Bile acids derived from cholesterol are produced in the liver, and their analysis is difficult due to their complex natures and their low concentrations in biological fluids. Mixtures of various derivatives, created via conventional heating, are used for such analyses. Microwave radiation is proposed to accelerate the derivatization process. This paper presents a mass fragmentation study and microwave-assisted derivatization (MAD) for the silylation of bile acids (cholic and ursodesoxycholic) prior to gas chromatography and mass spectrometry analysis. The derivatization was performed using the two-step process of methoximation and silylation. The reaction time, power, and quantity of N,O-bis-(trimethylsilyl) trifluoroacetamide (BSTFA) + 1% trimethylchlorosilane (TCMS) were optimized to improve the derivatization. The optimized derivatization conditions required 210？W for 3 min. The MAD method exhibited linearity with respect to cholic acid between 0.78 and 20.0？μg mL？1 with an LOQ of 0.23？μg mL？1 and a precision ranging from 1.08% to 9.32% CV. This optimized derivatization method is valid for the analysis of bile acids in different matrices. 1. Introduction Bile acids (BAs) are steroidal compounds synthesized in the liver during cholesterol metabolism, and their major structural components include a steroid nucleus with a side chain and carboxyl groups [1–3]. BAs are predominantly present in biological fluids in their ionized form. The composition of BA in serum and urine varies with different physicochemical properties and the rate of intestinal absorption by the liver [2, 4]. Its hepatic and intestinal metabolism can also be influenced by liver and gastrointestinal diseases. Therefore, modifications to the hepatic synthesis, intracellular metabolism, hepatic uptake, and biliary excretion can result in a disturbance in the metabolism of bile acids [1, 4, 5]. An increase in blood bile salts appears to result from changes in their hepatocellular uptake that are induced by chemical substances and can serve as a biological marker for the detection of liver damage [1, 4–6]. Bile acids are present in serum and urine at millimolar levels, and due to large differences in their chemical properties, such as lipophilicity and polarity, their separation and identification require accurate and sensitive methods. Several analytical methods based on chromatography techniques have been reported for detecting bile acids in biological fluids [1, 7–9]. Liquid chromatography (LC) coupled with evaporative light-scattering detection (ELSD) or conventional UV-Vis detection
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