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Simple Analysis of Bile Acids by GC-MS

30 Jun 2016

Bile acids are a group of steroidal acids with carboxyl and hydroxyl groups on the side. They are the major metabolic products of cholesterol. Bile acids play important roles as biomarkers for early diagnosis and therapeutic monitoring of many diseases, especially liver and intestinal diseases. For instance, high levels of intestinal bile acids, in particular, deoxcholic acid, is an indication of colon carcinogenesis.[1] Below are structures of some of the most common bile acids.


LC-MS and LC-MS/MS are commonly used for bile acid analysis in biological fluids without derivatization. Disadvantages of LC-based methods include relatively high carry-over on LC columns and low peak capacity.

GC is a robust, simple and inexpensive lab tool for volatile or semi-volatile compound analysis. The only drawback of GC versus HPLC for bile acid analysis is polar functional groups (carboxyl and hydroxyl) need to be derivatized into GC-detectable forms. Trimethylsilylating reagents, such as N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and N-methyl-N(trimethylsilyl)trifluoroacetamide (MSTFA), are commonly used for derivatization of both hydroxyl and carboxyl groups. Choosing the right reagents can be tricky. Kumar et al.[2] compared four types of derivatization mixtures: MSTFA:NH4I:DTE (500:4:2, 40 lL, 60 C, 30 min), MSTFA:TMSI:TMCS (100:2:5, 40 lL, 60 C, 30 min), MSTFA:TMCS (100:1, 40 lL, 60 C, 30 min), and BSTFA:TMCS (100:1, 40 lL, 60 C,30 min). Test results showed that MSTFA:NH4I:DTE (500:4:2, v/w/w) has the highest efficiency for bile acid derivatization. However, the mixture must be freshly prepared and thus is not convenient for fast analysis. Fortunately, my colleague Amanda Rigdon developed a one-step method for cannabinoid acids derivatization using BSTFA+1% TMCS, and the reaction is only 30 min.

Here, the same derivatization method was applied to bile acids, and the preliminary results are very promising. Basically, this reagent simply replaces the hydrogen in the –OH or –COOH groups with a trimethylsilyl group (see the picture below). The procedure is simple and straightforward.

  • Place 100 µL of bile acid standards (~100 ppm) into an interlock vial
  • Evaporate the solvent under nitrogen
  • Add 50 µL of ethyl acetate and 50 µL of BSTFA+1% TMCS
  • Incubate for 30 min at 70 °C
  • Cool down and dilute with ethyl acetate if necessary


The derivatized bile acid standards were analyzed by GC-MS under full scan mode using an Rxi-5ms 30 m × 0.25 mm × 0.25 µm column. The chromatogram below shows good separation of closely related bile acids (isomers and enantiomers), such as HDCA and UDCA.


The reaction and GC conditions (reagent/bile acid amount ratio, reaction temperature, time, column dimensions, etc.) can be further optimized. Hopefully, this preliminary work could open the door for the fast GC analysis of bile acids. Keep in mind there are some limitations on conjugated bile acids.

GC analysis time too long? EZGC can help! Here is one of the modulation solutions using shorter column. The total analysis time is less than 10 min.

ezgc modulation1.xlsx


[1] (a) Jensen, R. G.; Clark, R. M.; deJong, F. A.; Hamosh, M.; Liao, T. H.; Mehta, N. R., The lipolytic triad: human lingual, breast milk, and pancreatic lipases: physiological implications of their characteristics in digestion of dietary fats. Journal of pediatric gastroenterology and nutrition 1982, 1 (2), 243-55; (b) Reddy, B. S., Diet and excretion of bile acids. Cancer research 1981, 41 (9 Pt 2), 3766-8.

[2] Kumar, B. S.; Chung, B. C.; Lee, Y. J.; Yi, H. J.; Lee, B. H.; Jung, B. H., Gas chromatography-mass spectrometry-based simultaneous quantitative analytical method for urinary oxysterols and bile acids in rats. Anal. Biochem. 2011, 408 (2), 242-52.