High Precision Bio-analytical Facility, Department of Ocular Pharmacology and Pharmacy, Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India.
Department of Biochemistry and Cell Biology, Sankara Nethralaya, 18, College Road, Nungambakkam, Chennai 600006, TN, India.
J Chromatogr B Analyt Technol Biomed Life Sci. 2014 Jan 1;944:49-54. doi: 10.1016/j.jchromb.2013.11.011. Epub 2013 Nov 14.
The present study demonstrates the development and validation of a sensitive method for the quantification of homocysteine thiolactone (HCTL) in human plasma using the technique of LC-MS/MS. The gradient elution of HCTL was achieved within 5min using ZIC HILIC column having acetonitrile with 0.1% formic acid and water with 0.1% formic acid. The method was validated for the linearity, sensitivity, accuracy, precision, recovery, matrix effect and stability. A good linearity was found within a range of 0.5-32.5nmol/ml. Quantification was performed using multiple reaction monitoring (MRM) mode based on the molecular/fragment ion transitions for HCTL (118/56) and homatropine (276.1/142.2) as internal standard. Generally, HCTL levels in plasma were found to be highly unstable. In order to verify the stability of the HCTL levels in plasma for a longer period, the samples were extracted immediately and stored at -86°C. Using the above method it was found to be stable for a period of 1 month. The method was well applied for quantification of HCTL in plasma of healthy human volunteers.
本研究展示了一种使用 LC-MS/MS 技术定量检测人血浆中同型半胱氨酸硫内酯 (HCTL) 的灵敏方法的开发和验证。使用 ZIC HILIC 柱，在乙腈中含有 0.1%甲酸和水中含有 0.1%甲酸的条件下，HCTL 的梯度洗脱在 5 分钟内完成。该方法对线性、灵敏度、准确性、精密度、回收率、基质效应和稳定性进行了验证。在 0.5-32.5nmol/ml 的范围内发现了良好的线性关系。采用基于 HCTL（118/56）和阿托品（276.1/142.2）作为内标物的分子/碎片离子跃迁的多重反应监测 (MRM) 模式进行定量。通常，发现血浆中的 HCTL 水平极不稳定。为了验证更长时间内血浆中 HCTL 水平的稳定性，立即提取样品并储存在-86°C。使用上述方法，发现其在 1 个月内稳定。该方法很好地应用于定量检测健康人类志愿者血浆中的 HCTL。
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