Development and validation of LC-MS/MS method for the determination of bile acids from biological samples

Bile acid is an atypical steroid molecule synthesised from cholesterol by liver enzymes and intestinal bacteria. It is divided into primary and secondary bile acids. Bile acids become conjugated in the synthesis steps and form different bile acids. Today, liquid chromatography/mass spectrometry (LC-MS/MS), which is used in many different analyses in many different fields, especially in analytical chemistry, is frequently used in biological sample analysis due to its high sensitivity. LC-MS/MS parameters and different stationary phases were optimised for bile acid determination in biological samples using twenty-three different bile acids and one internal standard. For optimisation, MS parameters such as fragmentation voltages, fragmentation energies, ion source, ionising agent, and ion mode were optimised for each bile acid, and parameters such as mobile phase, temperature, flow rate, and stationary phases were tested. According to the obtained optimisation parameters, the analysis was performed in negative ionisation mode with an electrospray ion source, 100 x 3.00 mm, 1.8 µm, 95 Å, using C18 stationary phase at a temperature of 60 °C, with water containing 0.5% formic acid and methanol mobile phase containing 20% acetonitrile, and validated according to the ICH M10 bioanalytical validation guideline under optimum operating conditions. Blood, urine, and stool samples were analysed by applying the preparation procedure for quantitative analysis. The analysis results were evaluated within the limits specified in the ICH M10 bioanalytical validation guideline.