中国临床药理学杂志

UHPLC-MS/MS法测定人血浆中褪黑素浓度

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黄冉 ¹^,² , 刘海艳 ³ , 曾阳 ³ , 段思美 ² , 李专 ¹

中国临床药理学杂志 | 研究方法 2025,41(18): 2655-2660

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中国临床药理学杂志 |研究方法 2025 , 41 (18) : 2655 -2660

UHPLC-MS/MS法测定人血浆中褪黑素浓度

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黄冉¹^,², 刘海艳³, 曾阳³, 段思美², 李专¹

作者信息

^1.湖南师范大学医学院，湖南长沙 410081

^2.长沙晶易医药科技股份有限公司，湖南长沙 410221

^3.湖南科锐斯医药科技有限公司，湖南长沙 410221

通讯作者:

李专，教授，博士生导师 Tel: （0731）88912403 E-mail: zhuanli@hunnu.edu.cn

作者简介:

黄冉（1997-），女，硕士研究生，主要从事药物分析研究

Determination of melatonin concentration in human plasma by UHPLC-MS/MS

Ran HUANG¹^,², Hai-yan LIU³, Yang ZENG³, Si-mei DUAN², Zhuan LI¹

Affiliations

^1.College of Medicine, Hunan Normal University, Changsha 410081, Hunan Province, China

^2.Changsha Jing Yi Pharmaceutical Technology Co., Ltd., Changsha 410221, Hunan Province, China

^3.Hunan Creates Med Technology Co., Ltd., Changsha 410221, Hunan Province, China

出版时间: 2025-09-28 doi: 10.13699/j.cnki.1001-6821.2025.18.015

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摘要

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目的

建立高效灵敏准确的超高效液相质谱联用法（UHPLC-MS/MS）测定人血浆中褪黑素（MLT）浓度。

方法

用UHPLC-MS/MS法测定人血浆中褪黑素浓度。血浆样品用乙腈进行蛋白沉淀前处理，以其同位素褪黑素-d4（MLT-d4）做内标。用ACQUITY UPLC BEH C₁₈（2.1 mm×50.0 mm，1.7 μm）反向色谱柱，以0.05 mM氟化铵-乙腈为流动相进行梯度洗脱，流速为0.4 mL·min^-1，采集时间为3 min。用电喷雾离子源（ESI+），正离子模式，多反应监测（MRM）扫描方式进行监测。待测化合物褪黑素，离子反应为m/z 233.0→174.0，内标（褪黑素-d4）离子对为m/z 237.2→178.2。该方法应用于一项单中心、随机、开放、单次口服给药药代动力学试验，空腹及餐后各入组12例受试者，给药剂量为2 g（含有4 mg褪黑素），用WinNonlin 8.3或以上版本或SAS 9.4或以上版本对药代动力学参数C_max（ΔC_max）、AUC_0-t（ΔAUC_0-t）和AUC_0-∞（ΔAUC_0-∞）分别进行统计分析。

结果

人血浆中褪黑素定量下限为50.00 pg·mL^-1；在50.00~50 000.00 pg·mL^-1呈现较好的线性y=2.00x+1.12×10^-4（r²>0.999 4）；批内和批间精密度结果显示，不同浓度相对标准偏差（RSD）均不超过5.77%，准确度在92.11%~102.01%之间。低、中、高浓度质控样品提取回收率分别为103.35%、113.75%和131.21%，变异系数为12.13%，无基质效应。通过12例健康人体药代动力学研究首次发现，服用相同剂量的褪黑素后，餐后的C_max约为空腹的50%，但AUC基本一致，说明食物可能影响褪黑素在体内的吸收及代谢。

结论

该方法专属性强、精密度好、灵敏度高，能快速准确测定人血浆中褪黑素含量，并用于褪黑素的人体药代动力学研究。

关键词

褪黑素 / 失眠 / 超高效液相质谱联用 / 药代动力学

Abstract

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Objective

To establish a highly efficient, sensitive, and accurate ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS/MS) method to determine the concentration of melatonin (MLT) in human plasma with heparin sodium.

Methods

The concentration of melatonin in plasma was measured using UHPLC-MS/MS. Plamsa sample preparation was done by protein precipitation using acetonitrile, its isotope melatonin-d4 (MLT-d4) was used as the internal standard (IS). Using UPLC BEH C₁₈ (2.1 mm×50.0 mm, 1.7 μm) reverse phase chromatography column, gradient elution was performed with a mobile phase consisting of acetonitrile-water (0.05 mM Ammonium fluoride) at a flow rate of 0.4 mL·min^-1 for 3 min. MLT was monitored using positive electrospray triple quadrupole mass spectrometer via multiple reaction monitoring (MRM) mode. The monitored transitions were set at m/z 233.0→174.0 for MLT. Moreover, the IS was set at m/z 237.2→178.2. This method was applied in a single-center, randomized, open-label, single oral dose pharmacokinetic trial, with 12 subjects in each group for fasting and after meals, administered a dose of 2 g (containing 4 mg of melatonin). Statistical analysis of pharmacokinetic parameters C_max (ΔC_max), AUC_0-t (ΔAUC_0-t), and AUC_0-∞ (ΔAUC_0-∞) using WinNonlin version 8.3 or above or SAS version 9.4 or above.

Results

The achieved lower limit of quantification was 50.00 pg·mL^-1. The validated method had an excellent linearity in the range of 50.00-50 000.00 pg·mL^-1 for MLT y=2.00x+1.12×10^-4 (r²>0.999 4). The results of the intra-batch and inter-batch precision showed that the relative standard deviation (RSD) at different concentrations did not exceed 5.77%, and the accuracy was between 92.11% - 102.01% . The extraction recoveries of low, medium and high quality control samples of MLT were 103.35%, 113.75% and 131.21%, respectively. The coefficient of variation was 12.13%. No significant matrix effect was found. Through the pharmacokinetic study of 12 healthy humans, it was found for the first time that after taking the same dose of melatonin, the postprandial C_max was about 50% of that of fasting, but the AUC was very similar, indicating that food might affect the absorption and metabolism of melatonin in the body.

Conclusion

This method has strong specificity, good precision and high sensitivity, which can quickly and accurately determine the content of melatonin in human plasma with heparin sodium, and is used for the detection of human blood drug concentration and pharmacokinetics of melatonin.

Key words

melatonin / insomnia / ultra-high performance liquid chromatography-mass spectrometry / pharmacokinetics

引用本文

黄冉, 刘海艳, 曾阳, 段思美, 李专. UHPLC-MS/MS法测定人血浆中褪黑素浓度. 中国临床药理学杂志, 2025 , 41 (18) : 2655 -2660 . DOI: 10.13699/j.cnki.1001-6821.2025.18.015

Ran HUANG, Hai-yan LIU, Yang ZENG, Si-mei DUAN, Zhuan LI. Determination of melatonin concentration in human plasma by UHPLC-MS/MS[J]. Chinese Journal of Clinical Pharmacology, 2025 , 41 (18) : 2655 -2660 . DOI: 10.13699/j.cnki.1001-6821.2025.18.015

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2025年第41卷第18期

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文章信息

doi: 10.13699/j.cnki.1001-6821.2025.18.015

接收时间：2025-06-11
首发时间：2026-08-05
出版时间：2025-09-28

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收稿日期：2025-06-11

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作者信息

^1.湖南师范大学医学院，湖南长沙 410081

^2.长沙晶易医药科技股份有限公司，湖南长沙 410221

^3.湖南科锐斯医药科技有限公司，湖南长沙 410221

通讯作者:

李专，教授，博士生导师 Tel: （0731）88912403 E-mail: zhuanli@hunnu.edu.cn

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https://castjournals.cast.org.cn/joweb/zglcylxzz/CN/10.13699/j.cnki.1001-6821.2025.18.015

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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
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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