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To establish a quantitative analysis method for serum vitamin D (VD) based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) and evaluate its clinical application value in pediatric nutritional support.
Serum samples were processed with acetonitrile, using 25-hydroxyvitamin D2-d6 (25-OH-VD2-d6) and 25-hydroxyvitamin D3-d6 (25-OH-VD3-d6) as internal standards. The chromatographic column was Kinetex C18 (50.0 mm × 3.0 mm, 2.6 μm). The mobile phase consisted of 0.1% formic acid aqueous solution and 0.1% formic acid methanol solution, with gradient elution at a flow rate of 0.5 mL·min-1 and an injection volume of 10 μL. The column temperature was set to 30 ℃. Electrospray ionization was used in positive ion mode, with multiple reaction monitoring. The LC-MS/MS method was evaluated for specificity, calibration curves, lower limits of quantification, precision, recovery rate, stability, and matrix effects. The method was applied to detect VD levels in the serum of infants from the external group (oral 10 μg·kg-1 VD drops, qd) and the parenteral group [parenteral nutrition solution with injectable fat-soluble vitamin (Ⅰ) administered intravenously, it was equivalent to 10 μg·mL-1 VD, qd].
Both 25-OH-VD2 and 25-OH-VD3 showed good linearity in the range of 5-100 ng·mL-1, with the 25-OH-VD2 standard curve of y=1.00x+2.34×10-2 (R=0.999 8), and a lower limit of quantification (LLOQ) of 0.5 ng·mL-1; 25-OH-VD3 standard curve of y=1.00x+5.6×10-3 (R=0.999 8), and LLOQ of 1 ng·mL-1. The recovery rate of 25-OH-VD2 quality control samples was 95.59%-105.38%, with intra- and inter-day precision ≤ 4.75% and ≤ 4.68%, respectively. The recovery rate of 25-OH-VD3 was 98.38%-113.63%, with intra- and inter-batch precision ≤5.64% and ≤7.83%, respectively. Stability of 25-OH-VD2 and 25-OH-VD3 samples at different concentrations was good under various conditions, with matrix effects ranging from 85%-110% and 88%-112%, respectively. After enteral or parenteral VD supplementation, the serum levels of 25-OH-VD2 and 25-OH-VD3 in both groups of preterm infants significantly increased, with the parenteral group showing significantly higher concentrations of 25-OH-VD2 and 25-OH-VD3 compared to the enteral group (P<0.05). The daily weight gain rate of preterm infants in the parenteral group was also significantly higher than those in the enteral group (all P<0.05).
The quantitative analysis method of serum VD based on LC-MS/MS has strong specificity and high sensitivity, which is suitable for the monitoring of VD level in premature infants.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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