Derivatization Reagent For Carboxylic Acids Research Articles

Profiling of chiral and achiral carboxylic acid metabolomics: synthesis and evaluation of triazine-type chiral derivatization reagents for carboxylic acids by LC-ESI-MS/MS and the application to saliva of healthy volunteers and diabetic patients.

Novel triazine-type chiral derivatization reagents, i.e., (S)-1-(4,6-dimethoxy-1,3,5-triazin-2-yl)pyrrolidin-3-amine (DMT-3(S)-Apy) and (S)-4,6-dimethoxy-N-(pyrrolidin-3-yl)-1,3,5-triazin-2-amine (DMT-1(S)-Apy), were developed for the highly sensitive and selective detection of chiral carboxylic acids by UPLC-MS/MS analysis. Among the synthesized reagents, DMT-3(S)-Apy was a more efficient chiral reagent for the enantiomeric separation of chiral carboxylic acids in terms of separation efficiency by reversed-phase chromatography and detection sensitivity by ESI-MS/MS. The DMT-3(S)-Apy was used for the determination of 13 carboxylic acids in human saliva of healthy volunteers and diabetic patients. Various biological carboxylic acids including chiral carboxylic acids, and mono- and di-carboxylic acids were clearly identified in the saliva of healthy persons and diabetic patients. The concentrations of carboxylic acids detected in the saliva of diabetic patients were relatively higher than those in the healthy persons. Furthermore, the concentration of D-lactic acid (LA) and the ratio of D/L-LA in the diabetic patients were significantly higher than those in the healthy persons. The low ratio of D/L-LA in healthy persons was also identified to be independent of age and sex. These results suggest that the determination of the D/L-LA ratio in saliva might be applicable for the diagnosis of diabetes. Based on these observations, DMT-3(S)-Apy seems to be a useful chiral derivatization reagent for the determination not only of chiral carboxylic acids but also achiral ones. In conclusion, the proposed method using DMT-3(S)-Apy is useful for the carboxylic acid metabolomics study of various specimens.

Compounds having thiourea moiety as derivatization reagents in liquid chromatography/electrospray ionization–tandem mass spectrometry (LC/ESI‐MS/MS): synthesis of derivatization reagents for carboxylic acids

The derivatization reagents for carboxylic acids, N-(Pyridin-3-yl)hydrazinecarbothioamide, N-[4-(dimethylamino)phenyl]hydrazinecarbothioamide, 1-(2-aminoethyl)-3-(pyridin-3-yl)thiourea, 1-(2-aminoethyl)-3-[4-(dimethylamino)phenyl]thiourea and 4-(2-aminoethyl)-N-phenylpiperazine-1-carbothioamide were synthesized. These reagents reacted with carboxylic acids at 60°C for 45 min in the presence of the condensation reagents. The generated derivatives were favorably separated on the reversed-phase column and sensitively detected by electrospray ionization tandem mass spectrometry. These reagents enhanced the electrospray ionization response of the analyte and generated a particular product ion efficiently by collision-induced dissociation, and thus they were suitable for MS/MS detection.

Rapid UPLC-MS/MS method for routine analysis of plasma pristanic, phytanic, and very long chain fatty acid markers of peroxisomal disorders

Quantification of pristanic acid, phytanic acid, and very long chain fatty acids (i.e., hexacosanoic, tetracosanoic, and docosanoic acids) in plasma is the primary method for investigateing a multitude of peroxisomal disorders (PDs). Typically based on GC-MS, existing methods are time-consuming and laborious. In this paper, we present a rapid and specific liquid chromatography tandem mass spectrometric method based on derivatization with 4-[2-(N,N-dimethylamino)ethylaminosulfonyl]-7-(2-aminoethylamino)-2,1,3-benzoxadiazole (DAABD-AE). Derivatization was undertaken to improve the poor mass spectrometric properties of these fatty acids. Analytes in plasma (20 mul) were hydrolyzed, extracted, and derivatized with DAABD-AE in approximately 2 h. Derivatives were separated on a reverse-phase column and detected by positive-ion electrospray ionization tandem mass spectrometry with a 5 min injection-to-injection time. Calibration plots were linear over ranges that cover physiological and pathological concentrations. Intraday (n = 12) and interday (n = 10) variations at low and high concentrations were less than 9.2%. Reference intervals in normal plasma (n = 250) were established for each compound and were in agreement with the literature. Using specimens from patients with established diagnosis (n = 20), various PDs were reliably detected. In conclusion, this method allows for the detection of at least nine PDs in a 5 min analytical run. Furthermore, this derivatization approach is potentially applicable to other disease markers carrying the carboxylic group.

6‐Oxy‐(acetyl ethylenediamine) fluorescein, a novel fluorescent derivatization reagent for carboxylic acids and its application in HPLC

A novel fluorescent derivatization reagent for carboxylic acids, 6-oxy-(acetyl ethylenediamine) fluorescein (AEF), was well designed, synthesized, and applied to HPLC. The derivatization reaction with 12 fatty acids, including n-valeric acid (C5), n-hexanoic acid (C6), n-heptanoic acid (C7), n-octanoic acid (C8), n-nonanoic acid (C9), n-decanoic acid (C10), lauric acid (C12), myristic acid (C14), palmitic acid (C16), stearic acid (C18), oleic acid (C18:1), and linoleic acid (C18:2), was completed at 55 degrees C within 40 min. The derivatives of fatty acids were separated on a C18 RP column and detected by fluorescence detection. The LODs attained were 0.4-1.2 nM (S/N of 3). It has been demonstrated that AEF is a prominent derivatization reagent for carboxylic acids which is suitable for HPLC.

Design and synthesis of a novel fluorescent reagent, 6-oxy-(ethylpiperazine)-9-(2′-methoxycarbonyl) fluorescein, for carboxylic acids and its application in food samples using high-performance liquid chromatography

A novel derivatization reagent for carboxylic acids, 6-oxy-(ethylpiperazine)-9-(2′-methoxycarbonyl)fluorescein (EPMF) has been well designed and synthesized. It was used to label 13 fatty acids, n-butyric acid (C4), n-valeric acid (C5), n-hexanoic acid (C6), n-heptanoic acid (C7), n-octanoic acid (C8), n-nonanoic acid (C9), n-decanoic acid (C10), lauric acid (C12), myristic acid (C14), palmitic acid (C16), stearic acid (C18), oleic acid (C18:1) and linoleic acid (C18:2), successfully. The derivatization reaction with fatty acids was completed at 50 °C, 50 min. The derivatives of fatty acids were separated on a C18 reversed-phase column with gradient elution and fluorescence detection at λ ex/ λ em = 469/518 nm. The detection limits obtained were 0.4–2 nmol L −1 (signal-to-noise ratio of 3). The proposed method has been applied to the quantification of fatty acids in edible oil with recoveries over 93%. It has been demonstrated that EPMF is a prominent derivatization reagent for fatty acids and is suitable for HPLC.

Synthesis of benzofurazan derivatization reagents for carboxylic acids in liquid chromatography/electrospray ionization–tandem mass spectrometry

The applicability of benzofurazan derivatization regents to carboxylic acids analysis in LC/ESI-MS/MS (high-performance liquid chromatography/electrospray ionization tandem mass spectrometry) was examined. The product ion spectra of DAABD-AE {4-[2-(N,N-dimethylamino)ethylaminosulfonyl]-7-(2-aminoethylamino)-2,1,3-benzoxadiazole}, DAABD-PZ {4-[2-(N,N-dimethylamino)ethylaminosulfonyl]-7-N-piperazino-2,1,3-benzoxadiazole}, DAABD-PiCZ {4-[4-carbazoylpiperidin-1-yl]-7-[2-(N,N-dimethylamino)ethylaminosulfonyl]-2,1,3-benzoxadiazole}, DAABD-ProCZ {4-[2-carbazoylpyrrolidin-1-yl]-7-[2-(N,N-dimethylamino) ethylaminosulfonyl]-2,1,3-benzoxadiazole} and DAABD-Apy {4-[2-(N,N-dimethylamino)ethylaminosulfonyl]-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole}, and their acetylated compounds were obtained. An intense fragment ion at m/z 151 corresponding to (dimethylamino)ethylaminosulfonyl moiety was observed in each spectra, suggesting that these reagents were suitable for ESI-MS/MS analysis. DAABD-AE, DAABD-APy and DAABD-PZ were applied to the analysis of octanoic acid and it was found that DAABD-AE and DAABD-APy gave high signal intensity suitable for LC/ESI-MS/MS.

Rapid measurement of deuterium-labeled long-chain fatty acids in plasma by HPLC-ESI-MS

Imbalanced fatty acid metabolism contributes significantly to the increased incidence of metabolic disorders. Isotope-labeled fatty acids (2H, 13C) provide efficient means to trace fatty acid metabolism in vivo. This study reports a new and rapid method for the quantification of deuterium-labeled fatty acids in plasma by HPLC-MS. The sample preparation protocol developed required only hydrolysis, neutralization, and quenching steps followed by high-performance liquid chromatography-electrospray ionization-mass spectrometry analysis in negative ion mode using single ion monitoring. Deuterium-labeled stearic acid (d7-C18:0) was synthesized to reduce matrix interference observed with d5 analog, which improved the limit of detection (LOD) significantly, depending on the products analyzed. Linearity > 0.999 between the LOD (100 nM) and 30 microM, accuracy > 90%, precision > 88%, and adequate recovery in the dynamic range were obtained for d7-C18:0 and d7-oleic acid (C18:1). Upon oral dosing of d7-C18:0 in rats, the parent compound and its desaturation and beta-oxidation products, d7-C18:1 and d7-C16:0, were circulating with a maximal concentration ranging from 0.6 to 2.2 microM, with significant levels of d7-fatty acids detected for up to 72 h.

Improvement of Fluorescence Characteristics of Coumarins: Syntheses and Fluorescence Properties of 6‐Methoxycoumarin and Benzocoumarin Derivatives as Novel Fluorophores Emitting in the Longer Wavelength Region and Their Application to Analytical Reagents.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Synthesis of the isotope‐labeled derivatization reagent for carboxylic acids, 7‐(N,N‐dimethylaminosulfonyl)‐4‐(aminoethyl)piperazino‐2,1,3‐benzoxadiazole (d6) [DBD‐PZ‐NH2 (D)], and its application to the quantification and the determination of relative amount of fatty acids in rat plasma samples by high‐performance liquid chromatography/mass spectrometry

The isotope-labeled benzofurazan derivatization reagent for carboxylic acids, 7-(N,N-dimethylaminosulfonyl)-4-(aminoethyl)piperazino-2,1,3-benzoxadiazole (d6) [DBD-PZ-NH2 (D)] was synthesized. DBD-PZ-NH2 (D) was used for the accurate quantification of fatty acids by liquid chromatography/mass spectrometry (LC/MS). The standard fatty acids were derivatized with DBD-PZ-NH2 (D) to the stable isotope-labeled compounds for the fatty acids derivatives of DBD-PZ-NH2 and used for the internal standards. The obtained calibration curves for fatty acids were linear over the range 0.1-200 microM (r2 > 0.999). Fatty acids in plasma samples were determined after derivatization with DBD-PZ-NH2 and analyzed by LC/MS using standard fatty acid DBD-PZ-NH2 (D) derivatives as internal standards. Furthermore, the relative amounts of fatty acids in two plasma samples were determined after derivatization with DBD-PZ-NH2 and DBD-PZ-NH2) (D). The isotope-labeled derivatization reagent was useful for accurate quantification and the determination of relative amounts of the metabolites in biological samples having the target functional group.

Synthesis of benzofurazan derivatization reagents for carboxylic acids and its application to analysis of fatty acids in rat plasma by high-performance liquid chromatography-electrospray ionization mass spectrometry

The derivatization regents for carboxylic acids, DAABD-AE (4-[2-(N,N-dimethylamino)ethylaminosulfonyl]7-(2-aminoethylamino)-2,1,3-benzoxadiazole), MePZBD-AE ([4-(4-N-methyl)piperazinosulfonyl]-7-(2-aminoethylamino)-2,1,3-benzoxadiazole) and APZBD-NHMe ([4-(4-N-aminoethyl)piperazinosulfonyl]-7-methylamino-2,1,3-benzoxadiazole) were developed for electrospray ionization-mass spectrometry (ESI-MS). The derivatization reaction with fatty acids was completed at 60 degrees C within 30 min. The derivatives of fatty acids were separated on a reversed-phase column and detected with ESI-MS. The detection limits attained for fatty acids were femtomol range and the calibration curves were linear over the range from 0.1 to 100 pmol (r2 > 0.992) for DAABD-AE and MePZBD-AE. DAABD-NHMe was applied to the analysis of fatty acids in rat plasma samples.

Improvement of Fluorescence Characteristics of Coumarins: Syntheses and Fluorescence Properties of 6-Methoxycoumarin and Benzocoumarin Derivatives as Novel Fluorophores Emitting in the Longer Wavelength Region and Their Application to Analytical Reagents

To improve the fluorescence characteristics, especially emission wavelength, of coumarins, various 3-substituted-6-methoxycoumarin derivatives were synthesized, and then benzocoumarin derivatives were also synthesized in expectation of the shift to the longer wavelength region by the extension of the conjugated system. Their fluorescence properties were investigated spectrophotometrically in acetonitrile and evaluated from the viewpoint of the intramolecular charge transfer (ICT) between push- and pull-substituents in the ground and the excited states. Among them, benzocoumarin derivatives especially fluoresced in the longer wavelength around 540 nm with remarkably large Stokes shifts beyond 10,000 cm(-1). Using such fluorophores, some novel fluorescence derivatization reagents for carboxylic acids, alcohols, phenols, and amines were preliminarily prepared as an example, and their derivatized products were also found to fluoresce in the longer wavelength region with large Stokes shifts.

HPLC analysis of aliphatic and aromatic dicarboxylic acid cross-linkers hydrolyzed from carbohydrate polyesters for estimation of the molar degree of substitution

Quantitative analysis of carboxylic acids in hydrophilic matrixes remains a challenge despite the introduction of derivatization reagents for carboxylic acids nearly three decades ago. Analysis of many bifunctional ester forming polymer cross-linkers are additionally complicated by the their amphiphatic character. We report a sensitive and selective HPLC method to quantify organic dicarboxylic acids resulting from the hydrolytic degradation of cross-linked carbohydrate polyesters. An important group of cross-linkers encompassing C 7–C 10 aromatic and non-chromophoric aliphatic diacid chlorides were investigated. Specific validation is reported for terephthalic acid, diethylmalonic acid and sebacic acid. Linear calibration curves were obtained in the 0.5–15.0 μg/ml range corresponding to a mass fraction of 0.05–1.5% diacid to polyester. The accuracy and precision in this range was 94.4–114.1 and 6.1–24.8% RSD, respectively. This method is suitable for the routine determination of the molar or mass degree of substitution in carbohydrate polyesters.

A study of the relationship between the chemical structures and the fluorescence quantum yields of coumarins, quinoxalinones and benzoxazinones for the development of sensitive fluorescent derivatization reagents.

To develop new fluorescent derivatization reagents, we investigated the relationship between the chemical structures and the fluorescence quantum yields (phi(f)) of coumarins, quinoxalinones and benzoxadinones. Forty-six compounds were synthesized and their fluorescence spectra were measured in n-hexane, ethyl acetate, methanol and water. The energy levels of these compounds were calculated by combination of the semi-empirical AM1 and INDO/S (CI = all) methods. The deltaE(Tn(n,pi*), S1(pi,pi*)) (the energy gap between the Tn(n,pi*) and S1(pi,pi*) states) values were well correlated with the phi(f) values, which enables us to predict the phi(f) values from their chemical structures. Based on this relationship, 3-phenyl-7-N-piperazinoquinoxalin-2(1H)-one (PQ-Pz) and 7-(3-(S)-aminopyrrolidin-1-yl)-3-phenylquinoxalin-2-(1H)-one (PQ-APy) were developed as fluorescent derivatization reagents for carboxylic acids. The derivatives of the carboxylic acids with PQ-Pz and PQ-APy showed large phi(f) values even in polar solvents, suggesting that these reagents are suitable for the microanalysis of biologically important carboxylic acids by reversed phase HPLC.

Design and synthesis of a hydrophilic fluorescent derivatization reagent for carboxylic acids, 4-N-(4-N-aminoethyl)piperazino-7-nitro-2,1,3-benzoxadiazole (NBD-PZ-NH2), and its application to capillary electrophoresis with laser-induced fluorescence detection.

A hydrophilic fluorescent derivatization reagent for fatty acids, 4-N-(4-N-aminoethyl)piperazino-7-nitro-2,1,3-benzoxadiazole (NBD-PZ-NH(2)), was designed and synthesized. NBD-PZ-NH(2) possesses not only a fluorophore and a reacting group but also a positive charge group and, thus, was hydrophilic and suitable for application to capillary electrophoresis. NBD-PZ-NH(2) reacted with fatty acids in the presence of triphenylphosphine (TPP) and 2,2'-dipyridyl disulfide (DPDS) at room temperature within 10 min. The derivatives were strongly fluoresced and were positively charged at pH below 3. The derivatives of C4-C20 fatty acids were separated within 10 min in 50% acetonitrile in water containing 30 mM ammonium acetate and 1.0 M acetic acid by capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection. The detection limits attained were 6.5 nM (signal-to-noise ratio of 3). It is proposed that NBD-PZ-NH(2) is a prominent derivatization reagent for fatty acids which is suitable for CE-LIF application.

Electrogenerated chemiluminescence derivatization reagents for carboxylic acids and amines in high-performance liquid chromatography using tris(2,2'-bipyridine)ruthenium(II).

2-(2-Aminoethyl)-1-methylpyrrolidine and N-(3-aminopropyl)pyrrolidine (NAPP) were found to be selective and sensitive derivatization reagents for carboxylic acid by high-performance liquid chromatography (HPLC) with electrogenerated chemiluminescence detection using tris(2,2'-bipyridine)ruthenium(II). Free fatty acids and ibuprofen were used as model compounds of carboxylic acids, and the derivatization conditions were optimized with myristic acid as a representative of free fatty acids. All the fatty acids tested were reacted with NAPP to produce highly sensitive derivatives under the mild reaction conditions of room temperature for 30 min in acetonitrile containing 2-bromo-1-ethylpyridinium tetrafluoroborate and 9-methyl-3,4-dihydro-2H-pyrido[1,2-a]pyrimidin-2-one. The chemiluminescence intensities were similar for all fatty acids. The derivatives obtained from 10 free fatty acids were completely separated by reversed-phase chromatography under isocratic elution conditions. The on-column detection limits (signal-to-noise ratio of 3) with proposed HPLC separation and chemiluminescence detection were 70 and 45 fmol for myristic acid and ibuprofen, respectively. The free fatty acids in human plasma were successfully determined using the present method. Histamine, a model compound of primary amines, was also determined after precolumn derivatization with 3-(diethylamino)propionic acid at room temperature for 60 min in acetonitrile containing N,N'-dicyclohexylcarbodiimide and 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine with the detection limit of 70 fmol.

6,7-Methylenedioxy-1-methyl-2-oxo-1,2-dihydroquinoxalin-3-ylpropionohydrazide as a Highly Sensitive Fluorescence Derivatization Reagent for Carboxylic Acids in High-Performance Liquid Chromatography

6,7-Methylenedioxy-1-methyl-2-oxo-1,2-dihydroquinoxalin-3-ylpropionohydrazide as a Highly Sensitive Fluorescence Derivatization Reagent for Carboxylic Acids in High-Performance Liquid Chromatography

N-(4-nitro-2,1,3-benzoxadiazoyl-7-yl)- N-methyl-2-aminoacetohydrazide (NBD-CO-Hz) as a precolumn fluorescent derivatization reagent for carboxylic acids in high-performance liquid chromatography

A new fluorescent reagent for carboxylic acids, N-(4-nitro-2,1,3-benzoxadiazoyl-7-yl)- N-methyl-2-aminoacetohydrazide (NBD-CO-Hz) was synthesized and its applicability as a precolumn derivatization reagent in high-performance liquid chromatography was examined. NBD-CO-Hz reacted with 2-arylpropionic acids (2-APAs), a group of non-steroidal antiinflammatory drugs (NSAIDs) in the presence of a condensing agent, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and pyridine at room temperature for 2 h to give fluorescent adducts. The reaction solution was subjected to a reversed phase or a chiral stationary phase HPLC and the derivatives were detected fluorometrically at a wavelength of 530 nm with an exitation of 475 nm. The detection limits were in the fmol range on column.

4- N, N-Dimethylaminosulfonyl-7- N-(2-aminoethyl)amino-benzofurazan as a new precolumn fluorescence derivatization reagent for carboxylic acids (fatty acids and drugs containing a carboxyl moiety) in liquid chromatography

A new fluorescent reagent for carboxylic acids, 4- N, N-dimethylaminosulfonyl-7- N-(2-aminoethyl) amino-2, l,3-benzoxadiazole (DBD-ED) was synthesized from 4- N, N-dimethylaminosulfonyl-7-chloro-2,1,3-benzoxadiazole (DBD-Cl) and 1,2-diaminoethane. DBD-Cl is also a new compound described in this work for the first time. The applicability of DBD-ED as a precolumn derivatization reagent in liquid chromatography (LC) was examined. DBD-ED reacted with saturated fatty acids and non-steroidal anti-inflammatory drugs (NSAIDs) in the presence of triphenylphosphine and 2,2′-dipyridyl disulfide at room temperature for 30 min for NSAIDs and 1 h for fatty acids to give fluorescent products. The reaction solutions were subjected to LC and the derivatized products were detected spectrofluorimetrically at 560 nm with excitation at 450 nm. The detection limits were in the fmol range

Highly Sensitive Isomeric Determination of Beraprost Sodium in Plasma Using a Fluorescent Chiral Derivatization Reagent.

(S)-(+)-2-(Anthracene-2,3dicarboximido)-1-propyl trifluoromethanesulfonate (AP-OTf) was synthesized as a chiral fluorescence derivatization reagent for carboxylic acids. Beraprost sodium (BPS), which is composed of four optical isomers, was derivatized with this reagent, and the derivatives were separated into each isomer by an HPLC using a silica gel column. This method was applied to determine BPS in human plasma. By the method, the determination range of the four isomers of BPS were 6 - 3000 fmol (0.5 -250 ng/ml) and their detection limits were 3-4 fmol (1.3-1.7 pg, signal-to-noise ratio =3). This method made it possible to detect 0.25-0.33 ng/ml of each isomer of BPS in plasma.

4-(N-hydrazinoformylmethyl-N-methyl)-amino-7-N, N-dimethylaminosulphonyl-2,1,3-benzoxadiazole (DBD-CO-Hz) as a precolumn fluorescence derivatization reagent for carboxylic acids in high-performance liquid chromatography.

A new fluorescent reagent for carboxylic acids, 4-(N-hydrazinoformylmethyl-N-methyl)amino-7-N,N-dimethylaminosu lphonyl-2, 1,3-benzoxadiazole (DBD-CO-Hz) was synthesized and its applicability as a precolumn derivatization reagent in high-performance liquid chromatography (HPLC) was examined. DBD-CO-Hz reacted with saturated fatty acids in the presence of a condensing agent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and pyridine at room temperature for 30 min to give fluorescent products. The reaction solution was subjected to a reversed-phase HPLC and detected fluorometrically at a wavelength of 550 nm with an excitation of 440 nm. The detection limits of the derivatives were at low fmol range on column.