Yuan Ying-Shi, Liao Jia-Min, Kang Chun-Min, Li Bing-Ling, Lei Xu-Ri, Yu Ke-Wei, Chen Lu, Dong Heng, Ke Pei-Feng, Xiao Yao, Huang Xian-Zhang, Zhao Bei-Bei
Department of Laboratory Medicine, The Second Clinical College of Guangzhou University of Chinese Medicine, Guangzhou, Guangdong 510120, P.R. China.
Department of Laboratory Medicine, Guangdong Provincial Hospital of Chinese Medicine, Guangzhou, Guangdong 510120, P.R. China.
Exp Ther Med. 2023 May 23;26(1):342. doi: 10.3892/etm.2023.12041. eCollection 2023 Jul.
With time, the number of samples in clinical laboratories from therapeutic drug monitoring has increased. Existing analytical methods for blood cyclosporin A (CSA) monitoring, such as high-performance liquid chromatography (HPLC) and immunoassays, have limitations including cross-reactivity, time consumption, and the complicated procedures involved. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has long been considered the reference standard owing to its high accuracy, specificity, and sensitivity. However, large numbers of blood samples, multi-step preparation procedures, and longer analytical times (2.5-20 min) are required as a consequence of the different technical strategies, to ensure good analytical performance and routine quality assurance. A stable, reliable, and high throughput detection method will save personnel time and reduce laboratory costs. Therefore, a high throughput and simple LC-MS/MS method was developed and validated for the detection of whole-blood CSA with CSA-d12 as the internal standard in the present study. Whole blood samples were prepared through a modified one-step protein precipitation method. A C18 column (50x2.1 mm, 2.7 µm) with a mobile phase flow rate of 0.5 ml/min was used for chromatographic separation with a total running time of 4.3 min to avoid the matrix effect. To protect the mass spectrometer, only part of the sample after LC separation was allowed to enter the mass spectrum, using two HPLC systems coupled to one mass spectrometry. In this way, throughput was improved with detection of two samples possible within 4.3 min using a shorter analytical time for each sample of 2.15 min. This modified LC-MS/MS method showed excellent analytical performance and demonstrated less matrix effect and a wide linear range. The design of multi-LC systems coupled with one mass spectrometry may play a notable role in the improvement of daily detection throughput, speeding up LC-MS/MS, and allowing it to be an integral part of continuous diagnostics in the near future.
随着时间的推移，临床实验室中来自治疗药物监测的样本数量有所增加。现有的用于血液中环孢素A（CSA）监测的分析方法，如高效液相色谱法（HPLC）和免疫测定法，存在包括交叉反应性、耗时以及操作程序复杂等局限性。液相色谱 - 串联质谱法（LC - MS/MS）长期以来因其高准确性、特异性和灵敏度而被视为参考标准。然而，由于不同的技术策略，需要大量血液样本、多步制备程序以及较长的分析时间（2.5 - 20分钟），以确保良好的分析性能和常规质量保证。一种稳定、可靠且高通量的检测方法将节省人力时间并降低实验室成本。因此，在本研究中开发并验证了一种以CSA - d12作为内标物用于检测全血CSA的高通量且简便的LC - MS/MS方法。全血样本通过改良的一步蛋白沉淀法进行制备。使用C18柱（50x2.1 mm，2.7 µm），流动相流速为0.5 ml/min进行色谱分离，总运行时间为4.3分钟以避免基质效应。为保护质谱仪，仅允许LC分离后的部分样品进入质谱，使用两个HPLC系统与一个质谱联用。通过这种方式，提高了通量，能够在4.3分钟内检测两个样品，每个样品的分析时间缩短至2.15分钟。这种改良的LC - MS/MS方法显示出优异的分析性能，基质效应较小且线性范围宽。多LC系统与一个质谱联用的设计可能在提高日常检测通量、加快LC - MS/MS分析速度以及使其在不久的将来成为连续诊断的一个组成部分方面发挥显著作用。
