BMN-673_SDS_MedChemExpress

超高效液相色谱-串联质谱法测定人血浆中精氨酸及衍生物含量田晔;江骥;胡蓓;薛金萍;王洪允【摘要】建立了超高效液相色谱-串联质谱(UPLC-MS/MS)法同时测定使用艾普拉唑后人血浆中二甲基精氨酸(ADMA)、对称二甲基精氨酸(SDMA)、单甲基精氨酸(NMMA)、瓜氨酸(Cit)和L-精氨酸(L-Arg)的浓度.采用HILIC亲水相互作用色谱和非衍生化的蛋白沉淀法进行分离分析,色谱柱选取Waters Atlantic HILIC柱(2.1 mm×50 mm×3μm),流动相由乙腈(含0.5％乙酸和0.025％三氟乙酸)-水(含0.5％乙酸和0.025％三氟乙酸)(85:15,v/V)组成,流速0.25 mL/min.采用多反应离子监测(MRM)模式,以电喷雾离子源(ESI)正离子方式检测.结果显示,ADMA、SDMA、NMMA、L-Arg和Cit的线性关系良好,相关系数r均大于0.994 0;ADMA、SDMA和NMMA的线性范围为0.1～5 mmol/L,L-Arg和Cit的线性范围为10～250 mmol/L;5种氨基酸的日内、日间精密度均小于15％,准确度在85％～115％之间.该方法快速、简便、灵敏,可为相关疾病的临床诊断提供一种高效的检测手段.【期刊名称】《质谱学报》【年(卷),期】2016(037)005【总页数】7页(P446-452)【关键词】超高效液相色谱-串联质谱(UPLC-MS/MS);艾普拉唑;蛋白沉淀法;亲水性色谱【作者】田晔;江骥;胡蓓;薛金萍;王洪允【作者单位】福州大学化学学院,福建省功能材料工程研究中心,福建省光动力治疗药物与诊疗工程技术研究中心,福建福州350108;中国医学科学院北京协和医院临床药理中心,北京100730;中国医学科学院北京协和医院临床药理中心,北京100730;中国医学科学院北京协和医院临床药理中心,北京100730;福州大学化学学院,福建省功能材料工程研究中心,福建省光动力治疗药物与诊疗工程技术研究中心,福建福州350108;中国医学科学院北京协和医院临床药理中心,北京100730【正文语种】中文【中图分类】O657.63一氧化氮是人体重要的信使分子，L-精氨酸(L-Arg)在一氧化氮全酶(NOS)的催化下，产生一氧化氮(NO)和瓜氨酸(Cit)[1-2]。

肝素-琼脂糖凝胶6FF的制备及其在抗凝血酶III分离纯化中
的应用
穆成华;姚红娟;马光辉;连宾
【期刊名称】《过程工程学报》
【年(卷),期】2005(5)5
【摘要】以自制琼脂糖凝胶6FF为基质,肝素为配基,利用还原胺化方法制备了肝素-琼脂糖凝胶6FF介质.考察了肝素偶联反应的影响因素,并对其进行了优化,得到肝素配基的含量为2.7mg/mL.用所制介质从人血浆中分离出抗凝血酶III,从血浆开始计算的抗凝血酶III活性回收率为31.76%,与商品Heparin-SepharoseCL-6B的分离效果相当.
【总页数】5页(P545-549)
【关键词】亲和层析;肝素;抗凝血酶Ⅲ
【作者】穆成华;姚红娟;马光辉;连宾
【作者单位】中国科学院过程工程研究所生化工程国家重点实验室;贵州大学化学与生物工程学院
【正文语种】中文
【中图分类】O657
【相关文献】
1.镍化磁性琼脂糖凝胶微球的制备及其在蛋白纯化中的应用研究 [J], 梁媛媛;张海利;杨鲁萍;焦艳华
2.一种新型的琼脂糖疏水层析介质开发及其在重组乙肝表面抗原(CHO-HBsAg)分离纯化中的应用 [J], 王阳木;闭静秀;赵岚;周卫斌;李岩;黄永东;张焱;林海;苏志国
3.琼脂糖凝胶电泳分离纯化和制备质粒DNA [J], 郭三堆
4.肝素—琼脂糖凝胶4B的制备及在肝生长因子提取纯化中的… [J], 姜世明;徐朝晖
5.羧甲基琼脂糖微球的制备及其在国产碱性蛋白酶纯化中的应用 [J], 刘微;王勇尊;许慧;李优鑫;包建民
因版权原因，仅展示原文概要，查看原文内容请购买。

SDS是阴离子去污剂，能断裂分子内和分子间的氢键，破坏蛋白分子的二、三级结构。

作用有四：去蛋白质电荷、解离蛋白质之间的氢键、取消蛋白分子内的疏水作用、去多肽折叠。

而强还原剂如巯基乙醇，二硫苏糖醇能使绊胱氨酸残基间的二硫键断裂。

在样品和凝胶中加入还原剂和SDS后，分子被解聚成多肽链，解聚后的氨基酸侧链和SDS结合成蛋白- SDS 胶束，所带的负电荷大大超过了蛋白原有的电荷量，这样就消除了不同分子间的电荷差异和结构差异。

SDS-PAGE一般采用的是不连续缓冲系统，浓缩胶是由AP催化聚合而成的大孔胶，凝胶缓冲液为pH6.7的Tris-HC1。

分离胶是由AP催化聚合而成的小孔胶，凝胶缓冲液为pH8.9 Tris-HC1。

电极缓冲液是pH8.3 Tris-甘氨酸缓冲液。

2种孔径的凝胶、2种缓冲体系、3种pH值使不连续体系形成了凝胶孔径、pH值、缓冲液离子成分的不连续性，这是样品浓缩的主要因素。

当样品液和浓缩胶选TRIS/HCL缓冲液，电极液选TRIS/甘氨酸。

电泳开始后，HCL解离成氯离子，甘氨酸解离出少量的甘氨酸根离子。

蛋白质带负电荷，因此一起向正极移动，其中氯离子最快，甘氨酸根离子最慢，蛋白居中。

电泳开始时氯离子泳动率最大，超过蛋白，因此在后面形成低电导区，而电场强度与低电导区成反比，因而产生较高的电场强度，使蛋白和甘氨酸根离子迅速移动，形成以稳定的界面，使蛋白聚集在移动界面附近，浓缩成一中间层。

过硫酸铵（AP）为催化剂，四甲基乙二胺（TEMED）为加速剂。

在聚合过程中，TEMED 催化过硫酸铵产生自由基，后者引发丙烯酰胺单体聚合，同时甲叉双丙烯酰胺与丙烯酰胺链间产生甲叉键交联，从而形成三维网状结构。

当分子量在15KD到200KD之间时，蛋白质的迁移率和分子量的对数呈线性关系，符合下式：logMW=K-bX，式中：MW为分子量，X为迁移率，k、b均为常数，若将已知分子量的标准蛋白质的迁移率对分子量对数作图，可获得一条标准曲线，未知蛋白质在相同条件下进行电泳，根据它的电泳迁移率即可在标准曲线上求得分子量。

柳氮磺吡啶肠溶片人体生物等效性研究梁燕;王凌;王蕊;常臻;钟延强;邹豪【摘要】目的柳氮磺吡啶肠溶片(sulfasalazine enteric-coated tablet,SECT)属于口服不易吸收的药物,其人体药动学和相对生物利用度研究存在一定难度.本研究通过评价不同厂家生产的两种制剂的生物等效性,建立SECT的研究方法.方法对24名健康男性志愿者按随机交叉自身对照的方法,给予单剂量口服500 mgSECT 受试制剂和参比制剂,两次试验间隔为1周,分别测定血浆柳氮磺吡啶(sulfasalazine,SS)、磺胺吡啶(sulfapyridine,SP)、5-氨基水杨酸(5-aminosalicylic acid,5-ASA)浓度.结果血浆中SS、SP、5-ASA的药动学参数如下:以AUC0→t计算,受试制剂SECT中SS相对生物利用度为(108.8±24.1)％;SP相对生物利用度为(105.0±25.0)％;5-ASA相对生物利用度为(94.1±22.0)％.以AUCo→∞计算,受试制剂SECT中SS相对生物利用度为(106.9±23.4)％;SP相对生物利用度为(104.1±25.0)％;5-ASA相对生物利用度为(93.6±22.2)％.受试制剂与参比制剂的血药浓度-时间曲线基本一致,受试制剂与参比制剂主要药动学参数无显著性差异(P＞0.05).结论两家厂家生产的SECT,受试制剂与参比制剂在健康中国人体内具有生物等效性.【期刊名称】《药学实践杂志》【年(卷),期】2013(031)006【总页数】5页(P424-427,466)【关键词】柳氮磺吡啶;磺胺吡啶;5-氨基水杨酸;生物等效性【作者】梁燕;王凌;王蕊;常臻;钟延强;邹豪【作者单位】第二军医大学药学院药剂学教研室,上海200433;四川大学华西药学院,成都610041;四川大学华西药学院,成都610041;上海信谊药厂有限公司药物研究所,上海201206;上海信谊药厂有限公司药物研究所,上海201206;第二军医大学药学院药剂学教研室,上海200433;第二军医大学药学院药剂学教研室,上海200433【正文语种】中文【中图分类】R969.1柳氮磺吡啶肠溶片(sulfasalazine enteric-coated tablet,SECT)临床应用广泛，可用于治疗克隆病和溃疡性结肠炎，目前也是类风湿关节炎(RA)的治疗主要用药，并可用于治疗强直性脊柱炎、瑞氏综合征、反应性关节炎等，近年来柳氮磺吡啶(sulfasalazine,SS)亦报道用于治疗皮肤病。

第35卷第3期 长治医学院学报2021 年 6 月JOURNAL OF CHANGZHI MEDICAI COLLEGE167Vol. 35 No. 3Jun. 2021高效液相色谱法测定注射用美罗培南的有关物质李金格禹玉洪**作者单位山西医科大学药学院药剂教研室(030001)* 通信作者(E-mail ：3024546064@ qq. com)摘要目的：探讨优化注射用美罗培南杂质A 、B 的测定方法。

方法：运用高效液相色谱法(HPLC) 进行检测，色谱柱以十八烷基硅烷键合硅胶为填充剂；流动相A ：20. 0 mmol-L'1磷酸二氢钠-甲醇(89 ：11, V/V)，流动相B ：甲醇，流速1.0 mL-min 1,检测波长220 nm,柱温30 P 。

结果：主成分峰与杂质峰可实现基线分离，杂质A 检测限和定量限分别为1. 62,5.15 ng,杂质B 检测限和定量限分别为0. 85,2. 51 ng ；1.2~24.0 ixg-mL -1的杂质A 具有良好的线性关系(r=0. 999 9) ,0.7-14.0 ixg-mL 1的杂质B 具有良好的线性 关系(r=0. 999 9)；杂质A 平均加样回收率为101.2%(RSD= 1.38%,“ = 9),杂质B 平均加样回收率为100.2%(RSD=1.29%,n = 9)o 经破坏性试验，美罗培南可能的降解杂质A 、B 均不干扰美罗培南主峰的测定。

结论:检测限及定量限、精密度、稳定性、耐用性试验结果均符合HPLC 有关物质测定的方法学验证要求。

本HPLC 法专属性良好，可用于美罗培南的主要杂质A 、B 的定量控制。

关键词美罗培南；有关物质；高效液相色谱法中图分类号R97&1文献标识码 A 文章编号1006(2021)03-167-05Determination of Related Substances of Meropenem for Injection by High Performance Liquid ChromatographyLI Jinge , YU YuhongDepartment of Pharmacy , School of Pharmacy , Shanxi Medical UniversityAbstract Objective : To explore and optimize the determination method of impulity A andB of meropenem for injection. Meth ­ods :Using the high performance liquid chromatography ( HPLC ) to detection , Octadecylsilane-bonded silica gel was used as the fi ­ler ； The mobile phase A : 20. 0 mmol * L -1 sodium dihydrogen phosphate-methanol ( 89 ： 11, V/V) . The mobile phase B : methanol ,the flow rate was 1. 0 mL *m in _1 and the detection wavelength was set at 220 nm. The column temperature was set at 30 % . Re ­sults :The principal component peak and impurity peak could achieve baseline separation. The detection limit and quantitative limit of impurity A were 1. 62 ng and 5. 15 ng respectively , and the detection limit and quantitative lim 让 of impurity B were 0. 85 ng and 2. 51 ng respectively. There was A good linear relationship between impurity A (r = 0. 999 9) and impurity B ( r= 0. 999 9 ) in therange of 1. 2-24. 0 |xg *m L _1 and 0. 7 ~ 14. 0 jig * mL -1. The average recovery of impurity A was 101. 2% ( RSD = 1. 38% , n = 9),and that of impurity B was 100. 2% ( RSD = 1. 29% , n= 9). After stressing test, both of impurities A and B of meropenem didn * tinterfere w 让h the determination of meropenem main peak. Conclusion : The test results of detection lim 让 and quant N ative lim 让，pre ­cision ,stabil 让y and durability all meet the methodological verification requirements of HPLC related substance determination. The HPLC method has good specificity and can be used for the quant N ative control of major impurities A and B.Key words meropenem ； related substances ； HPLC注射用美罗培南(Meropenem, C ”H25弘0申) 是由日本住友制药公司与英国ICI 制药公司共同 开发的第二代碳青霉烯抗生素，通过干扰细菌细胞壁的合成发挥杀菌作用，具有广谱耐酶的特 点[1_4]o 在美罗培南原料中常检测出杂质A 及杂质B,杂质A(C 17H 27N 3O 6S)为美罗培南四元内酰 胺环结构发生水解反应而形成，系美罗培南的降 解产物；杂质B(C 34H 50N 6O 10S 2)为美罗培南与杂质A 发生聚合反应而形成，系美罗培南的二聚体X 。

活性氧在乳香挥发油抑制结肠癌细胞增殖中的作用发表时间：2015-10-14T16:42:06.120Z 来源：《河南中医》2015年5月供稿作者：郝雪微1 阮莹2 刘欣宇1 顾园竹1 李金桐1[导读] 1哈尔滨医科大学大庆校区医学检验与技术学院2哈尔滨医科大学药学院黑龙江大庆所有实验结果均是以3次独立实验数据分析所得。

采用SPSS13.0统计学软件进行t检验及方差分析。

郝雪微1 阮莹2 刘欣宇1 顾园竹1 李金桐1（1哈尔滨医科大学大庆校区医学检验与技术学院黑龙江大庆 163000）（2哈尔滨医科大学药学院黑龙江大庆 163319）【摘要】目的：研究活性氧在乳香挥发油抑制结肠癌细胞增殖中的作用。

方法：用MTT法检测乳香挥发油对结肠癌细胞SW480的抑制率，用流式细胞术检测细胞内ROS含量及细胞的凋亡率。

结果：乳香挥发油对结肠癌细胞有抑制作用，同时使细胞内ROS含量增加，凋亡率增加，加入GSH后可以减少ROS含量，减少凋亡率。

结论：乳香挥发油可以抑制结肠癌细胞的增殖及诱导凋亡，GSH可以通过减少活性氧含量减少细胞凋亡。

【关键词】乳香挥发油；人结肠癌细胞SW480；活性氧；凋亡【中图分类号】R34 【文献标识码】B 【文章编号】1003-5028（2015）5-0235-021 实验材料药品与试剂：乳香挥发油为课题组之前制备[2]，成分大多为单萜、倍半萜、醇及酯类等，其中含量最高的成分为乙酸辛酯、橙花叔醇异丁酯、3,7,11-三甲基-1,6,10-十二碳三烯-3-醇甲酸酯及β-榄香烯，含量分别为34.660%、18.290%、9.612%和5.614%。

左旋谷胱甘肽(L-Glutathione, GSH)购于Sigma公司。

MTT，活性氧(Reactive Oxygen Species, ROS)检测试剂盒、Annexin V-FITC细胞凋亡检测试剂盒购于碧云天生物技术公司。

细胞及培养：人结肠癌细胞SW480细胞为本室保存, 接种在含10%胎牛血清、2mmol/L谷氨酰胺、100U/ml青霉素和100μg /ml链霉素的RPMI-1640培养液中，在37℃，5%的C02环境下培养。

第42 卷第 11 期2023 年11 月Vol.42 No.111469~1478分析测试学报FENXI CESHI XUEBAO（Journal of Instrumental Analysis）超高效液相色谱-串联质谱法测定化妆品中15种N-亚硝胺化合物汪毅1，梁文耀1，何国山1，陈张好2，周智明2，吴谦1，席绍峰1，谭建华1*（1．广州质量监督检测研究院，国家化妆品质量检验检测中心（广州），广东广州511447；2．广东省药品检验所，广东广州510663）摘要：采用超高效液相色谱-串联质谱（UPLC-MS/MS）建立了化妆品中15种痕量N-亚硝胺化合物的分析方法。

水剂样品以水或乙腈分组超声提取，膏霜乳液样品采用亚铁氰化钾-乙酸锌溶液沉淀大分子或者饱和氯化钠-乙腈盐析分组处理后，以Agilent Poroshell 120 SB-Aq（100 mm×3.0 mm，2.7 μm）色谱柱分离，经大气压化学电离源（APCI）电离，多反应监测模式检测，以同位素内标法定量。

结果表明，15种N-亚硝胺化合物在相应质量浓度范围内线性关系良好（r2＞0.995），检出限和定量下限分别为5~15 ng/g和15~45 ng/g。

水、乳、膏霜3种化妆品基质在25、50、100 ng/g加标水平下的平均回收率为88.0%~111%，相对标准偏差（RSD，n=6）为1.4%~9.8%。

该方法用于市售化妆品检测，发现13批次样品检出N-亚硝基二乙醇胺（NDELA），其中1批次超限量值。

方法的专属性强，灵敏度高，精密度好，解决了N-亚硝胺化合物稳定性差、易被干扰等问题，适用于化妆品中15种N-亚硝胺化合物的痕量测定。

关键词：N-亚硝胺化合物；化妆品；超高效液相色谱-串联质谱法（UPLC-MS/MS）；大气压化学电离源中图分类号：O657.63；O623.732文献标识码：A 文章编号：1004-4957（2023）11-1469-10 Determination of Fifteen N-nitrosamine Compounds in Cosmetics by Ultra Performance Liquid Chromatography-TandemMass SpectrometryWANG Yi1，LIANG Wen-yao1，HE Guo-shan1，CHEN Zhang-hao2，ZHOU Zhi-ming2，WU Qian1，XI Shao-feng1，TAN Jian-hua1*（1．Guangzhou Quality Supervision and Testing Institute，National Quality Supervision and Testing Center for Cosmetics（Guangzhou），Guangzhou　511447，China；2．Guangdong Institute for Drug Control，Guangzhou　510663）Abstract：An ultra performance liquid chromatography-tandem mass spectrometric（UPLC-MS/MS）method was established for detecting 15 trace N-nitrosamine compounds in cosmetics. The final estab⁃lished method involved ultrasonic extraction of cosmetics using water or acetonitrile for different com⁃pounds. The samples were treated with potassium ferrocyanide-zinc acetate solution for precipitating macromolecules or saturated sodium chloride-acetonitrile for salting out.An Agilent Poroshell 120 SB-Aq（100 mm × 3.0 mm，2.7 μm） chromatography column was used for separation，followed by atmospheric pressure chemical ionization（APCI） source and multiple reaction monitoring mode detec⁃tion in the isotope internal standard method for quantification. The result showed good linearity（r2> 0.995） for the 15 N-nitrosamine compounds in their respective concentration ranges，with detection and quantitation limits of 5-15 ng/g and 15-45 ng/g，respectively.The average recoveries for the three cosmetic matrices（aqueous，emulsion，cream） at spiked levels of 25，50，100 ng/g were be⁃tween 88.0% and 111%，with relative standard deviations（RSD，n=6） of 1.4%-9.8%. The method was applied to the detection of commercial cosmetics and N-nitrosodiethanolamine（NDELA） was de⁃tected in 13 batches，with one batch exceeding the limit. The strong specificity，high sensitivity，and good precision made the method could solve the problems of poor stability and easy interference ofdoi：10.19969/j.fxcsxb.23051602收稿日期：2023－05－16；修回日期：2023－06－10基金项目：广东省药品监督管理局化妆品风险评估重点实验室专项（2021ZDZ03）；广东省市场监督管理局科技项目（2022CZ06）∗通讯作者：谭建华，博士，正高级工程师，研究方向：色谱－质谱检测技术研究，E-mail：tanjianhua0734@第 42 卷分析测试学报N-nitrosamine compounds，and was suitable for the trace determination of 15 N-nitrosamine com⁃pounds in cosmetics.Key words：N-nitrosamine compounds；cosmetics；ultra performance liquid chromatography-tan⁃dem mass spectrometry（UPLC-MS/MS）；atmospheric pressure chemical ionization（APCI） sourceN-亚硝胺化合物是一类具有N-亚硝基结构的化合物，因取代基的不同，形成了种类繁多的同系物，目前已发现超过300种［1］。

Guidance for Industry Bioanalytical Method ValidationU.S. Department of Health and Human ServicesFood and Drug AdministrationCenter for Drug Evaluation and Research (CDER)Center for Veterinary Medicine (CVM)May 2001BPGuidance for Industry Bioanalytical Method ValidationAdditional copies are available from:Drug Information Branch (HFD-210)Center for Drug Evaluation and Research (CDER)5600 Fishers Lane, Rockville, MD 20857 (Tel) 301-827-4573Internet at /cder/guidance/index.htmorCommunications Staff (HFV-12)Center for Veterinary Medicine (CVM)7500 Standish Place, Rockville, MD 20855 (Tel) 301–594-1755Internet at /cvmU.S. Department of Health and Human ServicesFood and Drug AdministrationCenter for Drug Evaluation and Research (CDER)Center for Veterinary Medicine (CVM)May 2001BPTable of ContentsI.INTRODUCTION (1)II.BACKGROUND (1)A.F ULL V ALIDATION (2)B.P ARTIAL V ALIDATION (2)C.C ROSS-V ALIDATION (3)III.REFERENCE STANDARD (4)IV.METHOD DEVELOPMENT: CHEMICAL ASSAY (4)A.S ELECTIVITY (4)B.A CCURACY, P RECISION, AND R ECOVERY (5)C.C ALIBRATION/S TANDARD C URVE (5)D.S TABILITY (6)E.P RINCIPLES OF B IOANALYTICAL M ETHOD V ALIDATION AND E STABLISHMENT (8)F.S PECIFIC R ECOMMENDATIONS FOR M ETHOD V ALIDATION (10)V.METHOD DEVELOPMENT: MICROBIOLOGICAL AND LIGAND-BINDING ASSAYS (11)A.S ELECTIVITY I SSUES (11)B.Q UANTIFICATION I SSUES (12)VI.APPLICATION OF VALIDATED METHOD TO ROUTINE DRUG ANALYSIS (13)A CCEPTANCE C RITERIA FOR THE R UN (15)VII.DOCUMENTATION (16)A.S UMMARY I NFORMATION (16)B.D OCUMENTATION FOR M ETHOD E STABLISHMENT (17)C.A PPLICATION TO R OUTINE D RUG A NALYSIS (17)D.O THER I NFORMATION (19)GLOSSARY (20)GUIDANCE FOR INDUSTRY1Bioanalytical Method ValidationI.INTRODUCTIONThis guidance provides assistance to sponsors of investigational new drug applications (INDs), new drug applications (NDAs), abbreviated new drug applications (ANDAs), and supplements in developing bioanalytical method validation information used in human clinical pharmacology, bioavailability (BA), and bioequivalence (BE) studies requiring pharmacokinetic (PK) evaluation. This guidance also applies to bioanalytical methods used for non-human pharmacology/toxicology studies and preclinical studies. For studies related to the veterinary drug approval process, this guidance applies only to blood and urine BA, BE, and PK studies.The information in this guidance generally applies to bioanalytical procedures such as gas chromatography (GC), high-pressure liquid chromatography (LC), combined GC and LC mass spectrometric (MS) procedures such as LC-MS, LC-MS-MS, GC-MS, and GC-MS-MS performed for the quantitative determination of drugs and/or metabolites in biological matricessuch as blood, serum, plasma, or urine. This guidance also applies to other bioanalytical methods, such as immunological and microbiological procedures, and to other biological matrices, such as tissue and skin samples.This guidance provides general recommendations for bioanalytical method validation. The recommendations can be adjusted or modified depending on the specific type of analytical method used. II.BACKGROUND1 This guidance has been prepared by the Biopharmaceutics Coordinating Committee in the Center for Drug Evaluation and Research (CDER) in cooperation with the Center for Veterinary Medicine (CVM) at the Food and Drug Administration.This guidance has been developed based on the deliberations of two workshops: (1) Analytical Methods Validation: Bioavailability, Bioequivalence, and Pharmacokinetic Studies (held on December 3B5, 19902 ) and (2) Bioanalytical Methods Validation C A Revisit With a Decade of Progress (held on January 12B14, 20003).Selective and sensitive analytical methods for the quantitative evaluation of drugs and their metabolites (analytes) are critical for the successful conduct of preclinical and/or biopharmaceutics and clinical pharmacology studies. Bioanalytical method validation includes all of the procedures that demonstrate that a particular method used for quantitative measurement of analytes in a given biological matrix, such as blood, plasma, serum, or urine, is reliable and reproducible for the intended use. The fundamental parameters for this validation include (1) accuracy, (2) precision, (3) selectivity, (4) sensitivity, (5) reproducibility, and (6) stability. Validation involves documenting, through the use of specific laboratory investigations, that the performance characteristics of the method are suitable and reliable for the intended analytical applications. The acceptability of analytical data corresponds directly to the criteria used to validate the method.Published methods of analysis are often modified to suit the requirements of the laboratory performing the assay. These modifications should be validated to ensure suitable performance of the analytical method. When changes are made to a previously validated method, the analyst should exercise judgment as to how much additional validation is needed. During the course of a typical drug development program, a defined bioanalytical method undergoes many modifications. The evolutionary changes to support specific studies and different levels of validation demonstrate the validity of an assay’s performance. Different types and levels of validation are defined and characterized as follows:A.Full Validation•Full validation is important when developing and implementing a bioanalytical method for the first time.•Full validation is important for a new drug entity.• A full validation of the revised assay is important if metabolites are added to an existing assay for quantification.B.Partial ValidationPartial validations are modifications of already validated bioanalytical methods. Partial validation can range from as little as one intra-assay accuracy and precision determination to a nearly full2 Workshop Report: Shah, V.P. et al., Pharmaceutical Research: 1992; 9:588-592.3 Workshop Report: Shah, V.P. et al., Pharmaceutical Research: 2000; 17:in press.validation. Typical bioanalytical method changes that fall into this category include, but are not limited to:•Bioanalytical method transfers between laboratories or analysts•Change in analytical methodology (e.g., change in detection systems)•Change in anticoagulant in harvesting biological fluid•Change in matrix within species (e.g., human plasma to human urine)•Change in sample processing procedures•Change in species within matrix (e.g., rat plasma to mouse plasma)•Change in relevant concentration range•Changes in instruments and/or software platforms•Limited sample volume (e.g., pediatric study)•Rare matrices•Selectivity demonstration of an analyte in the presence of concomitant medications•Selectivity demonstration of an analyte in the presence of specific metabolitesC.Cross-ValidationCross-validation is a comparison of validation parameters when two or more bioanalytical methods are used to generate data within the same study or across different studies. An example of cross-validation would be a situation where an original validated bioanalytical method serves as thereference and the revised bioanalytical method is the comparator. The comparisons should be done both ways.When sample analyses within a single study are conducted at more than one site or more than one laboratory, cross-validation with spiked matrix standards and subject samples should be conducted at each site or laboratory to establish interlaboratory reliability. Cross-validation should also be considered when data generated using different analytical techniques (e.g., LC-MS-MS vs.ELISA4) in different studies are included in a regulatory submission.All modifications should be assessed to determine the recommended degree of validation. The analytical laboratory conducting pharmacology/toxicology and other preclinical studies for regulatory submissions should adhere to FDA=s Good Laboratory Practices (GLPs)5 (21 CFR part 58) and to sound principles of quality assurance throughout the testing process. The bioanalytical method for human BA, BE, PK, and drug interaction studies must meet the criteria in 21 CFR 320.29. The analytical laboratory should have a written set of standard operating procedures (SOPs) to ensure a complete system of quality control and assurance. The SOPs should cover all aspects of analysis from the time the sample is collected and reaches the laboratory until the results of the analysis are reported. The SOPs also should include record keeping, security and chain of sample custody4 Enzyme linked immune sorbent assay5 For the Center for Veterinary Medicine, all bioequivalence studies are subject to Good Laboratory Practices.(accountability systems that ensure integrity of test articles), sample preparation, and analytical tools such as methods, reagents, equipment, instrumentation, and procedures for quality control and verification of results.The process by which a specific bioanalytical method is developed, validated, and used in routine sample analysis can be divided into (1) reference standard preparation, (2) bioanalytical method development and establishment of assay procedure, and (3) application of validated bioanalytical method to routine drug analysis and acceptance criteria for the analytical run and/or batch. These three processes are described in the following sections of this guidance.III.REFERENCE STANDARDAnalysis of drugs and their metabolites in a biological matrix is carried out using samples spiked with calibration (reference) standards and using quality control (QC) samples. The purity of the reference standard used to prepare spiked samples can affect study data. For this reason, an authenticated analytical reference standard of known identity and purity should be used to prepare solutions of known concentrations. If possible, the reference standard should be identical to the analyte. When this is not possible, an established chemical form (free base or acid, salt or ester) of known purity can be used. Three types of reference standards are usually used: (1) certified reference standards (e.g., USP compendial standards); (2) commercially supplied reference standards obtained from a reputable commercial source; and/or (3) other materials of documented purity custom-synthesized by an analytical laboratory or other noncommercial establishment. The source and lot number, expiration date, certificates of analyses when available, and/or internally or externally generated evidence of identity and purity should be furnished for each reference standard.IV.METHOD DEVELOPMENT: CHEMICAL ASSAYThe method development and establishment phase defines the chemical assay. The fundamental parameters for a bioanalytical method validation are accuracy, precision, selectivity, sensitivity, reproducibility, and stability. Measurements for each analyte in the biological matrix should be validated. In addition, the stability of the analyte in spiked samples should be determined. Typical method development and establishment for a bioanalytical method include determination of (1) selectivity, (2) accuracy, precision, recovery, (3) calibration curve, and (4) stability of analyte in spiked samples.A.SelectivitySelectivity is the ability of an analytical method to differentiate and quantify the analyte in thepresence of other components in the sample. For selectivity, analyses of blank samples of theappropriate biological matrix (plasma, urine, or other matrix) should be obtained from at leastsix sources. Each blank sample should be tested for interference, and selectivity should be ensured at the lower limit of quantification (LLOQ).Potential interfering substances in a biological matrix include endogenous matrix components, metabolites, decomposition products, and in the actual study, concomitant medication and other exogenous xenobiotics. If the method is intended to quantify more than one analyte, each analyte should be tested to ensure that there is no interference.B.Accuracy, Precision, and RecoveryThe accuracy of an analytical method describes the closeness of mean test results obtained by the method to the true value (concentration) of the analyte. Accuracy is determined by replicate analysis of samples containing known amounts of the analyte. Accuracy should be measured using a minimum of five determinations per concentration. A minimum of three concentrations in the range of expected concentrations is recommended. The mean value should be within 15% of the actual value except at LLOQ, where it should not deviate by more than 20%. The deviation of the mean from the true value serves as the measure of accuracy.The precision of an analytical method describes the closeness of individual measures of an analyte when the procedure is applied repeatedly to multiple aliquots of a single homogeneous volume of biological matrix. Precision should be measured using a minimum of five determinations per concentration. A minimum of three concentrations in the range of expected concentrations is recommended. The precision determined at each concentration level should not exceed 15% of the coefficient of variation (CV) except for the LLOQ, where it should not exceed 20% of the CV. Precision is further subdivided into within-run, intra-batch precision or repeatability, which assesses precision during a single analytical run, and between-run, inter-batch precision or repeatability, which measures precision with time, and may involve different analysts, equipment, reagents, and laboratories.The recovery of an analyte in an assay is the detector response obtained from an amount of the analyte added to and extracted from the biological matrix, compared to the detector response obtained for the true concentration of the pure authentic standard. Recovery pertains to the extraction efficiency of an analytical method within the limits of variability. Recovery of the analyte need not be 100%, but the extent of recovery of an analyte and of the internal standard should be consistent, precise, and reproducible. Recovery experiments should be performed by comparing the analytical results for extracted samples at three concentrations (low, medium, and high) with unextracted standards that represent 100% recovery.C.Calibration/Standard CurveA calibration (standard) curve is the relationship between instrument response and known concentrations of the analyte. A calibration curve should be generated for each analyte in thesample. A sufficient number of standards should be used to adequately define the relationship between concentration and response. A calibration curve should be prepared in the same biological matrix as the samples in the intended study by spiking the matrix with known concentrations of the analyte. The number of standards used in constructing a calibration curve will be a function of the anticipated range of analytical values and the nature of theanalyte/response relationship. Concentrations of standards should be chosen on the basis of the concentration range expected in a particular study. A calibration curve should consist of a blank sample (matrix sample processed without internal standard), a zero sample (matrix sample processed with internal standard), and six to eight non-zero samples covering the expected range, including LLOQ.1.Lower Limit of Quantification (LLOQ)The lowest standard on the calibration curve should be accepted as the limit ofquantification if the following conditions are met:C The analyte response at the LLOQ should be at least 5 times the responsecompared to blank response.C Analyte peak (response) should be identifiable, discrete, and reproducible witha precision of 20% and accuracy of 80-120%.2.Calibration Curve/Standard Curve/Concentration-ResponseThe simplest model that adequately describes the concentration-response relationshipshould be used. Selection of weighting and use of a complex regression equation should be justified. The following conditions should be met in developing a calibration curve:C#20% deviation of the LLOQ from nominal concentrationC#15% deviation of standards other than LLOQ from nominal concentrationAt least four out of six non-zero standards should meet the above criteria, including the LLOQ and the calibration standard at the highest concentration. Excluding thestandards should not change the model used.D.StabilityDrug stability in a biological fluid is a function of the storage conditions, the chemical properties of the drug, the matrix, and the container system. The stability of an analyte in a particular matrix and container system is relevant only to that matrix and container system and should not be extrapolated to other matrices and container systems. Stability procedures should evaluate the stability of the analytes during sample collection and handling, after long-term (frozen at theintended storage temperature) and short-term (bench top, room temperature) storage, and after going through freeze and thaw cycles and the analytical process. Conditions used in stability experiments should reflect situations likely to be encountered during actual sample handling and analysis. The procedure should also include an evaluation of analyte stability in stock solution.All stability determinations should use a set of samples prepared from a freshly made stock solution of the analyte in the appropriate analyte-free, interference-free biological matrix. Stock solutions of the analyte for stability evaluation should be prepared in an appropriate solvent at known concentrations.1.Freeze and Thaw StabilityAnalyte stability should be determined after three freeze and thaw cycles. At least three aliquots at each of the low and high concentrations should be stored at the intendedstorage temperature for 24 hours and thawed unassisted at room temperature. Whencompletely thawed, the samples should be refrozen for 12 to 24 hours under the sameconditions. The freeze–thaw cycle should be repeated two more times, then analyzedon the third cycle. If an analyte is unstable at the intended storage temperature, thestability sample should be frozen at -700C during the three freeze and thaw cycles.2.Short-Term Temperature StabilityThree aliquots of each of the low and high concentrations should be thawed at roomtemperature and kept at this temperature from 4 to 24 hours (based on the expectedduration that samples will be maintained at room temperature in the intended study) and analyzed.3.Long-Term StabilityThe storage time in a long-term stability evaluation should exceed the time between the date of first sample collection and the date of last sample analysis. Long-term stabilityshould be determined by storing at least three aliquots of each of the low and highconcentrations under the same conditions as the study samples. The volume of samples should be sufficient for analysis on three separate occasions. The concentrations of allthe stability samples should be compared to the mean of back-calculated values for the standards at the appropriate concentrations from the first day of long-term stabilitytesting.4.Stock Solution StabilityThe stability of stock solutions of drug and the internal standard should be evaluated at room temperature for at least 6 hours. If the stock solutions are refrigerated or frozenfor the relevant period, the stability should be documented. After completion of thedesired storage time, the stability should be tested by comparing the instrumentresponse with that of freshly prepared solutions.5.Post-Preparative StabilityThe stability of processed samples, including the resident time in the autosampler, should be determined. The stability of the drug and the internal standard should be assessedover the anticipated run time for the batch size in validation samples by determiningconcentrations on the basis of original calibration standards.Although the traditional approach of comparing analytical results for stored samples with those for freshly prepared samples has been referred to in this guidance, other statistical approaches based on confidence limits for evaluation of an analyte=s stability in abiological matrix can be used. SOPs should clearly describe the statistical method andrules used. Additional validation may include investigation of samples from dosedsubjects.E.Principles of Bioanalytical Method Validation and Establishment•The fundamental parameters to ensure the acceptability of the performance of a bioanalytical method validation are accuracy, precision, selectivity, sensitivity,reproducibility, and stability.• A specific, detailed description of the bioanalytical method should be written. This can be in the form of a protocol, study plan, report, and/or SOP.•Each step in the method should be investigated to determine the extent to which environmental, matrix, material, or procedural variables can affect the estimation of analyte in the matrix from the time of collection of the material up to and including the time ofanalysis.•It may be important to consider the variability of the matrix due to the physiological nature of the sample. In the case of LC-MS-MS-based procedures, appropriate steps should be taken to ensure the lack of matrix effects throughout the application of the method,especially if the nature of the matrix changes from the matrix used during method validation.• A bioanalytical method should be validated for the intended use or application. All experiments used to make claims or draw conclusions about the validity of the methodshould be presented in a report (method validation report).•Whenever possible, the same biological matrix as the matrix in the intended samples should be used for validation purposes. (For tissues of limited availability, such as bone marrow, physiologically appropriate proxy matrices can be substituted.)•The stability of the analyte (drug and/or metabolite) in the matrix during the collection process and the sample storage period should be assessed, preferably prior to sampleanalysis.•For compounds with potentially labile metabolites, the stability of analyte in matrix from dosed subjects (or species) should be confirmed.•The accuracy, precision, reproducibility, response function, and selectivity of the method for endogenous substances, metabolites, and known degradation products should beestablished for the biological matrix. For selectivity, there should be evidence that thesubstance being quantified is the intended analyte.•The concentration range over which the analyte will be determined should be defined in the bioanalytical method, based on evaluation of actual standard samples over the range,including their statistical variation. This defines the standard curve.• A sufficient number of standards should be used to adequately define the relationship between concentration and response. The relationship between response and concentration should be demonstrated to be continuous and reproducible. The number of standards used should be a function of the dynamic range and nature of the concentration-responserelationship. In many cases, six to eight concentrations (excluding blank values) can define the standard curve. More standard concentrations may be recommended for nonlinear than for linear relationships.•The ability to dilute samples originally above the upper limit of the standard curve should be demonstrated by accuracy and precision parameters in the validation.•In consideration of high throughput analyses, including but not limited to multiplexing, multicolumn, and parallel systems, sufficient QC samples should be used to ensure control of the assay. The number of QC samples to ensure proper control of the assay should be determined based on the run size. The placement of QC samples should be judiciously considered in the run.•For a bioanalytical method to be considered valid, specific acceptance criteria should be set in advance and achieved for accuracy and precision for the validation of QC samples over the range of the standards.F.Specific Recommendations for Method Validation•The matrix-based standard curve should consist of a minimum of six standard points, excluding blanks, using single or replicate samples. The standard curve should cover the entire range of expected concentrations.•Standard curve fitting is determined by applying the simplest model that adequately describes the concentration-response relationship using appropriate weighting and statistical tests for goodness of fit.•LLOQ is the lowest concentration of the standard curve that can be measured with acceptable accuracy and precision. The LLOQ should be established using at least five samples independent of standards and determining the coefficient of variation and/orappropriate confidence interval. The LLOQ should serve as the lowest concentration on the standard curve and should not be confused with the limit of detection and/or the low QC sample. The highest standard will define the upper limit of quantification (ULOQ) of an analytical method.•For validation of the bioanalytical method, accuracy and precision should be determined using a minimum of five determinations per concentration level (excluding blank samples).The mean value should be within ±15% of the theoretical value, except at LLOQ, where it should not deviate by more than ±20%. The precision around the mean value should not exceed 15% of the CV, except for LLOQ, where it should not exceed 20% of the CV.Other methods of assessing accuracy and precision that meet these limits may be equally acceptable.•The accuracy and precision with which known concentrations of analyte in biological matrix can be determined should be demonstrated. This can be accomplished by analysis ofreplicate sets of analyte samples of known concentrations C QC samples C from anequivalent biological matrix. At a minimum, three concentrations representing the entire range of the standard curve should be studied: one within 3x the lower limit of quantification (LLOQ) (low QC sample), one near the center (middle QC), and one near the upperboundary of the standard curve (high QC).•Reported method validation data and the determination of accuracy and precision should include all outliers; however, calculations of accuracy and precision excluding values that are statistically determined as outliers can also be reported.•The stability of the analyte in biological matrix at intended storage temperatures should be established. The influence of freeze-thaw cycles (a minimum of three cycles at twoconcentrations in triplicate) should be studied.•The stability of the analyte in matrix at ambient temperature should be evaluated over a time period equal to the typical sample preparation, sample handling, and analytical run times.•Reinjection reproducibility should be evaluated to determine if an analytical run could be reanalyzed in the case of instrument failure.•The specificity of the assay methodology should be established using a minimum of six independent sources of the same matrix. For hyphenated mass spectrometry-basedmethods, however, testing six independent matrices for interference may not be important.In the case of LC-MS and LC-MS-MS-based procedures, matrix effects should beinvestigated to ensure that precision, selectivity, and sensitivity will not be compromised.Method selectivity should be evaluated during method development and throughout methodvalidation and can continue throughout application of the method to actual study samples.•Acceptance/rejection criteria for spiked, matrix-based calibration standards and validation QC samples should be based on the nominal (theoretical) concentration of analytes.Specific criteria can be set up in advance and achieved for accuracy and precision over therange of the standards, if so desired.V.METHOD DEVELOPMENT: MICROBIOLOGICAL AND LIGAND-BINDING ASSAYSMany of the bioanalytical validation parameters and principles discussed above are also applicable to microbiological and ligand-binding assays. However, these assays possess some unique characteristics that should be considered during method validation.A.Selectivity IssuesAs with chromatographic methods, microbiological and ligand-binding assays should be shown to be selective for the analyte. The following recommendations for dealing with two selectivity issues should be considered:1.Interference From Substances Physiochemically Similar to the Analyte•Cross-reactivity of metabolites, concomitant medications, or endogenouscompounds should be evaluated individually and in combination with the analyteof interest.•When possible, the immunoassay should be compared with a validated reference method (such as LC-MS) using incurred samples and predetermined criteria foragreement of accuracy of immunoassay and reference method.。

超高效液相色谱-串联质谱法同时测定鸡肉中的6种磺胺类药物王红梅（山东省食用植物油质量检验中心山东省莒南县检验检测中心山东临沂276000）试验旨在建立同时快速检测鸡肉中6种磺胺类药物含量的超高效液相色谱串联质谱法。

鸡肉样品用乙腈超声提取，经Thermo Fisher Scientific Hyperis11Gold (100×2.1mm ，1.9μm)色谱柱分离，流动相A ：乙腈；流动相B ：0.1%甲酸水溶液作为流动相梯度洗脱。

电喷雾离子源正离子多反应监测模式定量分析。

结果表明，6种磺胺类药物在浓度10～500ng/mL 范围内线性关系很好，相关系数R 2≥0.9998。

6种磺胺类药物在含量为8和60μg/kg 的样品中，回收率为73%～88%，相对标准偏差RSD 为0.9%～6%。

综上，该方法高效、快速、高分辨，可以用于鸡肉样品中磺胺类药的定性、定量分析。

肉；磺胺类药物；超高效液相色谱-串联质谱磺胺类药物(sulfonamides ，SAs)是指一类具有对氨基苯磺酰胺结构化学药物的总称，用于预防和治疗全身性细菌感染性疾病，是当前畜禽生产中常用的抗菌、抗原虫药物，具有抗菌谱广、价格低、化学性质稳定、高效、低毒、使用方便等优点。

其低剂量使用时，可提高饲料的利用率，促进畜禽的生长，但不正确的用药量、用药时间、方式以及用药部位会导致磺胺在畜禽体内及其排泄物的过量残留，导致磺胺类在人体中蓄积，因此产生过敏反应、泌尿系统损害、抑制白细胞生成等危害，重者导致急性血管性水肿、休克甚至死亡[1]。

因此，我国农业部第235号公告《动物性食品中兽药最高残留限量》对SAs 总量及单个SAs 残留量做了明确规定，均不得超过100μg/kg 。

目前，测定动物源性食品中磺胺类药物的方法不止一种，但超高效液相色谱－串联质谱法背景干扰少，能高效、快速、高分辨的定性、定量检测和自动分析微量物质，为高灵敏测定磺胺类药物提供了支持。