食糜样本中11种生物胺同步检测方法的建立

doi:10.11843/j.issn.0366-6964.2020.015

摘要/Abstract

摘要： 旨在建立一种利用液质联用（liquid chromatography mass spectrometry，LC-MS）技术实现对猪肠道食糜中多种生物胺进行同步检测的方法。试验首先优化了丹磺酰氯（dansyl chloride，DNS）对生物胺的衍生条件，并进一步确定了肠道食糜样品中生物胺的最佳净化方法。结果显示，衍生条件在衍生剂浓度为8 mg·mL^-1，衍生温度为40℃的条件下，衍生90 min为最佳。生物胺净化方法确定为先用乙酸乙酯进行提取，衍生结束后再用乙酸乙酯进行萃取的组合方式提取生物胺的效果最好；11种生物胺在5～1 000 μg·L^-1的质量浓度范围内定量结果呈良好的线性关系（R²>0.99），检出限和定量限分别为0.01～0.23 μg·L^-1和0.03～0.76 μg·L^-1；各生物胺的日内相对标准偏差在0.73%～ 7.92%，日间相对标准偏差在0.07%～8.67%。最后采用本方法对仔猪回肠、盲肠、结肠和直肠食糜样品中的生物胺进行检测，初步确定了11种生物胺在不同肠段食糜中的含量分布。通过对仔猪回肠、盲肠、结肠和直肠中的食糜样品进行检测，发现食糜中的生物胺主要为亚精胺、腐胺、尸胺、精胺和组胺，而酪胺、1，7-二氨基庚烷、5-羟色胺和胍基丁胺4种生物胺含量较少，色胺和苯乙胺未检出。综上所述，本试验建立的方法具有结果准确、灵敏度高、重现性好等优点，可用于仔猪不同肠道段食糜中多种生物胺的同步检测。

关键词: 肠道食糜, 生物胺, 检测方法, 液质联用

Abstract: This experiment aimed to establish a method for simultaneous detection of multiple biogenic amines in pig intestinal chyme using liquid chromatography mass spectrometry (LC-MS). The experiment firstly optimized the conditions for derivatization of biogenic amines by dansyl chloride (DNS), and further determined the best method for purifying biogenic amines in intestinal chyme samples. The results showed that the derivatization conditions were optimal when the derivatization agent concentration was 8 mg·mL^-1 and the derivatization temperature was 40℃ for 90 min. The biogenic amine purification method was determined to be the best combination of extraction with ethyl acetate first and extraction with ethyl acetate after derivatization. The quantification results of 11 biogenic amines in the mass concentration range of 5-1 000 μg·L^-1 showed a good linear relationship (R²>0.99). The detection limit and limit of quantification were 0.01 - 0.23 μg·L^-1 and 0.03 - 0.76 μg·L^-1, respectively. The intra-day relative standard deviation of each bioamine was 0.73%-7.92%, and the inter-day relative standard deviation was 0.07%-8.67%. Finally, this method was used to detect biogenic amines in chyme samples from piglets' ileum, cecum, colon and rectum. The content distribution of 11 biogenic amines in different intestinal chyme was preliminarily determined. Detection of chyme samples in piglets' ileum, cecum, colon and rectum found that the biogenic amines in the chyme were mainly spermidine, putrescine, cadaverine, spermine and histamine. Tyramine, 1,7-diaminoheptane,serotonin, and agmatine were detected in very small amounts, and no tryptamine and phenethylamine were detected. In summary, the method established in this test has accurate results, high sensitivity, and good reproducibility, and can be used to detect multiple biogenic amines in chyme of different intestinal sections of piglets simultaneously.

Key words: intestinal chyme, biogenic amine, detection method, LC-MS

中图分类号:

S828.9

任梦雅, 冯潇慧, 解竞静, 文晓宾, 孟庆石, 刘丽莉. 食糜样本中11种生物胺同步检测方法的建立[J]. 畜牧兽医学报, 2020, 51(6): 1320-1331.

REN Mengya, FENG Xiaohui, XIE Jingjing, WEN Xiaobin, MENG Qingshi, LIU Lili. Establishment of Simultaneous Detection of 11 Biogenic Amine in Chyme Samples[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(6): 1320-1331.

参考文献 32

[1]	慕春龙.日粮蛋白质水平对大鼠和仔猪机体代谢和肠道微生物的影响[D].南京:南京农业大学,2016.MU C L.Effects of dietary protein content on host metabolism and gut microbioto in rats and piglets[D].Nanjing:Nanjing Agricultural University,2016.(in Chinese)
[2]	BLACHIER F,DAVILA A M,BENAMOUZIG R,et al.Channelling of arginine in NO and polyamine pathways in colonocytes and consequences[J].Front Biosci,2011,16:1331-1343.
[3]	BODMER S,IMARK C,KNEUBÜHL M.Biogenic amines in foods:histamine and food processing[J].Inflamm Res,1999,48(6):296-300.
[4]	MOHAMMED G I,BASHAMMAKH A S,ALSIBAAI A A,et al.A critical overview on the chemistry,clean-up and recent advances in analysis of biogenic amines in foodstuffs[J].Trac Trends Anal Chem,2016,78:84-94.
[5]	KELLY M T,BLAISE A,LARROQUE M.Rapid automated high performance liquid chromatography method for simultaneous determination of amino acids and biogenic amines in wine,fruit and honey[J].J Chromatogr A,2010,1217(47):7385-7392.
[6]	杨姗姗.食品中生物胺液相色谱检测方法的建立及应用[D].天津:天津科技大学,2016.YANG S S.The development and application of high performance liquid chromatographic of biogenic amines in foods[D].Tianjin:Tianjin University of Science & Technology,2016.(in Chinese)
[7]	SWANN L M,FORBES S L,LEWIS S W.A capillary electrophoresis method for the determination of selected biogenic amines and amino acids in mammalian decomposition fluid[J].Talanta,2010,81(4-5):1697-1702.
[8]	温永柱,范文来,徐岩.GC-MS法定性白酒中的多种生物胺[J].酿酒,2013,40(1):38-41.WEN Y Z,FAN W L,XU Y.Identification of a variety of biogenic amines by GC-MS method in China's liquor[J].Liquor Making,2013,40(1):38-41.(in Chinese)
[9]	CELANO G V,CAFARCHIA C,BUJA F,et al.Biogenic amines determination in cheese[J].Ind Aliment,1992,31(3):764-768.
[10]	HUANG J,GAN N,LV F Y,et al.Environmentally friendly solid-phase microextraction coupled with gas chromatography and mass spectrometry for the determination of biogenic amines in fish samples[J].J Separat Sci,2016,39(22):4384-4390.
[11]	曾立威,蔡翔宇,吴玉杰,等.超高效液相色谱法同时快速测定多种动物源食品中9种生物胺的含量[J].食品安全质量检测学报,2017,8(3):968-974.ZENG L W,CAI X Y,WU Y J,et al.Rapid and simultaneous determination of 9 kinds of biogenic amines in various animal-derived foodstuffs by ultra performance liquid chromatography[J].Journal of Food Safety & Quality,2017,8(3):968-974.(in Chinese)
[12]	刘辰麒,丁卓平,王锡昌.生物胺的检测方法评价[J].现代科学仪器,2006(4):89-91.LIU C Q,DING Z P,WANG X C.Appraisal of biogenic amines determination[J].Modern Scientific Instruments,2006(4):89-91.(in Chinese)
[13]	CASAL S,OLIVEIRA M B P P,FERREIRA M A.Determination of biogenic amines in coffee by an optimized liquid chromatographic method[J].J Liq Chromatogr Relat Technol,2002,25(16):2535-2549.
[14]	PALEOLOGOS E K,KONTOMINAS M G.On-line solid-phase extraction with surfactant accelerated on-column Derivatization and Micellar liquid chromatographic separation as a tool for the determination of biogenic amines in various food substrates[J].Anal Chem,2004,76(5):1289-1294.
[15]	CHEN H C,HUANG Y R,HSU H H,et al.Determination of histamine and biogenic amines in fish cubes (Tetrapturus angustirostris) implicated in a food-borne poisoning[J].Food Control,2010,21(1):13-18.
[16]	ANLI R E,VURAL N,YILMAZ S,et al.The determination of biogenic amines in Turkish red wines[J].J Food Compost Anal,2004,17(1):53-62.
[17]	DUGO G M,VILASI F,LA TORRE G L,et al.Reverse phase HPLC/DAD determination of biogenic amines as dansyl derivatives in experimental red wines[J].Food Chem,2006,95(4):672-676.
[18]	HERNÁNDEZ-BORGES J,D'ORAZIO G,ATURKI Z,et al.Nano-liquid chromatography analysis of dansylated biogenic amines in wines[J].J Chromatogr A,2007,1147(2):192-199.
[19]	YEČIN S,ÜREN A.Biogenic amine content of boza:a traditional cereal-based,fermented Turkish beverage[J].Food Chem,2008,111(4):983-987.
[20]	杨贤庆,翟红蕾,郝淑贤,等.高效液相色谱法测定生物胺衍生条件的优化研究[J].南方水产科学,2012,8(1):49-53.YANG X Q,ZHAI H L,HAO S X,et al.Optimization for determining derivative conditions of biogenic amines by HPLC[J].South China Fisheries Science,2012,8(1):49-53.(in Chinese)
[21]	丁涛,吕辰,柳菡,等.高效液相色谱-四极杆/静电场轨道阱高分辨质谱检测葡萄酒中8种生物胺[J].分析测试学报,2014,33(1):27-32.DING T,LÜ C,LIU H,et al.Determination of eight biogenic amines in red wines by liquid chromatography-quadrupole/electrostatic field orbit trap mass spectrometry[J].Journal of Instrumental Analysis,2014,33(1):27-32.(in Chinese)
[22]	程卫,王艳伟,陈培云,等.HPLC测定虾皮中不同类型的生物胺[J].食品工业,2016,37(2):293-296.CHENG W,WANG Y W,CHEN P Y,et al.Determination of different species of biogenic amines in shrimps by HPLC[J].The Food Industry,2016,37(2):293-296.(in Chinese)
[23]	KUNG H F,LEE Y C,HUANG Y R,et al.Biogenic amines content,histamine-forming bacteria,and adulteration of pork and poultry in tuna dumpling products[J].Food Control,2010,21(7):977-982.
[24]	IGARASHI K,KASHIWAGI K.Modulation of cellular function by polyamines[J].Int J Biochem Cell Biol,2010,42(1):39-51.
[25]	SHAH P,SWIATLO E.A multifaceted role for polyamines in bacterial pathogens[J].Mol Microbiol,2008,68(1):4-16.
[26]	MOUILLÉ B,MOREL E,ROBERT V,et al.Metabolic capacity for L-citrulline synthesis from ammonia in rat isolated colonocytes[J].Biochim Biophys Acta Gen Sub,1999,1427(3):401-407.
[27]	WEI F S,XU X L,ZHOU G H,et al.Irradiated Chinese Rugao ham:changes in volatile N-nitrosamine,biogenic amine and residual nitrite during ripening and post-ripening[J].Meat Sci,2009,81(3):451-455.
[28]	GLORIOSO J M,MAO S A,RODYSILL B,et al.Pivotal preclinical trial of the spheroid reservoir bioartificial liver[J].J Hepatol,2015,63(2):388-398.
[29]	王光强,俞剑燊,胡健,等.食品中生物胺的研究进展[J].食品科学,2016,37(1):269-278.WANG G Q,YU J S,HU J,et al.Progress in research on biogenic amines in foods[J].Food Science,2016,37(1):269-278.(in Chinese)
[30]	余苗,李贞明,王刚,等.黑水虻幼虫粉对育肥猪盲肠食糜主要微生物数量和代谢产物的影响[J].畜牧兽医学报,2020,51(2):299-310.YU M,LI Z M,WANG G,et al.Effects of Hermetia illucens larvae meal on the number of main microbes and metabolites in the cecal digesta of finishing pigs[J].Acta Veterinaria et Zootechnica Sinica,2020,51(2):299-310.(in Chinese)
[31]	YEN G C,KAO H H.Antioxidative effect of biogenic amine on the peroxidation of linoleic acid[J].Biosci Biotechnol Biochem,1993,57(1):115-116.
[32]	YANG H, HUANG X, FANG S, et al. Uncovering the composition of microbial community structure and metagenomics among three gut locations in pigs with distant fatness[J]. Sci Rep, 2016, 6:27427.