中国临床药理学杂志

用LC-MS/MS法检测血清中维生素D的含量

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施卉 ^1a , 李海娟 ^1a , 顾玲琪 ^1a , 张小华 ^1b , 金霞 ^1b , 顾锦华 ^1a

中国临床药理学杂志 | 研究方法 2025,41(6): 866-871

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中国临床药理学杂志 |研究方法 2025 , 41 (6) : 866 -871

用LC-MS/MS法检测血清中维生素D的含量

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施卉^1a, 李海娟^1a, 顾玲琪^1a, 张小华^1b, 金霞^1b, 顾锦华^1a

作者信息

^1a.南通大学附属妇幼保健院，药学部，江苏南通 226000

^1b.南通大学附属妇幼保健院，儿科，江苏南通 226000

通讯作者:

顾锦华，主任药师，硕士生导师 Tel: （0513）59008153 E-mail: jhgu@ntu.edu.cn

作者简介:

施卉（1986-），女，主管药师，主要从事儿童治疗药物监测方向的研究

Determination the contents of vitamin D in serum by LC-MS/MS

Hui SHI^1a, Hai-juan LI^1a, Ling-qi GU^1a, Xiao-hua ZHANG^1b, Xia JIN^1b, Jin-hua GU^1a

Affiliations

^1a.Department of Pharmacy, Affiliated Maternity and Child Health Care Hospital of Nantong University, Nantong 226000, Jiangsu Province, China

^1b.Department of Pediatrics, Affiliated Maternity and Child Health Care Hospital of Nantong University, Nantong 226000, Jiangsu Province, China

出版时间: 2025-03-28 doi: 10.13699/j.cnki.1001-6821.2025.06.022

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摘要

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目的

建立一种基于液相色谱-串联质谱（LC-MS/MS）的血清维生素D（VD）定量分析方法并评估其在儿科营养支持中的临床应用价值。

方法

血清样品经乙腈处理，以25-羟基维生素D₂-d₆（25-OH-VD₂-d₆）与25-羟基维生素D₃-d₆（25-OH-VD₃-d₆）作为内标。色谱柱：Kinetex C18（50.0 mm×3.0 mm，2.6 μm）；流动相：0.1%甲酸水溶液和0.1%甲酸甲醇溶液梯度洗脱，流速为0.5 mL·min^-1，进样量为10 μL，柱温30 ℃。用电喷雾离子化源，正离子模式，多重反应监测，评估LC-MS/MS检测方法学的专属性、标准曲线及定量下限、精密度及回收率、稳定性和基质效应。应用建立的方法检测肠内组（口服10 μg·kg^-1 VD滴剂，qd）和肠外组[肠外营养液中加入注射用脂溶性维生素（Ⅰ）经静脉注射相当于10 μg·kg^-1 VD，qd]早产儿血清中VD水平。

结果

25-OH-VD₂与25-OH-VD₃均在5～100 ng·mL^-1呈良好的线性关系，25-OH-VD₂标准曲线为y=1.00x+2.34×10^-2（R=0.999 8），定量下限为0.5 ng·mL^-1；25-OH-VD₃标准曲线为y=1.00x+5.6×10^-3（R=0.999 8），定量下限为1 ng·mL^-1。精密度及回收率：25-OH-VD₂的质控样本回收率为95.59%～105.38%，批内和批间精密度分别≤ 4.75%和≤ 4.68%，25-OH-VD₃回收率为98.38%～113.63%，批内和批间精密度分别≤ 5.64%和≤ 7.83%；不同浓度25-OH-VD₂和25-OH-VD₃样品在不同条件下稳定性良好，基质效应分别为85%～110%和88%～112%。经肠内或肠外VD补充后，2组早产儿血清中25-OH-VD₂和25-OH-VD₃浓度均显著升高，其中肠外组25-OH-VD₂和25-OH-VD₃浓度均显著高于肠内组（均P<0.05）；肠外组早产儿每日体质量增加率均显著高于肠内组（均P<0.05）。

结论

基于LC-MS/MS的血清VD定量分析方法专属性强、灵敏度高，适用于早产儿VD水平监测。

关键词

维生素D / 液相色谱-串联质谱 / 定量分析 / 早产儿 / 营养支持

Abstract

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Objective

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.

Methods

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].

Results

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).

Conclusion

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.

Key words

vitamin D / liquid chromatography tandem mass spectrometry / quantitative analysis / preterm infants / nutritional support

引用本文

施卉, 李海娟, 顾玲琪, 张小华, 金霞, 顾锦华. 用LC-MS/MS法检测血清中维生素D的含量. 中国临床药理学杂志, 2025 , 41 (6) : 866 -871 . DOI: 10.13699/j.cnki.1001-6821.2025.06.022

Hui SHI, Hai-juan LI, Ling-qi GU, Xiao-hua ZHANG, Xia JIN, Jin-hua GU. Determination the contents of vitamin D in serum by LC-MS/MS[J]. Chinese Journal of Clinical Pharmacology, 2025 , 41 (6) : 866 -871 . DOI: 10.13699/j.cnki.1001-6821.2025.06.022

基金

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南通市药物政策与药学服务研究基金资助项目(2023NTPA11)

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2025年第41卷第6期

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文章信息

doi: 10.13699/j.cnki.1001-6821.2025.06.022

接收时间：2024-09-09
首发时间：2026-08-04
出版时间：2025-03-28

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收稿日期：2024-09-09

基金

南通市药物政策与药学服务研究基金资助项目(2023NTPA11)

作者信息

^1a.南通大学附属妇幼保健院，药学部，江苏南通 226000

^1b.南通大学附属妇幼保健院，儿科，江苏南通 226000

通讯作者:

顾锦华，主任药师，硕士生导师 Tel: （0513）59008153 E-mail: jhgu@ntu.edu.cn

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https://castjournals.cast.org.cn/joweb/zglcylxzz/CN/10.13699/j.cnki.1001-6821.2025.06.022

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2种不同金属材料的力学参数

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
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红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
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