Quantitative Analysis of l-Arginine, Dimethylated Arginine Derivatives, l-Citrulline, and Dimethylamine in Human Serum Using Liquid Chromatography-Mass Spectrometric Method

Abstract: Nitric oxide (NO) is a small molecule involved in the regulation of many physiological processes. It plays a crucial role in the regulation of nervous system, immune and inflammatory responses, and blood flow. NO is synthesized by nitric oxide synthase (NOS) during two-step oxidation of l-arginine to l-citrulline. Intermediates and derivatives of NO metabolism, such as l-arginine, l-citrulline, asymmetrical dimethylarginine (ADMA), symmetrical dimethylarginine (SDMA), and dimethylamine (DMA), are investigated as potential biomarkers. In this article, we present a novel analytical method that allowed for simultaneous analysis of l-arginine, ADMA, SDMA, l-citrulline, and DMA, in a single-step extraction and derivatization using benzoyl chloride. In brief, aliquots of serum were mixed with internal standard solution mixture (50 µM D6-DMA, 20 µM D7-ADMA, and 100 µM D7-arginine) and 0.025 M borate buffer, pH 9.2 (10:1:5). The derivatization process was performed at 25 °C for 5 min using 10% benzoyl chloride. A reverse phase column was used for chromatographic separation. Quantitation was performed using following ions (m/z): 279.1457, 286.1749, 307.1717, 314.2076, 280.1297, 150.0919, and 156.1113 for l-arginine, D7-arginine, ADMA, SDMA, D7-ADMA, l-citrulline, DMA, and D6-DMA, respectively. The method was validated, and its assay linearity, accuracy and precision, recovery, and limits of detection (1.7 µM l-arginine, 0.03 µM ADMA, 0.02 µM SDMA, 0.36 µM l-citrulline, 0.06 µM DMA) and quantification (3.2 µM l-arginine, 0.08 µM ADMA, 0.05 µM SDMA, 1.08 µM l-citrulline, 0.19 µM DMA) were determined. The method is sensitive, reliable, repeatable, and reproducible. It can be applied in the routine clinical/diagnostic laboratory.