Department of Pharmaceutical Sciences, University of Perugia, Via Fabretti 48, 06123, Perugia, Italy.
Department of Pharmacy and Biotechnology (FaBiT), Alma Mater Studiorum, University of Bologna, Via Belmeloro 6, 40126, Bologna, Italy.
Anal Chim Acta. 2021 Oct 2;1180:338928. doi: 10.1016/j.aca.2021.338928. Epub 2021 Aug 9.
Single enantiomers of mandelic acid (1), 3-phenyllactic acid (2), and 3-(4-hydroxyphenyl)lactic acid (3) are the subject of many fields of investigation, spanning from the pharmaceutical synthesis to that of biocompatible and biodegradable polymers, while passing from the interest towards their antimicrobial activity to their role as biomarkers of particular pathological conditions or occupational exposures to specific xenobiotics. All above mentioned issues justify the need for accurate analytical methods enabling the correct determination of the individual enantiomers. So far, all the developed liquid chromatography (LC) methods were not or hardly compatible with mass spectrometry (MS) detection. In this paper, a commercially available Cinchona-alkaloid derivative zwitterionic chiral stationary phase [that is, the CHIRALPAK® ZWIX(-)] was successfully used to optimize the enantioresolution of compounds 1-3 under polar-ionic (PI) conditions with a mobile phase consisting of an acetonitrile/methanol 95/5 (v/v) mixture with 80 mM formic acid. With the optimized conditions, enantioseparation and enantioresolution values up to 1.46 and 4.41, respectively, were obtained. In order to assess the applicability of the optimized enantioselective chromatography conditions in real-life scenarios and on MS-based systems, a proof-of-concept application was efficiently carried out by analysing dry urine spot samples spiked with 1 by means of a LC-MS system. The (S)<(R) enantiomer elution order (EEO) was established for compounds 1 and 2 by analysing a pure enantiomeric standard of known configuration. This was not possible for 3 because not commercially available. For this compound, the same EEO was identified applying a procedure based on ab initio time-dependent density-functional theory simulations coupled to electronic circular dichroism analyses. Moreover, a molecular dynamics simulation unveiled the role of the phenolic OH in compound 3 in the retention mechanism.
扁桃酸（1）、3-苯乳酸（2）和 3-（4-羟基苯基）乳酸（3）的单一对映异构体是许多领域研究的主题，涵盖了从药物合成到生物相容性和可生物降解聚合物的合成，从对其抗菌活性的兴趣到作为特定病理条件或职业暴露于特定外源性物质的生物标志物的作用。所有上述问题都证明了需要准确的分析方法来正确确定各个对映异构体。到目前为止，所有开发的液相色谱（LC）方法都不兼容或几乎不兼容质谱（MS）检测。在本文中，成功地使用了一种市售的金鸡纳生物碱衍生物两性离子手性固定相[即 CHIRALPAK® ZWIX（-）]，在极性离子（PI）条件下，使用包含乙腈/甲醇 95/5（v/v）混合物与 80 mM 甲酸的流动相来优化化合物 1-3 的对映体分辨率。在优化条件下，对映体分离度和对映体分辨率分别达到 1.46 和 4.41。为了评估优化的手性色谱条件在实际情况和基于 MS 的系统中的适用性，通过 LC-MS 系统分析了用 1 掺杂的干尿斑样品，进行了概念验证应用。通过分析已知构型的纯对映体标准品，确定了化合物 1 和 2 的（S）<（R）对映体洗脱顺序（EEO）。对于 3，由于不可用，无法进行此操作。对于该化合物，通过应用基于从头算时间相关密度泛函理论模拟与电子圆二色分析相结合的程序，确定了相同的 EEO。此外，分子动力学模拟揭示了化合物 3 中酚羟基在保留机制中的作用。
