目的：建立简便灵敏的液相色谱-串联质谱（LC-MS/MS）法测定人血浆中普拉克索浓度,并应用于2种缓释制剂生物等效性研究。方法：血浆样本加入内标,加入氢氧化钠后用乙酸乙酯进行液液萃取,取上清液氮气吹干,复溶离心后进样检测。采用Waters ACQUITY UPLC® HSS T3 色谱柱（2.1 mm×50 mm,1.8 μm）,柱温40 ℃,以20 mmol·L-1 甲酸铵水溶液（A）-乙腈（B）为流动相,梯度洗脱,流速0.3 mL·min-1,以正离子MRM 模式测定普拉克索（m/z 212.2 → 153.0）的浓度,普拉克索-d5二盐酸盐（m/z 217.0 → 153.0）作为内标,离子源为ESI 源。结果：建立并验证了简便灵敏的UPLC-MS/MS 法测定人血浆中普拉克索浓度,并应用于2种缓释制剂生物等效性研究。方法线性范围为5~1 000 pg·mL-1,定量下限为5 pg·mL-1,质控样品批内、批间精密度（RSD）<8.5%,准确度相对偏差在标示值-7.67%~7.35% 范围内,提取回收率、专属性、基质效应、稳定性等各项指标均符合国家药品监督管理局的技术指导原则。本法被成功应用于健康受试者单剂口服0.375 mg 盐酸普拉克索缓释片的生物等效性研究,参比制剂空腹及餐后给药状态下平均Cmax 分别为396.50 和502.76 pg·mL-1,受试制剂空腹及餐后给药状态下平均Cmax 分别为412.56 和472.36pg·mL-1。结论：该方法具有前处理过程简单,灵敏度高,色谱峰形好的优点。盐酸普拉克索缓释片与其参比制剂相比,具有生物等效性。
Objective: To establish a fast and sensitive LC-MS/MS method for determination of pramipexole in human plasma and to investigate the bioequivalence between 2 formulations of sustained release tablets. Methods: After addition of internal standard and sodium hydroxide,plasma was extracted by ethyl acetate. The supernatant was transferred and evaporated to dryness under a stream of nitrogen. The residue was re-dissolved,and centrifuged before sample injection. UPLC-MS/MS was performed on an Waters ACQUITY UPLC® HSS T3（2.1 mm×50 mm, 1.8 μm） column with the mobile phase consisting of 20 mmol·L-1 ammonium formate-water（A） and acetonitrile （B） in gradient elution. The flow rate was controlled at 0.3 mL·min-1 and the column temperature was set at 40 ℃ . A mass spectrometer equipped with electrospray ionization source was used and pramipexole（m/z 212.2 → 153.0） was monitored in positive ion MRM mode,with pramipexole-d5dihydrochloride（m/z 217. 0 → 153.0） as internal standard. Results: A fast and sensitive UPLC-MS/MS method for determination of pramipexole in human plasma was established and validated and was applied to bioequivalence study of 2 kinds of sustained release tablets. The standard curves of pramipoxole were linear from 5 to 1 000 pg·mL-1 and the lower limit of quantification was 5 pg·mL-1. The intra-day and inter-day relative standard deviation of qunlity-control samples was less than 8.5%, and the accuracy was in the range of -7.67% and 7.35% in terms of relative error. Recovery,specificity,matrix effect and stability met the guiding principles and criteria of NMPA. The method was successfully applied to a bioequivalence study of pramipexole dihydrochloride sustained release tablets containing 0.375 mg in healthy volunteers. The average Cmax under fasting and fed condition of the reference tablet were 396.50 and 502.76 pg·mL-1. The average Cmax under fasting and fed condition of the test tablet were 412.56 and 472.36 pg·mL-1,separately. Conclusion: The method is sensitive and simple in process,producing well chromatographic performance. The test pramipexole tablet is bioequivalent to the reference tablets.
[1] JANKOVIC J.Parkinson's disease:clinical features and diagnosis[J].J Neurol Neurosurg Psychiatry,2008,79(4):368
[2] 孔岳南,谢惠君.多巴胺系统基因和帕金森病的相关性[J].中国临床康复,2003,7(19):2730
KUN YN,XIE HJ.Correlation between genes of dopamine system amd Parkinson's disease[J].Chin J Clin Rehabil,2003,7(19):2730
[3] 曾丽莉,陈生弟.普拉克索在帕金森病治疗中的临床应用[J].中国现代神经疾病杂志,2007,7(1):44
ZENG LL,CHEN SD.Clinical application of pramipexol in the treatment of Parkinson's disease[J].Chin J Contemp Neurol Neurosurg,2007,7(1):44
[4] 张淑丽,杨海龙,汤宇.非麦角碱类多巴胺受体激动剂缓释制剂的研究进展[J].现代药物与临床. 2020,35(6):1279
ZHANG SL,YANG HL,TANG Y.Research progress on sustainedrelease preparations of non-ergoline dopamine receptor agonists[J].Drugs Clin,2020,35(6):1279
[5] BHARATHI DV,HOTHA KK,SAGAR PVV,et al.Development and validation of a sensitive LC-MS/MS method with electrospray ionization for quantitation of pramipexole in human plasma:application to a clinical pharmacokinetic study[J].Biomed Chromatogr,2009,23(2):212
[6] 李华,陈笑艳,施孝金,等.高灵敏度液相色谱-串联质谱法测定人血浆中普拉克索[J].药物分析杂志,2011,31(2):330
LI H,CHEN XY,SHI XJ,et al.LC-MS/MS determination of pramipexole in human plasma[J].Chin J Pharm Anal,2011,31(2):330
[7] 刘明周,陈平,汤进,等.HPLC-MS/MS 法测定健康人血浆中普拉克索浓度[J].中国临床药理学杂志,2013,29(5):378
LIU MZ,CHEN P,TANG J,et al.Determination of pramipexol hydrochloride sustained-release tablets in healithy human plasma by HPLC-MS/MS[J].Chin J Clin Pharmacol,2013,29(5):378
[8] 许静,林翠鸿,刘亦伟,等.UPLC-MS/MS 法同时测定人血浆中普拉克索和金刚烷胺浓度[J].药物分析杂志,2016,36(5):783
XU J,LIN CH,LIU YW,et al.Simutaneous determination of pramipexole and amantadine by UPLC-MS/MS[J].Chin J Pharm Anal,2016,36(5):783
[9] BHARATHI DV,HOTHA KK,SAGAR PVV,et al.Development and validation of a sensitive LC-MS/MS method with electrospray ionization for quantitation of pramipexole in human plasma:application to a clinical pharmacokinetic study[J].Biomed Chromatogr,2009,23(2):212
[10] PU TT,LI XH,SUN YM,et al.Development of a prolonged-release pramipexole transdermal patch:in vitro and in vivo evaluation[J].AAPS Pharm Sci Tch,2017,18(3):738
[11] NIROGI RVS,KANDIKERE V,SHRIVASTAVA W,et al.Quantification of pramipexole in human plasma by liquid chromatography tandem mass spectrometry using tamsulosin as internal standard[J].Biomed Chromatogr,2007,21(11):1151
[12] RAO R,SRAVAN B,RAMAKRISHNA K,et al.LC-MS/MS determination of pramipexole on rat dried blood spots:a pharmacokinetics study[J].J Chromatogr B,2013,932:34
[13] 向平,沈敏,卓先义.液相色谱-质谱分析中的基质效应[J].分析测试学报,2009,28(6):753
XIANG P,SHEN M,ZHUO XY.Matrix effect in liquid chromatographic-mass spectrometric analysis[J].J Instrum Anal,2009,28(6):753
[14] GUO WL,LI G,YANG YX,et al.LC-MS/MS analysis of pramipexole in mouse plasma and tissues:elimination of lipid matrix effects using weak cation exchange mode based solid-phase extraction[J].J Chromatogr B,2015,988:157
[15] 中华人民共和国药典2015 年版.四部[S].2015:363<9012>ChP 2015.Vol Ⅳ[S].2015:363<9012>
