Optimization of Pretreatment Method of Gas Chromatography-Mass Spectrometry for Determination of 37 Fatty Acids in Human Plasma

Single-factor and orthogonal experiments were used to optimize the catalytic reaction conditions of H₂SO₄, and the 37 fatty acids in the human plasma samples were determined by gas chromatography-mass spectrometry. The samples were thawed at 4℃, and an aliquot (100 μL) of the sample was added to 500 μL of CH₃OH solution containing 0.4 mol·L^-1 KOH. The mixture was vortexed for 30 s, and settled at room temperature for 10 min. n-Hexane of 2 mL was added, and the mixture was vortexed for 5 min. CH₃OH solution (1 mL) containing 7% (volume fraction) H₂SO₄ was added into the supernatant, and the mixed solution was blown to dry by N₂. After adding 200 μL of H₂O to promote reaction at 65℃ for 20 min, the solution was cooled down to room temperature, and mixed with 2 mL of n-hexane. The supernatant was introduced into gas chromatograph with flame ionization detector, and the targets were separated on Rt-2560 column with temperature program and detected with mass spectrometer equipped with electron impact ion source. It was shown that the catalytic reaction factors affecting the fatty acid determination were reaction temperature, volume fraction of H₂SO₄, reaction time, in which the reaction temperature had a significant effect (P ≤ 0.05). The 37 fatty acids could be separated by chromatography within 78 min, and linear relationships between values of peak area and mass concentration were found in the same range of 0.1-50 mg·L^-1, with detection limits (3S/N) of 0.003 0-0.411 0 mg·L^-1. Recovery tests were made on quality control samples at three concentration levels (1, 5, 10 mg·L^-1) by standard addition method, giving recoveries in the range of 80.0%-118%. Five repeated determinations and 5 d continuous determinations were made on quality control samples, and RSDs of the determined values for the inter-day and intra-day precision tests were found in the ranges of 0.27%-6.2% and 0.91%-8.7%. The proposed method was applied to the analysis of 1 913 plasma samples in 4 peculiar ethnic minorities in Yunnan, and it was found that the total content of fatty acids was higher in obese group compared to the healthy control group, and there were differences in the distribution of fatty acids in the plasma of the 4 ethnic minorities.