Vitamin D has a potential role in protecting against cardiovascular disease (CVD). Serum 25-hydroxyvitamin D (25D) is the most widely used indicator of vitamin D status in the human body. 25D is estimated as total of 25-hydroxyvitamin D2 (25D2) and 25-hydroxyvitamin D3 (25D3). However, the presence of 3-epi-25-hydroxyvitamin D3 (3epi25D3) can affect 25D measurement. In this research a novel validated UPLC–MS/MS technique was developed to measure three vitamin D metabolites, 25D2, 25D3 and 3epi25D3 in human plasma. A liquid–liquid extraction using hexane was applied for isolation of the analytes from the samples. A chromatographic separation was achieved in a Kinetex F5 analytical column with isocratic elution (water and methanol with 0.1% methanoic acid, 20:80 v/v). Mass spectrometry detection of the metabolites was performed in a triple-quadruple tandem mass spectrometer under positive ion mode. Concentrations of the analytes were estimated in plasma samples of 54 patients. Validation parameters of the UPLC-MS/MS method, including linearity, precision, accuracy, and stability, fulfilled the requirements for bioanalytical assays. The deficient concentration of 25D (<20 ng/mL) was stated in over 60% of patients. 3epi25D3 was present in 78% of samples and its relative amount ranged from 0 to 54.1% of 25D concentration. The analysis of 25D2, 25D3 and 3epi25D3 by the validated UPLC-MS/MS method in plasma of patients with CVD permitted the classification of the patients with insufficient levels of 25D. 3epi25D3 might be relevant in the classification of vitamin D status.

维生素D在预防心血管疾病（CVD）方面具有潜在作用。血清25-羟基维生素D（25D）是人体中维生素D状况最广泛使用的指标。估计25D为25-羟基维生素D2（25D2）和25-羟基维生素D3（25D3）的总和。但是，3-epi-25-羟基维生素D3（3epi25D3）的存在会影响25D测量。在这项研究中，开发了一种新颖的经过验证的UPLC-MS / MS技术来测量人体血浆中的三种维生素D代谢物25D2、25D3和3epi25D3。使用己烷进行液-液萃取，以从样品中分离分析物。在Kinetex F5分析柱中进行等度洗脱（水和甲醇，含0.1％甲磺酸，20:80 v / v），进行色谱分离。代谢物的质谱检测是在三重串联质谱仪中在正离子模式下进行的。估算了54位患者血浆样品中分析物的浓度。UPLC-MS / MS方法的验证参数（包括线性，精度，准确性和稳定性）满足了生物分析的要求。超过60％的患者中25D（<20 ng / mL）浓度不足。3epi25D3存在于78％的样品中，其相对量为25D浓度的0至54.1％。通过经过验证的UPLC-MS / MS方法对CVD患者血浆中的25D2、25D3和3epi25D3进行分析，可以对25D水平不足的患者进行分类。3epi25D3可能与维生素D状态的分类有关。