苯肼类衍生化结合LC-MS/MS定量分析生物样本中羰基代谢物的方法学的进展及应用

doi:10.12092/j.issn.1009-2501.2026.04.011

中国临床药理学与治疗学 ›› 2026, Vol. 31 ›› Issue (4): 517-526.doi: 10.12092/j.issn.1009-2501.2026.04.011

苯肼类衍生化结合LC-MS/MS定量分析生物样本中羰基代谢物的方法学的进展及应用

董文丽¹^,²^,³^,⁴(), 张若浩²^,³^,⁴, 郭蓓宁²^,³^,⁴, 张菁²^,³^,⁴, 徐宇虹¹, 彭保卫¹, 刘笑芬²^,³^,⁴^,*()

1. 大理大学药学院，大理 671000，云南
2. 复旦大学附属华山医院抗生素研究所/复旦大学附属华山医院临床药理研究中心，上海 200040
3. 国家卫生健康委抗生素临床药理重点实验室，上海 200040
4. 国家老年疾病临床医学研究中心（华山），上海 200040

收稿日期:2025-06-25 修回日期:2025-09-15 出版日期:2026-04-26 发布日期:2026-04-30
通讯作者: 刘笑芬 E-mail:1811675172@qq.com;liuxiaofen227@163.com
作者简介:董文丽，女，在读硕士研究生，研究方向：主要从事抗菌药物临床药理学和代谢组学研究。E-mail：1811675172@qq.com
基金资助:
科技部“重大新药创制”科技重大专项资助项目（2017ZX09304005）；复旦大学附属华山医院临床研究基金（2023-YN009）

Progress and application of phenylhydrazine derivatization combined with LC-MS/MS for quantitative analysis of carbonyl metabolites in biological samples

Wenli DONG¹^,²^,³^,⁴(), Ruohao ZHANG²^,³^,⁴, Beining GUO²^,³^,⁴, Jing ZHANG²^,³^,⁴, Yuhong XU¹, Baowei PENG¹, Xiaofen LIU²^,³^,⁴^,*()

1. College of Pharmacy, Dali University, Dali 671000, Yunnan, China
2. Institute of Antibiotics, Huashan Hospital, Fudan University/Clinical Pharmacology Center, Huashan Hospital, Fudan University, Shanghai 200040, China
3. Key Laboratory of Clinical Pharmacology of Antibiotics, National Health Commission of the People's Republic of China, Shanghai 200040, China
4. National Clinical Research Center for Aging and Medicine, Huashan Hospital, Fudan University, Shanghai 200040, China

Received:2025-06-25 Revised:2025-09-15 Online:2026-04-26 Published:2026-04-30
Contact: Xiaofen LIU E-mail:1811675172@qq.com;liuxiaofen227@163.com

摘要/Abstract

摘要：

酮类、醛类及羧酸类含羰基化合物作为重要的内源性代谢物，广泛分布于生物体血液和体液中。这类化合物具有挥发性强、化学稳定性差和生物基质中浓度低等特性，使其在复杂生物样本（血液、组织和粪便等）的痕量检测中存在显著局限性。通过对化合物进行柱前衍生化生成结构稳定且检测响应高的化合物，可以提高这类化合物的检测灵敏度，准确测定这些化合物的含量。本文综述了基于苯肼类化学衍生化策略和液相色谱串联质谱技术的检测方法，且该方法应用于测定的脂肪醛与短链脂肪酸等化合物在能量代谢、免疫应答等关键生理过程的作用。

关键词: 短链脂肪酸, 脂肪醛, 苯肼类衍生化, LC-MS/MS

Abstract:

Ketones, aldehydes and carboxylic acids containing carbonyl compounds are important endogenous metabolites, which are widely distributed in the blood and body fluids of organisms. However, these compounds have the characteristics of strong volatility, poor chemical stability and low concentration in biological matrix, which make them have limitations in the trace detection of complex biological samples ( blood, tissue, feces, etc. ). Pre-column derivatization of the compounds generates products with stable structures and high detection responses, which can improve the detection sensitivity and enable accurate quantification of these compounds. This paper reviews the method that based on the phenylhydrazine chemical derivatization strategy and combined with liquid chromatography tandem mass spectrometry, and this method is applied to the determination of the role of fatty aldehydes and short-chain fatty acids in key physiological processes such as energy metabolism and immune response.

Key words: short-chain fatty acids, aliphatic aldehyde, phenylhydrazine derivatization, LC-MS/MS

中图分类号:

R446.1
R965

董文丽, 张若浩, 郭蓓宁, 张菁, 徐宇虹, 彭保卫, 刘笑芬. 苯肼类衍生化结合LC-MS/MS定量分析生物样本中羰基代谢物的方法学的进展及应用[J]. 中国临床药理学与治疗学, 2026, 31(4): 517-526.

Wenli DONG, Ruohao ZHANG, Beining GUO, Jing ZHANG, Yuhong XU, Baowei PENG, Xiaofen LIU. Progress and application of phenylhydrazine derivatization combined with LC-MS/MS for quantitative analysis of carbonyl metabolites in biological samples[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2026, 31(4): 517-526.

图/表 4

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	Phenylhydrazines	Hydrazides	Hydroxylamines
Reaction mechanism	Condenses with carbonyls to form phenylhydrazones	Acid-catalyzed Schiff base formation	Oxime/nitrone formation via nucleophilic addition
Derivatization reagent	PH 2-NPH 3-NPH DNPH	Girard's T(GT) Girard's P(GP) tosylhydrazine dansylhydrazine	O-substituted hydroxylamine (-ONH₂), N-substituted hydroxylamine (-NHOH)
Reactivity	The reaction conditions are mild, the reaction rate is fast and the yield is high	Under acidic conditions, the reaction activity is higher	Neutral or weakly acidic conditions, longer reaction time
Sensitivity	High sensitivity and high ionization efficiency can significantly improve the signal-to-noise ratio	High sensitivity, high ionization efficiency	Ionization efficiency is low and sensitivity is low
Stability	The formed C = N double bond was stable and stored at room temperature or 4℃ in dark for several days to several weeks	The generated hydrazone is very stable, but it may hydrolyze in strong acid or alkali; Dansylhydrazine reagent is more sensitive to light	Relatively poor, hydrolysis may occur under acidic or alkaline conditions, not suitable for long-term storage
Selectivity	Carbonyl, aldehyde, carboxyl and phosphate groups can be derived at the same time, which simplifies the simultaneous analysis process of multi-class metabolites	It mainly forms covalent bonds with carbonyl-containing aldehydes or ketones	It is mainly labeled with carbonyl-containing steroid hormones, sugars, testosterone, furfural and fatty aldehydes
Application	LC-MS/MS HPLC-UV	HPLC-FLR LC-MS/MS	GC GC-MS
References	[1, 7, 43-52]

	Phenylhydrazines	Hydrazides	Hydroxylamines
Reaction mechanism	Condenses with carbonyls to form phenylhydrazones	Acid-catalyzed Schiff base formation	Oxime/nitrone formation via nucleophilic addition
Derivatization reagent	PH 2-NPH 3-NPH DNPH	Girard's T(GT) Girard's P(GP) tosylhydrazine dansylhydrazine	O-substituted hydroxylamine (-ONH₂), N-substituted hydroxylamine (-NHOH)
Reactivity	The reaction conditions are mild, the reaction rate is fast and the yield is high	Under acidic conditions, the reaction activity is higher	Neutral or weakly acidic conditions, longer reaction time
Sensitivity	High sensitivity and high ionization efficiency can significantly improve the signal-to-noise ratio	High sensitivity, high ionization efficiency	Ionization efficiency is low and sensitivity is low
Stability	The formed C = N double bond was stable and stored at room temperature or 4℃ in dark for several days to several weeks	The generated hydrazone is very stable, but it may hydrolyze in strong acid or alkali; Dansylhydrazine reagent is more sensitive to light	Relatively poor, hydrolysis may occur under acidic or alkaline conditions, not suitable for long-term storage
Selectivity	Carbonyl, aldehyde, carboxyl and phosphate groups can be derived at the same time, which simplifies the simultaneous analysis process of multi-class metabolites	It mainly forms covalent bonds with carbonyl-containing aldehydes or ketones	It is mainly labeled with carbonyl-containing steroid hormones, sugars, testosterone, furfural and fatty aldehydes
Application	LC-MS/MS HPLC-UV	HPLC-FLR LC-MS/MS	GC GC-MS
References	[1, 7, 43-52]

No.	Compounds	Derivatization Reagent	Specimen	References
1	Aliphatic aldehydes: formaldehyde, acetaldehyde, propanal, pentanal, butanal, hexanal, heptanal, octanal, nonanal, decanal, acrolein, crotonaldehyde	DNPH	Immunosuppressed rat serum and oxidative damage cells, human urine samples	[17, 54]
2	Acetaldehyde	DNPH	Hepatocellular carcinoma cell culture, rat blood and plasma, bovine blood	[7, 55]
3	Malonaldehyde	3-NPH	Human plasma	[4]
4	Formaldehyde, methylglyoxal, malondialdehyde, acetaldehyde	DNPH	Human liver tissue, rat urine	[56-57]
5	Short-chain fatty acids: acetic acid, propionic acid, L-lactic acid, crotonic acid, isobutyric acid, butyric acid, maleic acid, 2-methylbutyric acid, 3-methylcrotonic acid, isovaleric acid, valeric acid, hexanoic acid, isohexanoic acid	3-NPH, 2-NPH	Mouse bronchoalveolar lavage fluid,feces, serum, and lung tissue samples	[20, 53]
6	Acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid (6 SCFAs), and 18 fatty acids (C4-C26)	3-NPH, 2-NPH	Human plasma, serum, urine, feces	[13, 58]
7	Glycolic acid, glyoxylic acid	PH	Human urine	[59-61]
8	Central carbon metabolic carboxylic acids	3-NPH	Mouse heart tissue	[62]
9	Protein carbonyls: Sulfur-containing, aromatic, and aliphatic amino acids	DNPH	Plasma, cells, organ homogenates, isolated proteins and organelles	[63]
10	Carbonyl, carboxyl, and phosphoryl groups	3-NPH	Mouse hematopoietic stem cells	[46]
11	γ-Hydroxybutyric acid	3-NPH	Mammalian cell culture media	[52]
12	Sialic acid	3-NPH	Horse serum, crucian carp, and starfish samples	[27]
13	Carnitine	3-NPH	Whole blood	[64]

No.	Compounds	Derivatization Reagent	Specimen	References
1	Aliphatic aldehydes: formaldehyde, acetaldehyde, propanal, pentanal, butanal, hexanal, heptanal, octanal, nonanal, decanal, acrolein, crotonaldehyde	DNPH	Immunosuppressed rat serum and oxidative damage cells, human urine samples	[17, 54]
2	Acetaldehyde	DNPH	Hepatocellular carcinoma cell culture, rat blood and plasma, bovine blood	[7, 55]
3	Malonaldehyde	3-NPH	Human plasma	[4]
4	Formaldehyde, methylglyoxal, malondialdehyde, acetaldehyde	DNPH	Human liver tissue, rat urine	[56-57]
5	Short-chain fatty acids: acetic acid, propionic acid, L-lactic acid, crotonic acid, isobutyric acid, butyric acid, maleic acid, 2-methylbutyric acid, 3-methylcrotonic acid, isovaleric acid, valeric acid, hexanoic acid, isohexanoic acid	3-NPH, 2-NPH	Mouse bronchoalveolar lavage fluid,feces, serum, and lung tissue samples	[20, 53]
6	Acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid (6 SCFAs), and 18 fatty acids (C4-C26)	3-NPH, 2-NPH	Human plasma, serum, urine, feces	[13, 58]
7	Glycolic acid, glyoxylic acid	PH	Human urine	[59-61]
8	Central carbon metabolic carboxylic acids	3-NPH	Mouse heart tissue	[62]
9	Protein carbonyls: Sulfur-containing, aromatic, and aliphatic amino acids	DNPH	Plasma, cells, organ homogenates, isolated proteins and organelles	[63]
10	Carbonyl, carboxyl, and phosphoryl groups	3-NPH	Mouse hematopoietic stem cells	[46]
11	γ-Hydroxybutyric acid	3-NPH	Mammalian cell culture media	[52]
12	Sialic acid	3-NPH	Horse serum, crucian carp, and starfish samples	[27]
13	Carnitine	3-NPH	Whole blood	[64]

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苯肼类衍生化结合LC-MS/MS定量分析生物样本中羰基代谢物的方法学的进展及应用

Progress and application of phenylhydrazine derivatization combined with LC-MS/MS for quantitative analysis of carbonyl metabolites in biological samples

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