那西肽预混剂组分HPLC检测方法的建立

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【CN109580864B】一种动物性食品中那西肽残留的检测方法【专利】

(19)中华人民共和国国家知识产权局(12)发明专利(10)授权公告号 (45)授权公告日 (21)申请号 201910119392.6(22)申请日 2019.02.18(65)同一申请的已公布的文献号申请公布号 CN 109580864 A(43)申请公布日 2019.04.05(73)专利权人华南农业大学地址 510000 广东省广州市天河区五山路483号(72)发明人贺利民　谢景梦　曾振灵　汤有志　苏贻娟　(74)专利代理机构北京高沃律师事务所 11569代理人代芳(51)Int.Cl.G01N 30/88(2006.01)G01N 30/06(2006.01)审查员陈群霞 (54)发明名称一种动物性食品中那西肽残留的检测方法(57)摘要本发明提供了一种动物性食品中那西肽残留的检测方法，属于兽药残留检测领域。

本发明提供的动物性食品中那西肽残留的检测方法，包含以下步骤：将动物性食品的肌肉组织依次经碱溶液水解、除脂和调节pH值后，经固相萃取洗脱，得到洗脱液；将所述洗脱液依次经吹干、溶解和过滤，得到滤液；对所述滤液进行液相色谱-串联质谱分析检测4-羟甲基-3-甲基吲哚-2-甲酸的含量，根据所述那西肽标准对应的4-羟甲基-3-甲基吲哚-2-甲酸的含量计算得到所述动物性食品中那西肽残留的含量。

本发明提供的检测方法具有高选择性和高灵敏度的特点，能够高效检测出动物肌肉组织中那西肽残留的含量。

权利要求书1页说明书10页附图4页CN 109580864 B 2019.09.06C N 109580864B权　利　要　求　书1/1页CN 109580864 B1.一种动物性食品中那西肽残留的检测方法，其特征在于，包含以下步骤：将动物性食品的肌肉组织依次经碱溶液水解、除脂和调节pH值后，经固相萃取洗脱，得到洗脱液；将所述洗脱液依次经吹干、溶解和过滤，得到滤液；对所述滤液进行液相色谱-串联质谱分析检测4-羟甲基-3-甲基吲哚-2-甲酸的含量，根据所述那西肽标准对应的4-羟甲基-3-甲基吲哚-2-甲酸的含量计算得到所述动物性食品中那西肽残留的含量；所述液相色谱-串联质谱的参数包括：反相色谱柱C18分离，以乙腈和水为流动相进行梯度洗脱，采用电喷雾离子源电离，负离子在m/z 100～200amu扫描，用三重四极杆质谱仪监测分析；所述梯度洗脱的程序为：0～1min，流动相为乙腈和水按体积比5：95组成的混合液；1～10min，流动相为乙腈和水按体积比5：95～90：10组成的混合液；10～11min，流动相为乙腈和水按体积比90：10组成的混合液；11～11.5min，流动相为乙腈和水按体积比90：10～5：95组成的混合液；11.5～15min，流动相为乙腈和水按体积比5：95组成的混合液。

那西肽提取工艺研究

那西肽提取工艺研究作者：闵江娄燕刘正光王丹丹赵才兵梁景乐来源：《中国动物保健》2022年第08期摘要：优化那西肽提取工艺，80%四氢呋喃溶液为提取溶剂，提取那西肽，经过浓缩、结晶、重结晶，制备高含量那西肽产品。

得到最佳提取工艺条件是：浸提pH 7.0，浸提温度25℃，兩次浸提时间1h，一次浸提料液比1：7，二次浸提料液比1：4，粗精粉重结晶得到纯品，含量95%。

关键词：那西肽;提取工艺;四氢呋喃溶液那西肽（Nosiheptide），又称诺西肽，是由活跃链霉菌（Streptomyces actuosus）产生的，与Thiostrepton、Siomycin、Sporangiomycin、Thiopeptin、Althiomycin、Taitomycin等组成了一大类富含硫的多肽类抗生素，既能作为饲料添加剂，明显促进鸡、猪的生长[1]，低浓度时可抑菌，高浓度时能杀菌[2]，还具有抗病毒活性[3]。

那西肽纯品为黄色针状晶体，属于脂溶性抗生素[4]，主要存在于菌体细胞内，最有效的提取方法是有机溶剂萃取。

韩正枝等[5]发明了一种亚临界水提取菌丝体中那西肽的方法，发酵液进行过滤、亚临界水进行萃取，提取率达到93%以上，并未提及粗品含量。

张春颖[6]采用有机溶剂法、树脂分离、乙醇和水混合沉淀提取分离得到那西肽粗品，并进一步采用离子交换层析法分离纯化，提取率不低于85.1%，生物效价不低于750U/mg。

徐天华等[7]以那西肽菌丝体为原料，采用四氢呋喃为提取溶剂，用中性氧化铝进行初步纯化，经高速逆流色谱纯化，得到含量不低于95%的那西肽产品，回收率为86%。

徐天华等[8]采用大孔吸附树脂对那西肽提取工艺进行研究，比较8种大孔吸附树脂对那西肽粗品所含杂质的吸附能力，经混合树脂纯化后的那西肽产品为淡黄色粉末，纯度最高可以达到99%。

本文对那西肽提取工艺进行了系统研究，旨在简化提取工艺路线，保证产品质量，降低提取成本，提出切实可行的工业化生产方案。

那西肽预混剂质量分析

那西肽预混剂质量分析
于丽娜;韩宁宁;徐嫄;郝利华;戴青;赵晖
【期刊名称】《中国抗生素杂志》
【年(卷),期】2016(041)010
【摘要】通过对那西肽预混剂检验与研究,对其质量进行分析,并对现有标准进行修订.依据现有标准,对那西肽预混剂进行常规检验,对标准进行了修订并确定质量控制关键点;根据文献检索和调研等情况,开展了含量均匀度、那西肽组分A、含量测定等3个方面的探索研究.建议取消标准中关于性状的规定;调整了pH值的规定限度范围为6.0～8.0;补充建立了那西肽组分A的测定方法,拟规定限度为不小于88.0％;以N,N--二甲基甲酰胺为溶剂,修订了那西肽预混剂含量测定方法.
【总页数】4页(P767-770)
【作者】于丽娜;韩宁宁;徐嫄;郝利华;戴青;赵晖
【作者单位】中国兽医药品监察所,北京100081;中国兽医药品监察所,北京100081;中国兽医药品监察所,北京100081;中国兽医药品监察所,北京100081;中国兽医药品监察所,北京100081;中国兽医药品监察所,北京100081
【正文语种】中文
【中图分类】R917
【相关文献】
1.那西肽预混剂生产工艺研究 [J], 黄振锋;张永旺;陈文成
2.杆菌肽锌预混剂饲喂育肥羊效果分析 [J], 蔡洪波
3.那西肽预混剂组分HPLC检测方法的建立 [J], 韩宁宁;徐嫄;于丽娜;郝利华;赵晖
4.亚甲基水杨酸杆菌肽预混剂对鸡的安全性研究 [J], 孙晨明; 徐亚亚; 高清清; 袁华根
5.高效液相色谱-荧光检测法与抗生素微生物检定法测定那西肽预混剂含量的比较研究 [J], 林仙军;周芷锦;王彬
因版权原因，仅展示原文概要，查看原文内容请购买。

高效液相色谱法测定饲料中那西肽A

D O I：10.15906/11-2975/s.20181311高效液相色谱法测走饲料中那西肽A林仙军，陆春波，包爱情，王彬(浙江省兽药饲料监察所，浙江杭州311100)[摘要]为建立高效液相色谱测定饲料中那西肽A的方法，根据饲料样品多样性的特点，采用乙二胺四乙酸二钠溶液和N，N-二曱基曱酰胺超声提取饲料中的那西肽A。

采用C,8色谱柱，0.02%磷酸溶液+乙腈(60:40，V/V)为流动相，流速为1.0 mL/min，荧光检测器激发波长为327 nm，发射波长为521 nm。

那西肽A在浓度0.02耀10滋g/m L时线性良好，线性相关系数为0.9999。

当添加浓度为0.5耀500 mg/k g时，平均回收率为81.6%耀95.6%，相对标准偏差 (R SD)为1.6%耀9.2%。

方法的检测限为0.2 mg/kg，定量限为0.5 mg/kg。

该方法操作简便、结果准确、稳定性好，应用于实际样品检测，结果满意，表明该方法适用于饲料中那西肽A的测定。

[关键词]那西肽A;荧光检测器；饲料;前处理；高效液相色谱法[中图分类号]S816.17 [文献标识码]A那西肽（Nosiheptide)为浅黄绿褐色或黄绿褐色粉末，是一种带有5个噻唑环的含硫多肽类抗生素（孙小青，2007)其分子式为Csi^N^O,^，相对分子质量1222.36。

那西肽能促进猪(沈顺新，2008)、鸡生长(Cromwell，1984)，提高饲料效率（Benazet，1980a、b)。

按那西肽A计算，混饲每1000 k g饲料，猪2.5 ~ 20 g，鸡2.5 g (Y u，2009)，广泛应用于畜牧业生产中（Jiang，2015)。

饲料中添加浓度较低的那西肽即能促进畜禽生长（N iu，2011)，提高伺料效率(W ojtas，2016;W ang，2014;Pascal，1979)。

农牧函〔1998〕7号批准那西肽及那西肽预混剂为国家三类新兽药。

高效液相色谱法测定那西肽的纯度

ｓｏｌｕｔｉｏｎ色谱工作站；电子天平（感量０．１ｍｇ）。１．２药品与试剂那西肽原料（中国兽医药品监
察所委托企业制备）；乙腈与甲醇为色谱纯；磷酸二氢钾与磷酸氢二钾为分析纯；Ｍｉｌｌｉｐｏｒｅ去离子水。
ｐｅｒｆｏｍｅｒｄｏｎＣ１８ｃｏｌｕｍｎ（２５０ｍｍ ×４．６ｍｍ，５Ｉｘｍ）ｗｉｔｈａｍｏｂｉｌｅｐｈａｓｅｏｆｐＨ５．６ｐｈｏｓｐｈａｔｅｂｕｆｆｅｒ—
肽的纯度测定。
［关键词］那西肽；高效液相色谱；纯度ＤｅｔｅｒｍｉｎａｔｉｏｎｏｆＰｕｒｉｔｙｆｏｒＮｏｓｉｈｅｐｔｉｄｅｂｙＨＰＬＣ
ＺＨＡＮＧＸｉｕ—ｙｉｎｇ，ＬＵＬｉａｎ —ｓｈｏｕ，ＷＥＮＦａｎｇ，ＬＩＣｕｉ，ＤＡＩＺｈｉ —ｈｏｎｇ，ＪＩＡＮＧＨｕｉ，ＷＡＮＧＺａｉ —ｓｈｉ
ｈａｄｇｏｏｄｓｐｅｃｉｉｆｃｉｔｙ．Ｉｔｃａｎｂｅｕｓｅｄｏｒｆｔｈｅｄｅｔｅｍｉｒｎａｔｉｏｎｏｆｐｕｉｒｔｙｏｒｆｎｏｓｉｈｅｐｔｉｄｅ．Ｋｅｙｗｏｒｄｓ：ｎｏｓｉｈｅｐｔｉｄｅ；ＨＰＬＣ；ｐｕｉｒｔｙ
中国兽药杂志

那西肽检测方法

19 NosiheptideNNSS N ONHHNNSO HNH 2SN SNN HOOHSONH OOOHHN O O OHC 51H 43N 13O 12S 6 MW: 1222 CAS No.: 56377-79-8[Summary of nosiheptide]Nosiheptide (NH) is a polypeptide antibiotic obtained by the incubation of Streptomyces actuosus . For physicochemical properties, NH technical occurs as light-yellow grayish white to greenish light-yellow brown crystals or powder, and has no odor or slightly has a characteristic odor. It is soluble in cyclohexane, sparingly soluble in tetrahydrofuran, slightly soluble in acetone and in chloroform, very slightly soluble in ethanol, and practically insoluble in water.NH has a strong antibacterial effect on most part of Gram-positive bacteria and part of Gram-negative bacteria and a growth promoting effect on chickens (including broilers) and pigs.«Standards and specifications in the Act on Safety Assurance and Quality Improvement of Feeds»NH is a pure-grade and feed-grade antibiotic that was designated as a feed additive as of December 25, 1987. The specifications for feeds containing this ingredient are specified in Appended Table No.1, 1-(1)-C of the Standards and Specifications in the Act on Safety Assurance and Quality Improvement of Feeds.The amount of NH added to a commercial premix is roughly 1 to 10 g (potency)/kg.[Methods listed in the Feed Analysis Standards]1 Quantitative test method - Plate method1.1 Premix [Feed Analysis Standards, Chapter 9, Section 2, 19.1.1] Scope of application: Premix with copper sulfate content of 17 g/kg or lowerA. Reagent preparation1) Buffer solution: Buffer No.42) Diution solvent: Buffer No.4-acetone (4:1)3) Nosiheptide standard solution. Dry a suitable amount of nosiheptide working standard[1] under reducedpressure (not exceeding 0.67 kPa) at 60°C for 3 hours, weigh accurately not less than 40 mg, accurately add N,N-dimethylformamide and dissolve to prepare a nosiheptide standard stock solution with a concentration of 1 mg (potency)/mL[2].At the time of use, accurately dilute a quantity of the standard stock solution with the dilution solvent to prepare high- and low-concentration standard solutions with concentrations of 0.1 and 0.025 µg (potency)/mL, respectively[3].4) Culture medium: Medium F-75) Bacterial suspension and amount of addition. Use Micrococcus luteus ATCC 9341[4] as the testorganism. Add about 0.2 mL of a 10-fold diluted suspension of the test organism per 100 mL of the culture medium.6) Agar plate. Proceed by the agar well method.7) Potassium dihydrogen phosphate solution. Dissolve 52.5 g of potassium dihydrogen phosphate in 1,000mL of water and adjust the pH to 10.4 to 10.6 with a saturated solution of sodium hydroxide.8) Extracting solvent: A mixture of N,N-dimethylformamide and potassium dihydrogen phosphate solution(7:3)B. Preparation of sample solutionWeigh accurately 3 to 5 g of the analysis sample, place in a 200-mL stoppered Erlenmeyer flask, add 2 g of ethylenediaminetetraacetic acid tetrasodium salt[5] and 100 mL of the extracting solvent, and extract with stirring for 20 minutes. Transfer the extract to a 50-mL stoppered centrifuge tube, and centrifuge at 650×g for 15 minutes. Accurately dilute a quantity of the supernatant liquid with the dilution solvent to prepare high- and low-concentration sample solutions with concentrations of 0.1 and 0.025 µg (potency)/mL, respectively[6].C. Quantification[7]Proceed by the 2-2 dose method[8].«Summary of analysis method»This method is intended to determine the amount of NH in a premix by microbiological assay using a sample solution prepared by extracting with a mixture of N,N-dimethylformamide and potassium dihydrogen phosphate solution (7:3) containing ethylenediaminetetraacetic acid tetrasodium salt and diluting with a mixture of Buffer No.4 and acetone (4:1).This method is not applicable to a premix spiked with copper sulfate at a concentration exceeding17 g/kg.The flow sheet of this method is shown in Figure 9.2.19-1.Figure 9.2.19-1 Quantitative test method for nosiheptide (premix) References: Shoichi Yamatani, Kyoko Akimoto: Research Report of Animal Feed, 22, 116 (1997) History in the Feed Analysis Standards [19] New«Validation of analysis method»«Notes and precautions»[1] For the definition etc. of nosiheptide working standard, refer to «Notes and precautions» [9] inSection 1, 1 of this Chapter.[2] for the method of preparation for the standard stock solution, refer to «Notes and precautions» [10] inSection 1, 1 of this Chapter.Method of preparation: Example (when the weighed amount is 50 mg)When the labeled potency of the working standard is 945 µg (potency)/mg, 50 mg of the working standard contains 47,250 µg (potency) (i.e., 50 mg × 945 µg (potency)/mg). To prepare a standard stock solution with a concentration of 1,000 µg (potency)/mL, the required amount of solvent is thus calculated to be 47.25 mL (i.e., 47,250 µg (potency) / 1,000 µg (potency)/mL).Therefore, completely transfer 50 mg of the working standard to an Erlenmeyer flask containing 47.25 mL of N,N-dimethylformamide and dissolve to prepare the standard stock solution with a concentration of 1,000 µg (potency)/mL.[3] For the method of preparation for the standard solution, refer to «Notes and precautions» [8] in Section 1, 1 of this Chapter.An example method of preparation for nosiheptide standard solution is shown in Table 9.2.19-1.[4] For the number of bacteria, refer to «Notes and precautions» [33] in Section 1, 1 of this Chapter.[5] Ethylenediaminetetraacetic acid tetrasodium salt is intended as a chelating agent to remove the effect of minerals contained in the premix.[6] For the method of preparation for the sample solution, refer to «Notes and precautions» [8] in Section 1, 1 of this Chapter.An example method of preparation is shown in Table 9.2.19-1.Table 9.2.19-1 Method of preparation for nosiheptide standard solution and sample solution2) Method of preparation for sample solution (premix, example)When the analysis sample is collected in an amount equivalent to 10,000 µg (potency) of[7] An example standard response line for NH is shown in Figure 9.2.19-2.[8] Refer to «Notes and precautions» [53] to [60] in Section 1, 1 of this Chapter.Figure 9.2.19-2 Standard response line nosiheptide (premix, example)(Micrococcus luteus A TCC 9341, Medium F-7, Agar well method)2.1 Feed [Feed Analysis Standards, Chapter 9, Section 2, 19.2.1]Scope of application : Feeds excluding pelleted feeds, feeds for suckling period or feeds with NH contant less than 5 g (potency)/tA. Reagent preparation1) Buffer solution: Buffer No.42) Diution solvent: A mixture of Buffer No.4 and acetone (4:1)3) Nosiheptide standard solution. Dry a suitable amount of nosiheptide working standard under reduced pressure (not exceeding 0.67 kPa) at 60°C for 3 hours, weigh accurately not less than 40 mg, accurately add N ,N -dimethylformamide and dissolve to prepare a nosiheptide standard stock solution with a concentration of 1 mg (potency)/mL.At the time of use, accurately dilute a quantity of standard stock solution with the dilution solvent to prepare nosiheptide standard solutions with concentrations of 0.2, 0.1, 0.05, 0.025 and 0.0125 µg (potency)/mL [1].4) Culture medium: Medium F-75) Bacterial suspension and amount of addition. Use Micrococcus luteus ATCC 9341[2] as the test organism. Add about 0.2 mL of a 100-fold diluted suspension of the test organism per 100 mL of the culture medium.6) Agar plate. Proceed by the agar well method [3].B. Preparation of sample solutionExtraction. Weigh 10.0 g of the analysis sample, place in a 200-mL stoppered amber Erlenmeyer flask, add 100 mL of a mixture of Buffer No.4 and acetone (1:1)[4], extract with stirring for 20 minutes, and filter the extract through filter paper (No.5A). Transfer accurately 10 mL of the filtrate to a 50-mL amber volumetric flask [5], add water up to the marked line, and use as the sample solution subject to column treatment.Concentration of nosiheptide (µg(potency)/mL)2015100.00625 0.0125 0.025 0.05 0.1 0.2C o r r e c t e d i n h i b i t i o n z o n e d i a m e t e r (m m )Folumn treatment. Cover with aluminum foil the reservoir of an octadecylsilanized silica gel (trifunctional) minicolumn (400 mg)Note 1, and wash the minicolumn with 10 mL acetone and with 10 mL of water in this order.Transfer accurately 10 mL of the sample solution to the minicolumn, and inject under pressure to force to flow out Note 2. Add 20 mL of water[6] to the minicolumn, and inject under pressure to force to flow out Note 2 in the same manner.Place a 100-mL amber recovery flask under the minicolumn, add 10 mL of acetone and 20 mL of a mixture of acetone and chloroform (1:1) to the minicolumn in this order, and allow to flow out to elute NH. Condense the eluate to approximately 1 mL under reduced pressure in a water bath at 50°C, introduce nitrogen gas to evaporate into dryness, and add accurately 20 mL of the dilution solvent to dissolve the residue. Accurately dilute a quantity of this solution with the dilution solvent to prepare a sample solution with a concentration of 0.05 µg (potency)/mL.C. Quantification[7]Proceed by the standard response line method[8].Note 1. Use a Sep-Pak Plus tC18 Cartridge (Waters) connected with a reservoir with a sitable capacity or an equivalent.2. Set the flow rate to 0.6 to 0.8 mL/min.«Summary of analysis method»This method is intended to determine the amount of NH (pure-grade and feed-grade) in a feed by microbiological assay using a sample solution prepared by extracting with a mixture of Buffer No.4 and acetone (1:1) and purifying through an octadecylsilanized silica gel (trifunctional) (tC18) minicolumn. None of the antibacterial substances apploved for combined use with NH interfere with the quantification of NH.This method is not applicable to pelleted feed, feed for suckling period or feeds with NH contant less than 5 g (potency)/t.The flow sheet of this method is shown in Figure 9.2.19-3.Figure 9.2.19-3 Quantitative test method for nosiheptide (feed)References: Kyoko Akimoto, Y uji Fukumoto: Research Report of Animal Feed, 20, 81 (1995)History in the Feed Analysis Standards [17] New«Validation of analysis method»«Notes and precautions»[1] For the method of preparation for the standard solution, refer to «Notes and precautions» [8] inSection 1, 1 of this Chapter. An example method of preparation for nosiheptide standard solution is shown in Table 9.2.19-2.[2] For the number of bacteria, refer to «Notes and precautions» [33] in Section 1, 1 of this Chapter. [3] The agar well method is more sensitive to NH than the cylinder plate method. [4] Prepare at the time use. Stir for 30 minutes or longer with a stirrer before use.[5] As NH is unstable in light, use an amber container or cover the container with aluminum foil. [6] The addition of water shall be made by two 5-mL portions and one 10-mL portion, a total of 20 mL. [7] An example standard response line for NH is shown in Figure 9.2.19-4.Linearity is observed in the quantification range for NH (NH concentrations between 0.125 and 2 µg (potency)/mL). [8] Refer to «Notes and precautions» [53] to [57] and [61] in Section 1, 1 of this Chapter.Figure 9.2.19-4 Standard response line for nosiheptide (feed, example)(Micrococcus luteus A TCC 9341, Medium F-7, Agar well method)2 Trace quantitative test method - Microbioautography (Feed)[Feed Analysis Standards, Chapter 9, Section 2, 19.3.1]Scope of application : FeedA. Reagent preparation1) Nosiheptide standard solution. Dry a suitable amount of nosiheptide working standard under reducedRP0.05 µg (potency)/mLConcentration of nosiheptide (µg (potency)/mL)201510C o r r e c t e d i n h i b i t i o n z o n e d i a m e t e r (m m )0.00625 0.0125 0.025 0.05 0.1 0.2pressure (not exceeding 0.67 kPa) at 60°C for 3 hours, weigh accurately not less than 40 mg, accurately add N,N-dimethylformamideand dissolve to prepare a nosiheptide standard stock solution with a concentration of 1 mg (potency)/mL.At the time of use, accurately dilute a quantity of standard stock solution with methanol to prepare standard solutions with concentrations of 2, 1, 0.5, 0.25 and 0.125 µg (potency)/mL[1].2) Culture medium: Medium F-1113) Bacterial suspension and amount of addition. Use Micrococcus luteus ATCC 9341[2] as the test organism. Add about 0.5 mL of a 100-fold diluted suspension of the test organism per 100 mL of the culture medium.4) Developing solvent: A chloroform-methanol-ammonia reagent (20:13:5)Note 15) Sodium sulfate (anhydrous). Dry at 110 to 120°C for 2 hours and allow to cool in a desiccator.6) Chromigenic substrate. Dissolve 100 mg of 3-(4-iodophenyl)-2-(4-nitrophenyl)-5-phenyltetrazolium chloride in water to make 200 mL.B. Preparation of sample solutionExtraction. Weigh 40.0 g of the analysis sample, place in a 200-mL stoppered Erlenmeyer flask, add 100 mL of acetonitrile, extract with stirring for 30 minutes, and filter the extract through filter paper (No.5A). Transfer 50 mL of the filtrate to a 100-mL recovery flask, evaporate into dryness under reduced pressure in a water bath at 50°C, add 20 mL of a mixture of chloroform and ethyl acetate (9:1) to dissolve the residue, and use as the sample solution subject to column treatment.Column treatment. Wash a silica gel minicolumn (690 mg) with 10 mL of chloroform. Place a funnel loaded with approximately 40 g of sodium sulfate (anhydrous)[3] on the minicolumn, pour the sample solution into the funnel, and allow to flow down until the amount in the reservoir of the minicolumn reaches 1 mL[4]. Wash the recovery flask that contained the sample solution with 10 mL of a mixture of chloroform and ethyl acetate (9:1), add the washings to the funnel, and repeat this process three times.Wash the sodium sulfate remaining in the funnel with 10 mL of a mixture of chloroform and ethyl acetate (9:1), add the washings to the minicolumn, remove the funnel, add 20 mL of a mixture of chloroform and ethyl acetate (9:1) to wash the minicolumn.Place a 100-mL recovery flask under the minicolumn, add 30 mL of a mixture of chloroform and methanol (4:1) to elute NH. Evaporate the eluate into dryness under reduced pressure in a water bath at 50°C, add accurately 2 mL of methanol to dissolve the residue[5], and use as the sample solution.C. Quantification[6]Proceed as described in Section 1, 2-C[7], except that the developing solvent shall be placed in the developing chamber and allowed to stand for not less than 1 hour. The thin-layer plate shall be of silica gel Note 2, and developed at 20 to 25°C to a distance of 150 mm from the starting line.Note 1. Dilute ammonia solution with water to prepare a solution with ammonia content of 17%.2. Use a TLC plate Silica gel 60 (20×20 cm) (Merck) or an equivalent after drying at 110°C for 2hours.«Summary of analysis method»This method is intended to quantify and identify a trace of NH in a feed resulting fromcontamination due to carry-over etc., by microbioautography using a sample solution prepared by extracting with acetonitrile, purifying through a silica gel column, and dissolving in methanol.The flow sheet of this method is shown in Figure 9.2.19-5.Figure 9.2.19-5 Trace quantitative test method for nosiheptide (feed) References: Noriyuki Koyama: Research Report of Animal Feed, 17, 96 (1992)History in the Feed Analysis Standards [14] New, [17] Revision«Validation of analysis method»«Notes and precautions»[1]For the method of preparation for standard solution, refer to «Notes and precautions» [8] in Section 1,1 of this Chapter.An example method of preparation for nosiheptide standard solution is shown in Table 9.2.19-3.[2] For the number of bacteria, refer to «Notes and precautions» [33] in Section 1, 1 of this Chapter.[3] It is recommended to stuff a small plug of absorbent cotton at the top of the funnel stem and onwhich to place sodium sulfate (anhydrate).[4] When the flow is slow, it is permissible to inject under pressure useing the plunger of the syringe or adouble-baloon pump.[5] When the residue is difficult to dissolve, apply ultrasonic waves for 2 to 3 minutes.Table 9.2.19-3 Method of preparation for nosiheptide standard solution (trace quantitative test[6] An example standard response line for NH is shown in Figure 9.2.19-6.Figure 9.2.19-6 Standard response line for nosiheptide (trace quantitative test method, feed,example)(Micrococcus luteus A TCC 9341, Medium F-111, Microbioautography) [7] Refer to «Notes and precautions» [1] to [8] in Section 1, 2 of this Chapter.Concentration of nosiheptide (µg (potency)/mL)2520151050.016 0.032 0.063 0.125 0.25 0.5 1 2I n h i b i t i o n z o n e d i a m e t e r (m m )。

高效液相色谱法测定那西肽的纯度

高效液相色谱法测定那西肽的纯度张秀英;陆连寿;温芳;李翠;戴志红;蒋卉;王在时【期刊名称】《中国兽药杂志》【年(卷),期】2013(047)007【摘要】采用高效液相色谱法测定那西肽的纯度.以十八烷基硅烷键合硅胶为填充剂(250 mm×4.6 mm,5μ,m),pH 5.6磷酸盐缓冲液-乙腈-甲醇(54∶43∶3)为流动相；流速为0.7 mL/min；测定波长为330 nm.结果表明,在10.5～94.9 μg/mL浓度范围内峰面积与浓度呈良好的线性关系,定量限和检测限分别为0.25 μg/mL和0.12 μg/mL.该方法简便、快速、专属性强,适用于那西肽的纯度测定.【总页数】3页(P22-24)【作者】张秀英;陆连寿;温芳;李翠;戴志红;蒋卉;王在时【作者单位】中国兽医药品监察所,北京100081;中国兽医药品监察所,北京100081;中国兽医药品监察所,北京100081;中国兽医药品监察所,北京100081;中国兽医药品监察所,北京100081;中国兽医药品监察所,北京100081;中国兽医药品监察所,北京100081【正文语种】中文【中图分类】S859.83【相关文献】1.高效液相色谱法测定饲料中那西肽A [J], 林仙军;陆春波;包爱情;王彬2.QuEChERS样品前处理-高效液相色谱法测定动物组织中那西肽残留量 [J], 陈慧华;应永飞;杜旭奕;罗成江;周芷锦3.高效液相色谱法测定新型抗菌肽杆菌七肽的含量 [J], 张江;黄邦俊;李学莉;王旭;周晓容4.高效液相色谱-荧光检测法与抗生素微生物检定法测定那西肽预混剂含量的比较研究 [J], 林仙军;周芷锦;王彬5.反相高效液相色谱法测定枯草菌肽的含量及纯度 [J], 杨彩霞;吴金梅;何涛因版权原因，仅展示原文概要，查看原文内容请购买。

一种检测那西肽发酵液中那西肽含量的方法[发明专利]

(19)中华人民共和国国家知识产权局(12)发明专利申请(10)申请公布号 (43)申请公布日 (21)申请号 201810890529.3(22)申请日 2018.08.07(71)申请人河北圣雪大成制药有限责任公司地址 051430 河北省石家庄市栾城区圣雪路50号(72)发明人杜精精　马盼盼　曹建全　(74)专利代理机构石家庄君联专利代理事务所(特殊普通合伙) 13125代理人赵立军(51)Int.Cl.G01N 30/02(2006.01)G01N 30/06(2006.01)(54)发明名称一种检测那西肽发酵液中那西肽含量的方法(57)摘要本发明公开了一种检测那西肽发酵液中那西肽含量的方法，涉及检测技术领域，涉及那西肽含量的检测方法。

本发明包括如下步骤：a、制备那西肽的对照品溶液；b、取那西肽发酵液样品，用乙醇水混合溶剂作为浸提液定容后超声提取；制得那西肽的样品溶液；c、将对照品溶液和样品溶液依次注入HPLC进行测定，记录色谱图和峰面积；用对照品溶液中主成分那西肽A的质量浓度和峰面积计算校正因子，并用所得校正因子计算发酵液样品中那西肽A组分的含量，同时获得那西肽B组分的含量。

本发明方法简便快速、准确高效、低毒、低成本，可作为那西肽生产企业的发酵过程监控方法，及时为生产过程控制提供监测数据。

权利要求书1页说明书7页附图4页CN 109060980 A 2018.12.21C N 109060980A1.一种检测那西肽发酵液中那西肽含量的方法，其特征在于，包括如下步骤：a、制备那西肽的对照品溶液；b、取那西肽发酵液样品，用乙醇水混合溶剂作为浸提液定容后超声提取；制得那西肽的样品溶液；c、将对照品溶液和样品溶液依次注入HPLC进行测定，记录色谱图和峰面积；用对照品溶液中主成分那西肽A的质量浓度和峰面积计算校正因子，并用所得校正因子计算发酵液样品中那西肽A组分的含量，同时获得那西肽B组分的含量。

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那西肽预混剂组分HPLC检测方法的建立韩宁宁;徐嫄;于丽娜;郝利华;赵晖【摘要】为了建立那西肽预混剂组分测定的高效液相检查法,采用十八烷基键合硅胶为填充剂,以0.025％磷酸水溶液-乙腈(50: 50)为流动相,检测波长为241 nm,并以该色谱条件对来自7个厂家的14批不同规格的那西肽预混剂进行了测定.综合结果给出组分的建议限度为:那西肽组分A的峰面积不得少于那西肽组分A与组分B 峰面积之和的88.0％.该方法具有专属性强、耐用性好、操作简便等优点.【期刊名称】《中国兽药杂志》【年(卷),期】2015(049)010【总页数】5页(P27-31)【关键词】那西肽预混剂;组分;高效液相色谱法【作者】韩宁宁;徐嫄;于丽娜;郝利华;赵晖【作者单位】中国兽医药品监察所,北京,100081;中国兽医药品监察所,北京,100081;中国兽医药品监察所,北京,100081;中国兽医药品监察所,北京,100081;中国兽医药品监察所,北京,100081【正文语种】中文【中图分类】S859.796那西肽是一种含硫多肽类抗生素，我国于1998年批准其为国家三类新兽药，被农业部列为可在饲料中长期添加使用的饲料药物添加剂，对猪鸡有促进生长和提高饲料转化率的作用[1-2]。

那西肽预混剂工艺早期是由那西肽原料药与玉米淀粉、碳酸钙等配制而成，其质量标准收载于《兽药国家标准化学药品、中药卷》(第一册)[3]。

随着技术发展，国内那西肽预混剂生产厂家的生产工艺均已变更为那西肽发酵液的菌丝体干燥后与碳酸钙等配制而成。

而目前缺少针对发酵而得的那西肽预混剂的相关国家质量标准，相应的市场监督与质量控制也呈空白状态。

经文献检索，那西肽中含有一主组分A，同时含有一副组分B[4]。

《兽药国家标准化学药品、中药卷》(第一册)中收载的那西肽预混剂质量标准对组分B并没有相关的控制规定[3]。

根据兽药协会提供的资料，对国内7家那西肽预混剂生产企业内部控制标准中关于组分的相关规定进行了汇总，结果发现其中3家企业并无相关规定，剩余4家企业对组分B的限度规定从不得大于12%至不得大于15%不等。

由此可见，不同企业对组分B的控制缺乏一致性。

因此，根据文献中那西肽预混剂HPLC含量测定方法[5]，经过方法优化与验证，建立了那西肽预混剂组分的HPLC测定法。

高效液相色谱仪(HPLC)，配紫外检测器(UV)和二极管阵列检测器(PDA)(Waters 2695-2487/996)；XS205分析天平(Mettler Toledo)；色谱柱(COSMOSIL C18，5μm，250 mm×4.6 mm)。

色谱纯乙腈(Merck公司)；分析纯磷酸(国药集团)；去离子水(Millipore纯水机过滤)。

那西肽标准品由中国兽医药品监察所提供，批号K0431212，每1mg相当于928那西肽单位，以那西肽A组分计，含量为88.6%。

那西肽预混剂供试品共14批，由7家国内生产厂家提供，具体信息见表1。

2.1 色谱条件用十八烷基键合硅胶为填充剂，以乙腈-0.025%磷酸溶液(50∶50)为流动相，检测波长为241 nm，取标准品溶液20 μL，注入液相色谱仪，记录色谱图，出峰顺序为那西肽组分B、那西肽组分A。

理论板数按那西肽组分A峰计算不低于3000，主成分与相邻杂质峰的分离度应大于1.5。

2.2 对照品溶液与供试品溶液的制备取供试品适量，精密称定，置具塞锥形瓶中，精密加入磷酸盐缓冲溶液5 mL、二甲基甲酰胺50 mL，超声处理20 min，每隔5 min强力振摇一次，放至室温，制成每1 mL约含1000单位(规格：8%、4%、2%)或125单位(规格：1%、0.5%、0.25%)的溶液，滤过，精密量取续滤液适量，加二甲基甲酰胺 4 mL，加磷酸盐缓冲液-乙醇(85∶15)稀释至刻度，制成每1 mL 约含25单位的溶液。

对照品溶液同法配制。

供试品溶液应现配先用。

2.3 方法学验证2.3.1 专属性采用PDA检测器，在拟定方法下检测，获得标准品溶液和供试品溶液的那西肽组分光谱指数图(图1-图2)。

由图可见，溶剂峰对组分A与B均无干扰。

2.3.2 检测限取0.5单位/ mL的标准品溶液7 μL注入液相色谱仪，组分A的信噪比S/N=3。

以组分A的量(0.5单位/928单位/mg)×(7 μL/1000 μL)×88.6%×1000=0.003μg作为该方法的检测限(LOD)。

2.3.3 耐用性取那西肽标准品溶液，采用不同品牌色谱柱以及在不同柱温、不同流速、不同pH值下进行测定，对该方法的耐用性进行考察。

2.3.3.1 采用三款不同品牌色谱柱，流速为1.0 mL/min，柱温为30℃，考察方法耐用性，结果三款色谱柱均可满足方法系统适用性要求，参见表2。

2.3.3.2 用COSMOSIL C18(5 μm，4.6 mm×250 mm)色谱柱，流速为1.0mL/min，分别设定25℃、30℃和35℃的柱温，考察不同柱温的影响。

结果随着柱温升高，组分A与B的保留时间均略有提前，但组分A的理论塔板数及组分A与B的分离度基本不受影响，参见表3。

2.3.3.3 用COSMOSIL C18(5 μm，4.6 mm×250 mm)色谱柱，柱温为30℃，分别设定0.8、1.0、1.2 mL/min的流速，考察不同流速的影响。

结果见表4。

由结果可知，随着流速升高，组分A与B的保留时间均提前，组分A的理论塔板数及组分A与B的分离度均略有下降，但均仍符合拟定方法的要求。

2.3.3.4 用COSMOSIL C18(5 μm，4.6 mm×250 mm)色谱柱，柱温为30℃，流速为1.0 mL/min，将流动相中的水相分别设定为0.0125%磷酸溶液、0.025%磷酸溶液、0.05%磷酸溶液，考察不同pH值的影响。

结果三种不同浓度磷酸溶液的流动相对组分A的理论塔板数及组分A与B的分离度基本没有影响，参见表5。

2.3.4 被测溶液的稳定性取一份每1 mL约含25单位的供试品溶液，在室温放置0、1、2、4、8、12和24 h进样，考察供试品稳定性，结果见表6。

结果显示，随着室温放置时间的延长，组分A与B的峰面积均在逐渐减小，放置24 h后，二者峰面积均减少10%以上。

由于每1 mL约含25单位的供试品溶液稳定性较差，我们又另取一份每1 mL约含1000单位的供试品溶液，在室温放置0、1、2、4、8、24和48 h进样，考察其稳定性，结果见表6。

结果显示，随着室温放置时间的延长，虽然组分A的峰面积变化不大，二者总面积也变化不大，但是组分B的峰面积逐渐增大(增大了60%左右)。

因此，在拟定方法中规定供试品溶液现配现用。

2.4 供试品测定采用拟定方法对14批不同规格那西肽预混剂的组分进行测定。

测定结果见表7。

2.5 结果分析由结果可知，不同生产企业组分A的差距较大，从98.3%至83.9%不等，根据中国兽药协会提供的相关资料可查，企业内各批次间的组分也有较大差异，但约90%的产品均可符合组分A不小于88.0%这一限度，再结合各企业的内部控制标准相关规定，拟将那西肽预混剂组分A的限度规定为不小于88.0%。

3.1 溶媒的选择文献方法[5]中，供试品溶液配制第一步溶解与提取过程与效价含量测定方法基本一致，而第二步稀释过程采用甲醇。

将第二步也更改为与效价测定方法一致，即采用二甲基甲酰胺4 mL及磷酸盐缓冲液-乙醇(85：15)稀释。

这一改变可以使组分测定直接采用效价测定项下每1 mL约含25单位的供试品溶液，简化实验操作，节省试剂消耗。

将溶媒更改前与更改后的那西肽标准品色谱进行了比较，发现除溶剂峰稍有差别外，那西肽组分A的理论塔板数及A与B的分离度均不受影响。

3.2 色谱柱的选择文献方法中，填充剂类型有苯基键合硅胶[4,6]和十八烷基键合硅胶[5]2种。

将2种色谱柱类型进行了比较，结果发现那西肽组分A的理论塔板数及A与B的分离度均可符合要求，因此拟采用实验室更为通用的十八烷基键合硅胶柱进行测定。

3.3 检测波长的选择采用PDA检测器对那西肽组分A与B进行了全波长扫描，结果发现二者均在217和333 nm附近有最大吸收。

我们尝试将检测波长更改为217 nm，结果发现溶剂峰影响增强，甚至将组分B部分覆盖。

而将检测波长更改为333 nm，虽然溶剂峰的影响会大大降低，但会使组分A与B的响应均减弱。

因此仍采用241 nm作为检测波长。

3.4 流动相的选择尝试将流动相中的乙腈更换为甲醇，结果发现在100 min内那西肽组分A与B均未出峰，将甲醇换回乙腈后才可将组分A与B冲出。

这表明甲醇对组分A与B保留过强，不适宜作本方法的流动相。

而后尝试将0.025%的磷酸水溶液更换为水，结果发现会导致组分A与B的分离度显著降低(其他色谱条件相同的情况下，组分A与B的分离度由5.0降低至2.3)。

因此仍采用参考方法[5]中的流动相：乙腈-0.025%磷酸水溶液(50∶50)。

3.5 那西肽组分B与组分A相对保留时间的确定考察了不同品牌色谱柱及不同流速那西肽组分B相对A的保留时间。

结果组分B相对A的保留时间为0.80或0.74。

考虑到不同品牌色谱柱对相对保留时间的影响较大，因此采用标准品溶液定位的方法确定组分B相对A的保留时间。

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