Development and validation of a highly sensitive and robust LC-MS/MS with electrospray ionization method for simultaneous quantitation of itraconazole and hydroxyitraconazole in human plasma: Application to a bioequivalence study

摘要

A highly sensitive and specific LC-MS/MS method has been developed for simultaneous estimation of itraconazole (IT-Z) and hydroxyitraconazole (OH-ITZ) with 500 mu L of human plasma using fluconazole as an internal standard (IS). The API-4000 LC-MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique. Solid phase extraction process was used to extract ITZ, OH-IT-Z and IS from human plasma. The total run time was 3.0 min and the elution of ITZ, OH-ITZ and IS occurred at 2.08 min, 1.85 min and 1.29 min, respectively; this was achieved with a mobile phase consisting ofo.2% (v/v) ammonia solution: acetonitrile (20:80, vlv) at a flow rate of 0.50 mL/min on a HyPurity C-18 (50 mm x 4.6 mm, 5 mu m) column. The developed method was validated in human plasma with a lower limit of quantitation of 0.50 ng/mL for both IT-Z and OH-ITZ. A linear response function was established for the range of concentrations 0.5-263 ng/mL (r > 0.998) for both ITZ and OH-ITZ. The intraand inter-day precision values for ITZ a nd OH-ITZ met the acceptance as per FDA guidelines. ITZ and OH-ITZ were stable in the battery of stability studies, viz., bench-top, auto-sampler, dry extract and freeze/thaw cycles. The developed assay method was applied to an oral bioequivalence study in humans.