A Pyridinium Derivatization Reagent for Highly Sensitive Detection of Poly(carboxylic acid)s Using Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry.

Short-chain fatty acids are difficult to analyze with high sensitivity using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) owing to the high polarity of their carboxyl groups. Various derivatization methods have been developed; however, most are effective only for monocarboxylic acids and not for those having multiple carboxyl groups. Therefore, we successfully attempted to synthesize a derivatization reagent that could analyze both mono- and poly(carboxylic acid)s with high sensitivity. We optimized our derivatization reagent by modifying the structure of the reaction site, hydrophobicity of the derivatized compound, and linker structure connecting the reaction site to the permanently charged substructure. The reactivity toward carboxyl groups was improved by employing a piperidine moiety as the reaction site, and the ESI efficiency was improved by the highly hydrophobic and permanently charged triphenylpyridinium group. Furthermore, the incorporation of an alkyl linker enabled polylabeling. When the optimized reagent was applied to mono-, di-, tri-, and tetracarboxylic acids, the ESI efficiency increased with polylabeling; thus, our derivatization reagent outperforms existing derivatization methods and enables the analysis of poly(carboxylic acid)s with high sensitivity. Since this derivatization reagent can be applied to most carboxyl-containing compounds, it can be widely used for lipidomics, proteomics, and metabolomics.

Quantification of Casein in Baked Food Products by Selective Analysis of Phosphorylated Peptides Using Fluorous Derivatization with Liquid Chromatography-Tandem Mass Spectrometry Method.

Casein is one of the allergen proteins present in milk. Therefore, a quantification method for the selective analysis of casein using fluorous derivatization with LC-tandem mass spectrometry (LC-MS/MS) was developed. After two allergen proteins (αS1-casein and ß-casein) extracted from baked sugar cookies were tryptic digested, the obtained phosphorylated peptides were selectively derivatized by ß-elimination with Ba(NO3)2 under basic condition and Michael addition with perfluoroalkylthiol (1H,1H,2H,2H-perfluorooctanethiol, PFOT). In this study, YKVPQLEIVPN(pSer)AQQR (104-119 fragment from αS1-casein) and FQ(pSer)EEQQQTEDELQDK (33-48 fragment from ß-casein) obtained by tryptic digestion were selected as target peptides. The phosphorylated serine residue in each peptide was converted to a perfluoroalkyl group by derivatization. The obtained fluorous-derivatized peptides were analyzed by LC-MS/MS, to which a fluorous LC column was connected. Therefore, it was possible to analyze casein without being affected by the matrix components in the baked food sample. When the present method was applied to cookies with arbitrary amounts of αS1-casein and ß-casein, the obtained quantification values were in good agreement with the arbitrary amounts spiked. The quantification limits of αS1- and ß-casein in cookie analysis were 246 and 152 ng/g, respectively. Hence, this method can be used to analyze trace amounts of allergen proteins present in the baked food.

Multiple phosphorylated protein selective analysis via fluorous derivatization and liquid chromatography-electrospray ionization-mass spectrometry analysis.

Post-translational modification of proteins is involved in protein function and higher-order structure. Among such modification, phosphorylation is an important intracellular signal transduction pathway. Many studies on phosphorylated protein analysis using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) have been developed. However, there are few reports on the analysis of highly phosphorylated proteins because of their handling difficulty. Hence, we developed an analytical method that converts multiple phosphate groups contained in the peptides into perfluoroalkyl groups for selective analysis using fluorous affinity. Here, tryptic digested ß-casein fragment peptides [RELEELNVPGEIVE(pSer)L(pSer)(pSer)(pSer)EESITR and FQ(pSer)EEQQQTEDELQDK] were used as model phosphorylated peptides. 1H,1H,2H,2H-Perfluorooctanethiol (PFOT) and 2,2,2-trifluoroethanethiol (TFET) were used as derivatization reagents for mono-phosphorylated peptides and multi-phosphorylated peptides, respectively, to derivatize via ß-elimination/Michael addition. The derivatives were analyzed by LC-ESI-MS. A fluorous LC column is typically used to selectively retain the fluorous-derivatized peptides, which are expected to be separated from contaminants and non-phosphorylated peptides. When this method was applied to ß-casein, TFET- and PFOT-derivatized peptides were strongly retained in the fluorous LC column and clearly separated from non-phosphorylated peptides on the chromatogram. Therefore, the developed method enables quantification of mono- and multi-phosphorylated peptides and is suitable for application in proteomics.

Selective analysis of the okadaic acid group in shellfish samples using fluorous derivatization coupled with liquid chromatography-tandem mass spectrometry.

Okadaic acid (OA) group are diarrheal shellfish poison that accumulates in the midgut glands of shellfish. It is difficult to remove these poisons by normal cooking because they are thermally stable and hydrophobicity. Therefore, in order to prevent foodborne disease due to shellfish poison, analysis by liquid chromatography (LC)-tandem mass spectrometry (MS/MS) before shipment is necessary. Herein the selective analytical method for OA group in shellfish sample using fluorous derivatization coupled with LC-MS/MS was developed. OA group were derivatized with the fluorous alkylamine reagent by condensing agent, and the obtained derivatives were separated with fluorous LC column (Fluofix-II 120E, 250 × 2.0 mm i.d., 5 µm, Fujifilm Wako Pure Chemical). The derivatized OA group were selective retained by fluorous LC column and accurate analysis was enabled. The present method was applied to the analysis of OA and dinophysistoxin-1 (DTX-1) in scallop midgut gland which is the certified reference material provided by national metrology institute of Japan. As a result of analysis using the present method with DTX-2 as the internal standard, the quantitative value were in agreement with the certified value.

Assessment method for deamidation in proteins using carboxylic acid derivatization-liquid chromatography-tandem mass spectrometry.

An analytical method for the degree of protein deamidation has been developed by using carboxy group derivatization and liquid chromatography-tandem mass spectrometry (LCMS/MS). The fragment peptides (LGEYGFQNALIVR and YNGVFQECCQAEDK) obtained by digesting bovine serum albumin (BSA) with trypsin and their asparagine deamidated peptides (LGEYGFQDALIVR and YDGVFQECCQAEDK) were selected as model peptides, and their carboxy groups were derivatized with ethylamine. This derivatization enabled a clear distinction between natural peptides and deamidated peptides by mass, allowing for facile distinction by LCMS/MS before and after deamidation. Good linearity was confirmed for four peptides used in this study via isotope dilution mass spectrometry, showing that protein deamidation can be evaluated by the present method. To confirm the validity of this method for the evaluation of deamidation, natural peptides and deamidated peptides were mixed in arbitrary ratios, and degree of deamidation in these solution was analyzed. This confirmed that accurate evaluation was possible at deamidation degree values of ca. 10 %, 5 %, 2.5 %, and 1 %. Additionally, an accelerated storage test of BSA demonstrated that the deamidation of asparagine at position 404 of BSA progressed by 4 % in 9 weeks at 40 °C and pH 8 in the dark, and that the deamidation process can be traced over time.

Three-Dimensional High-Performance Liquid Chromatographic Determination of Asn, Ser, Ala, and Pro Enantiomers in the Plasma of Patients with Chronic Kidney Disease.

The concentrations of several d-amino acids have been reported to significantly increase in the plasma of patients with chronic kidney disease (CKD). However, the amounts of these d-amino acids are low (around 1% of the l-form or lower), and their analyses were complicated by various interfering compounds in many clinical samples. A highly selective analytical method is thus required to perform the accurate and precise determination of these d-amino acids in the plasma of CKD patients. In the present study, a selective 3D-HPLC system equipped with reversed-phase, anion-exchange, and enantioselective columns has been designed and developed for the determination of the asparagine, serine, alanine and proline enantiomers. For the sensitive analysis, amino acids were precolumn derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole and detected by their fluorescence. The system was validated by using human plasma in addition to the standard amino acids, and results with a sufficient linearity, precision, and accuracy were obtained. The 3D-HPLC system was applied to the plasma of patients with different stages of CKD and all of the target d-amino acids were clearly observed without interferences for all 25 tested patients. Good correlations were shown between the kidney function of the patients and the %d values of the target analytes, especially d-Asn and d-Ser, indicating that the present 3D-HPLC method is useful for the sensitive diagnosis of CKD.

Enantioselective and simultaneous determination of lactate and 3-hydroxybutyrate in human plasma and urine using a narrow-bore online two-dimensional high-performance liquid chromatography system.

For the enantioselective and simultaneous analysis of lactate and 3-hydroxybutyrate, a validated online two-dimensional high-performance liquid chromatography system using 4-nitro-7-piperazino-2,1,3-benzoxadiazole as a fluorescent derivatization reagent has been developed. For the reversed-phase separation in the first dimension, a Capcell Pak C18 ACR column (1.5 × 250 mm, particle size 3 µm) was used, and the target fractions were isolated by their hydrophobicity. In the second dimension, a polysaccharide-coated enantioselective column, Chiralpak AD-H (2.0 × 250 mm, 5 µm), was used. The system was validated by the calibration curve, intraday precision, interday precision, and accuracy using standards and real human samples, and satisfactory results were obtained. The present method was applied to human plasma and urine, and in the plasma, trace amounts of d-lactate (8.4 µM) and l-3-hydroxybutyrate (1.0 µM), besides high levels of l-lactate (860.9 µM) and d-3-hydroxybutyrate (59.4 µM), were successfully determined. In urine, trace levels of d-lactate (3.7 µM), d-3-hydroxybutyrate (2.3 µM), and l-3-hydroxybutyrate (3.3 µM) in addition to a relatively large amount of l-lactate (15.4 µM) were observed. The present online two-dimensional high-performance liquid chromatography system is useful for the simultaneous determination of all the lactate and 3-hydroxybutyrate enantiomers in human physiological fluids, and further clinical applications are ongoing.

Mouse d-Amino-Acid Oxidase: Distribution and Physiological Substrates.

d-Amino-acid oxidase (DAO) catalyzes the oxidative deamination of d-amino acids. DAO is present in a wide variety of organisms and has important roles. Here, we review the distribution and physiological substrates of mouse DAO. Mouse DAO is present in the kidney, brain, and spinal cord, like DAOs in other mammals. However, in contrast to other animals, it is not present in the mouse liver. Recently, DAO has been detected in the neutrophils, retina, and small intestine in mice. To determine the physiological substrates of mouse DAO, mutant mice lacking DAO activity are helpful. As DAO has wide substrate specificity and degrades various d-amino acids, many d-amino acids accumulate in the tissues and body fluids of the mutant mice. These amino acids are d-methionine, d-alanine, d-serine, d-leucine, d-proline, d-phenylalanine, d-tyrosine, and d-citrulline. Even in wild-type mice, administration of DAO inhibitors elevates D-serine levels in the plasma and brain. Among the above d-amino acids, the main physiological substrates of mouse DAO are d-alanine and d-serine. These two d-amino acids are most abundant in the tissues and body fluids of mice. d-Alanine derives from bacteria and produces bactericidal reactive oxygen species by the action of DAO. d-Serine is synthesized by serine racemase and is present especially in the central nervous system, where it serves as a neuromodulator. DAO is responsible for the metabolism of d-serine. Since DAO has been implicated in the etiology of neuropsychiatric diseases, mouse DAO has been used as a representative model. Recent reports, however, suggest that mouse DAO is different from human DAO with respect to important properties.

Enantioselective determination of citrulline and ornithine in the urine of d-amino acid oxidase deficient mice using a two-dimensional high-performance liquid chromatographic system.

Two-dimensional high-performance liquid chromatographic (2D-HPLC) and 2D-HPLC-mass spectrometric (2D-HPLC-MS) systems have been designed and developed for the determination of the citrulline (Cit) and ornithine (Orn) enantiomers. Several d-amino acids have already been identified as novel physiologically active molecules and biomarkers, and the enantioselective evaluation of the amounts, distributions and metabolisms of non-proteinogenic amino acids gain as well increasing interest. In the present study, highly selective analytical methods were developed using a capillary monolithic ODS column (0.53mm i.d. x 1000mm) for the reversed-phase separation of the target analytes from the matrix compounds in the first dimension, and a narrowbore-Pirkle type enantioselective column, KSAACSP-105S (1.5mm i.d. x 250mm), was used for the enantiomer separation in the second dimension. The amino acids were analyzed after pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and detected by the fluorescence detector and MS. The systems were applied to the urine of d-amino acid oxidase (DAO) deficient B6DAO- mice and control C57BL mice to evaluate the presence and metabolism of the Cit and Orn enantiomers in mammals. As a result, all of the 4 target enantiomers (d-Cit, l-Cit, d-Orn, l-Orn) were found in the urine of both strains. The %D value of Cit (d-Cit/Cit×100) increased about 3-fold in the urine of the DAO deficient mice and that of Orn also tended to increase with the DAO deficiency. These results were definitely confirmed by a 2D-HPLC-MS detection system. Further investigations about the biological significance of these d-isomers are currently ongoing.

Changes in D-aspartic acid and D-glutamic acid levels in the tissues and physiological fluids of mice with various D-aspartate oxidase activities.

D-Aspartic acid (D-Asp) and D-glutamic acid (D-Glu) are currently paid attention as modulators of neuronal transmission and hormonal secretion. These two D-amino acids are metabolized only by D-aspartate oxidase (DDO) in mammals. Therefore, in order to design and develop new drugs controlling the D-Asp and D-Glu amounts via regulation of the DDO activities, changes in these acidic D-amino acid amounts in various tissues are expected to be clarified in model animals having various DDO activities. In the present study, the amounts of Asp and Glu enantiomers in 6 brain tissues, 11 peripheral tissues and 2 physiological fluids of DDO(+/+), DDO(+/-) and DDO(-/-) mice were determined using a sensitive and selective two-dimensional HPLC system. As a result, the amounts of D-Asp were drastically increased with the decrease in the DDO activity in all the tested tissues and physiological fluids. On the other hand, the amounts of D-Glu were almost the same among the 3 strains of mice. The present results are useful for designing new drug candidates, such as DDO inhibitors, and further studies are expected.

Enantioselective two-dimensional high-performance liquid chromatographic determination of N-methyl-D-aspartic acid and its analogues in mammals and bivalves.

A validated and fully automated chiral 2D-HPLC system was developed for the simultaneous determination of N-methyl-d-aspartic acid (NMDA) analogues by combining a long microbore-monolithic ODS column (0.53 mm i.d.× 1,000 mm) and narrowbore-enantioselective columns (1.5mm i.d.×150 or 250 mm). The target analytes, enantiomers of N-methylaspartic acid (NMA) and N-methylglutamic acid (NMG), were precolumn-derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and detected by their fluorescence. The values of the lower limit of quantification for these enantiomers were 2.5 fmol. In the tissues and plasma of rats, neither NMA nor NMG were detected. On the other hand, in the mantle and foot of Scapharca broughtonii, a large amount of NMDA was present (170.1 and 43.5 nmol/g), and the enantiomers of NMG were also observed. Meretrix lusoria contained NMDA (29.3 nmol/g) and NMLG (13.8 nmol/g), and Ruditapes philippinarum contained only NMLG (2.6 nmol/g). The obtained results were confirmed using three different enantioselective columns and also using a 2D-HPLC-MS/MS system. These results indicated that neuroactive d-amino acid, NMDA, and its analogues were present in animals, and their physiological significance is expected to be clarified.

HPLC analysis of naturally occurring free D-amino acids in mammals.

D-amino acids are currently recognized as naturally occurring physiologically active substances and biomarkers in mammals. The progress of analytical technologies, mostly high resolution chromatographic or electrodriven separation methods, has significantly contributed to the advances in D-amino acid research in real biological matrices. In this review, we would like to describe the D-amino acid research, from the discovery of appreciable amounts of free D-amino acids in mammals to the current metabolomics study focusing on amino acid enantiomers. The liquid phase enantioselective analytical methods utilized for the determination of D-amino acids in mammals including human beings will be discussed.