Simultaneous quantification of daptomycin and rifampicin in plasma by ultra performance liquid chromatography: Application to a pharmacokinetic study

A rapid and simple method based on ultra performance liquid chromatography (UPLC) with ultra violet detection has been developed for the determination of daptomycin (DPT) and rifampicin (RFM) in rabbit plasma using 4-nitrophenol as internal standard (IS). Sample preparation involved protein precipitation with an acetonitrile:methanol mixture and centrifugation. Chromatographic separation was achieved on an Acquity BEH C18 column (100 mm × 2.1 mm, 1.7 μm) using gradient elution with methanol and 0.1% aqueous TFA. The total analysis time was 4.5 min with DPT and RFM eluting at 1.9 and 2.1 min, respectively. The method was fully validated with a lower limit of quantitation (LLOQ) of 2 μg mL⁻¹ for both DPT and RFM. The intra- and inter-day precision, measured as % relative standard deviation, were less than 12.1 for DPT and 10.7 for RFM, respectively. This validated method was successfully applied to a pharmacokinetic study involving intravenous administration of 14 mg kg⁻¹ DPT and 30 mg kg⁻¹ RFM to rabbits.     

Development and validation of an ultra performance liquid chromatography-tandem mass spectrometry method for the quantification of daptomycin in human plasma

A rapid, simple and accurate analytical method based on ultra performance liquid chromatography (UPLC) combined with electrospray ionization (ESI) tandem mass spectrometry (MS/MS) on a hybrid q TOF instrument has been developed and fully validated for the quantification of daptomycin (DPT) in human plasma. The samples were analyzed after simple pretreatment involving protein precipitation, while chromatographic separation of DPT and the internal standard (reserpine) was achieved on an Acquity BEH C18 column (100 mm × 2.1 mm, 1.7 μm) using gradient elution with 0.1% aqueous formic acid (FA) and acetonitrile with 0.1% FA (with DPT eluting at 2.60 min). The method presented good fit (r > 0.999) over the quantification range of 0.01-10 μg mL⁻¹ with the lower limit of quantitation (LLOQ) being 0.01 μg mL⁻¹ of human plasma for DPT. The intra- and inter-day precision, measured as % relative standard deviation, was less than 11% for DPT. The validation results showed that the developed method demonstrated adequate selectivity, sensitivity, precision and accuracy and therefore was successfully applied to the analysis of clinical samples following intravenous (iv) administration of 5.4 mg kg⁻¹ DPT to patients suffering from post-traumatic osteomyelitis induced by methicillin-resistant Staphylococcus aureus (MRSA). The developed methodology is the first report of an accurate mass tandem MS method for the analysis of this potent antibiotic in human plasma and can be used to further study pharmacokinetic, bioequivalence and even metabolic aspects related to this drug.     

Isolation and Characterization of an Acetylated Impurity in Escherichia coli-Derived Recombinant Human Interleukin-10 (IL-10) Drug Substance

Pharmaceutical Research -     

Volatiles with antimicrobial activity from the roots of Greek Paeonia taxa

The traditional uses of the roots of Paeonia taxa are described. The volatile constituents, obtained by steam distillation of the roots of Paeonia clusii Stern subsp. clusii, Paeonia mascula L. subsp. hellenica and Paeonia parnassica, were studied by GC/MS. Salicylaldehyde, paeonol, methyl salicylate and benzoic acid were found to be the major components among the twelve constituents identified. The evaluation of the in vitro antimicrobial activity of the volatile compounds against six bacteria and three fungi is also reported.     

Isolation and Characterization of a Monomethioninesulfoxide Variant of Interferon α-2b

Purpose. To isolate and characterize a monomethioninesulfoxide variant of the commercially available therapeutic protein interferon -2b. Methods. The methionine (Met)-oxidized variant was isolated by reverse-phase high performance liquid chromatography and characterized by SDS-PAGE, peptide mapping and mass spectrometric analysis of the trypsin/V8-generated peptide fragments. The biological and immunological activities of the isolated variant were also evaluated. Results. The rHuIFN -2b variant was found to contain a Met sulfoxide residue at position 111 of the rHuIFN -2b molecule. The far-UV CD spectra showed a slight loss of -helical content and an increase in the -sheet contribution. The CD spectra indicate that both chromatographic conditions and Met oxidation contribute to the observed secondary structure changes. Both interferon -2b main component and its methionine-oxidized variant showed different reactivity to monoclonal antibodies employed in immunoassays for the protein. Conclusions. A monomethioninesulfoxide rHuIFN -2b variant was found to be present in the rHuIFN -2b bulk drug substance in solution. The Met¹¹¹ residue was identified as Met sulfoxide by comparative tryptic/V8 mapping and mass spectrometric analysis. Nevertheless, the oxidation of the Met¹¹¹ residue did not seem to have a detectable effect on the biological activity of the molecule.