Profiling of bile acids in bovine follicular fluid by fused-core-LC-MS/MS

Bile acids (BAs) are present in follicular fluid (FF) from humans and cattle. This fact has triggered an interest on the role BAs might play in folliculogenesis and their possible association with fertility. To achieve a better understanding about this subject, new methods are needed to provide reliable information about concentrations of the most important BAs in FF. In this context, liquid chromatography-tandem mass spectrometry (LC-MS/MS) offers high specificity with a relatively simple sample workup. We developed and validated a new assay for the quick profiling of the 9 most abundant BAs in follicular fluid from cattle. The method uses 200 μl of FF and can quantify cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA) and their glycine (G) and taurine (T) conjugates. Lithocholic acid (LCA), its conjugates GLCA and TLCA, and sulfated forms, were present in some samples, but their concentration was low compared to other BAs (in average, below 60 ng/ml for LCA, GLCA or TLCA and below 20 ng/ml for their corresponding sulfates). Method performance was studied at three quality controls for each compound in consonance with their physiological concentration. Excellent linearity and recovery were found for all compounds at every control level. Intra-day and between-day precisions (%CV) and accuracies (relative errors) were below 15% for all the compounds. Matrix effects were negligible for most of the analytes. Samples undergoing freeze-thaw showed no degradation of their BAs. The method makes use of a fused-core phenyl column coupled to a triple quadrupole tandem mass spectrometer to achieve chromatographic separation within 5 min. We quantified BAs grouped in four different follicle sizes (3-5 mm, 6-8 mm, 9-14 mm, >15 mm), obtaining a similar relative BA profile for all the sizes, with CA always in higher concentration, ranging between 1600 and 18000 ng/ml, approximately, followed by its conjugate glycocholic acid, GCA, which ranged between 800 and 9000 ng/ml. The highest concentration in CA, DCA or CDCA was always detected in FF stemming from follicles of 6-8 mm. To our knowledge, this is the first report in which BAs subspecies have been detected and quantified in bovine follicular fluid.