Determination of bronchoalveolar lavage bile acids by solid phase microextraction liquid chromatography-tandem mass spectrometry in combination with metabolite profiling: comparison with enzymatic assay

Abstract

A thin-film solid-phase microextraction (SPME) method coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was developed for high-throughput determination of bronchoalveolar lavage bile acids. The proposed method was validated according to the bioanalytical method validation guidelines. LOQ and LLOQ were 0.007 and 0.02 μmol/L, respectively. The accuracy and the precision were <7 and <4%, respectively. The performance of the proposed method was also compared with an optimized enzymatic cycling assay. Results showed a weak correlation between total concentration of bile acid (BAs) obtained with enzymatic assay and cumulative concentration of specific BAs determined with SPME-LC-MS/MS. This discrepancy was probably due to the presence of other BAs in bronchoalveolar lavage fluid (BALF) samples. Metabolites profiling of BALF samples extracts using a high-resolution mass spectrometer (HRMS) revealed the presence of additional BAs, which were not included in the proposed method. After considering these additional BAs, a strong correlation was found between the LC-MS method and the enzymatic assay. Unsupervised statistical analysis conducted on HRMS data also showed clear separation within BALF samples, depending on the presence of BAs and other lipids. SPME-LC-MS-based metabolites profiling may provide additional information for diagnosis occurrence and severity of gastric reflux/aspiration in lung transplant patients.

Keywords: Bile acids; Bronchoalveolar lavage; Enzymatic assay; LC-MS; Metabolomics; SPME.