Quantitation of lanosterol in the vitreous humor of rabbits after ocular administration of lanosterol/thermogel formulation by ultra high performance liquid chromatography–tandem mass spectrometry with the electrospray ionization mode

Abstract

Cataracts are the most common cause of blindness worldwide affecting tens of millions of people. Here, we report a simple, rapid, sensitive and specific method by ultra performance liquid chromatography–tandem mass spectrometry with the electrospray ionization mode (UPLC–ESI-MS/MS) for quantitation of lanosterol, a possible effective drug for cataracts, in the vitreous humor of rabbits after ocular administration. The injected lanosterol was prepared by dispersing lanosterol molecules into the poly-(dl-lactic acid-co-glycolic acid)–poly(ethylene glycol)–poly-(dl-lactic acid-co-glycolic acid) (PLGA–PEG–PLGA) thermogel solution. The analyte and internal standard (IS, panaxadiol) were extracted by the simple protein precipitation with methanol. The chromatographic separation used an Agilent RRHD SB-C18 column with a methanol mobile phase containing 50mM of ammonium acetate aqueous solution (with 0.1% formic acid) (95:5, v/v). The protonated analyte was quantitated in positive ionization by multiple reaction monitoring (MRM) with a mass spectrometer. The mass transitions m/z 443.5→235 and m/z 461→127 were used to measure the analyte and IS, respectively. The assay exhibited a linear dynamic range of 1–1250ngmL−1 for lanosterol in vitreous samples. The lower limit of quantitation (LLOQ) was 1ngmL−1 with a relative standard deviation (RSD) of less than 15%. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. A run time of 5min per sample offered a throughput of more than 200 samples per day. This validated method was used to analyze vitreous samples of New Zealand white rabbits for pharmacokinetic studies. The results provided useful information on pharmacological action mechanism of lanosterol and were meaningful for cataract treatment among the elderly population.