Department of Pharmaceutics, Semmelweis University, Hőgyes Endre utca 7, Budapest H-1092, Hungary.
MS Proteomics Research Group, Institute of Organic Chemistry, Research Centre for Natural Sciences of the Hungarian Academy of Sciences, Magyar tudósok körútja 2, Budapest H-1117, Hungary.
J Pharm Biomed Anal. 2020 Feb 20;180:113018. doi: 10.1016/j.jpba.2019.113018. Epub 2019 Nov 28.
Altered serotonergic neurotransmission is a key factor in several neurologic and psychiatric disorders such as migraine. Human and animal studies suggest that chronically low interictal serotonin levels of plasma and brain may facilitate increased activity of the trigeminovascular pathway, and may contribute to development of repeated migraine attacks. However, brain serotonin synthesis is affected by the concentration of tryptophan, its metabolites and a number of amino acids. In this work a simple and robust LC-MS/MS method for the quantitative determination of valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serotonin and kynurenine in human plasma has been developed and validated. Sample preparation was achieved by protein precipitation, using trifluoroacetic acid. Chromatographic separation was carried out on a Supelco Ascentis® Express C18 column (3.0 mm i.d. × 150 mm, 2.7 μm) equipped with an Agilent Zorbax Eclipse XDB C8 guard-column under isocratic conditions at a flow rate of 0.4 mL/min, over a 6.5 min run time. Mobile phase was 0.2% trifluoroacetic acid - acetonitrile (85:15, v/v). The eight analytes and two internal standards were ionized by positive electrospray ionization and detected in multiple reaction monitoring mode. A "fit-for-purpose" validation approach was adopted using surrogate matrix for the preparation of calibration samples. The calibration curves of all analytes showed excellent linearities with a correlation coefficient (r) of 0.998 or better. Spiked surrogate matrix samples and pooled human plasma were used as quality control samples. Intra-day and inter-day precisions were less than 11.8% and 14.3%, and accuracies were within the ranges of 87.4-114.3% and 87.7-113.3%, respectively. Stability of the components in standard solutions, surrogate matrix, pooled plasma and processed samples were found to be acceptable under all relevant conditions. No significant carryover effect was observed. The surrogate matrix behaved parallel to human plasma when assessed by standard addition method and diluting the authentic matrix with surrogate matrix. The method was successfully applied for analysis of 800 human plasma samples to support a clinical study.
血清素能神经递质的改变是偏头痛等几种神经和精神疾病的一个关键因素。人类和动物研究表明，慢性低间歇性血浆和大脑中的血清素水平可能促进三叉血管通路的活动增加，并可能导致反复发作的偏头痛发作。然而，大脑中血清素的合成受到色氨酸、其代谢物和一些氨基酸的浓度的影响。在这项工作中，建立并验证了一种简单而强大的 LC-MS/MS 方法，用于定量测定人血浆中的缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸、酪氨酸、色氨酸、血清素和犬尿氨酸。样品制备采用三氟乙酸进行蛋白沉淀。色谱分离在 Supelco Ascentis®Express C18 柱（3.0mm id×150mm，2.7μm）上进行，采用 Agilent Zorbax Eclipse XDB C8 保护柱，在等度条件下以 0.4mL/min 的流速，6.5min 运行时间。流动相为 0.2%三氟乙酸-乙腈（85:15，v/v）。八种分析物和两种内标物通过正电喷雾电离进行离子化，并在多重反应监测模式下进行检测。采用替代基质制备校准样品的“适合目的”验证方法。所有分析物的校准曲线均具有优异的线性，相关系数（r）均大于 0.998。使用加标替代基质样品和混合人血浆作为质控样品。日内和日间精密度均小于 11.8%和 14.3%，准确度分别在 87.4%-114.3%和 87.7%-113.3%的范围内。在所有相关条件下，均发现标准溶液、替代基质、混合血浆和处理样品中的各成分稳定性可接受。未观察到明显的交叉污染效应。通过标准加入法和用替代基质稀释真实基质评估，替代基质与人类血浆表现出平行行为。该方法成功应用于 800 个人血浆样品的分析，以支持一项临床研究。
