多肽类药物“截短型GLP-1”检测方法相关关键性问题探索
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摘要
目的:建立可行的sGLP-1的溶解方法和标准化操作;考察标准溶液在室温20℃、4℃冷藏保存、-20℃冰冻保存三个储存条件下的稳定性;建立sGLP-1的HPLC-UV检测方法及可行的高浓度sGLP-1生物基质样本SPE前处理方法;初步探索sGLP-1的HPLC-MS/MS检测方法及HPLC-MS/MS检测时低浓度sGLP-1生物基质样本前处理方法;对生物基质中sGLP-1定量检测方法进行探索性研究。
研究方法:根据sGLP-1的一级结构,分别用纯水、纯乙腈、乙腈-水(50:50,v/v)、1 M醋酸钠、0.01 M NaOH溶液、0.1 M NaOH溶液、高温蒸汽灭菌的PBS缓冲溶液溶解药物。考察不同溶解方法的色谱峰,并最终确定药物溶解方法及标准化操作流程。
sGLP-1标准溶液3份分别放置于室温20℃、4℃、-20℃保存,分别在新配制(0 h)、24 h、48 h、72 h、一周、两周、一个月取样,每个条件下做3个平行样本, HPLC-UV检测方法测定。每项下的样本的峰面积均与每次实验前(0 h)对照品溶液峰面积作比较,进行稳定性研究。
通过优化HPLC-UV检测的初始条件中的流动相梯度,确定HPLC-UV检测最终条件并考察线性。考察超滤和固相萃取两种生物基质样本前处理方法,并对固相萃取的各种淋洗液和洗脱液进行评价,选择最优萃取效果的淋洗液和洗脱液。初步探索HPLC-MS/MS检测方法。依次优化一级质谱参数fragment、离子源参数(即氮气干燥气温度、氮气干燥气流速、雾化气压力、毛细管电压),二级质谱参数collision energy、电子倍增器(EMV)、灵敏度参数MS1 Res及MS2 Res质谱参数。优化色谱条件,包括色谱柱的选择、流速、柱温、甲酸浓度、流动相梯度。采用HPLC-MS/MS检测方法优化结果,连续三天测定浓度分别为1 ng/mL、5 ng/mL、10 ng/mL、50 ng/mL、100 ng/mL、300 ng/mL、500 ng/mL的标准系列溶液,对测试峰面积与浓度进行1/x加权线性回归计算标准曲线及r值。分别采用蛋白沉淀法和固相萃取法对生物基质中sGLP-1定量检测方法进行探索性研究。
结果:使用0.1 M NaOH溶液溶解sGLP-1后,再用0.1 M HCl调pH至5.71,此标准储备液溶解方法的色谱峰最好,将其方法进行标准化验证,制定为sGLP-1标准品溶解的标准化方案。经实验考察此法配置的标准储备液在室温20℃时至少稳定放置72 h、4℃冷藏保存时至少稳定2周、-20℃冰冻保存时至少稳定1个月。
实验建立了sGLP-1的HPLC-UV检测方法,最低检测限2μg/mL。同时建立了可行的高浓度时生物基质样本中sGLP-1的固相萃取前处理方案。
优化sGLP-1的HPLC-MS/MS检测方法的质谱参数和色谱条件,对HPLC-MS/MS检测方法进行初步探索。采用HPLC-MS/MS检测方法优化结果,连续测定三天浓度分别为1 ng/mL、5 ng/mL、10 ng/mL、50 ng/mL、100 ng/mL、300 ng/mL、500 ng/mL的标准系列溶液,结果显示在1~500 ng/mL的浓度范围内线性较好。但试验中期更换同一型号色谱柱后再进行相同试验,结果相差较大,经多次重现后均没有得到相同结果,表明HPLC-MS/MS检测sGLP-1的重现性较差,虽然研究中HPLC-MS/MS检测sGLP-1标准溶液方法重现性不佳,检测sGLP-1生物样本存在明显的内源性干扰,但本研究的探索对后续别人研究提供了参考。
结论:本研究建立了可行的sGLP-1的溶解方法和标准化操作;实验证明sGLP-1标准溶液在室温20℃时至少稳定放置72 h、4℃冷藏保存时至少稳定2周、-20℃冰冻保存时至少稳定1个月,因此在标准溶液样本配置和测定过程中没有稳定性问题的干扰;建立了sGLP-1的HPLC-UV检测方法及可行的高浓度sGLP-1生物基质样本SPE前处理方法;初步探索了sGLP-1的HPLC-MS/MS检测方法及HPLC-MS/MS检测时低浓度sGLP-1生物基质样本前处理方法。
AIM: To establish feasible dissoluble method and standard procedure of sGLP-1; to investigate the stability of standard solution under three stored conditions of 20℃, 4℃and -20℃; to establish detected method of HPLC-UV and feasible SPE pre-processing method of sGLP-1 with high concentration in biologic matrix; to research for detected method of HPLC-MS/MS and pre-processing method of sGLP-1 with low concentration in biologic matrical samples; to make a exploratory study of quantitative detected method of sGLP-1 in biologic base matrix.
METHOD: According to primary structure of sGLP-1, a bottle of sGLP-1 powder is dissolved by water, acetonitrile, acetonitrile-water(50:50,v/v), 1 M sodium acetate, 0.01 M sodium hydroxide, 0.1 M sodium hydroxide, PBS buffer solution for pyro-steam sterilization. To inspect different chromatographic peaks of different dissolved methods and to determinate the final dissolved method of the drug and the standard operated procedure.
The standard solution of sGLP-1 is stored at 20℃、4℃、-20℃respectively, and is measured at the moment of preparation, 24 h, 48 h, 72 h, one week, two weeks and one month. Three samples of every condition are derected by HPLC-UV. Every area of peek of drug is compared to the area of peek of control article for stability research.
The final detected conditions of HPLC-UV are determinated through optimizing the mobile phase gradient of HPLC-UV initial condition. To investigate ultrafiltration and solid phase extraction for biologic metrical sample, meanwhile to establish every elutriants and eluants in order to select the most effective elutriants and eluants.
The HPLC-MS/MS method is explored initially. To optimize the first class mass spectrum parameter fragment, ion source parameter (gas temperature, gas flow, nebulizer, capillary), the second class mass spectrum parameter collision energy, EMV, sensitivity parameter MS1 Res & MS2 Res one by one. To optimize chromatographic condition including selection of chromatographic column, flow rate, column temperature, concentration of formic acid, mobile phase gradient. Seven serial standard solutions for three continuous days by adopting the optimal result of HPLC-MS/MS method are measured. They are1 ng/mL、5 ng/mL、10 ng/mL、50 ng/mL、100 ng/mL、300 ng/mL、500 ng/mL. 1/x weighting linear regression of peak area and concentration is used to calculate the standard curve and r value. The method of quantitating sGLP-1 in biologic matrix is researched for the first time using precipitation of protein and solid phase extraction.
RESULT: The peak of the standard reserved solution dissolved by 0.1 M NaOH and 0.1 M HCl (pH=5.71) is best. This dissolving method is the standard dissolving schedule. The result of essay shows: the standard reserved solution can be stored for 72 h at 20℃, at lest two weeks at 4℃and one month at -20℃.
The HPLC-UV method is established with the limit of detection of 2μg/mL, as while as feasible SPE pre-processing method of sGLP-1 with high concentration in biologic matrix.
The mass spectrum parameter and chromatographic parameter of HPLC-MS/MS have been optimized. After determinated seven serial standard solutions, the results demonstrate that the linearity is good among 1~500 ng/mL. But during the research the column was changed for another one with the same tipe to do and repeat the same experiments, the results are different from the former. It show that the reproducibility of HPLC-MS/MS method is not good. And there are endogenous interferes in the sGLP-1 biologic metrical samples. After all, this investigation has provided references for further research.
CONCLUSIONS: The research has established the dissolving method of sGLP-1 and the standard dissolving schedule. The result of essay shows: the standard reserved solution can be stored for 72 h at 20℃, at lest two weeks at 4℃and one month at -20℃. So the concentration of sGLP-1 can be kept stable while preparing standard solutions and measuring them. The HPLC-UV method and feasible SPE pre-processing method of sGLP-1 with high concentration in biologic matrix have been established. HPLC-MS/MS method and pre-processing method of sGLP-1 with low concentration in biologic matrical samples have been explored.
研究方法:根据sGLP-1的一级结构,分别用纯水、纯乙腈、乙腈-水(50:50,v/v)、1 M醋酸钠、0.01 M NaOH溶液、0.1 M NaOH溶液、高温蒸汽灭菌的PBS缓冲溶液溶解药物。考察不同溶解方法的色谱峰,并最终确定药物溶解方法及标准化操作流程。
sGLP-1标准溶液3份分别放置于室温20℃、4℃、-20℃保存,分别在新配制(0 h)、24 h、48 h、72 h、一周、两周、一个月取样,每个条件下做3个平行样本, HPLC-UV检测方法测定。每项下的样本的峰面积均与每次实验前(0 h)对照品溶液峰面积作比较,进行稳定性研究。
通过优化HPLC-UV检测的初始条件中的流动相梯度,确定HPLC-UV检测最终条件并考察线性。考察超滤和固相萃取两种生物基质样本前处理方法,并对固相萃取的各种淋洗液和洗脱液进行评价,选择最优萃取效果的淋洗液和洗脱液。初步探索HPLC-MS/MS检测方法。依次优化一级质谱参数fragment、离子源参数(即氮气干燥气温度、氮气干燥气流速、雾化气压力、毛细管电压),二级质谱参数collision energy、电子倍增器(EMV)、灵敏度参数MS1 Res及MS2 Res质谱参数。优化色谱条件,包括色谱柱的选择、流速、柱温、甲酸浓度、流动相梯度。采用HPLC-MS/MS检测方法优化结果,连续三天测定浓度分别为1 ng/mL、5 ng/mL、10 ng/mL、50 ng/mL、100 ng/mL、300 ng/mL、500 ng/mL的标准系列溶液,对测试峰面积与浓度进行1/x加权线性回归计算标准曲线及r值。分别采用蛋白沉淀法和固相萃取法对生物基质中sGLP-1定量检测方法进行探索性研究。
结果:使用0.1 M NaOH溶液溶解sGLP-1后,再用0.1 M HCl调pH至5.71,此标准储备液溶解方法的色谱峰最好,将其方法进行标准化验证,制定为sGLP-1标准品溶解的标准化方案。经实验考察此法配置的标准储备液在室温20℃时至少稳定放置72 h、4℃冷藏保存时至少稳定2周、-20℃冰冻保存时至少稳定1个月。
实验建立了sGLP-1的HPLC-UV检测方法,最低检测限2μg/mL。同时建立了可行的高浓度时生物基质样本中sGLP-1的固相萃取前处理方案。
优化sGLP-1的HPLC-MS/MS检测方法的质谱参数和色谱条件,对HPLC-MS/MS检测方法进行初步探索。采用HPLC-MS/MS检测方法优化结果,连续测定三天浓度分别为1 ng/mL、5 ng/mL、10 ng/mL、50 ng/mL、100 ng/mL、300 ng/mL、500 ng/mL的标准系列溶液,结果显示在1~500 ng/mL的浓度范围内线性较好。但试验中期更换同一型号色谱柱后再进行相同试验,结果相差较大,经多次重现后均没有得到相同结果,表明HPLC-MS/MS检测sGLP-1的重现性较差,虽然研究中HPLC-MS/MS检测sGLP-1标准溶液方法重现性不佳,检测sGLP-1生物样本存在明显的内源性干扰,但本研究的探索对后续别人研究提供了参考。
结论:本研究建立了可行的sGLP-1的溶解方法和标准化操作;实验证明sGLP-1标准溶液在室温20℃时至少稳定放置72 h、4℃冷藏保存时至少稳定2周、-20℃冰冻保存时至少稳定1个月,因此在标准溶液样本配置和测定过程中没有稳定性问题的干扰;建立了sGLP-1的HPLC-UV检测方法及可行的高浓度sGLP-1生物基质样本SPE前处理方法;初步探索了sGLP-1的HPLC-MS/MS检测方法及HPLC-MS/MS检测时低浓度sGLP-1生物基质样本前处理方法。
AIM: To establish feasible dissoluble method and standard procedure of sGLP-1; to investigate the stability of standard solution under three stored conditions of 20℃, 4℃and -20℃; to establish detected method of HPLC-UV and feasible SPE pre-processing method of sGLP-1 with high concentration in biologic matrix; to research for detected method of HPLC-MS/MS and pre-processing method of sGLP-1 with low concentration in biologic matrical samples; to make a exploratory study of quantitative detected method of sGLP-1 in biologic base matrix.
METHOD: According to primary structure of sGLP-1, a bottle of sGLP-1 powder is dissolved by water, acetonitrile, acetonitrile-water(50:50,v/v), 1 M sodium acetate, 0.01 M sodium hydroxide, 0.1 M sodium hydroxide, PBS buffer solution for pyro-steam sterilization. To inspect different chromatographic peaks of different dissolved methods and to determinate the final dissolved method of the drug and the standard operated procedure.
The standard solution of sGLP-1 is stored at 20℃、4℃、-20℃respectively, and is measured at the moment of preparation, 24 h, 48 h, 72 h, one week, two weeks and one month. Three samples of every condition are derected by HPLC-UV. Every area of peek of drug is compared to the area of peek of control article for stability research.
The final detected conditions of HPLC-UV are determinated through optimizing the mobile phase gradient of HPLC-UV initial condition. To investigate ultrafiltration and solid phase extraction for biologic metrical sample, meanwhile to establish every elutriants and eluants in order to select the most effective elutriants and eluants.
The HPLC-MS/MS method is explored initially. To optimize the first class mass spectrum parameter fragment, ion source parameter (gas temperature, gas flow, nebulizer, capillary), the second class mass spectrum parameter collision energy, EMV, sensitivity parameter MS1 Res & MS2 Res one by one. To optimize chromatographic condition including selection of chromatographic column, flow rate, column temperature, concentration of formic acid, mobile phase gradient. Seven serial standard solutions for three continuous days by adopting the optimal result of HPLC-MS/MS method are measured. They are1 ng/mL、5 ng/mL、10 ng/mL、50 ng/mL、100 ng/mL、300 ng/mL、500 ng/mL. 1/x weighting linear regression of peak area and concentration is used to calculate the standard curve and r value. The method of quantitating sGLP-1 in biologic matrix is researched for the first time using precipitation of protein and solid phase extraction.
RESULT: The peak of the standard reserved solution dissolved by 0.1 M NaOH and 0.1 M HCl (pH=5.71) is best. This dissolving method is the standard dissolving schedule. The result of essay shows: the standard reserved solution can be stored for 72 h at 20℃, at lest two weeks at 4℃and one month at -20℃.
The HPLC-UV method is established with the limit of detection of 2μg/mL, as while as feasible SPE pre-processing method of sGLP-1 with high concentration in biologic matrix.
The mass spectrum parameter and chromatographic parameter of HPLC-MS/MS have been optimized. After determinated seven serial standard solutions, the results demonstrate that the linearity is good among 1~500 ng/mL. But during the research the column was changed for another one with the same tipe to do and repeat the same experiments, the results are different from the former. It show that the reproducibility of HPLC-MS/MS method is not good. And there are endogenous interferes in the sGLP-1 biologic metrical samples. After all, this investigation has provided references for further research.
CONCLUSIONS: The research has established the dissolving method of sGLP-1 and the standard dissolving schedule. The result of essay shows: the standard reserved solution can be stored for 72 h at 20℃, at lest two weeks at 4℃and one month at -20℃. So the concentration of sGLP-1 can be kept stable while preparing standard solutions and measuring them. The HPLC-UV method and feasible SPE pre-processing method of sGLP-1 with high concentration in biologic matrix have been established. HPLC-MS/MS method and pre-processing method of sGLP-1 with low concentration in biologic matrical samples have been explored.
引文
[1] 汤仲明主编. 生物技术药物药代动力学. 化学工业出版社. 2004, 第一版: 1-1.
[2] Schirra J, Katschinski M, Weidmann C, et al.Gastric emptying and release of incretin homones after glucose ingestion in humans. J Clin Invest, 1996, 97: 91-103.
[3] Drucker DJ. Minireview: the glucagons-like peptides. Endocrinology. 2001, 142(2): 521-527.
[4] Mentlein R, Gall witz B, Schmidt WE. Dipeptide-peptidase Ⅳ hydrolyses gastric inhibitory polypeptide glucagons-like peptide-1(7-36) amide peptide histidine methionin and is responsible for their degradation in human serum. Eur J Biochem, 1993, 214: 829-835.
[5] Reichert J M. Therapeutic monoclonal antibodies: trends in development and approval in the US.Curr Opin Mol Ther, 2002, 4(2): 110-118.
[6] Cossum PA, Dwyer KA,Roth M,et al. The disposition of a human relaxin (hRlx-2) inpregnant and nonpregnant rats[J]. Pharm Res, 1992, 9(3): 419-421.
[7] 戴舒佳, 廖智, 刘秀文,等.大鼠硬脊膜外和静脉注射[ 125 I] 虎纹毒素-1 后的药代动力学, 中国药理学与毒理学杂志,2004,18 (5): 344-347.
[8] 赵宁,张英起,王增禄,等.新型重组人肿瘤坏死因子在小鼠体内的药代动力学研究.第四军医大学学报,2002,23(12): 1065-1067.
[9] Piscitelli SC, Reiss WG, Figg WD, et al. Pharmacokinetic studies with recombinant cytokines. Scientific issues and practical considerations[J]. Clin Pharmacokinet, 1997, 32: 368-375.
[10] Tanswell P, Modi N, Combs D, et al. Pharmacokinetics and pharmacodynamics of tenecteplase in fibrinolytic therapy of acute myocardial infarction[J]. Clin Pharmacokinet, 2002, 41: 1229-1242.
[11] Iacona I, Lazzarino M, Avanzini MA, et al. Rituximab (IDEC-C2B8): Validation of a sensitive enzyme-linked immunoassay applied to a clinical pharmacokinetic study[J]. Ther Drug Monit, 2000, 22: 295-308.
[12] Cheung W, Minton N, Gunawardena K. Pharmacokinetics and pharmacodynamics of epoetin alfa once weekly and three times weekly[J]. Eur J Clin Pharmacol, 2001, 57: 411-433.
[13] Jerry Z. Yang, K. Chad Bastian, Randy D. Moore,et al. Quantitative analysis of a model opioid peptide and its cyclic prodrugs in rat plasma using high-performance liquid chromatography with fluorescence and tandem mass spectrometric detection. J Chromatogr B Analyt Technol Biomed Life Sci. 2002 Nov 25; 780(2):269-81.
[14] D. Chang, S.J. Kolis, K.H. Linderholm, et al. Bioanalytical method development and validation for a large peptide HIV fusion inhibitor (Enfuvirtide, T-20) and its metabolite in human plasma using LC–MS/MS. Journal of Pharmaceutical and Biomedical Analysis, 2005, 38:487-496.
[15] I. van den Broek, R.W. Sparidans, A.D.R. Huitema,et al. Development and validation of a quantitative assay for the measurement of two HIV-fusion inhibitors, enfuvirtide and tifuvirtide, and one metabolite of enfuvirtide (M-20) in human plasma by liquid chromatography–tandem mass spectrometry. Eur J Biochem, 1993, 214: 829-835.
[16] Wan Yong Feng, Kenneth K. Chan, Joseph M. Covey. Electrospray LC–MS/MS quantitation, stability, and preliminary pharmacokinetics of bradykinin antagonist polypeptide B201 (NSC 710295) in the mouse. Journal of Pharmaceutical and Biomedical Analysis, 2002, 28: 601-612.
[17] E. Stokvis, H. Rosing, L. López-Lázaro. Quantitative analysis of the novel depsipeptide anticancer drug Kahalalide F in human plasma by high-performance liquid chromatography under basic conditions coupled to electrospray ionization tandem mass spectrometry. Journal of Mass Spectrometry, 2002, 37(9):992-1000.
[2] Schirra J, Katschinski M, Weidmann C, et al.Gastric emptying and release of incretin homones after glucose ingestion in humans. J Clin Invest, 1996, 97: 91-103.
[3] Drucker DJ. Minireview: the glucagons-like peptides. Endocrinology. 2001, 142(2): 521-527.
[4] Mentlein R, Gall witz B, Schmidt WE. Dipeptide-peptidase Ⅳ hydrolyses gastric inhibitory polypeptide glucagons-like peptide-1(7-36) amide peptide histidine methionin and is responsible for their degradation in human serum. Eur J Biochem, 1993, 214: 829-835.
[5] Reichert J M. Therapeutic monoclonal antibodies: trends in development and approval in the US.Curr Opin Mol Ther, 2002, 4(2): 110-118.
[6] Cossum PA, Dwyer KA,Roth M,et al. The disposition of a human relaxin (hRlx-2) inpregnant and nonpregnant rats[J]. Pharm Res, 1992, 9(3): 419-421.
[7] 戴舒佳, 廖智, 刘秀文,等.大鼠硬脊膜外和静脉注射[ 125 I] 虎纹毒素-1 后的药代动力学, 中国药理学与毒理学杂志,2004,18 (5): 344-347.
[8] 赵宁,张英起,王增禄,等.新型重组人肿瘤坏死因子在小鼠体内的药代动力学研究.第四军医大学学报,2002,23(12): 1065-1067.
[9] Piscitelli SC, Reiss WG, Figg WD, et al. Pharmacokinetic studies with recombinant cytokines. Scientific issues and practical considerations[J]. Clin Pharmacokinet, 1997, 32: 368-375.
[10] Tanswell P, Modi N, Combs D, et al. Pharmacokinetics and pharmacodynamics of tenecteplase in fibrinolytic therapy of acute myocardial infarction[J]. Clin Pharmacokinet, 2002, 41: 1229-1242.
[11] Iacona I, Lazzarino M, Avanzini MA, et al. Rituximab (IDEC-C2B8): Validation of a sensitive enzyme-linked immunoassay applied to a clinical pharmacokinetic study[J]. Ther Drug Monit, 2000, 22: 295-308.
[12] Cheung W, Minton N, Gunawardena K. Pharmacokinetics and pharmacodynamics of epoetin alfa once weekly and three times weekly[J]. Eur J Clin Pharmacol, 2001, 57: 411-433.
[13] Jerry Z. Yang, K. Chad Bastian, Randy D. Moore,et al. Quantitative analysis of a model opioid peptide and its cyclic prodrugs in rat plasma using high-performance liquid chromatography with fluorescence and tandem mass spectrometric detection. J Chromatogr B Analyt Technol Biomed Life Sci. 2002 Nov 25; 780(2):269-81.
[14] D. Chang, S.J. Kolis, K.H. Linderholm, et al. Bioanalytical method development and validation for a large peptide HIV fusion inhibitor (Enfuvirtide, T-20) and its metabolite in human plasma using LC–MS/MS. Journal of Pharmaceutical and Biomedical Analysis, 2005, 38:487-496.
[15] I. van den Broek, R.W. Sparidans, A.D.R. Huitema,et al. Development and validation of a quantitative assay for the measurement of two HIV-fusion inhibitors, enfuvirtide and tifuvirtide, and one metabolite of enfuvirtide (M-20) in human plasma by liquid chromatography–tandem mass spectrometry. Eur J Biochem, 1993, 214: 829-835.
[16] Wan Yong Feng, Kenneth K. Chan, Joseph M. Covey. Electrospray LC–MS/MS quantitation, stability, and preliminary pharmacokinetics of bradykinin antagonist polypeptide B201 (NSC 710295) in the mouse. Journal of Pharmaceutical and Biomedical Analysis, 2002, 28: 601-612.
[17] E. Stokvis, H. Rosing, L. López-Lázaro. Quantitative analysis of the novel depsipeptide anticancer drug Kahalalide F in human plasma by high-performance liquid chromatography under basic conditions coupled to electrospray ionization tandem mass spectrometry. Journal of Mass Spectrometry, 2002, 37(9):992-1000.