Quantification of plasma bile acids and bilirubin

Quantification of plasma bile acids and bilirubin

1.     Quantification of bile acids

Blood sample collection:

1-2 ml whole blood was collected from patients or healthy donors by collaborative doctors using BD Vacutainer EDTA Tubes (BD Cat. No. 367841). The collected whole blood was immediately used for plasma separation or stored at 4°C until the following plasma separation step (within 12h).

Plasma separation:

Transfer 1 ml of collected whole blood into a 1.5 ml centrifuge tube, and centrifuge at 1000xg at 4°C for 10 min. Collect the upper layer plasma and store at -80°C until following sample preparation.

Sample preparation:

100 µl plasma sample was transferred to a 1.5 ml centrifuge tube, then add 400 ul acetonitrile (Acetonitrile, Optima™, for HPLC and GC, Fisher Chemical). Mix them thoroughly by vortexing for 10 seconds and left to sit at 4°C for 20 min. The mixture was centrifuged at 11000xg, at 4°C for 20 min, and the supernatant was transferred to a new 1.5 ml centrifuge tube and concentrated at 45°C until completely dry (Eppendorf Concentrator plus™). The dried residue was then re-dissolved in 100 ul 60% methanol (Methanol, Optima™ for HPLC, Fisher Chemical) for the following HPLC-MS/MS quantification or stored at -80°C for up to 3 months.

Quantification of plasma bile acids by HPLC-MS/MS:

The bile acid level in plasma samples was measured using HPLC-MS/MS (Agilent model LC1260 QQQ 6495). Chromatographic separation was performed in an ACQUITY UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm; Waters Corp.). In order to make the MS response fall within the linear range of standard curve for accurate quantification, two concentrations (no dilution and 1:100 dilution) for each sample was used for running HPLC/MS/MS. Before sample running, the chromatographic column was balanced in 10% acetonitrile for 20 min at 0.3 ml/min. Then 5 ul sample was loaded into the chromatographic column. The chromatographic condition (including mobile phase compositions, gradients, and running time) was shown in Table 1. Mass spectrometric was performed in negative ion mode. Two MRM identifiers were used for recognizing targeted bile acids (Table 2). The more intensive signals were used for quantitation, the weaker ones combining with the chromatographic retention times (Table 3) were used for auxiliary identification. 100 nM deoxycholic-2,2,4,4,11,11-d⁶ acid (Sigma-Aldrich, cat. # 809675) was added into each sample as an internal control to calibrate the matrix effect. For every batch of quantification, pure bile acid mix (CA, CDCA, DCA, LCA, GCA, GCDCA, GDCA, GLCA, TCA, TCDCA, TDCA and TLCA) with different concentrations (10 nM, 25 nM, 50 nM, 100 nM, 250 nM, 500 nM, 750 nM, 1000 nM) was used for making a standard curve for absolute quantification. The Agilent data analysis software was used for calculating bile acid concentration.

Table 1. Chromatographic conditions

Table 2. Mass spectrometric parameter for bile acid

Table 3. Retention time of each bile acid

2.     Quantification of bilirubin

Total bilirubin and direct bilirubin values were obtained from the patients’ hospital blood chemistry reports, measured by Beckman Coulter – AU5800 chemistry analyzer.