Technical Note: Improved Extraction Method with Hexane for Gas Chromatographic Analysis of Conjugated Linoleic Acids

Extraction properties of different solvents (chloroform/methanol, hexane/isopropanol, and hexane) were studied for the gas chromatographic analysis of conjugated linoleic acids (CLA) from probiotic bacteria grown in de Man, Rogosa, and Sharpe medium. As compared with chloroform/methanol and hexane/isopropanol, hexane showed comparable extraction efficiency for CLA from unspent de Man, Rogosa, and Sharpe medium, but showed minimal extraction of oleic acid originated from the emulsifier in broth. The extraction efficiency of CLA by hexane was influenced by the broth pH, showing the optimal pH of 7.0. Repeated extraction with hexane increased the yield. Extraction with hexane showed excellent recovery of spiked CLA from the spent broth with up to 97.2% (standard deviation of 1.74%). This represents the highest recovery of CLA from culture broth ever reported. The sample size was also successfully reduced to 0.5 mL to analyze CLA from the broth without impairment of analytical data. This smaller sample size in the 1.5-mL microcentrifuge tube using a small bench-top centrifuge reduced analytical time significantly.