Liquid chromatographic determination of dicarboxylic acids based on intramolecular excimer-forming fluorescence derivatization

A highly sensitive and selective fluorimetric determination method for dicarboxylic acids (C5-C12) has been developed. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, 4-(1-pyrene)butyric acid hydrazide (PBH), followed by reversed-phase liquid chromatography (LC). The carboxylic acids were converted to the corresponding dipyrene-labeled derivatives by reaction with PBH in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The derivatives afforded intramolecular excimer fluorescence (450-550 nm) which can clearly be discriminated from the normal fluorescence (370-420 nm) emitted from PBH and monopyrene-labeled derivatives of monocarboxylic acids. The structures of the derivatives and the emission of excimer fluorescence were studied by LC with mass spectrometry and with spectrofluorimetry, respectively. The PBH derivatives of the carboxylic acids could be separated by reversed-phase LC on an ODS column with isocratic elution. The detection limits (signal-to-noise ratio = 3) were 1.3 fmol to undetectable for a 20-microl injection.     

Liquid chromatographic determination of ornithine and lysine based on intramolecular excimer-forming fluorescence derivatization.

A highly sensitive and selective fluorometric determination method for ornithine and lysine has been developed. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester (PSE), followed by reversed-phase liquid chromatography (LC). The analytes, containing two amino moieties in a molecule, were converted to the corresponding dipyrene-labeled derivatives by reaction with PSE. The derivatives afforded intramolecular excimer fluorescence (450-550 nm) which can clearly be discriminated from the normal fluorescence (370-420 nm) emitted from PSE and monopyrene-labeled derivatives of monoamines. The structures of the derivatives and the emission of excimer fluorescence were confirmed by LC with mass spectrometry and with three-dimensional fluorescence detection system, respectively. The PSE derivatives of ornithine and lysine could be separated by reversed-phase LC on ODS column with isocratic elution. The detection limits (signal-to-noise ratio = 3) for ornithine and lysine were 3.5 and 3.7 fmol, respectively, for a 20-microl injection. Furthermore, this method had enough selectivity and sensitivity for the determination of ornithine and lysine in normal human urine.     

Simultaneous determination of histamine and histidine by liquid chromatography following intramolecular excimer-forming fluorescence derivatization with pyrene-labeling reagent.

A highly sensitive and selective fluorometric method for the determination of histamine and histidine has been developed. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent followed by reversed-phase liquid chromatography. The analytes, containing two amino moieties in a molecule, were converted to the corresponding dipyrene-labeled derivatives by derivatization. The derivatives afforded intramolecular excimer fluorescence (440 - 540 nm), which can clearly be discriminated from the normal fluorescence (360 - 420 nm) emitted from reagent blanks. The detection limits (signal-to-noise ratio = 3) were femto mole levels.     

Liquid chromatographic determination of polythiols based on pre-column excimer fluorescence derivatization and its application to α-lipoic acid analysis

We developed an LC method for the sensitive and selective fluorometric determination of polythiols. This method employs pre-column intramolecular excimer-forming fluorescence derivatization with N-(1-pyrene)iodoacetamide followed by LC separation. Polythiols were converted to the corresponding dipyrene-labeled derivatives, and the derivatives afforded intramolecular excimer fluorescence (440–540 nm). After the optimization using dithiothreitol and dimercaprol as model polythiols, α-lipoic acid (LA) and α-lipoamide were determined with high sensitivity and selectivity. The detection limits for polythiols were 0.6–3.5 fmol on column. Furthermore, this method could be successfully applied to the determination of LA in commercial dietary supplements and in human urine.     

Determination of methylmalonic acid in urine by HPLC with intramolecular excimer-forming fluorescence derivatization

We developed and validated an HPLC method with intramolecular excimer-forming fluorescence derivatization to determine methylmalonic acid, a unique biochemical marker for methylmalonic aciduria. Methylmalonic acid in urine and an internal standard were derivatized with pyrenebutyric hydrazide and separated on a C8 column. The derivatives were detected by monitoring the fluorescence at 475 nm (excitation wavelength 345 nm). At a signal-to-noise ratio of 3, the detection limit was 0.33 pmol on the column and the calibration curve was linear up to 1 mmol[sol ]L in urine. In a retrospective study on a relatively large number of known methylmalonic aciduria cases (n = 48), the method enabled us to differentiate methylmalonic aciduria cases from healthy controls (n = 52), regardless of age of patients at sampling or years of specimen storage. No interference was observed from isomeric or other dicarboxylic acids, or other urine constituents. As described, the method can be used retrospectively or prospectively for the diagnosis of methylmalonic aciduria and can be easily adopted by laboratories with no access to gas chromatography-mass spectrometry.     

Determination of catecholamines and indoleamines in human urine based on intramolecular excimer-forming derivatization and fluorescence detection.

A liquid chromatographic (LC) determination of catecholamines and indoleamines is described. This is based on intramolecular excimer-forming fluorescence derivatization with 4-(1-pyrene)butanoyl chloride, followed by reversed-phase LC. The analytes, containing an amino moiety and phenolic hydroxyl moieties in a molecule, were converted to the corresponding polypyrene-labeled derivatives by one-step derivatization. They afforded intramolecular excimer fluorescence, which can clearly be discriminated from the normal fluorescence emitted from reagent blanks. The detection limits (S/N = 3) for catecholamines and indoleamines were femto-mole levels per 20-microL injection. Furthermore, this method was applied to a urine assay.     

Liquid chromatographic determination of polyamines in human urine based on intramolecular excimer-forming fluorescence derivatization using 4-(1-pyrene)butanoyl chloride

A liquid chromatographic method for highly sensitive and selective fluorometric determination of polyamines (putrescine, cadaverine, spermidine and spermine) in human urine is described. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, 4-(1-pyrene)butanoyl chloride (PBC), followed by reversed-phase liquid chromatography. The method offers higher sensitivity for determination of spermidine and spermine than previously reported method utilizing 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester as a derivatization reagent. Samples containing free polyamines in diluted human urine were directly derivatized with PBC and separated on an octyl column. The derivatives were detected at excitation 345 and emission 475 nm wavelengths. For determination of total polyamine content, the conjugated polyamines were first hydrolyzed in 4 M HCl. The detection limits (signal-to-noise ratio = 3) for polyamines in urine were 1.1-3.4 pmol/mL. At optimized derivatization and chromatographic conditions, interferences such as biogenic monoamines gave no peaks or the peaks did not interfere with the peaks of polyamine derivatives. In conclusion, the present derivatization method allows direct determination of polyamines in human urine samples without the need for sample clean-up procedures.     

Liquid chromatographic determination of microcystins in water samples following pre-column excimer fluorescence derivatization with 4-(1-pyrene)butanoic acid hydrazide

A method to measure the concentrations of microcystins (MCs) in water samples has been developed by incorporating pre-column fluorescence derivatization and liquid chromatography (LC). A solid-phase extraction for pretreatment was used to extract the MCs in water samples. The MCs were derivatized with excimer-forming 4-(1-pyrene)butanoic acid hydrazide (PBH). The MCs could then be detected by fluorescence after separation with a pentafluorophenyl (PFP)-modified superficially porous (core shell) particle LC column. The derivatization reactions of MCs with PBH proceeded easily in the presence of 4,6-dimethoxy-1,3,5-triazin-2-yl-4-methylmorpholinium (DMT-MM) as a condensation reagent, and the resulting derivatives could be easily separated on the PFP column. The derivatives were selectively detected at excimer fluorescence wavelengths (440–540 nm). The instrument detection limit and the instrument quantification limit of the MCs standards were 0.4–1.2 μg L⁻¹ and 1.4–3.9 μg L⁻¹, respectively. The method was validated at 0.1 and 1.0 μg L⁻¹ levels in tap and pond water samples, and the recovery of MCs was between 67 and 101% with a relative standard deviation of 11%. The proposed method can be used to quantify trace amounts of MCs in water samples.     

Selective determination of tryptophan-containing peptides through precolumn derivatization and liquid chromatography using intramolecular fluorescence resonance energy transfer detection.

A selective and sensitive fluorometric determination method for native fluorescent peptides has been developed. This method is based on intramolecular fluorescence resonance energy transfer (FRET) detection in a liquid chromatography (LC) system following precolumn derivatization of the amino groups of tryptophan (Trp)-containing peptides. In this detection process, we monitored the FRET from the native fluorescent Trp moieties (donor) to the derivatized fluorophore (acceptor). From a screening study involving 10 fluorescent reagents, we found that o-phthalaldehyde (OPA) generated FRET most effectively. The OPA derivatives of the native fluorescent peptides emitted OPA fluorescence (445 nm) through an intramolecular FRET process when they were excited at the excitation maximum wavelength of the Trp-containing peptides (280 nm). The generation of FRET was confirmed through comparison with the analysis of a non-fluorescent peptide (C-reactive protein fragment (77 - 82)) performed using LC and a three-dimensional fluorescence detection system. We were able to separate the OPA derivatives of the Trp-containing peptides when performing LC on a reversed-phase column. The detection limits (signal-to-noise ratio = 3) for the Trp-containing peptides, at a 20-microL injection volume, were 41 - 180 fmol. The sensitivity of the intramolecular FRET-forming derivatization method is higher than that of the system that takes advantage of the conventional detection of OPA derivatives. Moreover, native non-fluorescent amines and peptides in the sample monitored at FRET detection are weaker than those of conventional fluorescence detection.     

A sensitive and selective determination method of histamine by HPLC with intramolecular excimer-forming derivatization and fluorescence detection

A highly sensitive, selective and simple method is described for the determination of histamine by high-performance liquid chromatography (HPLC) with fluorescence detection. The method is based on an intramolecular excimer-forming fluorescence derivatization of histamine with 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester (PSE), followed by reversed-phase HPLC. Histamine, having two amino moieties in a molecule, was converted to the dipyrene-labeled derivative by reaction with PSE. The derivative afforded intramolecular excimer fluorescence (450-540 nm), which can clearly be discriminated from the monomer fluorescence (370-420 nm) emitted from PSE. Typically, a 10 micro L sample solution was mixed with 100 micro L of derivatization reagent solution, which was a mixture of 0.5 mm PSE in acetonitrile and 0.5 mm potassium carbonate in water (8:2, v/v). The derivatization was carried out at 100 degrees C for 90 min. The PSE derivative of histamine could be separated by reversed-phase ODS column with isocratic elution using acetonitrile:water (82:18, v/v) containing 0.03% triethylamine. The detection limit (singnal-to-noise ratio = 3) of histamine was 0.5 fmol for a 30 micro L injection. The method was successfully applied to the determination of histamine in human urine, and had enough selectivity and sensitivity for urinary histamine quantification.     

Liquid chromatographic fluorescence determination of amino acids in plasma and urine after derivatization with phanquinone

Phanquinone (4,7-phenanthroline-5,6-dione) has been investigated as a pre-column derivatization fluorogenic reagent for liquid chromatographic determination of primary amino acids in biological samples. The derivatization reaction was carried out at 68 degrees C both in the presence of aqueous phosphate buffer (pH 8) for 30 min and without buffer for 60 min to allow the determination of basic amino acids (Orn, Lys, Arg). The resulting derivatives were separated under reversed-phase HPLC and detected at lambda(em) = 460 nm with lambda(ex) = 400 nm. The proposed method was validated and applied to the determination of a variety of amino acids directly in urine and after deproteinization with 5-sulfosalicylic acid in plasma samples. The detection and quantitation limits were found in the range 10-450 and 35-1400 fmol, respectively.     

Development of a sensitive fluorescent derivatization reagent 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC) and its application for determination of amino acids from seeds and bryophyte plants using high-performance liquid chromatography with fluorescence detection and identification with electrospray ionization mass spectrometry

A pre-column derivatization method for the sensitive determination of amino acids and peptides using the tagging reagent 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC) followed by high-performance liquid chromatography with fluorescence detection has been developed. Identification of derivatives was carried out by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS/MS). The chromophore of 2-(9-carbazole)-ethyl chloroformate (CEOC) reagent was replaced by 1,2-benzo-3,4-dihydrocarbazole functional group, which resulted in a sensitive fluorescence derivatizing reagent BCEOC. BCEOC can easily and quickly label peptides and amino acids. Derivatives are stable enough to be efficiently analyzed by high-performance liquid chromatography. The derivatives showed an intense protonated molecular ion corresponding m/z (M + H)⁺ under electrospray ionization (ESI) positive-ion mode with an exception being Tyr detected at negative mode. The collision-induced dissociation of protonated molecular ion formed a product at m/z 246.2 corresponding to the cleavage of CO bond of BCEOC molecule. Studies on derivatization demonstrate excellent derivative yields over the pH 9.0-10.0. Maximal yields close to 100% are observed with a 3-4-fold molar reagent excess. Derivatives exhibit strong fluorescence and extracted derivatization solution with n-hexane/ethyl acetate (10:1, v/v) allows for the direct injection with no significant interference from the major fluorescent reagent degradation by-products, such as 1,2-benzo-3,4-dihydrocarbazole-9-ethanol (BDC-OH) (a major by-product), mono-1,2-benzo-3,4-dihydrocarbazole-9-ethyl carbonate (BCEOC-OH) and bis-(1,2-benzo-3,4-dihydrocarbazole-9-ethyl) carbonate (BCEOC)₂. In addition, the detection responses for BCEOC derivatives are compared to those obtained with previously synthesized 2-(9-carbazole)-ethyl chloroformate (CEOC) in our laboratory. The ratios AC_BCEOC/AC_CEOC = 2.05-6.51 for fluorescence responses are observed (here, AC is relative fluorescence response). Separation of the derivatized peptides and amino acids had been optimized on Hypersil BDS C₁₈ column. Detection limits were calculated from 1.0 pmol injection at a signal-to-noise ratio of 3, and were 6.3 (Lys)-177.6 (His) fmol. The mean interday accuracy ranged from 92 to 106% for fluorescence detection with mean %CV < 7.5. The mean interday precision for all standards was <10% of the expected concentration. Excellent linear responses were observed with coefficients of >0.9999. Good compositional data could be obtained from the analysis of derivatized protein hydrolysates containing as little as 50.5 ng of sample. Therefore, the facile BCEOC derivatization coupled with mass spectrometry allowed the development of a highly sensitive and specific method for the quantitative analysis of trace levels of amino acids and peptides from biological and natural environmental samples.     

A sensitive liquid chromatographic method for the analysis of isovaleric and valeric acids in urine as fluorescent derivatives

A simple and sensitive isocratic liquid chromatographic method was developed for the analysis of isovaleric and valeric acids in human urine as biomarkers in metabolic acidosis. The method is based on the derivatization of isovaleric and valeric acids with a fluorescent reagent 2-(2-naphthoxy)ethyl-2-(piperidino)ethanesulfonate for labeling the analytes with the naphthoxy fluorophore. The resulting fluorescent derivatives of isovaleric and valeric acids were separated on a phenyl-hexyl column, using a mixed solvent of methanol-water-tetrahydrofuran (55:31:14, v/v) as the mobile phase. The separated derivatives were monitored with a fluorimetric detector (excitation at 225 nm and emission at 360 nm). The linear range of the method for the determination of isovaleric acid or valeric acid derivative was over 0.2 approximately 8.0 microM. The detection limit (signal to noise ratio=3 with 10 microl injected) of isovaleric acid or valeric acid was about 0.04 microM. Application of the method to the analysis of isovaleric acid in the urine of a patient with isovaleric acidemia proved feasible.     

血清中游离脂肪酸的液相色谱荧光测定及质谱鉴定

利用新型荧光试剂1,2 苯并 3,4 二氢咔唑 9 乙基对甲苯磺酸酯(BDETS)对19种游离脂肪酸(FFAs)进行柱前衍生,在EclipseXDB C8反相色谱柱上,采用梯度洗脱优化分离.90℃下在DMF溶剂中以K2CO3作催化剂,衍生反应30min获得稳定的荧光产物.激发和发射波长分别为λex=333nm,λem=390nm,采用大气压化学电离源(APCI)正离子模式进行柱后在线质谱定性.多数脂肪酸的线性回归系数大于0.9989,检测限为24.80～80.37fmol.实现了人体血清中长链脂肪酸的定性及相应含量测定.     

R(-)-4-(3-Isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole, a fluorescent chiral tagging reagent: sensitive resolution of chiral amines and amino acids by reversed-phase liquid chromatography

The usefulness of R(-)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole [R(-)-DBD-PyNCS], a fluorescent chiral tagging reagent, for the determination of racemic amines and amino acids, was studied. The reagent reacted with beta-blockers selected as representative secondary amines to produce corresponding fluorescent diastereomers (excitation at 460 nm and emission at 550 nm). The yields of the derivatization reaction were dependent on the stereostructure arround the NH group in beta-blockers. The resulting diastereomers were completely separated with single chromatographic run using linear gradient elutions by reversed-phase chromatography. R(-)-DBD-PyNCS was also applied to the determination of DL-amino acid, considered to be one of the primary amines, in human urine and foodstuffs. DL-amino acids tested equally reacted with the reagent, and the thiocarbamoyl derivatives were separated with an ODS column. The epimerization during the derivatization reaction was negligible judging from the resolution of opposite diastereomers on the chromatogram. The occurence of D-amino acids (D-Ala, D-Ser, D-Asp and/or D-Glu) was identified in the samples tested. The structures and the purities were elucidated with on-line HPLC-MS. The chiral reagent possessing an isothiocyanate group (-NCS) in the structure seems to be applicable to continuous sequential analysis of peptides containing D-amino acids. The thiocarbamoyl derivatives obtained from the reaction with DL-amino acids were converted to thiohydantoins via thiazolinones in acidic medium. The thiohydantoins produced from acidic, basic, neutral, hydroxyl and aromatic amino acids were completely separated with isocratic elutions using acidic mobile phase containing 0.1% TFA. The separations were sufficient for the identification of DL-amino acid in peptide sequences. Although the epimerization during the conversion reaction to thiohydantoins was not avoidable, the descrimination of D- and L-configuration was demonstrated with some commercially available peptides such as beta-lipotropin and [D-Ala2]-deltorphin II. The Edman degaradation method using R(-)-DBD-PyNCS was also adopted to autoanlaysis by gas-phase sequencer. The separation and the detection (UV 254 nm) conditions of the derivatives were used without any change from those for the Edman degradation method using PITC as the tagging reagent. The three DL-amino acid residues (Tyr, Ala and Gly) in [L-Ala2]-leucine-enkephalin and [D-Ala2]-leucine-enkephalin were perfectly identidied with the autoanalysis.     

LC-Fluorescence Detection Analysis of Amino Acids from Stellera chamaejasme L. Using 2-[2-(Dibenzocarbazole)-ethoxy] Ethyl Chloroformate as Labeling Reagent

A pre-column derivatization method for the sensitive determination of amino acids using the tagging reagent 2-[2-(dibenzocarbazole)-ethoxy] ethyl chloroformate (DBCEC) followed by liquid chromatography with fluorescence detection has been developed. Identification of DBCEC-amino acids derivatives was by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS–MS). DBCEC can easily and quickly label amino acids, and derivatives are stable enough to be efficiently analyzed by LC. Separation of the derivatized amino acids had been optimized on Hypersil BDS C₁₈ column. A perfect baseline separation for 20 amino acid derivatives was achieved with a ternary gradient elution program. The chromophore of dibenzocarbazole group, which comprise a large rigid planar structure with p–π conjugation system, resulted in a sensitive fluorescence detection for amino acid derivatives. The derivatized amino acids were detected with fluorescence detector with excitation maximum and emission maximum at 300 and 390 nm, respectively. Excellent linear responses were observed with coefficients of >0.9993, and detection limits were in the range of 0.78–5.13 fmol (signal-to-noise ratio of 3). The mean accuracy ranged from 83.4 to 98.7% for fluorescence detection. The mean inter-day precision for all standards was <4.2% of the expected concentration. Therefore, the proposed method was a highly sensitive and specific method for the quantitative analysis of amino acids from biological and natural environmental samples.     

花生油中游离脂肪酸的HPLC-FLD分析

采用柱前衍生-高效液相色谱荧光检测法(HPLC-FLD)分析了花生油中的游离脂肪酸.用荧光衍生试剂2-(11 H-苯[a]咔唑)乙基对甲苯磺酸酯(BCETS)作为柱前衍生化试剂对11种脂肪酸标准品(9种不饱和脂肪酸和棕榈酸、硬脂酸)进行衍生,经梯度洗脱实现了11种游离脂肪酸BCETS衍生物的完全分离,使用外标法定量,建...     

Analysis of organic acid markers relevant to inherited metabolic diseases by ultra-performance liquid chromatography/tandem mass spectrometry as benzofurazan derivatives

We describe a new approach applicable to the determination of organic acids that serve as diagnostic markers for several inherited metabolic disorders. We utilized liquid chromatography/tandem mass spectrometry for analysis of organic acid derivatives of a recently described benzofurazan reagent. The derivatization step was necessary to obtain organic acid derivatives suitable for analysis by reversed-phase liquid chromatography with high ionization efficiency for mass spectrometry in the positive-ion mode. In this work, a group of related dicarboxylic acid markers containing five or six carbon atoms were analyzed and validation was performed for glutaric and 3-hydroxyglutaric acids, the specific markers for glutaric acidemia type 1. Derivatization was achieved by reacting untreated urine with the derivatization reagent under mild conditions. The reaction mixture was analyzed on a C18 ultra-performance liquid chromatography (UPLC) column (50x2.1 mm, 1.7 microm) and detected in the multiple reaction monitoring mode in 5 min. Calibration curves were linear up to at least 1000 microM with detection limits for glutaric and 3-hydroxyglutaric acids of 0.025 and 0.02 microM, respectively (signal-to-noise ratio of 3). Intra-day (n=11) and inter-day (n=6) coefficients of variation were better than 11.2%. The assay was successfully applied to control (n=134) and glutaric acidemia type 1 (n=55) urine samples.     

Targeted LC–MS derivatization for aldehydes and carboxylic acids with a new derivatization agent 4-APEBA

Based on the template of a recently introduced derivatization reagent for aldehydes, 4-(2-(trimethylammonio)ethoxy)benzeneaminium dibromide (4-APC), a new derivatization agent was designed with additional features for the analysis and screening of biomarkers of lipid peroxidation. The new derivatization reagent, 4-(2-((4-bromophenethyl)dimethylammonio)ethoxy)benzenaminium dibromide (4-APEBA) contains a bromophenethyl group to incorporate an isotopic signature to the derivatives and to add additional fragmentation identifiers, collectively enhancing the abilities for detection and screening of unknown aldehydes. Derivatization can be achieved under mild conditions (pH 5.7, 10 °C). By changing the secondary reagent (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide instead of sodium cyanoborohydride), 4-APEBA is also applicable to the selective derivatization of carboxylic acids. Synthesis of the new label, exploration of the derivatization conditions, characterization of the fragmentation of the aldehyde and carboxylic acid derivatives in MS/MS, and preliminary applications of the labeling strategy for the analysis of aldehydes in urine and plasma are described.     

1,3,5,7-Tetramethyl-8-aminozide-difluoroboradiaza-s-indacene as a new fluorescent labeling reagent for the determination of aliphatic aldehydes in serum with high performance liquid chromatography

A BODIPY-based fluorescent derivatization reagent with a hydrazine moiety, 1,3,5,7-tetramethyl-8-aminozide-difluoroboradiaza-s-indacene (BODIPY-aminozide), has been designed for aldehyde labeling. An increased fluorescence quantum yield was observed from 0.38 to 0.94 in acetonitrile when it reacted with aldehydes. Twelve aliphatic aldehydes from formaldehyde to lauraldehyde were used to evaluate the analytical potential of this reagent by high performance liquid chromatography (HPLC) on C₁₈ column with fluorescence detection. The derivatization reaction of BODIPY-aminozide with aldehydes proceeded at 60 °C for 30 min to form stable corresponding BODIPY hydrazone derivatives in the presence of phosphoric acid as a catalyst. The maximum excitation (495 nm) and emission (505 nm) wavelengths were almost the same for all the aldehyde derivatives. A baseline separation of all the 12 aliphatic aldehydes (except formaldehyde and acetaldehyde) is achieved in 20 min with acetonitrile–tetrahydrofuran (THF)–water as mobile phase. The detection limits were obtained in the range from 0.43 to 0.69 nM (signal-to-noise = 3), which are better than or comparable with those obtained by the existing methods based on aldehyde labeling. This reagent has been applied to the precolumn derivatization followed with HPLC determination of trace aliphatic aldehydes in human serum samples without complex pretreatment or enrichment method.