Department of Physical Pharmacy and Pharmacokinetics, Poznan University of Medical Sciences, 6 Święcickiego Street, 60-781 Poznan, Poland.
Department of Physical Pharmacy and Pharmacokinetics, Poznan University of Medical Sciences, 6 Święcickiego Street, 60-781 Poznan, Poland.
J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Nov 30;1159:122350. doi: 10.1016/j.jchromb.2020.122350. Epub 2020 Aug 29.
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 具有预防心血管疾病 (CVD) 的作用。血清 25-羟维生素 D（25D）是人体维生素 D 状态最常用的指标。25D 估计为 25-羟维生素 D2（25D2）和 25-羟维生素 D3（25D3）的总和。然而，3-表-25-羟维生素 D3（3epi25D3）的存在会影响 25D 的测量。在这项研究中，开发了一种新的经过验证的 UPLC-MS/MS 技术，用于测量人血浆中的三种维生素 D 代谢物 25D2、25D3 和 3epi25D3。采用正己烷进行液-液萃取，从样品中分离分析物。在 Kinetex F5 分析柱上实现了色谱分离，采用等度洗脱（水和甲醇，含 0.1% 甲醇酸，20:80 v/v）。代谢物的质谱检测在正离子模式下的三重四极串联质谱仪中进行。在 54 名患者的血浆样本中估计了分析物的浓度。UPLC-MS/MS 方法的验证参数，包括线性、精密度、准确度和稳定性，满足生物分析测定的要求。超过 60%的患者存在 25D 不足浓度（<20ng/mL）。78%的样本中存在 3epi25D3，其相对量占 25D 浓度的 0 至 54.1%。通过验证的 UPLC-MS/MS 方法对 CVD 患者血浆中 25D2、25D3 和 3epi25D3 的分析，允许对 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。
