Establishment and preliminary clinical application of serum bile acid spectrum analysis by ultra high performance liquid chromatography-tandem mass spectrometry

Abstract

Objective To establish an ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for detecting the bile acid expression in serum and to explore the relationship between the bile acid spectrum and the obesity. Methods After pre-treatment through solid phase extraction, serum samples were chromatographed by gradient elution on an UPLC HSS T3 column, and then mass spectrometric analysis of bile acid was performed by multiple reaction monitoring (MRM) mode with negative electrospray ionization (ESI).And its methodology performance, including specificity, linearity, sensitivity, imprecision and recovery, were verified according to the guideline of State Food and Drug Administration (SFDA).Furthermore，serum samples from 10 simple obesity subjects and 10 healthy controls were analyzed with this established method, and rank sum test was used for statistical analysis. Results This established UPLC-MS/MS method could simultaneously quantify 14 bile acid subgroups in serum sample, its analytical linear range was 10-20 000 nmol/L.For different bile acid subgroups, the limit of detection (LOD) was 0.02-7.90 nmol/L, limit of quantitation (LOQ) was 0.07-44.20 nmol/L; and its within-day coefficient of variation (CV) was 0.35%-12.41%, between-day CV was 1.11%-13.04%; the relative recovery ratio of this method was 89.8%-114.6%.Some differences were observed in chromatogram between obesity and control subjects, and both free bile acid and conjugated bile acid concentrations in obesity subjects ［0.49 （0.45-1.90）, 1.44（0.84-3.72） μmol/L］ were lower than them in control subjects ［0.98（0.53-3.06）, 1.99（0.67-2.88） μmol/L］, but the difference was not significant (Z=-0.958, -0.801, P>0.05). Conclusions This established UPLC-MS/MS method can efficiently differentiate and quantify 14 bile acid subgroups, and is characterized with broad analytical measurement range, good analytical sensitivity and precision.This method can be applied for the free and conjugated bile acid analysis in clinical specimens.(Chin J Lab Med, 2012，35：253-256) Key words: Obesity; Bile acids and salts; Chromatography; high pressure liquid; Tandem mass spectrometry