Increased inosine levels in drug-free individuals with at-risk mental state: A serum metabolomics study.

AIM: At-risk mental state (ARMS) has been recently attracting attention with respect to the improvement of the management and outcome of psychiatric diseases, such as schizophrenia. Since only a few studies have reported on biological alterations in ARMS, serum metabolomics was carried out in ARMS subjects and healthy controls using liquid chromatography with high-resolution mass spectrometry. METHODS: Serum samples were collected from ARMS subjects (n = 24; male: 12; female 12) and age- and sex-matched healthy controls (n = 23 male: 11, female: 12). After serum pre-treatment, liquid chromatography with high-resolution mass spectrometry was performed. Multivariate analyses, such as orthogonal partial least-squares discriminant and volcano plot analyses, were performed. RESULTS: Serum inosine, lactate, taurine, 2,3-dihydroxypropanoate and glutamate levels differed between the two groups. A significant increase in inosine levels was detected in the positive- and negative-ion modes; however, significant differences were not observed in the levels of other purine-related metabolites (hypoxanthine, xanthine and urate) between the two groups. CONCLUSION: Increased inosine levels may serve as biological markers for ARMS, in addition to alterations in the levels of lactate and certain amino acids.

Separation of vigabatrin enantiomers using mixed-mode chromatography and its application to determine the vigabatrin enantiomer levels in rat plasma.

Mixed-mode chromatography-comprising a mixed phase with reversed and ionic phases, enabling hydrophobic and ion-exchange interactions simultaneously-was applied to identify vigabatrin enantiomers by HPLC with pre-column fluorescence derivatization with 2,5-dioxopyrrolidin-1-yl (4-(((2-nitrophenyl)sulfonyl)oxy)-6-(3-oxomorpholino)quinoline-2-carbonyl)prolinate (Ns-MOK-(S)-Pro-OSu). The MOK-(S)-Pro-vigabatrin enantiomers were efficiently separated within 12 min (total analysis time per sample: 28 min, including washing and equilibrium time for the column). The mobile phase was H2O/CH3OH/10 mM ammonium formate (pH 2.0) (20/20/60, v/v/v). Column temperature was maintained at 60℃. The proposed HPLC method could successfully monitor plasma vigabatrin enantiomer levels in rats administered (±)-vigabatrin (50 mg/kg, p.o.).

Development of a Derivatization Reagent with a 2-Nitrophenylsulfonyl Moiety for UHPLC-HRMS/MS and Its Application to Detect Amino Acids Including Taurine.

Taurine (Tau) has some important ameliorating effects on human health and is present in bivalve. For the selective analysis of Tau with other amino acids, we designed a derivatization reagent, 2,5-dioxopyrrolidin-1-yl(4-(((2-nitrophenyl)sulfonyl)oxy)-6-(3-oxomorpholino)quinoline-2-carbonyl)pyrrolidine-3-carboxylate (Ns-MOK-ß-Pro-OSu). After derivatization with Ns-MOK-ß-Pro-OSu, amino acids with Tau in Japanese littleneck clams were determined through ultra-high-performance-liquid chromatography with high-resolution tandem mass spectrometry (UHPLC-HRMS/MS) using an octadecyl silica column. We could detect 18 amino acids within 10 min. Tau, valine, glutamine, glutamic acid, and arginine in the clams were determined in the negative ion mode using the characteristic fragment ion, C6H4N1O5S, which corresponded to the 2-nitrobenzenesulfonylate moiety. The fragment ion, C6H4N1O5S, was recognized as a common feature regardless of the amino acid to be derivatized, and it was convenient for detecting amino acid derivatives with high selectivity and sensitivity. Therefore, highly selective quantification using UHPLC-HRMS/MS was possible using Ns-MOK-ß-Pro-OSu.

Development of derivatization reagents bearing chiral 4-imidazolidinone for distinguishing primary amines from other amino acids and application to the liquid chromatography-tandem mass spectrometric analysis of miso.

We designed and synthesized three novel derivatization reagents bearing chiral 4-imidazolidinone, namely succinimidyl 2-(3-((benzyloxy)carbonyl)-1-methyl, ethyl, and -phenyl-5-oxoimidazolidin-4-yl)acetates (CIMs), for use in liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The CIMs were able to discriminate primary amines from other compounds such as secondary amines and phenols, based on their unique m/z reduction of precursor ion to form product ion in MS/MS. As amino acid derivatization reagents, the CIMs were compared in terms of enantioseparation of amino acid and detection sensitivity. CIMa-OSu with 1-methyl-5-oxoimidazolidinone moiety gave the best optical resolution and detection sensitivity among the CIM reagents. Next, we applied (R)-CIMa-OSu to determine amino acids in miso by LC-triple-quadrupole MS. The proposed method achieved simultaneous determination of 20 l-amino acids and two d-amino acids (d-alanine and d-serine) in the sample with a high sensitivity (limits of detection 5-238 fmol, signal-to-noise ratio 3.3). After derivatization with CIMa-OSu, it was possible to determine whether each peak in the chromatogram was a component of primary amine or not, by using a high-resolution orbitrap MS instrument.

Fluorimetric determination of the enantiomers of vigabatrin, an antiepileptic drug, by reversed-phase HPLC with a novel diastereomer derivatization reagent.

Herein, determination of an antiepileptic drug, (±)-vigabatrin (VB), was performed by reversed-phase HPLC with fluorimetric detection using a newly designed and synthesized fluorescence derivatization reagent, 2,5-dioxopyrrolidin-1-yl (4-{[(2-nitrophenyl)sulfonyl]oxy}-6-(3-oxomorpholino)quinoline-2-carbonyl)prolinate [Ns-MOK-(R)- or (S)-Pro-OSu]. During the derivatization of VB with Ns-MOK-(R)-Pro-OSu at 60°C, the nosyl (Ns) group, which was introduced to protect a phenolic hydroxy group, was released within 30 min to produce MOK-(R)-Pro-VB, which was detected fluorimetrically at 448 nm with an excitation wavelength of 333 nm. The VB enantiomers were separated on an octadecylsilica (ODS) column with a resolution value of 5.57, because Ns-MOK-(R)-Pro-OSu bears an optically active D-proline structure. A complete separation of MOK-(R)-Pro-(R)- and -(S)-VB enantiomers was achieved on the ODS column within 40 min using stepwise gradient elution, and the detection limits were ~0.80 and 0.37 pmol on the column, respectively. The proposed HPLC with fluorimetric detection method was successfully used for determining VB enantiomers in VB-spiked human serum following solid-phase extraction with an anion-exchange cartridge.

LC-MS/MS Analysis of Thiol-Containing Amino Acids in Exosomal Fraction of Serum.

It has been suggested that thiol-containing amino acids could be used as biomarkers for diseases associated with oxidative stress. We investigated the thiol-containing amino acids, homocysteine (Hcy), cysteine (Cys), glutathione (GSH) and γ-glutamylcysteine (γ-GluCys), in commercial human serum by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) after precolumn derivatization with 4-fluoro-7-sulfobenzofurazan. This method was applied to determine the composition of thiol-containing amino acids in exosomes prepared from the serum. Hcy, Cys, GSH and γ-GluCys could be detected in the exosomal fraction, and the ratio of each thiol-containing amino acid was similar to those in the corresponding native serum. Cys (94.76%) was most enriched in the exosomal fraction, followed by GSH (2.97%), γ-GluCys (1.59%) and Hcy (0.68%). These findings suggest that thiol-containing amino acids, Hcy, Cys, GSH and γ-GluCys, are included in exosomes in human serum.

Serum d- and l-lactate, pyruvate and glucose levels in individuals with at-risk mental state and correlations with clinical symptoms.

AIM: Little information exists on the peripheral metabolite levels in individuals with at-risk mental state who meet the criteria for a high-risk state of psychosis. Here, we aimed to investigate serum levels of glucose, pyruvate and d- and l-lactate, which may act as a signalling molecule for learning and memory in neuronal cells. METHODS: High performance liquid chromatography or commercial kits were used to assess serum metabolites in individuals with attenuated psychosis symptoms of at-risk mental state (n = 24, men = 12) who were not receiving antipsychotics. The metabolite levels of these individuals were compared with those of age- and sex-matched healthy individuals (controls, n = 23, men = 11). Correlations between the metabolites and clinical symptoms of at-risk mental state were also examined. RESULTS: Individuals with at-risk mental state had higher serum glucose levels than did controls (P = 2.18 × 10-3 ), while no significant difference in pyruvate levels were observed between the groups. Individuals with at-risk mental state had significantly lower serum l-lactate levels than did controls (P = 6.31 × 10-5 ), while no differences in d-lactate levels were observed. Furthermore, a negative correlation was identified between serum l-lactate levels and Positive and Negative Syndrome Scale negative symptoms scores (r = -0.5651, P = 4.01 × 10-3 ) in individuals with at-risk mental state. CONCLUSIONS: We found that, compared with controls, individuals with at-risk mental state have reduced serum l-lactate levels, which may predate psychosis onset, and may be involved in the related negative symptoms.

Alteration in plasma docosahexaenoic acid levels following oral administration of ethyl icosapentate to rats.

OBJECTIVES: Ethyl icosapentate, a prodrug of eicosapentaenoic acid (EPA), has been prescribed to not only hyperlipidemia, but also psychotic patients. We have examined the impact of an orally administered polyunsaturated fatty acid (PUFA), ethyl icosapentate, on the plasma concentrations of seven other types of fatty acids and one metabolite (3-hydroxybutyrate, 3-HB) using rats. DESIGN: and Methods: A commercial omega-3 PUFA, EPA, formulation (ethyl icosapentate, Epadel®) was administered orally to Sprague-Dawley rats (15, 50, 100 mg/kg, n = 4-8) and changes in the plasma fatty acid concentrations were investigated by HPLC using fluorescence detection. RESULTS: The concentration of an n-3 PUFA, docosahexaenoic acid (DHA), was significantly increased from 11.6 ±â€¯1.45 (0 h) to 25.9 ±â€¯6.54 µM (6 h) in rat plasma (n = 8, p = 1.88 × 10-2) at a dose of 100 mg/kg, as was the EPA concentration from 2.58 ±â€¯0.16 (0 h) to 6.03 ±â€¯2.20 µM (1 h) (n = 8, p = 2.09 × 10-2), whereas concentrations of other fatty acids, such as α-linolenic acid, palmitoleic acid, arachidonic acid, linolenic acid, and oleic acid, were not significantly changed. In addition, the concentration of the ultimate fatty acid metabolite, 3-hydroxybutyrate (3-HB), was significantly increased (from 94.6 ±â€¯10.2 to 217 ±â€¯43.4, p = 5.41 × 10-3) 12 h after oral administration of ethyl icosapentate (n = 8, 100 mg/kg). CONCLUSIONS: This result suggests that intake of the EPA formulation contributed not only to an increase in EPA concentration, but also to increases in DHA and 3-HB concentrations in vivo.

Alterations in methionine to homocysteine ratio in individuals with first-episode psychosis and those with at-risk mental state.

BACKGROUND: Disturbance of the methionine (Met) cycle, which produces Met from homocysteine (Hcy), is suggested to be involved in several diseases, including psychiatric disorders. This study was aimed to investigate both levels of Met and Hcy in serum from individuals with first-episode psychosis (FEP) and individuals with at-risk mental state (ARMS). METHOD: We measured serum Met and Hcy levels in individuals with FEP (n = 13) and ARMS (n = 30) using HPLC with fluorescence detection and LC-ESI-MS/MS. Met and Hcy levels in healthy controls (n = 41) were also measured. Differences between the 3 groups were analyzed by one-way analysis of variance (ANOVA) with Bonferroni correction. RESULTS: Serum Met levels were decreased (p = 0.038) and Hcy levels were increased (p = 0.017) in the FEP group. Hcy levels were also significantly increased compared to the ARMS group (p = 0.016), while Met levels were not significantly different between the FEP and ARMS groups. A significant decrease in the Met to Hcy ratio (Met/Hcy) was observed in the FEP group compared to both the control (p = 4.58 × 10-4) and ARMS (p = 8.07 × 10-3) groups. Furthermore, Met/Hcy ratio was correlated with Positive and Negative Syndrome Scale, especially positive scores (p = 5.90 × 10-5). CONCLUSION: Taken together, these data indicate that a decrease in the serum Met/Hcy ratio may be a risk factor for developing psychosis during the transition from ARMS to FEP, and may prove to be a useful marker of the phase between ARMS and FEP.

Succinimidyl (3-[(benzyloxy)carbonyl]-5-oxo-1,3-oxazolidin-4-yl)acetate on a triazole-bonded phase for the separation of dl-amino-acid enantiomers and the mass-spectrometric determination of chiral amino acids in rat plasma.

Changes in the levels of amino-acid enantiomers are associated with some serious diseases; consequently, amino acid monitoring in peripheral blood can be used to diagnose and predict the onset of disease. Herein, we report the design and synthesis of a new chiral derivatization reagent, namely succinimidyl (4S)-(3-[(benzyloxy)carbonyl]-5-oxo-1,3-oxazolidin-4-yl)acetate ((S)-COXA-OSu), for the separation of dl-amino-acid enantiomers. The usefulness of (S)-COXA-OSu was examined as a derivatization reagent for LC-MS/MS following certification of its total optical purity (>99%). The enantiomeric separations of amino-acid derivatives tagged with the reagent were examined using a triazole-bonded phase. (S)-COXA-OSu enabled the simultaneous enantiomeric separation of more than 40 α-amino acids. (S)-COXA-amino-acid derivatives were efficiently converted into their product ions, from which formaldehyde (CH2O) was eliminated [M-30] from the oxazolidinone moiety of COXA by collision-induced dissociation during LC-MS/MS. Limits of detection were in the 0.0138-0.518 pmol/injection range. For precise and accurate quantitation, we synthesized and used a stable-isotope-labeled (S)-COXA-OSu that was used as an internal standard in LC-MS/MS-determination experiments. Finally, changes in plasma amino-acid levels in rats, following administration of S-methyl-l-cysteine, an alanine-serine-cysteine transporter-1 (Asc-1) inhibitor, were successfully detected by LC-MS/MS using (S)-COXA-OSu.

Amino acid analyses of the exosome-eluted fractions from human serum by HPLC with fluorescence detection.

OBJECTIVES: Amino acid levels in serum or plasma are used for early detection and diagnosis of several diseases. The objective of this study was to analyze amino acid levels in serum exosomes, which have not been previously reported. DESIGN AND METHODS: We investigated the amino acid composition of exosomes from human serum using HPLC with fluorescence detection. RESULTS: The composition ratios of His, Arg, Glu, Cys-Cys, Lys, and Tyr were significantly increased in the exosomes compared with those in the corresponding native serum. d-Ser, an endogenous co-agonist of the N-methyl-d-aspartate receptor, was also enriched in the exosome-eluted fraction. CONCLUSIONS: Our results suggest that certain amino acids are enriched in the exosome-eluted fraction from human serum. These differences could have future diagnostic potential.

Liquid chromatography-mass spectrometry with triazole-bonded stationary phase for N-methyl-D-aspartate receptor-related amino acids: development and application in microdialysis studies.

We aimed to monitor changes in the levels of amino acid neurotransmitters or neuromodulators simultaneously at the synaptic clefts of experimental animals. We developed a method for the simultaneous determination of the levels of amino acids, such as D-Ser, Gly, and L-Glu, which were involved in neurotransmission via the N-methyl-D-aspartate (NMDA) receptor, and other protein-constituted amino acids in a rat brain microdialysis (MD) sample. We used a liquid chromatography-mass spectrometry (LC-MS)/MS device equipped with a triazole-bonded column. The determination was achieved without using stable isotope-labeled compounds. We instead used suitable amino acid analogues as internal standards (ISs). We examined various analyte-IS combinations to improve reproducibility. We found a positive correlation (r = 0.720, **p < 0.0001) between relative standard deviation (%) of the area ratio and the analyte-IS retention time differences. Using the proposed method, we were able to accurately analyze trace amounts of amino acids in MD samples using ISs that were structurally similar to the analytes. Furthermore, we observed that the peripheral administration of S-methyl-L-cysteine, which was an inhibitor of the amino acid transporter Asc-1, caused some amino acid level changes in the rat brain. The proposed LC-MS/MS method can be applied in vivo to study the effects of novel therapeutic agents with monitoring the levels of amino acid neuromodulators, such as Glu, Gly, GABA, and D-Ser, in the brain. Graphical abstract LC-MS/MS analysis of amino acid enantiomers in microdialysis samples from rat striatum using triazole-bonded stationary phase.

Alteration in plasma and striatal levels of d-serine after d-serine administration with or without nicergoline: An in vivo microdialysis study.

AIMS: d-Serine (d-Ser), a co-agonist of N-methyl-d-aspartate receptor (NMDAR), is effective for treating schizophrenia. The present study investigated changes in plasma and striatal d-Ser levels in Sprague-Dawley (SD) rats after intraperitoneal d-Ser administration alone or together with nicergoline (Nic), a commercial cerebral ameliorating drug, using in vivo microdialysis (MD) to explore the function of Nic. MAIN METHODS: Phosphate-buffered saline (PBS) or Nic (0, 1.0, or 3.0 mg/kg) followed by d-Ser (5.0, 10.0, 20.0, and 50.0 mg/kg for PBS or 20.0 mg/kg for Nic) was administered intraperitoneally to male SD rats, and the profiles of d-Ser levels in plasma and striatal MD samples were examined by high-performance liquid chromatography (HPLC) with fluorescence detection. The area under the curve (AUC) for the MD and plasma samples was also calculated and statistically compared among groups. KEY FINDINGS: AUC values of d-Ser increased in a d-Ser dose-dependent manner in plasma samples, while a proportional increase in the AUC values of striatal MD samples was only observed in d-Ser doses up to 20 mg/kg. The Nic co-administered group showed a significant increase in the AUC of plasma d-Ser in a Nic dose-dependent manner, but the AUC in striatal d-Ser significantly decreased with increasing Nic doses suggesting that Nic may prevent excess d-Ser from penetrating the central nervous system (CNS). SIGNIFICANCE: Nic may prevent an excessive distribution of exogenous d-Ser, such as that from a dietary origin, into the CNS by suppressing excitatory neurotransmission through NMDAR.

Chromatographic profiles of tryptophan and kynurenine enantiomers derivatized with (S)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole using LC-MS/MS on a triazole-bonded column.

d- and l-Tryptophan (Trp) and d- and l-kynurenine (KYN) were derivatized with a chiral reagent, (S)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole (DBD-PyNCS), and were separated enantiomerically by high-performance liquid chromatography (HPLC) equipped with a triazole-bonded column (Cosmosil HILIC) using tandem mass spectrometric (MS/MS) detection. Effects of column temperature, salt (HCO2 NH4 ) concentration, and pH of the mobile phase in the enantiomeric separation, followed by MS detection of (S)-DBD-PyNCS-d,l-Trp and -d,l-KYN, were investigated. The mobile phase consisting of CH3 CN/10 mM ammonium formate in H2 O (pH 5.0) (90/10) with a column temperature of 50-60 °C gave satisfactory resolution (Rs) and mass-spectrometric detection. The enantiomeric separation of d,l-Trp and d,l-KYN produced Rs values of 2.22 and 2.13, and separation factors (α) of 1.08 and 1.08, for the Trp and KYN enantiomers, respectively. The proposed LC-MS/MS method provided excellent detection sensitivity of both enantiomers of Trp and KYN (5.1-19 nM).

Effect of risperidone on plasma d-serine concentration in rats post-administered with d-serine.

AIMS: Risperidone (Ris) is a second-generation antipsychotic (SGA) used to treat patients with schizophrenia. Additional interventions that increase plasma d-serine (d-Ser) levels could provide improved amelioration of the negative symptoms of schizophrenia. In the present study, we studied whether Ris pretreatment altered the concentration of plasma d-Ser administered intraperitoneally. In addition, the effects of Ris and its main metabolite, 9-hydroxyrisperidone (9-OHRis), on rat d-amino acid oxidase (DAO) activity were examined in vitro. MATERIALS AND METHODS: Ris (0, 0.5, 1.0, or 3.0mg/kg), followed by d-Ser (20mg/kg), were administered intraperitoneally (i.p.) to male Sprague-Dawley rats, and the time-courses of plasma d-Ser, Ris, and 9-OHRis concentrations were examined. Inhibition of DAO activity in rat cerebellar and kidney preparations by Ris and 9-OHRis were measured spectrophotometrically. KEY FINDINGS: Significant increases in plasma d-Ser levels were observed in rats treated with both Ris and d-Ser. This effect occurred in a Ris dose-dependent manner, and the areas under the plasma d-Ser concentration-time curves were similar in rats treated with Ris (1.0mg/kg) and with a commercial DAO inhibitor, 3-methylpyrazole-5-carboxylic acid (1.0mg/kg). Rat plasma analyses showed that 9-OHRis was rapidly produced from Ris; however, high concentrations of Ris and 9-OHRis produced weak DAO inhibition in vitro, suggesting that some other pharmacological effect of Ris and/or 9-OHRis might contribute to its effects on plasma d-Ser levels. SIGNIFICANCE: The combined administration of Ris and d-Ser may increase plasma d-Ser levels, suggesting that this approach could reduce the dose of d-Ser required for these patients.

Determination of l-tryptophan and l-kynurenine derivatized with (R)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole by LC-MS/MS on a triazole-bonded column and their quantification in human serum.

The concentrations of l-tryptophan (Trp) and the metabolite l-kynurenine (KYN) can be used to evaluate the in-vivo activity of indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO). As such, a novel method involving derivatization of l-Trp and l-KYN with (R)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole (DBD-PyNCS) and separation by high-performance liquid chromatography (HPLC) with tandem mass spectrometric (MS/MS) detection on a triazole-bonded column (Cosmosil HILIC®) was developed to determine their concentrations. The optimized mobile phase, CH3 CN/10 mm ammonium formate in H2 O (pH 5.0) (90:10, v/v) eluted isocratically, resulted in satisfactory separation and MS/MS detection of the analytes. The detection limits of l-Trp and l-KYN were approximately 50 and 4.0 pm, respectively. The column temperature affected the retention behaviour of the Trp and KYN derivatives, with increased column temperatures leading to increased capacity factors; positive enthalpy changes were revealed by van't Hoff plot analyses. Using the proposed LC-MS/MS method, l-Trp and l-KYN were successfully determined in 10 µL human serum using 1-methyl-l-Trp as an internal standard. The precision and recovery of l-Trp were in the ranges 2.85-9.29 and 95.8-113%, respectively, while those of l-KYN were 2.51-16.0 and 80.8-98.2%, respectively. The proposed LC-MS/MS method will be useful for evaluating the in vivo activity of IDO or TDO. Copyright © 2016 John Wiley & Sons, Ltd.

Determination of D-serine in human serum by LC-MS/MS using a triazole-bonded column after pre-column derivatization with (S)-4-(3-isothiocyanatopyrrolidin-1-yl)-7- (N, N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole.

An LC-MS/MS-based method for determining D-serine (Ser), an endogenous co-agonist of the N-methyl-D-aspartate receptor, in human serum, was developed and validated using a triazole-bonded silica-packed column after pre-column fluorescence derivatization with a chiral labeling reagent, (S)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole. Enantiomeric separation of the D- and L-Ser derivatives occurred in the triazole-bonded column (R s: 1.85) with CH3CN/100 mM HCO2NH4 in H2O (95.5:4.5) as the mobile phase with isocratic elution. The ln(capacity factor of D-Ser) in the van't Hoff plot gradually decreased with the inverse of temperature, suggesting enhanced hydrophilic interactions with the triazole-bonded stationary phase with increasing column temperature, owing to decrease in the partition coefficient to the mobile phase. Multiple reaction monitoring (m/z 457.10 > 409.00) by triple quadrupole mass spectrometry was used for quantifying D-Ser in human serum. The presence of D-Ser in the serum was confirmed by treatment with commercial D-amino acid oxidase. A linear calibration curve was constructed in the D-Ser concentration range of 0.5-5.0 µM (r (2) = 0.999, n = 3) using D-homoserine as the internal standard. The precision and recovery values were adequate for quantification. The detection limit for D-Ser was 1.1 fmol/injection (signal-to-noise ratio = 3), owing to the high CH3CN content in the mobile phase. The proposed LC-MS/MS method showed few fluctuations in the retention times of D- and L-Ser, and R s was stable until the 40th injection of serum without column washing, and thus can be useful for D-Ser determination in human serum in clinical research.

A high-performance liquid chromatography assay with a triazole-bonded column for evaluation of d-amino acid oxidase activity.

Elution profiles of kynurenic acid (KYNA) and 7-chlorokynurenic acid (Cl-KYNA) were examined by high-performance liquid chromatography (HPLC) using a triazole-bonded stationary phase column (Cosmosil® HILIC) under isocratic elution of a mobile phase consisting of CH3 CN-aqueous 10 mm ammonium formate between pH 3.0 and 6.0. The capacity factors of KYNA and Cl-KYNA varied with both the CH3 CN content and the pH of the mobile phase. The elution order of KYNA and Cl-KYNA was reversed between the CH3 CN- and H2 O-rich mobile phases, suggesting that hydrophilic interactions and anion-exchange interactions caused retention of KYNA and Cl-KYNA in the CH3 CN- and H2 O-rich mobile phases, respectively. The present HPLC method using a triazole-bonded column and fluorescence detection (excitation 250 nm, emission 398 nm) was applied to monitor in vitro production of KYNA from d-kynurenine (d-KYN) by d-amino acid oxidase (DAO) using Cl-KYNA as an internal standard. A single KYNA peak was clearly observed after enzymatic reaction of d-KYN with DAO. Production of KYNA from d-KYN was suppressed by the addition of commercial DAO inhibitors. The present HPLC method can be used to evaluate DAO activity and DAO inhibitory effects in candidate drugs for the treatment of schizophrenia.

Determination of sex-based differences in serum γ-linolenic [corrected] acid and dihomo-γ-linolenic [corrected] acid using gas chromatography-mass spectrometry.

Because serum unsaturated fatty acids can provide useful information on disease diagnosis, the simultaneous determination of several fatty acids in small volumes of human serum would be beneficial for clinical applications. In the present study, serum fatty acids were extracted with n-heptane/chloroform from 10µL of serum collected from 26 healthy Japanese subjects (11 men, ages 23-37 years; 15 women, ages 18-37 years) after deproteinization with perchloric acid, derivatization to their methyl ester using p-toluenesulfonic acid as an acid catalyst, and subsequent separation and measurement by gas chromatography-mass spectrometry (GC-MS) in the selected ion monitoring mode. Nine types of fatty acids (palmitoleic acid [PLA], oleic acid [OA], linoleic [corrected] acid [LA], γ-linolenic acid [GLA], α-linolenic acid [ALA], dihomo-GLA [DGLA], arachidonic acid [AA], eicosapentaenoic acid [EPA], and docosahexaenoic acid [DHA]) were analyzed in the serum within 35 min by GC-MS. The concentrations of these fatty acids in serum ranged from 3.64±0.38µM (GLA) to 413±26.3 µM (LA). Among these nine fatty acids, GLA and DGLA levels were significantly lower in women than in men (p=0.0034 and 0.0012, respectively), suggesting that there may be sex-based differences in the biosynthetic production or metabolic processes of GLA and DGLA in humans.