首页> 外文期刊>Analytica chimica acta >High-performance liquid-chromatographic tandem-mass spectrometric methods for atropinesterase-mediated enantioselective and chiral determination of R- and S-hyoscyamine in plasma
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High-performance liquid-chromatographic tandem-mass spectrometric methods for atropinesterase-mediated enantioselective and chiral determination of R- and S-hyoscyamine in plasma

机译:高效液相色谱-串联质谱法测定阿托品酯酶介导的对映体的手性对映体的选择性和手性测定

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摘要

S-hyoscyamine (S-hyo) is a toxic tropane alkaloid from plants of the solanacea family, which is extracted for pharmaceutical purposes thereby undergoing racemization (atropine). Merely the S-hyo enantiomer acts as an antagonist of muscarinic receptors (MR). Nevertheless, racemic atropine is clinically administered in e.g. ophthalmology and for symptomatic therapy of acute poisoning with organophosphorus compounds (OPCs, e.g. pesticides, nerve agents). However, very limited data are available of comparative pharmacokinetics of S- and R-enantiomers in humans or other species. Therefore, we developed an enantioselective LC-ES1-MS/MS assay making use of rabbit serum containing atropinesterase (AtrE, EC 3.1.1.10) which is suitable for stereospecific hydrolysis of S-hyo into tropine and tropic acid while R-hyo is unaffected. For sample preparation plasma was incubated with human serum (not containing AtrE, procedure A) and with rabbit serum (procedure B). Afterwards, hyoscyamines were quantified by a validated previously published non-chiral LC-ESI-MS/MS method. Following procedure A the concentration of total hyo and following procedure B remaining R-hyo were determined. S-hyo was calculated by the difference between these concentrations. This assay design allowed reproducible, precise(RSD2-9%), accurate (93-101 %) and selective determination of total and individual hyoscyamines. Potential therapeutics for OPC poisoning (carbamates, oximes) and thiono-pesticides did not interfere with the assay whereas some oxon-pesticides inhibited S-hyo hydrolysis. A control experiment was designed allowing to be aware of such interferences thus avoiding the use of false results. To validate this assay, results were compared to those from a novel isocratic chiral LC-ESI-MS/MS method. Separation of S-hyo (t_R 31.1 ±0.2min) and R-hyo (t_R 33.4±0.2min) was achieved on α-glycoprotein (AGP) chiral stationary phase at 40 °C (selectivity factor α 1.07). Ammoniumformate (0.01 M, pH 8.0) with 3.75% (v/v) acetonitrile served as mobile phase (300 μLmin~(-1)). Hyoscyamines were detected in the positive multiple reaction monitor mode. The enantioselective assay was applied to the analysis of atropine degradation in diluted rabbit serum in vitro as well as to human in vivo plasma samples from a pesticide-poisoned patient treated with atropine.
机译:S-hycycyamine(S-hyo)是茄科植物的有毒托烷生物碱,其被提取用于药物用途,从而进行消旋(阿托品)。 S-hyo对映异构体仅充当毒蕈碱受体(MR)的拮抗剂。然而,外消旋阿托品在临床上以例如阿司匹林施用。眼科和有机磷化合物(OPC,例如杀虫剂,神经毒剂)急性中毒的对症治疗。但是,关于人类或其他物种中S和R对映异构体的比较药代动力学的数据非常有限。因此,我们开发了一种对映选择性LC-ES1-MS / MS分析方法,该方法利用了含有阿托品酯酶(AtrE,EC 3.1.1.10)的兔血清,该化合物适用于S-hyo立体定向水解为tropine和原酸,而R-hyo不受影响。 。对于样品制备,将血浆与人血清(不含AtrE,步骤A)和兔血清(流程B)一起孵育。此后,通过验证的先前公开的非手性LC-ESI-MS / MS方法对hy胺进行定量。按照步骤A,确定总hyo的浓度,并按照步骤B,确定剩余的R-hyo。通过这些浓度之间的差计算S-hyo。该测定设计允许可重复,精确(RSD2-9%),准确(93-101%)以及选择性测定总和单独的菱胺。 OPC中毒的潜在疗法(氨基甲酸酯,肟)和硫代农药不会干扰测定,而某些氧农药会抑制S-hyo水解。设计了一个控制实验,可以意识到这种干扰,从而避免使用错误的结果。为了验证该测定,将结果与新型等度手性LC-ESI-MS / MS方法的结果进行了比较。在40°C(选择因子α1.07)的α-糖蛋白(AGP)手性固定相上实现S-hyo(t_R 31.1±0.2min)和R-hyo(t_R 33.4±0.2min)分离。甲酸铵(0.01 M,pH 8.0)和3.75%(v / v)乙腈用作流动相(300μLmin〜(-1))。在阳性多重反应监测器模式下检测到了丝胺。对映选择性测定可用于体外分析稀释的兔血清中阿托品的降解,以及来自使用阿托品治疗的农药中毒患者的人体内血浆样品。

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