Quantification of glycocholic acid in human serum by stable isotope dilution ultra performance liquid chromatography electrospray ionization tandem mass spectrometry

A rapid, accurate and sensitive stable isotope dilution ultra performance liquidchromatography electrospray ionization tandem mass spectrometry (ID-UPLC-ESI-MS/MS) method for the determination of glycocholic acid (GCA) in human serum was developed and validated. Serum samples were spiked with D5-glycocholic acid and then pretreated with protein precipitation. The analysis was performed on a Waters BEH C18 column (100 mm × 2.1 mm, 1.7 μm), followed by ESI-MS/MS detection in negative ion mode under multiple reaction monitoring mode. The calibration curves covered a concentration range from 0.2 to 400 ng/mL. The limit of detection and limit of quantification was 0.01 ng/mL and 0.05 ng/mL, respectively. The method showed satisfactory precision on intra-day (2.3-6.1%) and inter-day (2.4-4.6%) analyses and achieved good recovery at three spiked levels (103.7-114.3%). Moreover, this established method was successfully applied for quantification of GCA in serum samples from healthy volunteers, patients with hepatocellular carcinoma (HCC) and patients with other cancers. We demonstrated that the level of GCA in patients with HCC was significantly higher not only than that in healthy controls, but also than that in patients with other cancer, whereas no significant difference of GCA level was observed between healthy control group and other cancers group.