Study on the effect of calibration standards prepared with different matrix on the accuracy of bile acid quantification using LC-MS/MS.

The transition from relative to absolute quantification of metabolites is the future development trend of mass spectrometry-based metabolomics research, which could fundamentally solve the problem of comparability of data between different laboratories. However, absolute quantification of endogenous molecules is largely hampered by the lack of analyte-free matrix, leading to uncertainty and inconsistency in the preparation of calibration standards. Bile acids (BAs) are an important class of biomarkers that play a key role in disease progression. In this paper, the quantitative accuracy of calibration curves prepared in neat solvent (NSCCs), charcoal stripped matrix (SMCCs) and authentic matrix (AMCCs) were validated using quality control samples (QCs) prepared in authentic matrix. Results suggested that AMCCs could largely minimize the confidence interval (C.I.) and the deviation in accuracy compared with NSCCs and SMCCs when measured concentration is higher than 20% of the background level. In addition, experimental data demonstrated that two-step calibration strategy proposed here is a promising and reliable alternative strategy to quantify endogenous BAs in biological sample.