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The detection and quantification of insulin and its therapeutic analogs is important for medical, sports doping, and forensic applications. Synthetic variants contain slight sequence variations to affect bioavailability. To reduce sample handling bias, a universal extraction method is required for simultaneous extraction of endogenous and variant insulins with subsequent targeted quantification by LC-MS. A mass spectrometric immunoassay (MSIA), a multiplexed assay for intact insulin and its analogues that couples immunoenrichment with high resolution and accurate mass (HR/AM) spectrometric detection across the clinical range is presented in this report. The assay is sensitive, selective, semi-automated and can potentially be applied to detect new insulin isoforms allowing their further incorporation into second or third generation assays., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Ciclosporin (CsA) is metabolized exclusively by the hepatic cytochrome P-450 mixed function oxidase system. Ketoconazole (KC) is a potent inhibitor of this enzyme system. CsA was administered alone and in combination with five different doses of KC (1.25, 2.5, 5.0, 10.0, 20.0 mg/kg/day) under steady-state conditions to 7 adult mongrel dogs. KC produced a highly significant (p = 0.0001), dose-dependent decrease in CsA total body clearance [Cl(T)]. The critical KC dosage range for this to occur was found to be between 2.5 and 10 mg/kg/day. The reduction of CsA CL(T) was insignificant (p greater than 0.05) at a KC dose of less than 2.5 mg/kg/day, and the 92% reduction observed using 20 mg/kg/day KC was not significantly greater than the 85% reduction occurring after only 10 mg/kg/day KC (p greater than 0.05). The dose of concomitant KC was also highly correlated with a reduction in the whole blood CsA parent/parent + metabolite ratio as determined using high-performance liquid chromatography and polyclonal fluorescent polarization immunoassay for CsA measurement (r = 0.998, p less than 0.0001). The absolute oral bioavailability of CsA as well as the time required to reach its maximum concentration in the blood following oral administration did not change significantly over the course of the study (p greater than 0.05). We conclude from these new observations that the KC-induced decrease in CsA Cl(T) in the dog in vivo is dose-dependent and maximized within the KC dosage range of 2.5-10 mg/kg/day. The effect does not appear to involve a decrease in the rate of CsA oral absorption, and may be compensated for by an appropriate reduction in the concomitantly administered dose of CsA.