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We developed a highly sensitive and accurate differential method for analyzing chiral and achiral carboxylic acids in Japanese commercial beers, using ultra high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS), based on chiral derivatization combining three isotopic chiral reagents: 12 C-DMT-3(S)-Apy, 13 C 2 -DMT-3(S)-Apy, and d 6 -DMT-3(S)-Apy. By combining these reaction solutions and analyzing the resulting mixture, simultaneous comparative analyses of the three samples could be achieved while offsetting the matrix effects on the samples. Using this approach, it was possible to differentiate the beers according to the type (draft, low-malt, and non-alcoholic), manufacturer, and storage conditions (temperature and storage period), based on the concentrations of carboxylic acids in the beers. Furthermore, we identified α-ketoglutaric acid as a new marker for determining the storage condition of Japanese beers. The proposed method would provide insights into the identification of markers in various fields, such as in biological samples, as well as food and beverages., (Copyright © 2019 Elsevier B.V. All rights reserved.)

We propose a chiral metabolomics approach based on a data-dependent MS/MS analysis (DDA) using high-resolution quadrupole-time-of-flight mass spectrometry (Q-TOFMS) and 13 C-isotope coded derivatization (ICD) reagents, i.e., iDMT-( S)-A and iDMT-( S)-PO. The advantage of the method is the correction of all detected derivatives by parallel derivatization of the isotope-coded and noncoded reagents. The automatic data analysis platform using an MSDIAL and ICD discrimination program, called DINA, was also developed and used for the data analysis process. As a result, a 0.5-2.0% (d-/l-isomer) variation of the isomers was correctly recognized in the automatic data analysis step. Both the semiquantitative comparison and identification efficiency were improved as a result of the high resolution/accuracy of the MS and MS/MS spectra derived from the DDA analysis. This method was used for biomarker discovery in the cerebrospinal fluid (CSF) of patients with Alzheimer's disease (AD). Twenty-four biomarker candidates were successfully determined, including 8 chiral ones.

Liquid chromatography coupled with mass spectrometry (LC-MS) is one of the most prominent analytical techniques due to its inherent selectivity and sensitivity. LC-MS is currently the first choice for high-throughput bioanalysis due to the advancements in MS instruments and the analytical software. Based on this situation, we are developing various types of derivatization reagents, including chiral reagents for MS and/or MS/MS detection. These developed reagents are adopted for the detection of biomarker candidates related to diseases. The biomarker candidates include not only achiral molecules, but also chiral ones. Although determining the already-identified chiral molecules is relative easy, it is very difficult to identify and/or determine unknown enantiomer(s) in real samples. To solve this difficulty, we proposed a new strategy to identify unknown enantiomeric biomarkers related to diseases. This review paper deals with the development of derivatization reagents for amines and carboxylic acids in LC-MS analysis and their application to bioanalysis.

This paper describes the preparation of new dress-up columns featuring reproducibly removable and replaceable chiral stationary phases. After synthesizing perfluroalkylated quinine and quinidine derivatives as chiral stationary phase compounds (F-CSPs), we adsorbed them reversibly onto a fluorous LC column through pumping of their solutions. Using this dress-up chiral column and fluorophobic elution of aqueous ammonium formate/MeOH mixtures, we could enantioseparate four racemic N-acetyl amino acids, dichlorprop, and sixteen fluorescent 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC)-derivatized amino acids. Dressing and undressing of the coated F-CSPs could be controlled by varying the fluorophilicity and fluorophobicity of the eluent. The relative standard deviations of the retention times, the retention factors, the number of theoretical plates, the enantioseparation factors, and the resolutions of each of four preparations of such dress-up columns were all less than or equal to 5.26% (from 20 repeated analyses); the reproducibilities from four different preparations were all less than or equal to 10.6%. These columns also facilitated highly sensitive and selective analyses of AQC-amino acids when detected using LC-MS/MS., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A sensitive, versatile, and reproducible automatic analyzer for highly polar carboxylic acids based on a fluorescence derivatization-liquid chromatography (LC) method was developed. In this method, carboxylic acids were automatically and fluorescently derivatized with 4-(N,N-dimethylaminosulfonyl)-7-piperazino-2,1,3-benzoxadiazole (DBD-PZ) in the presence of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride by adopting a pretreatment program installed in an LC autosampler. All of the DBD-PZ-carboxylic acid derivatives were separated on the ODS column within 30 min by gradient elution. The peak of DBD-PZ did not interfere with the separation and the quantification of all the acids with the exception of lactic acid. From the LC-MS/MS analysis, we confirmed that lactic acid was converted to an oxytriazinyl derivative, which was further modified with a dimethoxy triazine group of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM). We detected this oxytriazinyl derivative to quantify lactic acid. The detection limits (signal-to-noise ratio = 3) for the examined acids ranged from 0.19 to 1.1 µm, which correspond to 95-550 fmol per injection. The intra- and inter-day precisions of typical, highly polar carboxylic acids were all <9.0%. The developed method was successfully applied to the comprehensive analysis of carboxylic acids in various samples, which included fruit juices, red wine and media from cultured tumor cells., (Copyright © 2014 John Wiley & Sons, Ltd.)

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.

Mass spectrometry has become a popular analytical tool because of its high sensitivity and specificity. The use of a chiral derivatization reagent for the mass spectrometry (MS) detection seems to be efficient for the enantiomeric separation of racemates. However, the number of chiral reagents for the liquid chromatography (LC)-MS/MS analysis is very limited. According to these observations, we are currently in the process of developing novel labeling reagents for chiral molecules in MS/MS analysis. The derivatization reagent that is effective for enhancing not only the electrospray ionization-MS/MS sensitivity but also the reversed-phase LC resolution of carboxylic acid enantiomers should have a highly proton-affinitive moiety and an asymmetric structure near the reactive functional group. Furthermore, the resulting derivative has to provide a characteristic product ion suitable for the selected reaction monitoring. Based upon these considerations, a series of prolylamidepyridines ((S)-N-pyrrolidine-2-carboxylic acid N-(pyridine-2-yl)amide (PCP2), (S)-N-pyrrolidine-2-carboxylic acid N-(pyridine-3-yl)amide, and (S)-N-pyrrolidine-2-carboxylic acid N-(pyridine-4-yl)amide) was synthesized as ideal labeling reagents for the enantioseparation of chiral carboxylic acids and evaluated in terms of separation efficiency and detection sensitivity by ultra-performance LC (UPLC)-MS/MS. Among the synthesized reagents, PCP2 was the most efficient chiral derivatization reagent for the enantioseparation of carboxylic acid. The Rs values and the detection limits of the derivatives of non-steroidal anti-inflammatory drugs, which were selected as the representative carboxylic acids, were in the range of 2.52-6.07 and 49-260 amol, respectively. The sensitive detection of biological carboxylic acids (detection limits, 32-520 amol) was also carried out by the proposed method using PCP2 and UPLC-MS/MS. The PCP2 was applied to the determination of carboxylic acids in human saliva. Several biological carboxylic acids, such as lactic acid (LA), 3-hydroxybutylic acid, maric acid, succinic acid, α-ketoglutalic acid, and citric acid, were clearly identified in the saliva of healthy persons and diabetic patients. Furthermore, the ratio of D-LA in diabetic patients was higher than that in normal subjects. Judging from these results, PCP2 seems to be a useful chiral derivatization reagent for the determination not only of chiral, but also achiral, carboxylic acids in real samples.

This paper reports the synthesis and the application of novel derivatization reagents possessing a pyridylthiourea structure for the enantiospecific determination of chiral amines and carboxylic acids in high-throughput LC-ESI-MS/MS. The novel reagents, i.e., (R)-N-(3-pyridylthiocarbamoyl)pyrrolidine-2-carboxylic acid (PyT-C) and (S)-3-amino-1-(3-pyridylthiocarbamoyl)pyrrolidine (PyT-N), were evaluated as chiral derivatization reagents for the enantiomeric determination of chiral amines and carboxylic acids, respectively, in terms of separation efficiency by reversed-phase chromatography and detection sensitivity by ESI-MS/MS. The chiral amines and carboxylic acids were easily labeled with PyT-C and PyT-N, respectively, at 60°C in 60min in the presence of 2,2'-dipyridyl disulfide (DPDS) and triphenylphosphine (TPP) as the activation reagents. The resulting diastereomers were completely separated by reversed-phase chromatography using a small particle (1.7μm) ODS column (Rs=3.54-6.00 for carboxylic acids and Rs=3.07-4.75 for amines). A highly sensitive detection at the sub-fmol level was also obtained from the SRM chromatograms at a single monitoring ion, m/z 137.0 (0.72-1.46fmol for carboxylic acids and 0.55-1.89fmol for amines). The proposed procedure using PyT-C and PyT-N was applied to the determination of chiral amines and carboxylic acids spiked into human saliva, as a model study of chiral metabonomics identification. dl-Amino acid methyl esters and N-acetyl dl-amino acids, which are the representatives as the chiral amines and carboxylic acids, in the saliva were clearly identified by the present method. Because the same product ion at m/z 137.0 was obtained from collision-induced dissociation (CID) of protonated molecular ions of all the derivatives, the proposed procedure using both reagents (i.e., PyT-C and PyT-N) seems to be useful for chiral metabolomics identification having selected functional groups (i.e., amines and carboxylic acids)., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A novel bromoquinolinium reagent, i.e. 1-(3-aminopropyl)-3-bromoquinolinium bromide (APBQ), was synthesized for the analysis of carboxylic acids. A simple and practical precolumn derivatization procedure using the APBQ in RP chromatography and MS (HPLC-MS) has been developed using bile acids and free fatty acids, as the representative carboxylic acids in biological samples. The APBQ efficiently reacted with carboxylic acids at 60°C for 60 min in the presence of N,N-dicyclohexylcarbodiimide and pyridine as the activation reagents. Because the APBQ possesses a bromine atom in the structure, the identification of a series of carboxylic acids was easily achieved due to the characteristic bromine isotope pattern in the mass spectra. The APBQ also has a quaternary amine structure, thus the positively charged derivatives are predominate for the highly sensitive detection of carboxylic acids. The APBQ was successfully applied to the selective determination of biological carboxylic acids in human plasma. The bile acids (chenodeoxycholic acid and deoxycholic acid) and several saturated (stearic acid and palmitic acid) and unsaturated free fatty acids (oleic acid and linoleic acid) were reasonably determined by HPLC-MS under the proposed procedure. Based on the results of analyses of human plasma and saliva, the proposed procedure using APBQ seems to be applicable for the qualitative and quantitative analyses of a series of carboxylic acids in biological samples., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A simple and practical derivatization procedure for increasing the detectability and enantiomeric separation of chiral carboxylic acids in LC/ESI-MS/MS has been developed. (S)-Anabasine (ANA) was used as the derivatization reagent and rapidly reacted with carboxylic acids [3-hydroxypalmitic acid (3-OH-PA), 2-(β-carboxyethyl)-6-hydroxy-2,7,8-trimethylchroman (γ-CEHC), and etodolac] in the presence of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholium chloride. The resulting ANA-derivatives were highly responsive in ESI-MS operating in the positive-ion mode and gave characteristic product ions during MS/MS, which enabled sensitive detection using selected reaction monitoring; the detection responses of the ANA-derivatives were increased by 20-160-fold over those of the intact carboxylic acids and the limits of detection were in the low femtomole range (1.8-11 fmol on the column). The ANA-derivatization was also effective for the enatiomeric separation of the chiral carboxylic acids; the resolution was 1.92, 1.75, and 2.03 for 3-OH-PA, γ-CHEC, and etodolac, respectively. The derivatization procedure was successfully applied to a biological sample analysis; the derivatization followed by LC/ESI-MS/MS enabled the separation and detection of trace amounts of 3-OH-PA in neonatal dried blood spot and γ-CEHC in human saliva with a simple pretreatment and small sample volume., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Mass spectrometry (MS) has become a popular analytical technique because of its high sensitivity and specificity. Therefore, the use of a chiral derivatization reagent for the MS detection seems to be efficient for the enantiomeric separation of racemates. However, the number of chiral reagents for the liquid chromatography (LC)-tandem mass spectrometry (MS/MS) analysis is very limited. The applicability of commercially available chiral amines as the derivatization reagents for the enantiomeric separation of chiral carboxylic acids is reported in this paper by using non-steroidal anti-inflammatory drugs (NSAIDs), i.e. ibuprofen, flurbiprofen, and loxoprofen. The efficiency of the chiral reagents was evaluated in terms of tagging easiness, separation by reversed-phase chromatography, and detection sensitivity by electrospray ionization (ESI)-MS/MS. Among the tested eight chiral amines, i.e. (R)-(+)-4-(3-aminopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole (DBD-APy), (S)-(+)-1-(2-pyrrolidinylmethyl)-pyrrolidine (PMP), L-prolinamide, (3R)-(-)-1-benzyl-3-aminopyrrolidine, (S)-(+)-1-cyclohexyl-ethylamine, (3R)-(+)-3-(trifluoroacetamido)-pyrrolidine (TFAP), (R)-(-)-1-aminoindan (AI), and (S)-(+)-tetrahydrofurfuryl-amine, DBD-APy, PMP, AI, and TFAP could be used as the chiral reagents for the enantiomeric separation of the NSAIDs. The Rs values and the detection limits of the derivatives were in the range of 1.29-3.85 and 0.57-0.96 fmol, respectively. These four reagents were applied for the determination of the NSAIDs in rat plasma., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A simple and practical derivatization procedure for increasing the detection responses of carboxylic acids in liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) has been developed. 2-Hydrazinopyridine (HP) and 2-picolylamine (PA) rapidly reacted with biologically and clinically important carboxylic acids [chenodeoxycholic acid, glycochenodeoxycholic acid, prostaglandin E2, 2-(-carboxyethyl)-6-hydroxy-2,7,8-trimethylchroman (gamma-CEHC),alpha-lipoic acid, homovanillic acid (HVA) and 5-hydroxyindole-3-acetic acid] in the presence of 2,2'-dipyridyl disulfide and triphenylphosphine. The resulting HP- and PA-derivatives were highly responsive in ESI-MS operating in the positive-ion mode and gave characteristic product ions during MS/MS, which enabled the sensitive detection using selected reaction monitoring. Among the two reagents, PA was of more practical use; the detection responses of the PA-derivatives were increased by 9-158-fold over the intact carboxylic acids and the limits of detection were in the low femtomole range (1.5-5.6 fmol on column). The PA-derivatization was successfully applied to a biological sample analysis; the derivatization followed by LC-ESI-MS/MS enabled the detection of trace amounts of bile acids, gamma-CEHC and HVA in human saliva with a simple pretreatment, small sample volume and short analysis time.

A solution of optically pure kynurenine (KYN), i.e., D-KYN or L-KYN, was administered intravenously to male Sprague-Dawley rats (10 mg kg(-1) ml(-1)). The time-course of changes in the concentrations of urinary monoamines and their metabolites such as 5-hydroxytryptamine (5-HT), 5-hydroxyindole acetic acid (5-HIAA), dopamine, and 3-methoxytyramine were investigated by reversed-phase high-performance liquid chromatography with electrochemical detection after precolumn derivatization with (2R)-2,5-dioxopyrrolidin-1-yl-2,5,7,8-tetramethyl-6-(tetrahydro-2H-pyran-2-yloxy)chroman-2-carboxylate (NPCA). We observed a stereoselective difference in the effects of the KYN enantiomers. Only D-KYN, not L-KYN, caused a significant increase in urinary 5-HT levels within 30 min after its administration. With regard to the metabolites, urinary 3-MT level was increased by D-KYN administration. On the other hand, no significant change in the DA level was observed after administration of either D-KYN or L-KYN. These results suggest that D-KYN could affect the activity of neuroactive amines, especially 5-HT, in vivo., (2009 Wiley-Liss, Inc.)

Enantiomeric separation of D- and L-serine on an octadecylsilica column was investigated using (2R)-2,5-dioxopyrrolidin-1-yl-2,5,7,8-tetramethyl-6-(tetrahydro-2H-pyran-2-yloxy)chroman-2-carboxylate (R-NPCA), which was developed for a pre-column derivatization reagent for electrochemical detection. In addition, (2S)-2,5-dioxopyrrolidin-1-yl-2,5,7,8-tetramethyl-6-(tetrahydro-2H-pyran-2-yloxy)chroman-2-carboxylate (S-NPCA) was newly synthesized from (S)-(-)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (S-alpha-CA), and the enantiomeric separation of D- and L-serine using S-NPCA was also examined. The enantiomeric separation of D,L-serine was achieved using the R- or S-NPCA as a chiral derivatization reagent, and the elution orders of the enantiomers were reversed between R- and S-NPCA. The elution orders of D- and L-serine unexpectedly reversed between the phosphate buffer at pH 4.0 and pH 2.2, both of which were used in the mobile phase. Separation factors obtained using R- and S-NPCA were similar-1.09 and 1.07, respectively. The detection limit was approximately 940 fmol on the column (signal-to-noise ratio 3) when the applied voltage was +650 mV., ((c) 2009 John Wiley & Sons, Ltd.)

A simultaneous determination method for the enantiomers of chiral carboxylic acids by the combination of ultraperformance liquid chromatography and mass spectrometry (UPLC-MS/MS) has been developed. ( S)(+)-1-(2-Pyrrolidinylmethyl)-pyrrolidine (S-PMP) was used as the derivatization reagent for the high-throughput determination of biological chiral carboxylic acids, i.e., lactic acid (LA) and 3-hydroxybutyric acid (HA). The S-PMP efficiently reacted with the carboxylic acids under mild conditions at room temperature in the presence of 2,2′-dipyridyl disulfide and triphenylphosphine. The resulting S-PMP derivatives were highly responsive in the electrospray ionization (ESI)-MS operating in the positive-ion mode and gave characteristic product ions during the MS/MS, which enabled the sensitive detection using selected reaction monitoring. The derivatization was effective for the enantiomeric separation of the chiral carboxylic acids, and the resolution values of dl-LA and dl-HA were 4.91 and 9.37, respectively. Furthermore, a rapid separation of the derivatives of dl-LA and dl-HA within 7 min was performed using the UPLC system. The limits of detection on the column were in the low femtogram range (5-12 fg). The proposed procedure was successfully applied for the determination of the d- and l-isomers of LA and HA in the saliva of diabetes mellitus (DM) patients and healthy volunteers. The d-LA in DM patients was clearly higher than that in normal subjects. The derivatization followed by UPLC-ESI-MS/MS enabled the enantiomeric separation and detection of trace amounts of LA and HA in human saliva with a simple pretreatment and small sample volume. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

The tagging methods utilizing FL detection reagents for physiologically important carboxylic acids including fatty acids in HPLC are reviewed in terms of the tagging conditions, handling, flexibility, sensitivity, selectivity and stabilities of the reagents and the corresponding derivatives. Emphasis is given to the applications of the tagging techniques to biologically important carboxylic acids in real samples. Although many UV-labels have been developed for the determination of carboxylic acids, the tagging methods are not included in this review because recent progress in detection-oriented tagging in trace analysis by HPLC has been mainly carried out with fluorometry, and the resulting FL derivatives are also possible to monitor with the absorbance in UV–VIS regions. [Copyright &y& Elsevier]