Identification of saturated and unsaturated fatty acids as microsomal degradation by-products by direct high resolution -ESI-MS/MS

Abstract

In vitro clearance in liver microsomes is routinely measured in drug discovery and development for understanding the disposition properties of new chemical entities. Literature reports indicate that long chain fatty acids such as arachidonic, linoleic and oleic acids may be released over a period of time during a microsomal incubation. These fatty acids are known to be substrates for conjugating enzymes like Uridine diphosphoglucuronosyl transferases (UGTs), thus potentially inhibiting microsomal clearance of UGT substrates. The present study was aimed at deciphering the fatty acids that may be released as by-products of microsomal degradation. LC-MS analytical methods were developed to characterize and qualitatively assess the fatty acids without chemical derivatization in rat, monkey and human liver microsomes. Additionally, incubations with UDPGA were utilized to trap the released fatty acids in their glucuronate ester form, also characterized and confirmed by high resolution LC-MS/MS. Our results indicate for the first time that eicosapentanoic, trans- and cis- eicosenoic, docosanoic, and nervonic acid were released as microsomal by-products in incubation conditions. Our results corroborate that palmitic, stearic, linoleic, and arachidonic acid were also formed as previously reported. Additionally, α- and γ-linolenic, eicosapentaenoic, palmitoleic, linoleic, arachidonic, palmitic, oleic, and stearic acid were identified as their corresponding acyl-glucuronides in rat, monkey and human liver microsomes. Our investigation renders a deeper understanding about the fatty acids which may be released by degradation of liver microsomes