Development and validation of a method for the quantitative determination of monoamine neurotransmitters and their metabolites in rat brain tissue using HPLC-MS/MS

Acta Biomedica Scientifica Pub Date : 2024-03-27 DOI:10.29413/abs.2024-9.1.18

A. L. Khokhlov, I. I. Yaichkov, M. K. Korsakov, I. Kagramanyan, N. N. Volkhin, S. S. Petukhov, V. E. Zaikova

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Abstract

Background. Determining changes in the content of monoamine neurotransmitters and their metabolites in brain structures is a necessary part of studying the pharmacodynamics of antiparkinsonian drugs. A method for the joint determination of norepinephrine, adrenaline, dopamine, serotonin, 5-hydroxyindole-3-acetic acid, 3,4-dihydroxyphenylacetic acid, homovanillic acid, vanillylmandelic acid in rat brain tissue has not previously been developed.The aim of the study. To develop and to validate a method for the quantitative determination of norepinephrine, adrenaline, dopamine, serotonin, 5-hydroxyindole3-acetic acid, 3,4-dihydroxyphenylacetic acid, homovanillic acid, vanillylmandelic acid in rat brain tissue using high-performance liquid chromatography in combination with tandem mass spectrometry (HPLC-MS/MS).Materials and methods. A method for determining monoamine mediators and their metabolites was developed using the HPLC-MS/MS method. Brain tissue homogenates were prepared using a mechanical hand-operated homogenizer. The effect of various antioxidants on the stability of norepinephrine, adrenaline, dopamine and 3,4-dihydroxyphenylacetic acid in the test samples was studied.Results. Chromatographic separation of sample components was carried out using two Synergi Max RP (20 × 2.0 mm, 2.5 µm) and Synergi Fusion RP 80Å (250 × 4.6 mm, 4 µm) chromatographic columns. Elution was carried out in a gradient mode using a mobile phase based on methanol and a 0.1% solution of formic acid in water. To prepare homogenate batches, the samples were diluted with a solution of internal standards in methanol. A 5% aqueous solution of ascorbic acid was chosen as an antioxidant stabilizer.Conclusion. The developed methodology has been fully validated and meets the requirements of Russian and international guidelines. The chosen stabilization method allows samples of brain homogenates to be stored for 30 days after collection.

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利用 HPLC-MS/MS 技术开发和验证大鼠脑组织中单胺神经递质及其代谢物的定量测定方法

背景。测定脑结构中单胺神经递质及其代谢物含量的变化是研究抗帕金森病药物药效学的必要部分。此前尚未开发出一种联合测定大鼠脑组织中去甲肾上腺素、肾上腺素、多巴胺、5-羟基吲哚-3-乙酸、3,4-二羟基苯乙酸、高香草酸、香草醛酸的方法。采用高效液相色谱-串联质谱法（HPLC-MS/MS），建立并验证大鼠脑组织中去甲肾上腺素、肾上腺素、多巴胺、5-羟基吲哚-3-乙酸、3,4-二羟基苯乙酸、高香草酸、香草醛酸的定量检测方法。采用高效液相色谱-质谱/质谱法开发了一种测定单胺介质及其代谢物的方法。使用手动机械匀浆器制备脑组织匀浆。研究了各种抗氧化剂对测试样品中去甲肾上腺素、肾上腺素、多巴胺和 3,4-二羟基苯乙酸稳定性的影响。使用两根 Synergi Max RP（20 × 2.0 mm，2.5 µm）和 Synergi Fusion RP 80Å（250 × 4.6 mm，4 µm）色谱柱对样品成分进行色谱分离。以甲醇和 0.1% 甲酸水溶液为流动相进行梯度洗脱。为了制备均质物批次，样品用甲醇内标溶液稀释。抗坏血酸的 5%水溶液被选为抗氧化稳定剂。所开发的方法经过充分验证，符合俄罗斯和国际准则的要求。所选的稳定方法可使脑匀浆样本在采集后保存 30 天。

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Acta Biomedica Scientifica

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