Use of Commercial Mixed-Mode Stationary Phases and Sorbents in the High-Performance Liquid Chromatography Analysis and Solid-Phase Extraction of Ionized and Hydrophilic Bioactive Compounds.

Mixed-mode high-performance liquid chromatography (HPLC) is increasingly used for the analysis of ionic and highly hydrophilic drugs, which are difficult to separate by conventional single-mode HPLC. In the former case, chromatographic separation is achieved using one of the several commercially available mixed-mode stationary phases, typically combinations of reversed and ion-exchange phases. Moreover, mixed-mode stationary phases can be used as solid-phase extraction (SPE) sorbents. This review focuses on the recent applications of mixed-mode stationary phases in the chromatographic analysis of bioactive compounds, such as drugs, herbicides, and pesticides. Specifically, we briefly summarize HPLC methods utilizing mixed-mode stationary phases and SPE pretreatment procedures utilizing mixed-mode sorbents developed in the last decade, thus providing a reference work for overcoming the difficulties in analyzing ionized or hydrophilic drugs by conventional reversed-phase chromatography.

Substituted kynurenic acid derivatives as fluorophore-based probes for D- and L-amino acid oxidase assays and their in vitro application in eels.

High levels of D-amino acid oxidase (DAO) are associated with neurological and psychiatric disorders, while L-amino acid oxidase (LAO) exhibits antimicrobial and antitumor properties. The enzymatic conversion of the non-fluorescent kynurenine (KYN) into the endogenous weak fluorescent kynurenic acid (KYNA) by the action of DAO has previously been reported. However, the fluorescence of KYNA can be improved by changing the substituents on the aromatic rings. In this study, we prepared different 6-phenyl-substituted KYNA derivatives and investigated their fluorescence properties. Among them, 2-MePh-KYNA showed the maximum fluorescence quantum yield of 0.881 at 340 nm excitation and 418 nm emission wavelengths. The effects of solvent properties (dielectric constant, pKa, viscosity, and proticity) on the fluorescence intensity (FLI) of the KYNA derivatives were explored. The FLI of 2-MePh-KYNA was significantly large in protic solvents. Subsequently, 2-MePh-D-KYN and 2-MePh-L-KYN were prepared with high enantiopurity (>99.25%) for the enzymatic conversion. 2-MePh-D-KYN exhibited high sensitivity (∼19 times that of a commercial DAO substrate and ∼60 times that of the previously reported MeS-D-KYN) and high selectivity, as it was not cross-reactive towards LAO, while 2-MePh-L-KYN was also converted into 2-MePh-KYNA by LAO. Furthermore, the 2-MePh-D-KYN probe successfully detected DAO in eel liver, kidney, and heparin-anticoagulated plasma in the in vitro study.

Analyzing Citramalic Acid Enantiomers in Apples and Commercial Fruit Juice by Liquid Chromatography-Tandem Mass Spectrometry with Pre-Column Derivatization.

Optically active citramalic acid (CMA) is naturally present as an acidic taste component in fruits, such as apples. The absolute configuration of CMA in such fruits was investigated by high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) following pre-column derivatization with a chiral reagent, benzyl 5-(2-aminoethyl)-3-methyl-4-oxoimidazolidine-1-carboxylate. The developed LC-MS/MS method successfully separated the enantiomers of CMA using an octadecylsilica column with a resolution and separation factor of 2.19 and 1.09, respectively. Consequently, the R-form of CMA was detected in the peel and fruit of three kinds of apple at concentrations in the 1.24-37.8 and 0.138-1.033 mg/wet 100 g ranges, respectively. In addition, R- CMA was present in commercial apple juice, whereas no quantity was detected in commercial blueberry, perilla, or Japanese apricot juice.

Determination of d- and l-Amino Acids in Garlic Foodstuffs by Liquid Chromatography-Tandem Mass Spectrometry.

Black garlic is currently attracting interest as a health food and constituent of commercial supplements; however, no data regarding the d-amino acids within black garlic have been reported. Therefore, the amino acid compositions of methanol extracts from fresh and black garlic were compared herein. We investigated the contents of the d- and l-forms of amino acids in commercial fresh, black, and freeze-dried garlic foodstuffs by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a pre-column chiral derivatization reagent, succinimidyl 2-(3-((benzyloxy)carbonyl)-1-methyl-5-oxoimidazolidin-4-yl) acetate. Several d-amino acids, namely, the d-forms of Asn, Ala, Ser, Thr, Glu, Asp, Pro, Arg, Phe, Orn, Lys, and Tyr, were observed in the methanol extract of black garlic, whereas only d-Ala was detected in that of fresh garlic foodstuffs. These data suggest that several d-amino acids can be produced during fermentation for preparing black garlic.

Direct Fluorescence Evaluation of d-Amino Acid Oxidase Activity Using a Synthetic d-Kynurenine Derivative.

d-Amino acid oxidase (DAO) has been suggested to be associated with the central nervous system diseases, such as schizophrenia. We newly synthesized a nonfluorescent 5-methylthio-d-kynurenine (MeS-d-KYN), which was converted to blue-fluorescent 6-MeS-kynurenic acid (MeS-KYNA, λex = 364 nm, λem = 450 nm) through a one-step reaction by incubation with DAO. It was revealed that fluorescence intensity increased accompanied by commercial porcine kidney DAO activity (unit) with a good correlation (R2 = 0.9972), suggesting that the fluorometric evaluation of DAO activity using MeS-d-KYN is feasible. MeS-d-KYN was applied to fluorescent DAO imaging in cultured LLC-PK1 cells, and the blue fluorescence of MeS-KYNA overlapped considerably with the location of peroxisomes, which was suggested to be the location of DAO in the cells. Because fluorescence was diminished in the presence of 6-chloro-1,2-benzisoxazol-3(2H)-one (CBIO), a DAO inhibitor, it was considered that DAO activity in cells could be directly evaluated using MeS-d-KYN as the substrate.

Imatinib induces diastolic dysfunction and ventricular early-repolarization delay in the halothane-anesthetized dogs: Class effects of tyrosine kinase inhibitors.

Imatinib has been reported to induce heart failure and/or QTc prolongation. To better understand their underlying mechanisms, we assessed its effects on cardiohemodynamic, electrocardiographic and echocardiographic variables along with biomarkers of myocardial damage. Imatinib mesylate in doses of 1 and 10 mg/kg was intravenously administered to the halothane-anesthetized beagle dogs (n = 4). Effects of imatinib on each phase of isovolumetric contraction, ejection, isovolumetric relaxation and filling were studied, whereas its electrophysiological effects on early and late repolarization were analyzed by measuring J-Tpeak and Tpeak-Tend, respectively. The low and high doses of imatinib provided peak plasma concentrations of 3.23 and 17.39 µg/mL, reflecting clinically-relevant and supratherapeutic concentrations, respectively. Neither lethal ventricular tachyarrhythmia nor cardiohemodynamic collapse was observed. Imatinib decreased amplitude of peak -dP/dt, indicating suppression of isovolumetric relaxation, whereas no significant change was detected in the other phases. Imatinib prolonged QTc and J-Tpeakc without altering Tpeak-Tend, indicating increase of net inward current, which leads to intracellular Ca2+ overload. Thus, imatinib suppressed ventricular active relaxation and early repolarization, which may suggest the association of mitochondrial dysfunction-associated inhibition of ATP production. Since those findings were also reported for dasatinib, sunitinib and lapatinib, they could be common cardiac phenotype of tyrosine kinase inhibitors in vivo.

Simultaneous analyses of hemodynamic and electrophysiological effects of oseltamivir along with its pharmacokinetic profile using the canine paroxysmal atrial fibrillation model.

Since information of antiviral drug oseltamivir on the anti-atrial fibrillation (AF) property is still limited, we assessed it using the canine paroxysmal AF model. Oseltamivir in doses of 3 and 30 mg/kg/10 min was intravenously infused to the isoflurane-anesthetized, chronic atrioventricular block dogs (n = 6) with monitoring hemodynamic and electrophysiological variables, in which AF was induced by 10 s of burst pacing on atrial septum. Oseltamivir decreased AF incidence and AF duration, and prolonged AF cycle length in a dose-dependent manner. The low and high doses attained the peak plasma drug concentrations of 9.7 and 96.5 µg/mL, which were approximately 100 and 1000 times greater than those observed in human clinical cases, respectively. The low dose of oseltamivir decreased mean blood pressure without altering sinoatrial or idioventricular rate, whereas its high dose reduced each of them. Oseltamivir delayed inter-atrial conduction in dose- and frequency-dependent manners, whereas it prolonged atrial effective refractory period in dose-dependent but frequency-independent manners. The high dose prolonged ventricular effective refractory period, which was not detected with the low dose. These findings can be used for repurposing oseltamivir as an anti-AF drug candidate.

A review of chromatographic methods for bioactive tryptophan metabolites, kynurenine, kynurenic acid, quinolinic acid, and others, in biological fluids.

Kynurenine (KYN) is synthesized from an essential amino acid, tryptophan, by tryptophan 2,3-dioxygenase or indoleamine 2,3-dioxygenase via N-formyl-KYN in vivo. Subsequently, KYN acts as a precursor of some neuroactive metabolites such as kynurenic acid, quinolinic acid, and an important enzyme co-factor, nicotine adenine dinucleotide. These metabolites of tryptophan are a part of the 'kynurenine pathway.' In addition, KYN functions as an endogenous ligand for the aryl hydrocarbon receptor, which acts as a transcription factor. The levels of tryptophan metabolites are important for the assessment of the stage of neurological disorders, and therefore have garnered significant interest for clinical diagnosis. In this review, the detection of kynurenine, kynurenic acid, quinolinic acid, and other tryptophan metabolites performed via chromatographic methods such as HPLC using UV absorbance, fluorescence, and chromatographic-mass spectrometric detection is summarized.

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.

Increased Indoleamine 2,3-Dioxygenase Levels at the Onset of Sjögren's Syndrome in SATB1-Conditional Knockout Mice.

Sjögren's syndrome (SS) is a chronic autoimmune disease characterized by dysfunction of salivary and lacrimal glands, resulting in xerostomia (dry mouth) and keratoconjunctivitis sicca (dry eyes). Autoantibodies, such as anti-SSA and anti-SSB antibodies, are hallmarks and important diagnostic factors for SS. In our previous study, we demonstrated that SS-like xerostomia was observed in SATB1 conditional knockout (SATB1cKO) mice, in which the floxed SATB1 gene was specifically deleted in hematopoietic cells as early as 4 weeks of age. In these mice, autoantibodies were not detected until 8 weeks of age in SATB1cKO mice, although exocrine gland function reached its lowest at this age. Therefore, other markers may be necessary for the diagnosis of SS in the early phase. Here, we found that mRNA expression of the interferonγ (IFN-γ) gene and the IFN-responsive indoleamine 2,3-dioxygenase (IDO) gene is upregulated in the salivary glands of SATB1cKO mice after 3 and 4 weeks of age, respectively. We detected l-kynurenine (l-KYN), an intermediate of l-tryptophan (l-Trp) metabolism mediated by IDO, in the serum of SATB1cKO mice after 4 weeks of age. In addition, the upregulation of IDO expression was significantly suppressed by the administration of IFN-γ neutralizing antibodies in SATB1cKO mice. These results suggest that the induction of IFN-dependent IDO expression is an initial event that occurs immediately after the onset of SS in SATB1cKO mice. These results also imply that serum l-KYN could be used as a marker for SS diagnosis in the early phases of the disease before autoantibodies are detectable.

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.

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.

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).

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.

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.

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.

Anti-proliferative effect of Fe(III) complexed with 1-(2-hydroxy-3-methoxybenzaldehyde)-4-aminosalicylhydrazone in HepG2 cells.

We previously developed a chelating ligand, 1-(2-hydroxy-3-methoxybenzaldehyde)-4-aminosalicylhydrazone (HMB-ASH), which can chelate Fe(III) to form a complex. The HMB-ASH-Fe(III) complex exhibits a dose-dependent anti-proliferative effect in HepG2 cells, whereas the ligand, HMB-ASH, and Fe(III) alone had no considerable effect. The HMB-ASH-Fe(III) complex was composed of Fe(III):HMB-ASH (1:2), as determined by high-performance liquid chromatography with high-resolution mass spectrometry. The IC50 value was approximately 20 µM, which was comparable to those of the anti-cancer drugs oxaliplatin (OXP) and etoposide (ETP) under the same conditions. Similar to OXP and ETP, HMB-ASH-Fe(III) induced apoptosis in HepG2 cells, as revealed by terminal deoxynucleotidyl transferase fluorescein-12-dUTP nick end labeling assay.

Deletion of SHATI/NAT8L increases dopamine D1 receptor on the cell surface in the nucleus accumbens, accelerating methamphetamine dependence.

In a previous report, we identified a novel molecule, SHATI/NAT8L, having an inhibitory effect on methamphetamine (METH)-induced hyperlocomotion, sensitization, and conditioned place preference (CPP). SHATI/NAT8L attenuates the METH-induced increase in dopamine overflow in the nucleus accumbens (NAc) by promoting plasmalemmal and vesicular dopamine uptake. However, the biological functions of the protein remain unclear. In this study, we explored NAT8L-binding proteins using pull-down assays and identified a number of components of the adaptor protein (AP)-2 complex, which is a multimeric protein localized to the plasma membrane that functions to internalize cargo during clathrin-mediated endocytosis. To investigate whether NAT8L regulates the receptor localization to the cell surface, cell-surface dopamine D1 receptor in the NAc of Nat8l knockout (KO) mice was quantified. We found that dopamine D1 receptor on the cell surface was increased in the NAc of Nat8l KO mice compared with the wild type (WT) animals. Consistent with this finding, Nat8l KO mice showed higher basal locomotor activity and heightened sensitivity to D1 agonist compared with WT mice. In addition, METH-induced sensitization and CPP were enhanced in Nat8l KO mice. These results suggest that NAT8L might regulate the localization of cell-surface dopamine D1 receptor, thereby controlling basal behaviour and sensitivity to METH. Furthermore, we observed a single nucleotide polymorphism (SNP) in the human NAT8L gene related to reward dependence, a personality trait, and grey matter volume in the caudate nucleus in healthy subjects, suggesting that NAT8L might also affect human personality.

Enantiomeric separation of tolperisone and eperisone by reversed-phase HPLC with cellulose tris(3-chloro-4-methylphenylcarbamate)-coated chiral column.

Enantiomeric separations of centrally acting muscle relaxants, that is, tolperisone (TOL) and eperisone (EP), that are marketed as racemates were investigated by reversed-phase high-performance liquid chromatography (HPLC) on a polysaccharide-based chiral column. Both TOL and EP are basic drugs because they contain a tertiary amino group and have similar chemical structures with the exception of the p-methylphenyl and p-ethylphenyl groups in TOL and EP, respectively. A reversed-phase chiral column, that is, a Chiralcel OZ-RH column, which bears cellulose tris(3-chloro-4-methylphenylcarbamate) as the chiral moiety, was effective for the enantiomeric separation of TOL and EP enantiomers. The separation factor and resolution values obtained for TOL were 1.22 and 1.66, respectively, and those for EP were 1.21 and 2.24, respectively, using a 20 mm ammonium acetate in H2 O (pH 8.0 and 7.0, respectively)-CH3 CN (70:30) mobile phase. Using the proposed HPLC conditions, it was found that (R)-TOL eluted faster than (S)-TOL, as revealed by the optical rotation and circular dichroism spectroscopy. In contrast, EP was easily racemized under the experimental conditions, and hence, the elution order was not determined.

HPLC-fluorescence determination of thiol compounds in the serum of human male and female subjects using HILIC-mode column.

High-performance liquid chromatography-fluorescence detection using a hydrophilic interaction chromatography-mode column (ZIC®-HILIC) was used to determine four kinds of thiol compounds in human serum. Sera were obtained from 34 subjects for this study (17 male subjects aged 22-38 years and 17 female subjects aged 18-38 years). Serum cysteine, cysteinylglycine, glutathione, and γ-glutamylcysteine, derivatized with ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate, were separated on the ZIC®-HILIC column and quantified. The serum concentrations of cysteine, cysteinylglycine, glutathione and γ-glutamylcysteine were 226 ± 4.7, 23.4 ± 1.3, 3.7 ± 0.2 and 3.2 ± 0.1 µm, respectively. In addition, the concentrations of serum thiol compounds from male subjects were significantly higher than those of the female subjects (p < 0.05).