超高效液相色谱-串联质谱法同时检测肉鸡组织中4种异喹啉类生物碱

doi:10.16431/j.cnki.1671-7236.2022.12.032

摘要/Abstract

摘要： 【目的】试验旨在建立一种同时测定肉鸡组织中原阿片碱、别隐品碱、白屈菜红碱和血根碱含量的超高效液相色谱-串联质谱（UPLC-MS/MS）检测方法，以便更好地监测博落回散和博普总碱散活性成分在肉鸡组织中的分布与残留情况。【方法】对肉鸡组织进行前处理方法优化，以0.1%甲酸水和乙腈为流动相，在Agilent Zorbax SB-C₁₈上梯度洗脱，流速为0.3 mL/min，柱温为35℃，进样量为1 μL，在正离子模式下通过多反应监测模式采集数据，采用内标校正和外标法测定生物碱含量，并对方法线性、检测限与定量限、基质效应、回收率、批内与批间精密度、准确度及稳定性进行考察。【结果】白屈菜红碱、原阿片碱和别隐品碱的线性范围为0.5~200 μg/L，血根碱为2~200 μg/L，相关系数均≥0.999，线性关系良好；白屈菜红碱、原阿片碱和别隐品碱的检测限与定量限分别为0.2和0.5 μg/L，血根碱检测限与定量限分别为1和2 μg/L。4种生物碱在高、中、低浓度下方法回收率达到86.61%~111.17%，批内与批间精密度和准确度均≤14.93%，基质效应为81.73%~120.78%，稳定性的相对标准偏差均≤13.55%。【结论】本试验建立了同时检测肉鸡心脏、肝脏、肾脏、胸肌和腿肌中4种异喹啉类生物碱的超高效液相色谱-串联质谱法，方法符合各项方法学考察要求，具有专属性强、灵敏度高、方便快捷等特点，可实现对肉鸡组织中痕量生物碱的精确、快速定量。

关键词: 异喹啉类生物碱; 超高效液相色谱-串联质谱; 痕量分析

Abstract: 【Objective】 The purpose of the experiment was to establish an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for simultaneous determination of protopine, allocryptopine, chelerythrine and sanguinarine in broiler tissues, so as to better monitor the distribution and residue of active ingredients of Boluohui powder and Bopuzongjian powder in broiler tissues.【Method】 The pretreatment method of broiler tissues was optimized, with 0.1% formic acid water and acetonitrile as the mobile phase, gradient elution on Agilent Zorbax SB-C₁₈, the flow rate was 0.3 mL/min, the column temperature was 35 ℃, and the injection volume was 1 μL, multiple reaction monitoring mode for data acquisition in positive ion mode, the content was calculated by the external standard method, corrected by the internal standard, and the linearity, limit of detection and limit of quantification, recovery, intra-and inter-run precision, accuracy and stability of the method were investigated.【Result】 The results showed that the linear range of chelerythrine, protopine and allocryptopine were 0.5-200 μg/L, sanguinarine was 2-200 μg/L, the linear relationship was good, and the correlation coefficients were greater than 0.999.The detection and quantification limit of chelerythrine, protopine and allocryptopine were 0.2 and 0.5 μg/L respectively, and sanguinarine were 1 and 2 μg/L respectively.The recovery rate of the four alkaloids were 86.61%-111.17% at high, middle and low concentrations, the precision and accuracy were ≤14.93% within and between batches, and the matrix effect was 81.73%-120.78%, RSD of stability were all less than 13.55%.【Conclusion】 The established ultra-performance liquid chromatography-tandem mass spectrometry method for simultaneous detection of four isoquinoline alkaloids in the heart, liver, kidney, chicken breast and chicken thigh of broilers was consistent with various methodological investigations, and it was specificity, sensitivity, convenience and rapidness.It was suitable for accurate and rapid quantification of trace alkaloids in broiler tissues.

Key words: isoquinoline alkaloids; UPLC-MS/MS; trace analysis

中图分类号:

S859.84

刘梦婷, 胡南希, 邹湘霖, 杨子辉, 曾建国. 超高效液相色谱-串联质谱法同时检测肉鸡组织中4种异喹啉类生物碱[J]. 中国畜牧兽医, 2022, 49(12): 4843-4853.

LIU Mengting, HU Nanxi, ZOU Xianglin, YANG Zihui, ZENG Jianguo. Simultaneous Determination of 4 Isoquinoline Alkaloids in Broiler Tissues by Ultra-performance Liquid Chromatography-tandem Mass Spectrometry[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(12): 4843-4853.

参考文献

[1] 曾建国.博落回资源为示范的中药材全产业链研究与综合利用[J].中国现代中药, 2017, 19(10):1359-1366. ZENG J G.Applying the research of Macleaya cordata resources to a demonstration in the research and comprehensive utilization of traditional Chinese medicinal herbs full industry chain[J].Modern Chinese Medicine, 2017, 19(10):1359-1366.(in Chinese)
[2] LIN L, LIU Y C, HUANG J L, et al.Medicinal plants of the genus Macleaya (Macleaya cordata, Macleaya microcarpa):A review of their phytochemistry, pharmacology, and toxicology[J].Phytotherapy Research, 2018, 32(1):19-48.
[3] 陈伟, 张锐, 雷思敏, 等.博落回化学成分及其药理活性研究进展[J].中国实验方剂学杂志, 2020, 26(3):243-250. CHEN W, ZHANG R, LEI S M, et al.Advances in chemical constituents and bioactivities of Macleaya cordata[J].Chinese Journal of Experimental Traditional Medical Formulae, 2020, 26(3):243-250.(in Chinese)
[4] 侯博, 曾建国.血根碱的生物学活性及博落回提取物在动物生产中的应用[J].动物营养学报, 2018, 30(2):413-420. HOU B, ZENG J G.Biolological activities of sanguinarine and application of Macleaya cordata extract in animal production[J].Chinese Journal of Animal Nutrition, 2018, 30(2):413-420.(in Chinese)
[5] 欧阳龙, 盛利斌, 曾建国.博落回散在健康养殖中的应用前景[J].湖南饲料, 2015, 1:28-29. OUYANG L, SHENG L B, ZENG J G.Application prospect of Boluohui powder in healthy culture[J].Hunan Feed, 2015, 1:28-29.(in Chinese)
[6] 杨南松, 赵征, 唐昭山, 等.博普总碱散对种鸡生殖毒性试验研究[J].扬州大学学报(农业与生命科学版), 2019, 40(5):65-69. ZHAO N S, ZHAO Z, TANG Z S, et al.The reproductive toxicity test of Bopu Total Alkali Powder on breeding chickens[J].Journal of Yangzhou University, 2019, 40(5):65-69.(in Chinese)
[7] HUANG P, ZHANG Y, XIAO K, et al.The chicken gut metagenome and the modulatory effects of plant-derived benzylisoquinoline alkaloids[J].Microbiome, 2018, 6(1):211.
[8] XUE G D, WU S B, CHOCT M, et al.Impact of a Macleaya cordata-derived alkaloid extract on necrotic enteritis in broilers[J].Poultry Science, 2017, 96(10):3581-3585.
[9] LU Z, ALVENSLEBEN S V, FUSCONI G, et al.Safety evaluation of a standardized Macleaya cordata extract in a ninety day feeding study in weaned piglets[J].Open Journal of Animal Sciences, 2017, 7(2):213-231.
[10] DONG Z, LIU M T, ZOU X L, et al.Integrating network pharmacology and molecular docking to analyse the potential mechanism of action of Macleaya cordata (Willd.) R.Br.in the treatment of bovine hoof disease[J].Veterinary Sciences, 2021, 9(1):11.
[11] CHEN N Z, QI Y L, MA X, et al.Rediscovery of traditional plant medicine:An underestimated anticancer drug of chelerythrine[J].Frontiers in Pharmacology, 2022(13):906301
[12] 汪瑶, 燕鸽, 刘智慧, 等.马兜铃酸Ⅰ对大鼠五脏的毒性及功能影响[J].中国畜牧兽医, 2022, 49(7):2757-2767. WANG Y, YAN G, LIU Z H, et al.Study on the toxicity and function effects of aristolochi acid Ⅰ on the five viscera of rats[J].China Animal Husbandry & Veterinary Medicine, 2022, 49(7):2757-2767.(in Chinese)
[13] 耿响, 刘希望, 杨亚军, 等.乙酰氨基阿维菌素缓释注射剂中乙酰氨基阿维菌素含量与有关物质HPLC检测方法的建立[J].中国畜牧兽医, 2021, 48(12):4681-4689. GENG X, LIU X W, YANG Y J, et al.Establishment of HPLC method for determination of eprinomectin and related substances in eprinomectin sustained-release injection[J].China Animal Husbandry & Veterinary Medicine, 2021, 48(12):4681-4689.(in Chinese)
[14] 徐向月, 马文瑾, 安博宇, 等.固相萃取-高效液相色谱-紫外检测土壤中多西环素[J].中国畜牧兽医, 2020, 47(12):4174-4180. XU X Y, MA W J, AN B Y, et al.Detection of doxycycline in soil using soild phase extraction and high performance liquid chromatography-ultraviolet[J].China Animal Husbandry & Veterinary Medicine, 2020, 47(12):4174-4180.(in Chinese)
[15] 徐玉珍, 刘希望, 秦哲, 等.液相色谱-串联质谱法检测猪血浆中除虫脲的方法学研究[J].中国畜牧兽医, 2022, 49(7):2746-2756. XU Y Z, LIU X W, QIN Z, et al.Methodology study on determination of diflubenzuron in swine plasma by liquid chromatography-tandem mass spectrometry[J].China Animal Husbandry & Veterinary Medicine, 2022, 49(7):2746-2756.(in Chinese)
[16] 郑幸果, 郭灵安, 赵珊, 等.UPLC-MS/MS法测定动物源性食品中氯苯胍及其代谢物残留[J].中国畜牧兽医, 2020, 47(6):1961-1970. ZHENG X G, GUO L A, ZHAO S, et al.Determination of robenidine and its metabolites in animal derived food by UPLC-MS/MS[J].China Animal Husbandry & Veterinary Medicine, 2020, 47(6):1961-1970.(in Chinese)
[17] 张亚琛, 黄火强, 杜丽洁.药物分析中的生物样品前处理概况[J].中国民族民间医药, 2017, 26(19):42-45. ZHANG Y C, HUANG H Q, DU L J.Pretreatment of biological samples for pharmaceutical analysis[J].Chinese Journal of Ethnomedicine and Ethnopharmacy, 2017, 26(19):42-45.(in Chinese)
[18] 张换成.原阿片碱在鸡体内的药代动力学研究[D].长沙:湖南农业大学, 2017. ZHANG H C.Pharmacokinetics research of protopine in broiler chickens[D].Changsha:Hunan Agriculture University, 2017.(in Chinese)
[19] LUO X B, CHEN B, YAO S Z.High-performance liquid chromatography with electrospray mass spectrometry for rapid and sensitive determination of sanguinarine and chelerythrine in exogenously contaminated honey[J].Chromatographia, 2004, 60(5-6):347-351.
[20] HU N X, CHEN M, LIU Y S, et al.Pharmacokinetics of sanguinarine, chelerythrine, and their metabolites in broiler chickens following oral and intravenous administration[J].Journal of Veterinary Pharmacology and Therapeutics, 2018, 42(2):197-206.
[21] GUO C, JIANG Y, LI L, et al.Application of a liquid chromatography-tandem mass spectrometry method to the pharmacokinetics, tissue distribution and excretion studies of dactylicapnos scandens in rats[J].Journal of Pharmaceutical & Biomedical Analysis, 2013, 74:92-100.
[22] LIU P, LI W, LI Z H, et al.Comparisons of pharmacokinetic and tissue distribution profile of four major bioactive components after oral administration of Xiang-Fu-Si-Wu decoction effective fraction in normal and dysmenorrheal symptom rats[J].Journal of Ethnopharmacology, 2014, 154(3):696-703.
[23] DOU Z, LI K, WANG P, et al.Effect of wine and vinegar processing of Rhizoma corydalis on the tissue distribution of tetrahydropalmatine, protopine and dehydrocorydaline in rats[J].Molecules, 2012, 17(1):951-970.
[24] 刘梦婷, 吴聪明, 彭旋, 等.博落回提取物猪耐受性评价及其抗结肠炎机制[J].湖南农业大学学报(自然科学版), 2022, 48(3):326-334. LIU M T, WU C M, PENG X, et al.Evaluate on pigs'tolerance and anti-colitis mechanism of Macleaya cordata extract[J].Journal of Hunan Agricultural University (Natural Sciences), 2022, 48(3):326-334.(in Chinese)
[25] 陈梅.血根碱代谢物在鸡体内的药代动力学和组织残留研究[D].长沙:湖南农业大学, 2017. CHEN M.Pharmacokinetics and tissue residues of sanguinarine metabolites in broiler chickens[D].Changsha:Hunan Agricultural University, 2017.(in Chinese)
[26] 冯宋岗.苯并菲啶类生物碱及初级代谢物在猪体内的残留研究[D].长沙:湖南农业大学, 2014. FENG S G.Studied on the residues of quaternary benzo[c] phenanthridine alkaloids and primary metabolites in swine[D].Changsha:Hunan Agricultural University, 2014.(in Chinese)