A feasible protocol to profile bile acids in dried blood spots from rats using a UHPLC-MS/MS method combining a surrogate matrix

Dried blood spot (DBS) sampling is a promising method for microliter blood sample collection with the advantages of convenient transportation, storage and clinical operations. However, it is challenging to develop an analytical protocol to determine endogenous metabolites, such as bile acids (BAs) in DBSs, due to the low-blood-volume character of DBSs and the complex features of filter paper. Herein, we developed a method of fast ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) to profile and quantify BAs in DBSs. The pretreatment methods were optimized and a two-step solvent addition method, where a small amount of water was firstly added to moisten the DBS and then methanol was added, showed high extraction efficiency for multiple BAs in DBSs. The UHPLC-MS/MS conditions were optimized and 35BAs in different types could be profiled with good resolution and quantified with acceptable precision and accuracy. Preparation of a DBS surrogate matrix without endogenous BAs has been well developed using rat erythrocytes in BSA solution and showed good performance on both the signal suppression/enhancement percentage and parallelism assessment evaluation of three different stable-isotope-labeled (SIL) BAs. The established protocol was successfully applied to profile BAs in DBSs of intrahepatic cholestasis model and healthy control rats with good repeatability. To our knowledge, it is the first time that 35 BAs in DBSs could be well profiled and an appropriate DBS surrogate matrix has been developed. This protocol presents future-oriented applications of DBSs for relevant preclinical studies to profile BAs and probe biomarkers.