Shenzhen Kanghua Juntai Biotech Co. Ltd., B 215, Unit No.7, Shahe Rd W, Nanshan, Shenzhen, Guangdong Province 518063, China.
Physical Examination Center, Yuebei People's Hospital, Wu Jiang Qu, Shao Guan Shi, Guangzhou 512027, China.
J Pharm Biomed Anal. 2023 Aug 5;232:115336. doi: 10.1016/j.jpba.2023.115336. Epub 2023 Mar 11.
Rheumatoid arthritis (RA) is a chronic autoimmune disorder characterized by systemic inflammation of the joints and extra-articular tissues. The incidence of cardiovascular disease (CVD) remains the main cause of morbidity and mortality in patients with RA. Despite the development of new therapeutics targeting the articular manifestations, the relief of the cardiovascular burden is still an unmet medical need during the management of RA. So, the early prognosis of RA-associated CVD plays a crucial role in improving the clinical outcomes of RA patients. Recently, circulating dimethylarginines have gained attention as potential biomarkers for CVDs. Here, we present the development and validation of a high-throughput liquid chromatography-tandem mass spectrometric (LC/MS/MS) method for simultaneous quantification of creatinine, arginine, and dimethylarginines in human serum within 2 mins by isotope dilution mass spectrometry. This method employed a protein precipitation method for rapid sample preparation, trichloroacetic acid (TCA)-based ion pairing chromatography for fast analyte separation, and multiple reaction monitoring (MRM) with stable isotope-labeled internal standards (ISs) for simultaneous quantitation. To assure the quality, our method was validated against the FDA guidelines for lower limit of quantitation (0.2 µM), linearity (square of coefficient correlation>0.99), precision (intra-&inter-assay imprecision < 10 %), accuracy (intra-&inter-assay inaccuracy < 10 %), sample preparation recovery (recovery ≥ 90 %), stability (instability < 10 %), matrix effect (signal suppression < 55 %), and carryover ( < 0.01 %). Afterward, we applied the validated method to a retrospective cross-sectional study. We aimed to evaluate the utility of serological dimethylarginines as potential cardiovascular biomarkers in the development of RA-associated CVD. Our results revealed that the serological ratio of asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA), an indicator of physiological arginine methylation status, was significantly elevated in patients with RA. This finding might provide value in detecting CVD to improve clinical outcomes in RA management.
类风湿关节炎（RA）是一种慢性自身免疫性疾病，其特征为关节和关节外组织的全身性炎症。心血管疾病（CVD）的发病率仍然是 RA 患者发病率和死亡率的主要原因。尽管针对关节表现的新疗法不断发展，但在 RA 的管理中，缓解心血管负担仍然是未满足的医疗需求。因此，RA 相关 CVD 的早期预后对于改善 RA 患者的临床结局至关重要。最近，循环二甲基精氨酸作为 CVD 的潜在生物标志物受到关注。在这里，我们介绍了一种高通量液相色谱-串联质谱（LC/MS/MS）方法的开发和验证，该方法可在 2 分钟内通过同位素稀释质谱法同时定量人血清中的肌酐、精氨酸和二甲基精氨酸。该方法采用蛋白质沉淀法进行快速样品制备，基于三氯乙酸（TCA）的离子对色谱法进行快速分析物分离，以及采用稳定同位素标记内标（IS）的多重反应监测（MRM）进行同时定量。为了确保质量，我们的方法符合 FDA 对定量下限（0.2 µM）、线性（系数相关平方>0.99）、精密度（日内和日间不精密度 < 10 %）、准确度（日内和日间不准确度 < 10 %）、样品制备回收率（回收率≥90 %）、稳定性（不稳定性<10 %）、基质效应（信号抑制<55 %）和交叉污染（<0.01 %）的指南。之后，我们将验证后的方法应用于回顾性横断面研究。我们旨在评估血清二甲基精氨酸作为 RA 相关 CVD 潜在心血管生物标志物的效用。我们的结果表明，不对称二甲基精氨酸（ADMA）和对称二甲基精氨酸（SDMA）的血清比率，即生理精氨酸甲基化状态的指标，在 RA 患者中显著升高。这一发现可能有助于检测 CVD，以改善 RA 管理中的临床结局。
