Objective

Fat-soluble vitamins (A, D, E and K) are isoprene derived apolar molecules. While deficiencies of these vitamins have been associated with various diseases such as type 2 diabetes and cancer, high doses of Vitamin A and D can cause toxic effects. Accurate detection of serum levels of these vitamins have critical importance. In this study, it is aimed to develop and validate a sensitive and specific Liquid Chromatography Tandem Mass Spectrometry (LC–MS / MS) method that allows simultaneous analysis of fat-soluble vitamins.

Materials and Methods

Serum samples were deproteinized with methanol and chromatographic separation of analytes were performed by LC–MS/MS system (Agilent Technologies 6420 Triple Quadrapole LC–MS), Agilent Pursuit PFP column (100 mm × 3.0 mm; 3.0 μm), in gradient mode using Mobile phase A (milli-Q+0.1 % formic acid) and Mobile phase B (Methanol+0.1 % formic acid). Ion scan was performed in MRM (multiple reaction monitoring) mode with positive ion selectivity in ESI ion source.

Results

The retention times were 6.93 min, 6.94 min and 9.34 min while concentrations were linear in the ranges between 10−150 ng/mL, 3−90 μg /dL and 6−90 μg/mL for 25-hydroxy vitamin D₃ (25−OHD₃), Vitamin A and Vitamin E, respectively. Inter-day Coefficient Variation (CV%) values for Vitamin A, Vitamin E and 25−OHD₃ were; 9.08 %, 9.85 % and 3.07 % and intra-day CV% values were; 2.98 %, 5.05 % and 5.01 %. LOD and LOQ results were 2.11 μg/dL and 3.50 μg/dL for Vitamin A; 1.71 μg/mL and 2.45 μg/mL for Vitamin E; 1.47 ng/mL and 2.50 ng/mL for 25−OHD₃, respectively.

Conclusion

In this study, a LC–MS/MS method that can analyze fat soluble vitamins in 13 min was developed and validated. This method will be useful for clinical purposes by replacing low specificity immunoassay methods and High Performance Liquid Chromatography (HPLC) methods that can not allow simultaneous analysis.

中文翻译：

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液相色谱串联质谱法同时测定脂溶性维生素的开发和优化。

目的

脂溶性维生素（A，D，E和K）是异戊二烯衍生的非极性分子。尽管这些维生素的缺乏与多种疾病（例如2型糖尿病和癌症）有关，但高剂量的维生素A和D会引起毒性作用。准确检测这些维生素的血清水平至关重要。在本研究中，其目的是开发和验证一种灵敏而特异的液相色谱串联质谱法（LC-MS / MS），该方法可同时分析脂溶性维生素。

材料和方法

用甲醇对血清样品进行脱蛋白处理，并通过LC-MS / MS系统（Agilent Technologies 6420三重四极杆LC-MS），Agilent Pursuit PFP色谱柱（100 mm×3.0 mm; 3.0μm），以梯度模式使用流动相A（milli-Q + 0.1％甲酸）和流动相B（甲醇+ 0.1％甲酸）。离子扫描以MRM（多反应监测）模式进行，在ESI离子源中具有正离子选择性。

结果

保留时间分别为6.93分钟，6.94分钟和9.34分钟，而浓度在25-羟基维生素D ₃（10-150 ng / mL，3-90μg/ dL和6-90μg/ mL之间呈线性变化（25- OHD ₃），维生素A和维生素E。维生素A，维生素E和25-OHD ₃的日间系数变化（CV％）值为；日内CV％值分别为9.8％，9.85％和3.07％。2.98％，5.05％和5.01％。维生素A的LOD和LOQ结果为2.11μg/ dL和3.50μg/ dL；维生素E为1.71μg/ mL和2.45μg/ mL; 25-OHD ₃分别为1.47 ng / mL和2.50 ng / mL 。

结论

在这项研究中，开发并验证了可以在13分钟内分析脂溶性维生素的LC-MS / MS方法。该方法将替代不能同时进行分析的低特异性免疫分析方法和高效液相色谱（HPLC）方法，可用于临床。