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

doi:10.12092/j.issn.1009-2501.2026.04.011

Abstract

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

CLC Number:

R446.1
R965

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.

Figures/Tables 4

References 66

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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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