preclinical and clinical studies. Copyright © 2013 John Wiley & Sons, Ltd.     

The enthalpies of vaporisation of ionic liquids: new measurements and predictions

The enthalpies of vaporisation, Δ(vap)H(298), of seven ionic liquids (ILs) (four imidazoliums, a pyridinium, a phosphonium and an isouronium) have been determined by temperature programmed desorption using line of sight mass spectrometry. They were: 1-ethyl-3-methylimidazolium bis(pentafluoroethyl)phosphinate, [C(2)C(1)Im][PO(2)(C(2)F(5))(2)]; 1-butyl-3-methylimidazolium octylsulfate, [C(4)C(1)Im][C(8)OSO(3)]; 1-butyl-3-methylimidazolium tetrafluoroborate, [C(4)C(1)Im][BF(4)]; 1-hexyl-3-methylimidazolium tris(pentafluoroethyl)trifluorophosphate, [C(6)C(1)Im][FAP]; 1-butylpyridinium methylsulfate, [C(4)Py][C(1)OSO(3)]; trihexyl(tetradecyl)phosphonium tetrafluoroborate, [P(6,6,6,14)][BF(4)] and O-ethyl-N,N,N',N'-tetramethylisouronium trifluoromethanesulfonate, [C(2)(C(1))(4)iU][TfO]. The values were found to be consistent with a previously proposed, predictive, model in which Δ(vap)H(298) is decomposed into a Coulombic component (computable from the IL density) and van der Waals components from the anion and cation. Two previously predicted values of Δ(vap)H(298) were found to be within 6 kJ mol(-1) of the measured experimental values. Values for the van der Waals components are tabulated for eleven cations and twelve anions. Predictions are made for Δ(vap)H(298) for 13 ILs with as yet unmeasured Δ(vap)H(298) values (using experimental molar volumes), and for a further 44 ILs using estimated molar volumes.     

Evidence of zinc superoxide formation in the gas phase: comparisons in behaviour between ligated Zn(I/II) and Cu(I/II) with regard to the attachment of O2 or H2O

Singly and doubly charged atomic ions of zinc and copper have been complexed with pyridine and held in an ion trap. Complexes involving Zn(II) and Cu(I) (3d(10)) display a strong tendency to bind with H(2)O, whilst the Zn(I) (3d(10)4s(1)) complexes exhibit a strong preference for the attachment of O(2). DFT calculations show that this latter result can be interpreted as internal oxidation leading to the formation of superoxide complexes, [Zn(II)O(2)(-)](pyridine)(n), in the gas phase. The calculations also show that the oxidation of Zn(I) to form Zn(II)O(2)(-) is promoted by a mixing of the occupied 4s and vacant 4p orbitals on the metal cation, and that this process is facilitated by the presence of the pyridine ligands.     

Development of direct stereoselective and non-stereoselective assays in biological fluids for the enantiomers of a thieno[2,3-b]thiopyran-2-sulfonamide, a topically effective carbonic anhydrase inhibitor

A stereoselective assay for the optical isomers [(S) and (R)] of 5,6-dihydro-4-[(2-methylpropyl)amino]-4H-thieno[2,3-b]thiopyran-2- sulfonamide-7,7-dioxide in human whole blood has been developed. The assay is based on direct enantiomer separation on a chiral stationary phase column of bovine serum albumin attached to silica. The effect of pH, ionic strength, column length and organic modifier on chiral separation has been studied. The assay methodology, based on high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection (252 nm), has been fully validated in the concentration range 25-250 ng/ml of each enantiomer. Since no interconversion of the isomers was observed in vivo for the clinical studies involving the single (S)-enantiomer, a more sensitive (2.5 ng/ml), non-stereoselective assay has been developed. This method, also based on HPLC with UV detection, was fully validated in whole blood, plasma and urine in the concentration range 2.5-100 ng/ml. The details of these assays, together with some representative data from a pilot human study, are also presented.