Optimization of Pretreatment Method of Gas Chromatography-Mass Spectrometry for Determination of 37 Fatty Acids in Human Plasma
摘 要
采用单因素试验和正交试验优化硫酸催化反应条件,以气相色谱-质谱法测定人血浆样品中37种脂肪酸含量。样品于4℃融化,分取100 μL,加入500 μL含0.4 mol·L-1氢氧化钾的甲醇溶液,涡旋30 s,室温放置10 min。加入2 mL正己烷,离心5 min,在上清液中加入含7%(体积分数)硫酸(催化剂)的甲醇溶液1 mL,吹氮至干,加入200 μL水,于65℃反应20 min。冷却至室温,加入2 mL正己烷,分取上层清液注入附氢火焰离子化检测器的气相色谱仪,目标物经Rt-2560色谱柱固定后在程序升温条件下分离,以配电子轰击离子源的质谱仪检测。结果显示:影响脂肪酸测定的催化反应因素分别为反应温度、硫酸体积分数、反应时间,其中反应温度具有显著性影响(P≤0.05);37种脂肪酸可在78 min内完成色谱分离,其质量浓度均在0.1~50 mg·L-1内与其对应的峰面积呈线性关系,检出限(3S/N)为0.003 0~0.411 0 mg·L-1;对质控样品进行3个浓度水平(1,5,10 mg·L-1)的加标回收试验,回收率为80.0%~118%。对质控样品重复测定5次和连续测定5 d,所得测定值的相对标准偏差分别为0.27%~6.2%(日内精密度试验)和0.91%~8.7%(日间精密度试验)。方法用于分析云南4个特有少数民族人群1 913个血浆样品,发现肥胖组脂肪酸总含量高于正常组,且脂肪酸种类及含量在各民族血浆间分布存在差异。
Abstract
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.
中图分类号 O657.63 DOI 10.11973/lhjy-hx202205001
所属栏目 试验与研究
基金项目 国家自然科学基金(81360427)
收稿日期 2021/10/11
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备注钱映,硕士研究生,主要研究方向为营养与食品卫生学
引用该论文: QIAN Ying,LI Yanru,FENG Yuemei,WANG Songmei,ZHONG Dubo,YIN Jianzhong. Optimization of Pretreatment Method of Gas Chromatography-Mass Spectrometry for Determination of 37 Fatty Acids in Human Plasma[J]. Physical Testing and Chemical Analysis part B:Chemical Analysis, 2022, 58(5): 497~505
钱映,李艳茹,冯月梅,王松梅,钟读波,殷建忠. 用于测定人血浆中37种脂肪酸含量的气相色谱-质谱法前处理方法的优化[J]. 理化检验-化学分册, 2022, 58(5): 497~505
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【3】任晓莹,梁向艳,刘媛,等.游离脂肪酸受体4调控能量代谢的途径与分子机制[J].医学综述, 2020,26(4):636-640.
【4】HUANG X, SJÖGREN P, ÄRNLÖV J, et al. Serum fatty acid patterns, insulin sensitivity and the metabo-lic syndrome in individuals with chronic kidney disease[J]. Journal of Internal Medicine, 2014,275(1):71-83.
【5】WU J H, MICHA R, MOZAFFARIAN D. Dietary fats and cardiometabolic disease:Mechanisms and effects on risk factors and outcomes[J]. Nature Reviews Cardiology, 2019,16(10):581-601.
【6】GONZÁLEZ-BECERRA K, RAMOS-LOPEZ O, BARRÓN-CABRERA E, et al. Fatty acids, epigenetic mechanisms and chronic diseases:A systematic review[J]. Lipids in Health and Disease, 2019,18(1):178.
【7】SHARP G C, RELTON C L. Epigenetics and noncommunicable diseases[J]. Epigenomics, 2017,9(6):789-791.
【8】李珮芸.孕期血浆脂肪酸组成与妊娠糖尿病的关联性研究[D].武汉:华中科技大学, 2019.
【9】CHRISTINAT N, MORIN-RIVRON D, MASOODI M. High-throughput quantitative lipidomics analysis of nonesterified fatty acids in plasma by LC-MS[J]. Methods in Molecular Biology (Clifton, N.J.), 2017,1619:183-191.
【10】ZHANG H G, WANG Z Y, LIU O. Development and validation of a GC-FID method for quantitative analysis of oleic acid and related fatty acids[J]. Journal of Pharmaceutical Analysis, 2015,5(4):223-230.
【11】ECKER J, SCHERER M, SCHMITZ G, et al. A rapid GC-MS method for quantification of positional and geometric isomers of fatty acid methyl esters[J]. Journal of Chromatography B, 2012,897:98-104.
【12】LU Y H, WANG Y L, ONG C N, et al. Metabolic signatures and risk of type 2 diabetes in a Chinese population:An untargeted metabolomics study using both LC-MS and GC-MS[J]. Diabetologia, 2016,59(11):2349-2359.
【13】TAN B B, ZHANG Y, ZHANG T T, et al. Identi-fying potential serum biomarkers of breast cancer through targeted free fatty acid profiles screening based on a GC-MS platform[J]. Biomedical Chromatography, 2020,34(10):e4922.
【14】CHIU H H, KUO C H. Gas chromatography-mass spectrometry-based analytical strategies for fatty acid analysis in biological samples[J]. Journal of Food and Drug Analysis, 2020,28(1):60-73.
【15】ROBERTS L D, MCCOMBIE G, TITMAN C M, et al. A matter of fat:An introduction to lipidomic profiling methods[J]. Journal of Chromatography B, 2008,871(2):174-181.
【16】CIUCANU C I, VLAD D C, CIUCANU I, et al. Selective and fast methylation of free fatty acids directly in plasma for their individual analysis by gas chromatography-mass spectrometry[J]. Journal of Chromatography A, 2020,1624:461259.
【17】HAN L D, XIA J F, LIANG Q L, et al. Plasma esterified and non-esterified fatty acids metabolic profiling using gas chromatography-mass spectrometry and its application in the study of diabetic mellitus and diabetic nephropathy[J]. Analytica Chimica Acta, 2011,689(1):85-91.
【18】LIU L Y, LI Y, FENG R N, et al. Direct ultrasound-assisted methylation of fatty acids in serum for free fatty acid determinations[J]. Canadian Journal of Chemistry, 2010,88(9):898-905.
【19】CUI Y, CHEN X B, LIU L Y, et al. Gas chromatography-mass spectrometry analysis of the free fatty acids in serum obtained from patients with Alzheimer's disease[J]. Bio-Medical Materials and Engineering, 2015,26(S1):2165-2177.
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