基于超高效液相色谱法快速测定短链脂肪酸方法的建立
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摘要
建立了采用超高效液相色谱法全面快速分析粪便与发酵液中短链脂肪酸的方法。以InertSustain AQ-C18色谱柱对粪便与发酵液中短链脂肪酸进行分离,流速为0.5 mL/min,波长为210 nm,流动相为20 mmol/L NaH_2PO_4(pH 2.2)与乙腈进行梯度洗脱。结果表明:该方法能在10 min内快速分离11种短链脂肪酸,加标培养基中各短链脂肪酸回收率为95.15%～107.01%,精密度为0.58%～7.33%,加标粪便样本中各短链脂肪酸回收率为98.43%～105.14%,精密度为1.13%～5.20%。该方法可用于粪便、发酵液中短链脂肪酸的检测,具有快速、简单及全面等优点。
A rapid ultra-performance liquid chromatography method is developed to analyze the changes of short chain fatty acids in feces and fermentation broth. After a simple step of pretreatment,the feces and fermentation broth samples are analyzed by the UPLC column InertSustain AQ-C18 with the total flow rate 0.5 mL/min,at wavelength of 210 nm,20 mmol/L NaH_2PO_4 and acetonitrile(pH 2.2) are chosen as the mobile phase. A total of 11 short chain fatty acids are quickly detected. The results show that 11 SCFAs could be effectively separated in 10 minutes,adding standard recovery rate of spiked medium samples ranged from 95.15% to 107.01% and the precision was 0.58% to 7.33%. The adding standard recovery rate of spiked fecal samples ranged from 98.43% to 105.14% and the precision was 1.13% to 5.20%. This method is rapid,simple and comprehensive to provide a systematic understanding of the distribution of short chain fatty acids in the feces,and fermentation broth.
A rapid ultra-performance liquid chromatography method is developed to analyze the changes of short chain fatty acids in feces and fermentation broth. After a simple step of pretreatment,the feces and fermentation broth samples are analyzed by the UPLC column InertSustain AQ-C18 with the total flow rate 0.5 mL/min,at wavelength of 210 nm,20 mmol/L NaH_2PO_4 and acetonitrile(pH 2.2) are chosen as the mobile phase. A total of 11 short chain fatty acids are quickly detected. The results show that 11 SCFAs could be effectively separated in 10 minutes,adding standard recovery rate of spiked medium samples ranged from 95.15% to 107.01% and the precision was 0.58% to 7.33%. The adding standard recovery rate of spiked fecal samples ranged from 98.43% to 105.14% and the precision was 1.13% to 5.20%. This method is rapid,simple and comprehensive to provide a systematic understanding of the distribution of short chain fatty acids in the feces,and fermentation broth.
引文
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[12] DE B S,EECKHAUT V,STEPPE M,et al.Development of a HPLC-UV method for the quantitative determination of four short-chain fatty acids and lactic acid produced by intestinal bacteria during in vitro fermentation[J].J Pharm Biomed Anal,2013,80(3):107-115.
[13] DOSTAL A,BAUMGARTNER J,RIESEN N,et al.Effects of iron supplementation on dominant bacterial groups in the gut,faecal SCFA and gut inflammation:a randomised,placebo-controlled intervention trial in South African children[J].British J Nutr,2014,112(4):547-556.
[14] GAGNé J W,WAKSHLAG J J,SIMPSON K W,et al.Effects of a synbiotic on fecal quality,short-chain fatty acid concentrations,and the microbiome of healthy sled dogs[J].BMC Veter Res,2013,9(1):246.
[15] 王苗,蒋敏,李恒,等.乳酸菌对魔芋甘露低聚糖的降解与利用[J].食品与发酵工业,2016,42(11):20-24.
[2] 谭力,鞠熀先,黎介寿.生物样品中短链脂肪酸的提取与测定[J].色谱,2006,24(1):81-87.
[3] 许勤,吴文溪.短链脂肪酸的代谢及其在肠道外科中的应用[J].肠外与肠内营养,1999,6(4):218-223.
[4] 刘松珍,张雁,张名位,等.肠道短链脂肪酸产生机制及生理功能的研究进展[J].广东农业科学,2013,40(11):99-103.
[5] KACZMARCZYK M M,MILLER M J,FREUND G G.The health benefits of dietary fiber:beyond the usual suspects of type 2 diabetes mellitus,cardiovascular disease and colon cancer[J].Metab Clin Exp,2012,61(8):1058.
[6] 付文政,张春泽,贾岩峰,等.粪便中短链脂肪酸检测及临床意义[J].中国肛肠病杂志,2016,36(4):7-9.
[7] MOKARRAM P,NAGHIBALHOSSAINI F,SABERI FIROOZI M,et al.Methylenetetrahydrofolate reductase C677T genotype affects promoter methylation of tumor-specific genes in sporadic colorectal cancer through an interaction with folate/vitamin B12 status[J].World J Gastroenterol,2008,14(23):3662-3671.
[8] EWASCHUK J B,ZELLO G A,NAYLOR J M,et al.Metabolic acidosis:separation methods and biological relevance of organic acids and lactic acid enantiomers[J].J Chromatogr B,2002,781(1/2):39-56.
[9] 张萍,叶利民,肖晓蓉,等.短链脂肪酸的梯度高效液相色谱法分析研究[J].华西口腔医学杂志,2001,19(5):294-295.
[10] 刘娟,杨柳,马浩,等.离子排斥色谱法测定人体粪便中五种短链脂肪酸[J].分析试验室,2013(9):104-107.
[11] GARDANA C,DEL B C,SIMONETTI P.Validation and application of an ultrahigh-performance liquid chromatographic-orbitrap mass spectrometric method for the simultaneous detection and quantification of volatile and non-volatile organic acids in human faecal samples[J].J Pharm Biomed Anal,2017,141:46-51.
[12] DE B S,EECKHAUT V,STEPPE M,et al.Development of a HPLC-UV method for the quantitative determination of four short-chain fatty acids and lactic acid produced by intestinal bacteria during in vitro fermentation[J].J Pharm Biomed Anal,2013,80(3):107-115.
[13] DOSTAL A,BAUMGARTNER J,RIESEN N,et al.Effects of iron supplementation on dominant bacterial groups in the gut,faecal SCFA and gut inflammation:a randomised,placebo-controlled intervention trial in South African children[J].British J Nutr,2014,112(4):547-556.
[14] GAGNé J W,WAKSHLAG J J,SIMPSON K W,et al.Effects of a synbiotic on fecal quality,short-chain fatty acid concentrations,and the microbiome of healthy sled dogs[J].BMC Veter Res,2013,9(1):246.
[15] 王苗,蒋敏,李恒,等.乳酸菌对魔芋甘露低聚糖的降解与利用[J].食品与发酵工业,2016,42(11):20-24.