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. 2023 Nov 22;13(48):34157-34166.
doi: 10.1039/d3ra05700c. eCollection 2023 Nov 16.

A novel LC-MS/MS method combined with derivatization for simultaneous quantification of vitamin D metabolites in human serum with diabetes as well as hyperlipidemia

Xiaodi Wang  1   2 , Qian Qin  1 , Fasheng Li  1 , Ying Fu  1 , Na Liu  1
Affiliations

A novel LC-MS/MS method combined with derivatization for simultaneous quantification of vitamin D metabolites in human serum with diabetes as well as hyperlipidemia

Xiaodi Wang et al. RSC Adv. .

Abstract

Vitamin D plays an important role in calcium homeostasis. Recent studies indicate that vitamin D deficiency has become a major public health problem. In order to define vitamin D status, many analytical methods were used to quantify 25-hydroxyvitamin D (25OHD), as circulating 25OHD is regarded as the best indicator to evaluate vitamin D status. The current LC-MS/MS technology is internationally recognized as the "gold standard" for the detection of vitamin D and its metabolites. The impediment to the analysis of vitamin D metabolites is the low level of 25OHD and 1,25(OH)2D. Therefore, it is challenging to achieve the desired sensitivity and accuracy in the determination of trace vitamin D compounds in biological liquids. Here, a method based on liquid-liquid extraction in combination with derivatization, followed by liquid chromatography-electrospray/tandem mass spectrometry was developed for determination of the vitamin D metabolites, including 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, 1α,25-dihydroxyvitamin D2 and 1α,25-dihydroxyvitamin D3. The method was simple and rapid, and it was validated with good linearity (R2 > 0.998), excellent recovery (average value with 81.66-110.31%) and high precision of intra-day and inter-day (0.06-6.38% and 0.20-6.82%). The values of limit of detection (LOD) and limit of quantitation (LOQ) were as low as 0.3 ng mL-1 and 1.0 ng mL-1, respectively. Finally, the developed method was successfully applied to determination of the vitamin D metabolites from the human serum samples of healthy subjects and patients with diabetes as well as hyperlipidemia.

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Conflict of interest statement

There are no conflicts to declare.

Figures

Fig. 1
Fig. 1. Schematic diagram of the biotransformation pathway of vitamin D and the related metabolites.
Fig. 2
Fig. 2. Schematic diagram of the vitamin D analysis process of human serum samples.
Fig. 3
Fig. 3. MS intensity of vitamin D metabolites with protein precipitation by CH3OH and ACN (a), extracted by MTBE and CH2Cl2–CH3OH (b), extracted by MTBE for once and twice (c), and redissolved in CH3OH, ethanol and ACN (d).
Fig. 4
Fig. 4. The optimization of the amount of derivative reagent (a–d), derivatization time (e–h) and derivatization temperature (i–l) for the vitamin D metabolites.
Fig. 5
Fig. 5. Mass chromatogram response of vitamin D metabolites before (a) and after derivatization (b).
Fig. 6
Fig. 6. MRM chromatograms of the derivatives of the vitamin D metabolites obtained from standard mixture (a) and the human serum sample (b).
Fig. 7
Fig. 7. Univariate analysis of the vitamin D metabolites from normal subjects, diabetes (TDM) and hyperlipidemia (HLP) patients.
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