Constituents of Chimaphila japonica and Their Diuretic Activity.

Three new phenols (1-3), one new cyclohexanol (4), two known phenols (5-6), and six known flavonoids (7-12) were isolated from the n-butanol of the 75% ethanol extract of all plants of Chimaphila japonica Miq. Among them, compound 5 was named and described in its entirety for the first time, and compounds 9 and 10 were reported in C. japonica for the first time. The structures of all compounds were confirmed using a comprehensive analysis of 1D and 2D NMR and HRESIMS data. Biological results show that compounds 4, 7, and 11 exhibited potent diuretic activity. The modes of interaction between the selected compounds and the target diuretic-related WNK1 kinase were investigated in a preliminary molecular docking study. These results provided insight into the chemodiversity and potential diuretic activities of metabolites in C. japonica.

A duplex-specific nuclease (DSN) and catalytic hairpin assembly (CHA)-mediated dual amplification method for miR-146b detection.

A novel method for detecting miRNA has been developed using a combination of duplex-specific nuclease signal amplification (DSNSA) and a catalytic hairpin assembly (CHA). In this work, a biotinylated trigger release (BTR) probe with a biotin group at the 3'-end and a CHA reaction sequence trigger as an initiator (catalyst I) at the 5'-end was designed to hybridize target miRNA. The DSN enzyme was introduced to initiate the DSNSA. The miRNA was released to consume more BTR probes and amplify the signals. Subsequently, streptavidin-coated magnetic beads (SA-MBs) were added to the DSNSA reaction solution to remove excess BTR probes that did not hybridize with miRNA, which would then separate BTR probes and catalyst-I, to ensure detection with high selectivity and sensitivity. The catalyst-I remaining in the solution could trigger the CHA reaction to enable signal amplification in the second step. The developed method exhibits a sensitive detection limit and excellent selectivity in identifying a high sequence homology among family members.

Evaluation of chiral separation efficiency of a novel OTPTHE derivatization reagent: Applications to liquid-chromatographic determination of DL-serine in human plasma.

A novel chiral derivatization reagent, the N-[1-oxo-5-(triphenylphosphonium)pentyl]- (R)-1,3-thiazolidinyl-4-N-hydroxysuccinimide ester bromide salt (OTPTHE), was developed for the separation and selective detection of chiral DL-amino acids by RP-HPLC analysis. The OTPTHE reacted with DL-amino acids at 60°C maintained for 30 minutes in the presence of 100 mM borate buffer (pH 9.5). The separability of the diastereomeric derivatives was evaluated in terms of the resolution value (Rs) using 13 kinds of DL-amino acids, which were completely separated by reversed-phase chromatography using C18 column at 254 nm. The Rs of the DL-amino acids varied from 1.62 to 2.51. As for the application of the DL-amino acids, the determination of DL-Ser in the human plasma of healthy volunteers was performed based on our developed method. It was shown that linear calibrations were available with high coefficients of correlation (r2 > 0.9997). The limit of detection (S/N = 3) of the DL-Ser enantiomers was 5.0 pmol; the relative standard deviations of the intraday and interday variations were below 4.56%; the accuracy ranged between 95.40%-110.06% and 95.45%-109.80%, respectively; the mean recoveries (%) of the DL-Ser spiked in the human plasma were 99.49%-103.74%. The amounts of DL-Ser in the human plasma of healthy volunteers were determined.

Synthesis and Evaluation of 3-Substituted-4-(quinoxalin-6-yl) Pyrazoles as TGF-ß Type I Receptor Kinase Inhibitors.

The transforming growth factor-ß (TGF-ß), in which overexpression has been associated with various diseases, has become an attractive molecular target for the treatment of cancers. Thirty-two quinoxaline-derivatives of 3-substituted-4-(quinoxalin-6-yl) pyrazoles 14a⁻d, 15a⁻d, 16a⁻d, 17a⁻d, 18a⁻d, 19a⁻d, 25a, 25b, 25d, 26a, 26b, 26d, 27b, and 27d were synthesized and evaluated for their activin TGF-ß type I receptor kinase and p38α mitogen activated protein (MAP) kinase inhibitory activity in enzymatic assays. Among these compounds, the most active compound 19b inhibited TGF-ß type I receptor kinase phosphorylation with an IC50 value of 0.28 µM, with 98% inhibition at 10 µM. Compound 19b also had good selectivity index of >35 against p38α MAP kinase, with 9.0-fold more selective than clinical candidate, compound 3 (LY-2157299). A molecular docking study was performed to identify the mechanism of action of the synthesized compounds and their good binding interactions were observed. ADMET prediction of good active compounds showed that these ones possess good pharmacokinetics and drug-likeness behavior.

The great importance of normalization of LC-MS data for highly-accurate non-targeted metabolomics.

The non-targeted metabolomics analysis of biological samples is very important to understand biological functions and diseases. LC combined with electrospray ionization-based MS has been a powerful tool and widely used for metabolomic analyses. However, the ionization efficiency of electrospray ionization fluctuates for various unexpected reasons such as matrix effects and intraday variations of the instrument performances. To remove these fluctuations, normalization methods have been developed. Such techniques include increasing the sensitivity, separating co-eluting components and normalizing the ionization efficiencies. Normalization techniques allow simultaneously correcting of the ionization efficiencies of the detected metabolite peaks and achieving quantitative non-targeted metabolomics. In this review paper, we focused on these normalization methods for non-targeted metabolomics by LC-MS.

Dried Saliva Spot (DSS) as a Convenient and Reliable Sampling for Bioanalysis: An Application for the Diagnosis of Diabetes Mellitus.

This paper proposes the dried saliva spot (DSS) as a convenient sampling technique for bioanalysis. The analytical method with the DSS was used for the determination of D,L-lactic acid (D,L-LA) and the D/L ratio of diabetic patients and prediabetic persons for the simple screening of the disease. The D,L-LA in the DSS was labeled with a chiral reagent (DMT-3(S)-Apy) for carboxylic acids and determined by UPLC-ESI-MS/MS. The limits of detection (signal-to-noise ratio (S/N) = 3) for the DSS analysis were on the amol level (∼30 amol). Because good stability, recovery, accuracy, and precision of the D,L-LA for the DSS method was also obtained from the proposed procedure, the DSS method was applied to the determination of the D- and L-isomers of LA of diabetic patients, and prediabetic and healthy persons. The D/L-LA ratio by the present DSS method and the HbA1c value in blood were well-correlated to the serious diabetic patients, whereas the relation in the prediabetic persons was not very good. The reason seems to be due to the rough saliva sampling, and not to the DSS method, because strict regulation was not requested for the prediabetic and healthy persons. In order to have a successful DSS analysis, the stability of the target molecule, the detection sensitivity to the target molecule, and the validated determination method are important.

Stable isotope dilution HILIC-MS/MS method for accurate quantification of glutamic acid, glutamine, pyroglutamic acid, GABA and theanine in mouse brain tissues.

In this study, we developed the stable isotope dilution hydrophilic interaction liquid chromatography with tandem mass spectrometry (HILIC-MS/MS) technique for the accurate, reasonable and simultaneous quantification of glutamic acid (Glu), glutamine (Gln), pyroglutamic acid (pGlu), γ-aminobutyric acid (GABA) and theanine in mouse brain tissues. The quantification of these analytes was accomplished using stable isotope internal standards and the HILIC separating mode to fully correct the intramolecular cyclization during the electrospray ionization. It was shown that linear calibrations were available with high coefficients of correlation (r(2) > 0.999, range from 10 pmol/mL to 50 mol/mL). For application of the theanine intake, the determination of Glu, Gln, pGlu, GABA and theanine in the hippocampus and central cortex tissues was performed based on our developed method. In the region of the hippocampus, the concentration levels of Glu and pGlu were significantly reduced during reality-based theanine intake. Conversely, the concentration level of GABA increased. This result showed that transited theanine has an effect on the metabolic balance of Glu analogs in the hippocampus.

Uric acid quantification in fingernail of gout patients and healthy volunteers using HPLC-UV.

The presence of elevated uric acid (UA) levels is a sign of gout, that is, hyperuricemia. In this study the monitoring of the UA levels in less-invasive biological samples, such as the human fingernail, is suggested for the diagnosis and therapy of gout. Twenty-six healthy volunteers (HV) and 22 gout patients (GP) were studied. The UA was extracted from human fingernail samples, then separated on an Inertsil ODS-3 column (250 × 4.6 mm i.d., 4.0 µm, GL Sciences) by isocratic elution using methanol-74 mm phosphate buffer (pH 2.2) 2:98 (v/v). A UV detector was used to monitor the samples at 284 nm. Using the developed method, different UA concentrations were found in the GP and HV. When comparing the concentrations from GP with those from HV, a statistically significant correlation was observed between the UA (p < 0.01). In this study, the UA was confirmed as a potential biomarker for the diagnosis and therapy of gout. We have developed a novel sensitive, and simple method for the determination of UA in the fingernails of GP and HV. The human fingernail may serve as a noninvasive biosample for the diagnosis of gout. Copyright © 2016 John Wiley & Sons, Ltd.

Principal component analysis of molecularly based signals from infant formula contaminations using LC-MS and NMR in foodomics.

BACKGROUND: The challenge in developing analytical assessment of unexpected excess contaminations in infant formula has been the most significant project to address the widespread issue of food safety and security. Foodomics based on metabolomics techniques provides powerful tools for the detection of tampering cases with intentional contaminations. However, the safety and risk assessments of infant formula to reveal not only the targeted presence of toxic chemicals, but also molecular changes involving unexpected contaminations, have not been reported. In this study, a huge amount of raw molecularly based signals from infant formula was analysed using reversed phase and hydrophilic interaction chromatography with time-of-flight MS (LC-MS) and (1) H nuclear magnetic resonance (NMR) and then processed by a principal component analysis (PCA). RESULTS: PCA plots visualised signature trends in the complex signal-data batches from each excess contamination of detectable chemicals by LC-MS and NMR. These trends in the different batches from a portion of excess chemical contaminations such as pesticides, melamine and heavy metals and out-of-date products can be visualised from spectrally discriminated infant formula samples. CONCLUSION: PCA plots provide possible attempts to maximise the covariance between the stable lot-to-lot uniformity and excess exogenous contaminations and/or degradation to discriminate against the molecularly based signals from infant formulas. © 2015 Society of Chemical Industry.

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

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

Automatic analyzer for highly polar carboxylic acids based on fluorescence derivatization-liquid chromatography.

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.

Evaluation of a series of prolylamidepyridines as the chiral derivatization reagents for enantioseparation of carboxylic acids by LC-ESI-MS/MS and the application to human saliva.

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.

Evaluation of gardenia yellow using crocetin from alkaline hydrolysis based on ultra high performance liquid chromatography and high-speed countercurrent chromatography.

Gardenia yellow is globally the most valuable spice and food color. It is generally a mixture of water-soluble carotenoid glycosyl esters which consist of crocetin bis(gentiobiosyl) ester as the main component. Crocetin is a natural carotenoid dicarboxylic acid that may be a candidate drug for pharmaceutical development, however, it is either present in trace amounts or is absent in natural gardenia yellow products. We here propose that crocetin produced by alkaline hydrolysis can be used to qualitatively evaluate gardenia yellow products using an ultra high performance liquid chromatographic assay. A useful and efficient isolation technique for isolating high-purity crocetin from gardenia yellow using high-speed countercurrent chromatography is described. High-speed countercurrent chromatographic fractionation followed by an ultra high performance liquid chromatographic assay showed that trans-crocetin is easily converted to about 15% cis-crocetin (85% trans-crocetin). Crocetin in gardenia yellow was quantitatively evaluated. Our approach is based on the hydrolysis process for converting crocetin glycosyl esters to crocetin before evaluation and isolation using the ultra high performance liquid chromatographic and high-speed countercurrent chromatographic methods. The combination of hydrolysis and chromatographic methods allows evaluation of the purity and quantity of crocetin in gardenia yellow.

Rapid and sensitive determination of diacetylpolyamines in human fingernail by ultraperformance liquid chromatography coupled with electrospray ionization tandem mass spectrometry.

A rapid and sensitive ultraperformance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) method has been developed and validated for quantitatively determining diacetylpolyamines in the human fingernail. N(1),N(8)-diacetylspermidine (DiAct-Spd), N(1),N(12)- diacetylspermine (DiAct-Spm) and 1,6-diaminohexane (DAH) the [internal standard (IS)] were extracted from human fingernail samples by MeOH: 5 M HCl solution, followed by 4-(N,N-dimethylaminosulfonyl)-7-fluoro- 2,1,3-benzoxadiazole (DBD-F) derivatization, and then separated on an ACQUITY BEH C18 column with a gradient elution of acetonitrile and water containing 0.1% formic acid. The derivatives of the diacetylpolyamines were fully separated within a short run time (3.0 min). The triple quadrupole mass spectrometric detection was performed in the multiple reactions monitoring (MRM) mode by the UPLC-ESI- MS/MS system in the positive ionization mode. MRM using the fragmentation transitions of m/z 455.20→ 100.07, 737.25 → 100.07 and 567.10 → 479.07 in the positive ESI mode was performed to quantify DiAct-Spd, DiAct-Spm and IS, respectively. The calibration curve is between 0.04 ng mL(-1) for DiAct-Spd and DiAct-Spm. The detection limits (signal to noise ratio of five) were 5-10 pg mL(-1). A good linearity was achieved from the calibration curves (r(2) >0.9999), and the intra-day and inter-day assay precisions were less than 7.06%. Furthermore, the recoveries (%) of the diacetylpolyamines spiked in the human fingernails were 79.18-97.11. The present method proved that the high sensitivity is characterized by the specificity and feasibility of the sample analysis. Consequently, the proposed method was used to analyze human fingernail samples from 15 lung- cancer patients and 22 healthy volunteers. Diacetylpolyamines were detected from the fingernails of the lung- cancer patients for the first time. The concentration of DiAct-Spd in the lung-cancer patient group tended to be higher than those in the healthy volunteers.

A quantitative analysis of the polyamine in lung cancer patient fingernails by LC-ESI-MS/MS.

A quantitative analysis of polyamines in lung cancer patient fingernails by the combination of 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole derivatives and liquid chromatography-electrospray ionization tandem mass spectrometry is described. The reaction of the reagent with eight kinds of polyamines, that is, N(1) -acetylputrescine (N(1) -actPUT), N(8) -acetylspermidine, N(1) -acetylspermine, 1,3-diaminopropane, putrescine (PUT), cadaverine, spermidine and spermine (SPM) effectively occurs at 60 °C for 30 min. The detection limits (signal-to-noise ratio 5) were 5-100 fmol. A good linearity was achieved from the calibration curves, which was obtained by plotting the peak area ratios of the analytes relative to the internal standard (IS), that is, 1,6-diaminohexane, vs the injected amounts of polyamines (r(2) > 0.996), and the intra-day and inter-day assay precisions were <9.84%. Furthermore, the recoveries (%) of the polyamines spiked in the human fingernails were 89.14-110.64. The present method was applied to human fingernail samples from 17 lung cancer patients and 39 healthy volunteers. The polyamine concentration was different based on the gender, that is, the N(1) -actPUT and PUT contents were 3.10 times and 2.56 times higher in healthy men than in women, respectively. Additionally, in the lung cancer patient group, as compared with the healthy volunteers, the concentrations of SPM had a statistically significant (p < 0.05) correlation. Therefore, because the proposed method provides a good mass accuracy and the trace detection of the polyamines in human fingernails, this analytical technique could be a noninvasive technique to assist in the diagnosis and assessment of disease activity in lung cancer patients.

UPLC/ESI-MS/MS-based determination of metabolism of several new illicit drugs, ADB-FUBINACA, AB-FUBINACA, AB-PINACA, QUPIC, 5F-QUPIC and α-PVT, by human liver microsome.

The metabolism by human liver microsomes of several new illicit drugs, that is, N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-(4-fluorobenzyl)-1H-indazole-3- carboxamide (ADB-FUBINACA), N-(1-amino-3-methyl-1-oxobutan-2-yl)-1- (4-fluorobenzyl)-1H-indazole-3-carboxamide (AB-FUBINACA), N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-pentyl-1H-indazole-3-carboxamide (AB-PINACA), quinolin-8-yl 1-pentyl-(1H-indole)-3-carboxylate (QUPIC), quinolin-8-yl 1-(5-fluoropentyl)-(1H-indole)-3-carboxylate (5 F-QUPIC) and α-pyrrolidinovalerothiophenone (α-PVT), which have indole, indazole, quinolinol ester and thiophene structures, was investigated using reversed-phase chromatography and mass spectrometry. The present method is based upon the oxidation by cytochrome p450 superfamily enzymes in the microsomes. The oxidation of ADB-FUBINACA and AB-FUBINACA mainly occurred on the N-(1-amino-alkyl-1-oxobutan) moiety. However, the oxidation of AB-PINACA seemed to occur on the 1-pentyl moiety. On the other hand, QUPIC and 5 F-QUPIC, which have a quinolinol ester structure, predominantly underwent a cleavage reaction to produce indoleacetic acid type metabolites. In contrast, the metabolism reaction of α-PVT was different from that of the other tested drugs, and various oxidation products were observed on the chromatograms. The obtained metabolites are not in conflict with the results predicted by MetaboLynx software. However, the exact structures of the metabolites, except for 1-pentyl-1H-indole-3-carboxylic acid (QUPIC metabolite) and 1-(5-fluoropentyl)-1H-indole-3-carboxylic acid (5 F-QUPIC metabolite), are currently not proven, because we have no authentic compounds for comparison. The proposed approach using human liver microsome seems to provide a new technology for the prediction of possible metabolites occuring in humans.

Relative quantitation of chiral thiol compounds labeled based on isotope novel mass spectrometry probes: Monitoring of the dynamic changes of chiral thiol compounds in human urine during normal, exercise, and rest recovery states.

Monitoring changes in the content of chiral thiol compounds in the human body is crucial for the early diagnosis of oxidative stress-related diseases and the exploration of their pathogenesis. To address this, we synthesized a novel isotope mass spectrometry (MS) probe, denoted as (R)-(5-(3-isothiocyanato (13C) pyrrolidin-1-yl)-5-oxopentyl) triphenylphosphonium (N13CS-OTPP), with triphenylphosphine as its parent structure. In this study, we established a new ultra-high-performance liquid chromatography high-resolution mass spectrometry (UHPLCHRMS) relative quantitative method to monitor chiral thiol compounds in human urine under varying oxidative stress conditions. This method relies on the ratio of 12C/13C isotope-labeled peak areas. To assess the chiral separation efficiency of N13CS-OTPP, we employed three types of thiol compounds (D/L-GSH, D/L-Cys, and D/L-Hcy) and observed separation degrees (Rs) ranging from 1.82 to 1.89. We further validated the accuracy and feasibility of our relative quantitative methods using D/L-Cys-as a model compound. N12C/13CS-OTPP-Cys-exhibited excellent linearity (R2 = 0.9993-0.9994) across different molar ratios (D/L-Cys = 10:1, 4:1, 2:1, 1:1, 1:2, 1:4, 1:10) and achieved a low limit of detection (LOD) of 2.5 fmol. Additionally, we monitored the dynamic changes in urine D/L-Cys-and D/L-Hcy ratios in 12 healthy volunteers (six males and six females) under various oxidative stress states. We generated fitting curves and investigated the trends in chiral thiol compounds in vivo. This study introduces a novel method for the relative quantitative monitoring of chiral thiol compounds in different oxidative stress states within the human body. It also presents a new strategy for understanding the pathogenesis of related diseases resulting from the abnormal metabolism of thiol compounds.

Simultaneous analysis of natural and artificial sweeteners in sugar-free drinks and urine samples by column-switching UHPLC-charged aerosol detection method.

Sweeteners are considered an alternative to high-calorie foods or drinks and have been widely used globally. However, the simultaneous separation and detection of high-polarity natural and artificial sweeteners are challenging owing to their broad-spectrum physical and chemical properties. Herein, we developed a column-switching UHPLCCAD method and used it for detecting and quantitating 12 sweeteners, including natural sweeteners (erythritol, mannitol, xylitol, sorbitol and stevioside) and artificial sweeteners (acesulfame potassium, saccharin sodium salt, sodium cyclamate, sucralose, aspartame, alitame and neotame). The LOD and LOQ were 0.932-6.25 µg/mL and 3.10-20.83 µg/mL, respectively, and the method demonstrated excellent linearity (R² ≥ 0.9990), good precision (intraday and interday precision was 0.59-6.88 %), and high recovery (average recoveries were 85.16-108.64 %). This method was applied to determine the sweeteners in 15 sugar-free drinks purchased from the local Chinese supermarkets. What's more, natural sweetener erythritol and artificial sweetener acesulfame potassium were suspected over addition in sugar-free drinks. Meanwhile the method was applied to the sweeteners in various sugar-free drinks and the dynamic monitoring of transit and excretion in vivo after drinking. Those prove that the method can be used to the detection of sugar free drinks and quality control of the sweeteners. The study highlights the potential of UHPLC-charged aerosol detection technology in detection of multiple components in food industry.

Development of an on-site therapeutic drug monitoring method using a portable spectrometer.

We report on the development of an on-site therapeutic drug monitoring (TDM) method for vancomycin (VCM) utilizing a portable spectrometer and commercially available immunoturbidimetric assay reagents designed for automated clinical chemistry analyzers. The method enables the quantification of VCM in plasma samples within 10 min, with a good correlation between the measured values and the theoretical values (r2 = 0.995). The intra and inter-day precisions were found to be below 12.5% and 17.7%, respectively. Moreover, we established a correlation between the quantitative values using this method and those measured through HPLC-UV and automated clinical chemistry analyzers, showing good reliability (R2 = 0.970 and 0.951, respectively). This method allows anyone to rapidly perform TDM at the bedside and is expected to be used to evaluate appropriate drug therapy.

Synthesis and anti-liver fibrosis activity of imidazole and thiazole compounds containing amino acids.

Four series of imidazoles (15a-g, 20c, and 20d) and thiazoles (18a-g, 22a, and 22b) possessing various amino acids were synthesized and evaluated for activin receptor-like kinase 5 (ALK5) inhibitory activities in an enzymatic assay. Among them, compounds 15g and 18c showed the highest inhibitory activity against ALK5, with IC50 values of 0.017 and 0.025 µM, respectively. Compounds 15g and 18c efficiently inhibited extracellular matrix (ECM) deposition in TGF-ß-induced hepatic stellate cells (HSCs), and eventually suppressed HSC activation. Moreover, compound 15g showed a good pharmacokinetic (PK) profile with a favorable half-life (t1/2 = 9.14 h). The results indicated that these compounds exhibited activity targeting ALK5 and may have potential in the treatment of liver fibrosis; thus they are worthy of further study.