超滤法结合UPLC测定抗601合剂中黄芩苷、绿原酸在不同血浆中的蛋白结合率
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摘要
目的超滤法结合超高效液相色谱(UPLC)法同时检测抗601合剂中2种指标性成分——黄芩苷、绿原酸体外血浆蛋白结合率。方法建立UPLC法同时测定牛血清白蛋白(BSA)、大鼠空白血浆和正常人空白血浆超滤液中的黄芩苷、绿原酸含量,进行专属性、精密度、准确度、稳定性、超滤膜回收率等方法学考察;配制黄芩苷质量浓度分别为28.2、141.0、282.0 mg/L,绿原酸质量浓度为6.40、32.0、64.0 mg/L的抗601合剂溶液,分别与BSA溶液、大鼠空白血浆、人空白血浆混匀后,置于37℃水浴温育4 h,于超滤离心管中10 000 r/min离心20 min,取超滤液10μL注入UPLC分析测定,计算超滤液中黄芩苷和绿原酸浓度及血浆蛋白结合率。结果建立的UPLC法专属性、精密度、准确度、稳定性、超滤膜回收率均良好,能够满足定量分析测试要求;黄芩苷与BSA、大鼠空白血浆和正常人空白血浆的蛋白结合率分别为(73.70±1.65)%、(82.72±1.64)%、(87.33±1.84)%,绿原酸与3种血浆的蛋白结合率分别为(66.78±1.91)%、(74.18±2.01)%、(78.54±2.97)%,无浓度相关性,两成分与BSA溶液蛋白结合率显著低于与大鼠空白血浆、人空白血浆结合率(P<0.05)。结论抗601合剂中的黄芩苷、绿原酸与不同种属血浆均有较高的蛋白结合率,在考察范围内无浓度相关性,归类于中速处置类药物,体内消除情况与其血浆蛋白结合率可能密切相关。
Objective Ultrafiltration combined with ultra high performance liquid chromatography(UPLC) method were established for the simultaneous determination of plasma protein binding rate of two index components baicalin and chlorogenic acid in Anti-601 Mixture. Methods The UPLC method was established for the simultaneous determination of baicalin and chlorogenic acid in bovine serum albumin(BSA), blank plasma and normal human blank plasma ultrafiltration, and the methods of specificity, precision, accuracy, stability and ultrafiltration recovery rate were investigated. The Anti-601 Mixture of 3 concentrations was prepared with the concentration of baicalin was 28.2, 141 and 282 mg/L, and the concentration of chlorogenic acid was 6.40, 32.0 and 64.0 mg/L. After mixing the Anti-601 Mixture with BSA solution, rat blank plasma and human blank plasma respectively, the solutions were incubated at 37 C in water bath for 4 h, and placed in the ultrafiltration centrifuge tube for 10000 r/min centrifugation 20 min, and 10 μL ultrafiltration was injected into UPLC to determine the concentration of baicalin and chlorogenic acid in the ultrafiltration and the binding rate of plasma protein. Results The method had high sensitivity, good specificity and reproduction, with simple management in fulfilling the requirements. The plasma protein binding rate of baicalin with BSA, rat plasma and human plasma were(73.70 ± 1.65)%,(82.72 ± 1.64)%,(87.33 ± 1.84)% respectively, and the plasma protein binding rate of chlorogenic acid with BSA, rat plasma and human plasma were(66.78 ± 1.91)%,(74.18 ± 2.01)%,(78.54 ± 2.97)% respectively. No concentration correlation was observed. The binding rate of two components to BSA solution protein was significantly lower than that of rat blank plasma and human blank plasma(P < 0.05). Conclusion The two major compounds in Anti-601 Mixture showed a high binding power to plasma protein of different species in vitro, and it was independent of the investigated concentrations. It is classified as medium speed treatment drugs, elimination in vivo may be closely related to plasma protein binding rate.
Objective Ultrafiltration combined with ultra high performance liquid chromatography(UPLC) method were established for the simultaneous determination of plasma protein binding rate of two index components baicalin and chlorogenic acid in Anti-601 Mixture. Methods The UPLC method was established for the simultaneous determination of baicalin and chlorogenic acid in bovine serum albumin(BSA), blank plasma and normal human blank plasma ultrafiltration, and the methods of specificity, precision, accuracy, stability and ultrafiltration recovery rate were investigated. The Anti-601 Mixture of 3 concentrations was prepared with the concentration of baicalin was 28.2, 141 and 282 mg/L, and the concentration of chlorogenic acid was 6.40, 32.0 and 64.0 mg/L. After mixing the Anti-601 Mixture with BSA solution, rat blank plasma and human blank plasma respectively, the solutions were incubated at 37 C in water bath for 4 h, and placed in the ultrafiltration centrifuge tube for 10000 r/min centrifugation 20 min, and 10 μL ultrafiltration was injected into UPLC to determine the concentration of baicalin and chlorogenic acid in the ultrafiltration and the binding rate of plasma protein. Results The method had high sensitivity, good specificity and reproduction, with simple management in fulfilling the requirements. The plasma protein binding rate of baicalin with BSA, rat plasma and human plasma were(73.70 ± 1.65)%,(82.72 ± 1.64)%,(87.33 ± 1.84)% respectively, and the plasma protein binding rate of chlorogenic acid with BSA, rat plasma and human plasma were(66.78 ± 1.91)%,(74.18 ± 2.01)%,(78.54 ± 2.97)% respectively. No concentration correlation was observed. The binding rate of two components to BSA solution protein was significantly lower than that of rat blank plasma and human blank plasma(P < 0.05). Conclusion The two major compounds in Anti-601 Mixture showed a high binding power to plasma protein of different species in vitro, and it was independent of the investigated concentrations. It is classified as medium speed treatment drugs, elimination in vivo may be closely related to plasma protein binding rate.
引文
[1]中国药典(一部)[S].2015.
[2]陈少萍.绿原酸药代动力学研究进展[J].中成药,2010,32(4):645-648.
[3]郭宾,李川.药物与血浆蛋白结合的药理学基础及其研究进展[J].中国临床药理学与治疗学,2005,10(3):241-253.
[4]关瑾,丁爽,刘芷含.药物-血浆蛋白结合率测定方法的研究进展[J].中国新药杂志,2014,23(10):1149-1153.
[5]王绚,程莉华,徐进,等.UPLC法测定抗601合剂中黄芩苷、绿原酸的含量[J].南京中医药大学学报,2014,30(4):370-372.
[6]Beier H,Kaiser K,Langhans M,et al.Peritoneal microdialysis in freely moving rodents:an alternative to blood sampling for pharmacokinetic studies in the rat and the mouse[J].Eur J Pharm Sci,2007,30(1):75-83.
[7]Karla L S,Dennis O S,Craig E L,et al.Pharmacokinetic studies of aspirin in rats using in vivo microdialysis sampling[J].Anal Chem Acta,2008,246(1):1811.
[8]吴阳,张英丰.超滤法测定脱水穿心莲内酯的血浆蛋白结合率[J].中国实验方剂学杂志,2011,17(21):57-59.
[9]陈豆,涂星,张英丰.超滤法测定丹酚酸A的血浆蛋白结合率[J].中国实验方剂学杂志,2013,19(2):77-80.
[10]叶勇,黄秋洁,刘华钢.超滤法测定脱氢卡维丁的血浆蛋白结合率[J].中国医院药学杂志,2015,35(1):12-15.
[11]吴磊,陆超,戴国梁,等.清热养心颗粒中绿原酸在不同血浆中的蛋白结合率测定[J].中国药理学通报,2014,30(9):1331-1332.
[12]李晓天,褚延乐,吴凤娟,等.超滤法测定冬凌草乙素的血浆蛋白结合率[J].中国新药杂志,2013,22(20):2418-2422.
[13]周玲,居文政,刘子修,等.灯盏细辛注射液多成分血浆蛋白结合率测定[J].中国药理学通报,2011,27(5):719-722.
[14]童成亮,吴小英.牡荆素血浆蛋白结合率的测定[J].中国中药杂志,2012,37(14):2168-2170.
[15]Kang J,Liu Y,Xie M X,et al.Interactions of human serum albumin with chlorogenic acid and ferulic acid[J].Biochim Biophys Acta,2004,1674(2):205-214.
[2]陈少萍.绿原酸药代动力学研究进展[J].中成药,2010,32(4):645-648.
[3]郭宾,李川.药物与血浆蛋白结合的药理学基础及其研究进展[J].中国临床药理学与治疗学,2005,10(3):241-253.
[4]关瑾,丁爽,刘芷含.药物-血浆蛋白结合率测定方法的研究进展[J].中国新药杂志,2014,23(10):1149-1153.
[5]王绚,程莉华,徐进,等.UPLC法测定抗601合剂中黄芩苷、绿原酸的含量[J].南京中医药大学学报,2014,30(4):370-372.
[6]Beier H,Kaiser K,Langhans M,et al.Peritoneal microdialysis in freely moving rodents:an alternative to blood sampling for pharmacokinetic studies in the rat and the mouse[J].Eur J Pharm Sci,2007,30(1):75-83.
[7]Karla L S,Dennis O S,Craig E L,et al.Pharmacokinetic studies of aspirin in rats using in vivo microdialysis sampling[J].Anal Chem Acta,2008,246(1):1811.
[8]吴阳,张英丰.超滤法测定脱水穿心莲内酯的血浆蛋白结合率[J].中国实验方剂学杂志,2011,17(21):57-59.
[9]陈豆,涂星,张英丰.超滤法测定丹酚酸A的血浆蛋白结合率[J].中国实验方剂学杂志,2013,19(2):77-80.
[10]叶勇,黄秋洁,刘华钢.超滤法测定脱氢卡维丁的血浆蛋白结合率[J].中国医院药学杂志,2015,35(1):12-15.
[11]吴磊,陆超,戴国梁,等.清热养心颗粒中绿原酸在不同血浆中的蛋白结合率测定[J].中国药理学通报,2014,30(9):1331-1332.
[12]李晓天,褚延乐,吴凤娟,等.超滤法测定冬凌草乙素的血浆蛋白结合率[J].中国新药杂志,2013,22(20):2418-2422.
[13]周玲,居文政,刘子修,等.灯盏细辛注射液多成分血浆蛋白结合率测定[J].中国药理学通报,2011,27(5):719-722.
[14]童成亮,吴小英.牡荆素血浆蛋白结合率的测定[J].中国中药杂志,2012,37(14):2168-2170.
[15]Kang J,Liu Y,Xie M X,et al.Interactions of human serum albumin with chlorogenic acid and ferulic acid[J].Biochim Biophys Acta,2004,1674(2):205-214.