一种超灵敏 UHPLC-ESI-MS/MS 方法，辅以受控微波衍生反应，用于定量 COVID-19 患者的维生素 D3 及其主要代谢物。
An ultrasensitive UHPLC-ESI-MS/MS method augmented with a controlled microwave derivatization reaction for quantitation of vitamin D3 and its major metabolites in COVID-19 patients.
机构信息
Pharmacognosy and Pharmaceutical Chemistry Department, College of Pharmacy Taibah University, AlMadinah AlMunawarah, 30001, Saudi Arabia.
Clinical and Hospital Pharmacy Department, College of Pharmacy, Taibah University, AlMadinah AlMunawarah, 30001, Saudi Arabia.
出版信息
Talanta. 2022 Aug 15;246:123497. doi: 10.1016/j.talanta.2022.123497. Epub 2022 Apr 22.
It is established that vitamin D deficiency is correlated with the disease severity in COVID-19 patients. However, the reliable and sensitive quantitation of vitamin D3 (D3) and its metabolites remains a difficult challenge. Herein, a novel ultrasensitive and reliable UHPLC-ESI-MS/MS method was developed and validated for the quantitation of D3 and its major metabolites in COVID-19 patients. The mass spectral sensitivity was augmented via controlled microwave-assisted derivatization reaction (CMDR) with 2-nitrosopyridine (Pyr-NO) at 65 °C for 2 min. CMDR hyphenation with UHPLC-MS/MS improves detection sensitivity while shortening separation and derivatization reaction times. The precursor to product ion transitions for D3, 25-hydroxy D3 (25(OH)D3), 1,25-dihydroxy D3 (1,25-(OH)2D3) and calcipotriol (CPT) as an internal standard were m/z 493.4 → 231.3, m/z 509.4 → 231.3, m/z 525.4 → 247.3, and m/z 521.4 → 247.3; respectively. The separation of the formed derivatives was conducted using a gradient elution mode with mobile phase A: formic acid (0.1%) in water and mobile phase B: formic acid (0.1%) in acetonitrile. The elution started with 40% (v/v) of B for 0.3 min then increased linearly to 90% (v/v) at 2 min on an Agilent EclipsePlus C18 (50 × 2.1 mm, 1.8 μm) column at a flow rate of 0.3 mL min. The method was validated using FDA standards for bioanalytical method validation over a concentration range of 0.02-50 ng mL with correlation coefficient ≥0.9987 and the lower limit of quantitation (LLOQ) were 0.02-0.05 ng mL in human plasma. The developed method has demonstrated excellent comparability to a well-established chemiluminescent immunoassay (CLIA) method for the analysis of D3 metabolites in human samples. The developed UHPLC-ESI-MS/MS method was implemented for routine and reliable quantitation of D3 and its major metabolites in COVID-19 patients.
现已确定，维生素 D 缺乏与 COVID-19 患者的疾病严重程度相关。然而，可靠且灵敏地定量检测维生素 D3（D3）及其代谢物仍然是一个具有挑战性的问题。在此，我们开发并验证了一种新型的超灵敏且可靠的 UHPLC-ESI-MS/MS 方法，用于定量检测 COVID-19 患者的 D3 及其主要代谢物。通过在 65°C 下用 2-硝基吡啶（Pyr-NO）进行 2 分钟的受控微波辅助衍生化反应（CMDR），增强了质谱的灵敏度。CMDR 与 UHPLC-MS/MS 联用，可提高检测灵敏度，同时缩短分离和衍生化反应时间。D3、25-羟基维生素 D3（25（OH）D3）、1,25-二羟基维生素 D3（1,25-（OH）2D3）和卡泊三醇（CPT）作为内标物的前体到产物离子的跃迁分别为 m/z 493.4→231.3、m/z 509.4→231.3、m/z 525.4→247.3 和 m/z 521.4→247.3。衍生化产物的分离采用梯度洗脱模式，流动相 A：水中的甲酸（0.1%），流动相 B：乙腈中的甲酸（0.1%）。洗脱开始时，流动相 B 的体积比为 40%（v/v），持续 0.3 分钟，然后在 2 分钟内线性增加至 90%（v/v），在 Agilent EclipsePlus C18（50×2.1mm，1.8μm）柱上以 0.3mL·min 的流速洗脱。该方法使用 FDA 生物分析方法验证标准进行验证，在 0.02-50ng·mL 的浓度范围内，相关系数≥0.9987，人血浆中的定量下限（LLOQ）为 0.02-0.05ng·mL。与已建立的化学发光免疫分析法（CLIA）相比，所开发的方法在分析人样品中的 D3 代谢物时表现出良好的可比性。该开发的 UHPLC-ESI-MS/MS 方法已用于 COVID-19 患者中 D3 及其主要代谢物的常规和可靠定量。
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