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.

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.

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.

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.

Stable isotope labeling differential glycans discovery in the serum of acute myocardial infarction by ultrahigh-performance liquid chromatography-quadrupole-Orbitrap high resolution mass spectrometry.

Acute myocardial infarction (AMI) poses a grave threat to human life. However, most clinical biomarkers have limitations of low sensitivity and specificity. Therefore, screening novel glycan biomarkers with high sensitivity and specificity is crucial for the prevention and treatment of AMI. The novel method of ultrahigh-performance liquid chromatography coupled to quadrupole-Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) with d0/d5-BOTC probe labeling for relative quantification of glycans based on Pronase E digestion was established to screen novel glycan biomarkers in the serum of 34 AMI patients relative to healthy volunteers. The monosaccharide model D-glucosamine was used to investigate the effectiveness of the derivatization; the limit of detection (S/N = 3) was 10 amol. The accuracy was verified based on the consistency of different theoretical molar ratios (d0/d5 = 1:2, 2:1) and intensity ratios following digestion of glycoprotein ribonuclease B. Expressions of H4N4F3SA, H4N6F2, H4N6SA, H4N6F3 and H5N4FSA in the serum were significantly different (p < 0.0005) between AMI patients and healthy volunteers. The area under the receiver operating characteristic curve (AUC) for H4N6SA, H5N4FSA, and H4N6F2 was greater than 0.9039. Based on the proposed method, H4N6SA, H5N4FSA, and H4N6F2 in human serum showed high accuracy and specificity and may serve as potential glycan biomarkers, crucial for the diagnosis and treatment monitoring of AMI.

Development and evaluation of a novel fluorescent chiral derivatization reagent DBD-S-M-Pro: first observation of four chiral amino acids in human hair.

This study reports a novel fluorescent chiral derivatization reagent, 4-(N,N-dmethylaminosulfonyl)-2,1,3-benzoxadiazole-(2-succinimidoxy)-trans-2-methyl-L-proline (DBD-S-M-Pro), with a benzoxadiazole structure containing an N-hydroxysuccinimide activation group. DBD-S-M-Pro targets chiral amino-functional compounds under alkaline conditions without a condensation agent. Gradient elution was performed on a BEH C18 (100 × 2.1 mm, 1.7 µm) column with a mobile phase of 0.05% formic acid (FA) in 10 mM ammonium acetate (CH3COONH4) and 0.1% FA in acetonitrile or methanol. The efficiency of the chiral resolution was evaluated under excitation and emission wavelengths of 450 nm and 560 nm, respectively. The 19 chiral amino acids were separated in the range of 1.45-14.84. The resolutions of almost all DL-amino acids exceeded 1.5; the exceptions were serine (Ser) and lysine (Lys), with resolutions of 1.45 and 1.46, respectively. In addition, a new approach was devised for the simultaneous analysis of four chiral amino acids (DL-Glu, DL-Ala, DL-Val, and DL-Phe) in human hair. These amino acids were analyzed in the range of 12.5-400 pmol, with R2 ≥ 0.9990, limits of detection (S/N = 3) of 4-10 pmol, and intraday and interday precisions of 0.57-6.23%. The average spikes in the hair recoveries were 89.76-111.54%, and the matrix effects were 92.47-102.40%. Next, the contents of free chiral amino acids in the hair samples of 10 healthy volunteers (five males and five females) were analyzed with this method, and the differences were compared. The developed DBD-S-M-Pro provides a novel strategy for the sensitive determination of free chiral amino acids in living organisms.

A novel UHPLC-MS/MS method for the determination of four α-dicarbonyl compounds in wine and dynamic monitoring in human urine after drinking.

α-dicarbonyl compounds (α-DCs) serve as potential biomarkers for oxidative stress-related diseases but are difficult to detect.To study the metabolism of carbonyl compounds, we developed a new mass spectrometry probe, 3-benzyl-2-oxo-4λ3-thiazolidine-4-carbohydrazide (BOTC), containing hydrazyl groups for the targeted detection of carbonyl functional groups.In a novel approach, we used BOTC pre-column derivatization with ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) to simultaneously detect four kinds of α-DCs in red wine as well as in urine after drinking. The α-DCs were completely separated (R2 ≥ 0.9995), detection was sensitive (detection limit was 12.5-50 fmol), consistent (intraday and interday precision was 0.1-5.7 %), and efficient (average recoveries were 103.3-110.2 %). The method was applied to the analysis of α-DCs in different wines and the dynamic monitoring of transit and excretion in vivo after drinking. Our novel method provides a new strategy for the detection of α-dicarbonyl compounds in red wine and dicarbonyl compounds produced in oxidative stress-related diseases.

Development of a diphenyl sulfide structure derivatization reagent for amino acid enantiomers analysis: Application of dynamic monitoring in human urine after drinking wine.

We developed a novel chiral mass spectrometry derivatization reagent (S)-(3-(4-carboxythiazolidin-3-yl)-3-oxopropyl) diphenylsulfonium (CTOD) with a positively charged sulfur-containing structure for high-sensitivity detection of the chiral resolution of amino acid enantiomers. CTOD reacted with DL-amino acids at 60oC for 60 min to generate the corresponding diastereomers, fifteen chiral amino acid-derived products were separated. Resolution (Rs) values were of the range 1.54-4.36, except Asn 1.07, achieving good separation. A highly sensitive and selective UHPLC-MS/MS method for the simultaneous determination and chiral separation of five chiral amino acids (Pro, Ala, Glu, Asp, and Phe) based on CTOD derivatization was established and applied to the detection of chiral amino acids in different wines. The diastereomeric resolution of the five amino acids was 1.71-5.42, and an excellent linear relationship was obtained in the range of 0.25-500 pmol (R2 ≥0.9993). The detection limit was 0.05-0.25 pmol. The intra- and inter-day precisions were 0.51-5.76% and 0.78-5.18%, respectively, and the average recovery was 90.03-99.99%. In addition, the metabolic concentration of chiral amino acids was monitored after drinking red wine and white wine, and the fitting curve of metabolic concentration was drawn.

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.

Monitoring of salicylic acid content in human saliva and its relationship with plasma concentrations.

Aspirin is a widely used anti-inflammatory drug. It is reported that a relationship may exist between salicylic acid content in plasma and saliva after taking aspirin. This study established a rapid, convenient, and safe method to assess salicylic acid concentration in human saliva. A novel HPLC-ultraviolet detector was used to measure salicylic acid concentrations in human saliva and plasma. A C18 reversed-phase column with an aqueous solution of 0.1% trifluoroacetic acid (TFA)-acetonitrile mobile phase was used, and drug peaks were recorded at 303 nm. Salicylic acid was completely separated in saliva and plasma. Excellent linearity and correlation (r2 ≥ 0.9999) was observed between 0.1 and 2.0 µg/mL. The detection limit (S/N = 3) was 33 ng/mL, and intra- and inter-day recoveries were 103.5-113.3% and 101.1-109.5%, respectively. Salicylic acid was measured within nine hours after administration of acetylsalicylic acid tablets. A positive correlation between salicylic acid content in saliva and plasma was found (r = 0.867, p < 0.001). The proposed method was used successfully to measure salicylic acid concentration in human saliva. Meanwhile, we explored the relationship between salicylic acid levels in plasma and saliva. Saliva might replace blood for monitoring aspirin treatment. In addition, the research provides a reference for application to saliva samples.

Relative quantitation of glycans in cetuximab using ultra-high-performance liquid chromatography-high-resolution mass spectrometry by Pronase E digestion.

Glycans play important roles in the activity and function of monoclonal antibodies (mAbs). In this study, an isotope labeling method for the relative quantitative analysis of glycans in cetuximab, a chimeric human/mouse IgG1 monoclonal antibody that specifically targets epidermal growth factor receptor, via hydrophilic interaction LC-ultra-high-performance LC-HRMS was established based on Pronase E digestion. To this aim, novel isotope MS probes, i.e., 3-benzoyl-2-oxothiazolidine-4-carboxylic acid (d0-BOTC) and 3-(2,3,4,5,6-pentadeuterio-benzoyl)-2-oxothiazolidine-4-carboxylate acid (d5-BOTC), which include a carboxyl group to target the amino functional group in glycosylamine, were developed. The nonspecific Pronase E enzyme could simultaneously digest the peptide bound to the N- and O-glycans into glycosylamine having only one amino acid. Since the mass difference between the light- and heavy-labeled glycans was 5.0 Da, the relative abundance of their MS peaks was used to achieve the qualitative and relative quantitative analysis of glycans. Sialylglycopeptide was used as a complex glycan model to validate the accuracy of the method. The results demonstrated the good linearity (R2 ≥ 0.9994) between the experimentally detected MS intensity ratios and the theoretical molar ratios of the d0-BOTC to the corresponding d5-BOTC derivatives in the dynamic range of 0.03-10 and 0.03-20 of three orders magnitude for the d5-BOTC/d0-BOTC ratios. The reproducibility was between 0.16% and 10.70%, and the limit of detection was 13 fmol. The feasibility of the relative quantification method was investigated by analyzing the glycan content in cetuximab, finding good consistency between experimental and theoretical molar ratios (5:1, 3:1, 1:1, 1:3, 1:5) of d0/d5-BOTC-labeled glycans. Finally, 13 glycans were successfully identified in cetuximab by applying this method using an in-house Tracefinder database. This study provides a novel strategy for the high throughput analysis, identification, and functional study of glycans in mAbs.

Potential use of a dried saliva spot (DSS) in therapeutic drug monitoring and disease diagnosis.

In recent years, scientific researchers have increasingly become interested in noninvasive sampling methods for therapeutic drug monitoring and disease diagnosis. As a result, dried saliva spot (DSS), which is a sampling technique for collecting dried saliva samples, has been widely used as an alternative matrix to serum for the detection of target molecules. Coupling the DSS method with a highly sensitive detection instrument improves the efficiency of the preparation and analysis of biological samples. Furthermore, dried blood spots, dried plasma spots, and dried matrix spots, which are similar to those of the DSS method, are discussed. Compared with alternative biological fluids used in dried spot methods, including serum, tears, urine, and plasma, saliva has the advantage of convenience in terms of sample collection from children or persons with disabilities. This review aims to provide integral strategies and guidelines for dried spot methods to analyze biological samples by illustrating several dried spot methods. Herein, we summarize recent advancements in DSS methods from June 2014 to March 2021 and discuss the advantages and disadvantages of the key aspects of this method, including sample preparation and method validation. Finally, we outline the challenges and prospects of such methods in practical applications.

Determination of N-acetyl-DL-leucine in the saliva of healthy volunteers and diabetic patients using ultra-performance liquid chromatography with fluorescence detection.

BACKGROUND: Recent studies have indicated that N-acetyl-leucine (N-Ac-Leu) is a potential biomarker of diabetes. This study aimed to measure the levels of enantiomers of the chiral molecule N-Ac-DL-Leu in the saliva of patients with type 2 diabetes and further determine the potential association between them. METHOD: A novel validated method was established using ultra-performance liquid chromatography (UPLC) with fluorescence (FL) detection, in which precolumn derivatization of (R)-(-)-4-(N, N-dimethylaminosulfonyl)-7-(3-aminopyrrolidin-l-yl)-2,1,3-benzoxadiazole [(R)-(-)-DBD-APy] was used for the simultaneous determination and chiral separation of N-Ac-DL-Leu in human saliva. RESULTS: The labeled N-Ac-DL-Leu diastereomers were completely separated, with a resolution value of 1.93. Additionally, excellent linearity for N-Ac-DL-Leu was observed, with high coefficients of correlation (r2 ≥ 0.9999) in the range of 10-300 µM; the limit of quantitation (signal-to-noise ratio = 10) was 40-120 pmol/mL, and the mean recoveries of N-Ac-L-Leu and N-Ac-D-Leu were 102.48% and 104.68%, respectively. The levels of N-Ac-Leu in the saliva of diabetic patients and healthy volunteers were determined, and it was found that the levels of N-Ac-DL-Leu in the saliva of diabetic patients were significantly lower than those in healthy volunteers. (p < 0.01). CONCLUSIONS: The proposed method was successfully applied for the measurement of N-Ac-DL-Leu enantiomers in the saliva of diabetic patients and healthy volunteers.

Highly sensitive novel fluorescent chiral probe possessing (S)-2-methylproline structures for the determination of chiral amino compounds by ultra-performance liquid chromatography with fluorescence: An application in the saliva of healthy volunteer.

We developed a novel fluorescent chiral probe, DBD-trans-2-methyl-L-proline (DBD-M-Pro), which can be used to target recognition of amino functional groups using chiral resolution. To investigate the chiral resolution efficiency, 20 chiral amino enantiomers (19 DL-amino acids and phenylethylamine) were labeled using ultra-performance liquid chromatography (UPLC) with a fluorescence (FL) system. Diastereomers were formed by the reactions of DBD-M-Pro with enantiomers of amino functional groups at 60 °C for 60 min and detected on a BEH C18 column (100 × 2.1 mm, 1.7 µm). Gradient elution of 10 mM ammonium acetate with 0.05% formic acid (FA) aqueous solution and 0.1% FA acetonitrile or 0.1% FA methanol solution was performed at an excitation wavelength (Ex) 460 nm and emission wavelength (Em) 550 nm. Each resulting derivative of D- and L- type was effectively separated. The results showed that the resolution (Rs) of 17 amino acids and phenylethylamine (PEA) in the range of 1.59-24.11, except for histidine (His) (Rs = 1.32) and serine (Ser) (Rs = 1.47), achieved completely separation. The DBD-M-Pro chiral probe has a robust chiral selectivity for D-amino acids. Furthermore, a new method for the simultaneous determination of six DL-amino acids (Pro, Val, Trp, Phe, Leu, Lys) in human saliva was developed. The proposed method showed resolution values of 1.78-16.38, and an excellent linear relationship was obtained in the range of 2.5-500 pmol (R2 ≥ 0.9990). The limit of detection (S/N = 3) ranged from 0.5 to 3.75 pmol. The intra-day and inter-day coefficient of variation (CV) were within the range of 1.75-11.73%. The average addition recoveries in saliva ranged from 95.99 to 106.97%. The methodology was used to determine the content of DL-amino acids and the D/L-amino acid ratio in the saliva of 40 healthy volunteers (15 males and 25 females), as well as evaluating the differences between men and women. Our study suggests that the DBD-M-Pro chiral probe could be an effective tool for screening potential D-amino acid biomarkers in disease.

Development and validation of a simple and noninvasive method for salivary uric acid: potential applications for monitoring the salivary uric acid level in healthy volunteers and gout patients

Abstract Serum uric acid (UA) is a traditional biomarker in the clinical diagnosis of gout and hyperuricemia. However, serum treatment and storage are cumbersome, and wounds are susceptible to infection. Therefore, in this study, a simple and noninvasive method was developed to detect the UA in human saliva to monitor the gout. An Inertsil ODS-3 column was used for the analysis under the condition of isocratic elution with the mixed solution phosphate buffer (74 mM, pH=2.2): Methanol=98:2 (v:v) and the UV detection at 284 nm. Using salivary UA data from healthy volunteers (HVs) (n=68) and gout patients (GPs) (n=14), we examined the salivary UA difference in their content. The intra-and inter-day accuracy and precision (RSD %) were less than 2.56%, the limit of detection (LOD) of UA was 5.0 ng/mL, the mean recoveries of the corresponding compounds were 102.48%. Saliva levels of UA in HVs and GPs were 35.26±14.06 µg/mL and 91.96±23.90 µg/mL, respectively. The concentrations of salivary UA in GPs were significantly higher than those in HVs ( p < 0.001). This method was also expected to monitor the hyperuricemia and other metabolic disorders in the future

Simultaneous Determination of Chiral Thiol Compounds and Monitoring of Dynamic Changes in Human Urine after Drinking Chinese Korean Ethnic Rice Wine.

Chinese Korean ethnic rice wine, a traditional fermented wine made from rice or corn, has antioxidant and antihypertensive activities. Although the determination of amino acids and other nutrients in rice wine has been reported, the existence of chiral thiol compounds has not been published in the literature. Therefore, we established a highly sensitive and selective ultrahigh-performance liquid chromatography-high-resolution mass spectrometry method for simultaneous determination and chiral separation of dl-Cys-GSH, dl-Cys-Cys, and dl-Cys-Hcy based on (R)-(5-(3-isothiocyanatopyrrolidin-1-yl)-5-oxopentyl) triphenylphosphonium derivatization. Three thiol diastereomers were completely separated on a YMC Triart C18 (2.0 × 150 mm, 1.9 µm) column with a resolution value (Rs) ≥ 1.52. The correlation coefficients were ≥0.9996, limit of detection was 2.40-7.20 fmol, and mean recoveries were 83.33-98.59%. Furthermore, fitted curves for dynamic changes in three kinds of chiral thiols in 10 human urine samples after drinking rice wine were drawn. Meanwhile, the metabolic changes in d/l-thiol compounds in human urine were investigated.

Simultaneous determination of three endogenous chiral thiol compounds in serum from humans at normal and stress states using ultrahigh-performance liquid chromatography coupled to quadrupole-Orbitrap high resolution mass spectrometry.

Measurement of chiral thiol compounds such as glutathione (GSH), cysteine (Cys), and homocysteine (Hcy) in human serum plays an important role in the early diagnosis and warning of cardiovascular disease, neurodegenerative disease, and cancer. We developed a novel chiral mass spectrometry derivatization reagent, (R)-(5-(3-isothiocyanatopyrrolidin-1-yl)-5-oxopentyl) triphenylphosphonium (NCS-OTPP), with triphenylphosphine (TPP) as a basic structure carrying a permanent positive charge for the diastereomeric separation of chiral thiol compounds by ultrahigh-performance liquid chromatography coupled to quadrupole-Orbitrap high resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS). A novel method was developed for simultaneous determination of three kinds of chiral thiol compounds based on the NCS-OTPP derivatization method. Three kinds of chiral thiol compounds on a YMC Triart C18 (2.0 × 150 mm, 1.9 µm) column with Rs were 1.56-1.68. The protonated precursor to product ion transitions monitored for GSH was m/z 780.16→747.24/473.18, Cys was m/z 594.20→561.18/473.18, and Hcy was m/z 608.21→575.19/473.18. An excellent linearity for all the analytes with correlation coefficients ≥ 0.9995 and suitable precision with inter-day and intra-day coefficients of variation RSDs was 0.83-4.06% and 0.95-3.11%. Satisfactory accuracy with recoveries between 83.73 and 103.35% was observed. The limit of detection (S/N = 3) was 2.4-7.2 fmol. Furthermore, the method was successfully applied to the simultaneous determination of three kinds of free and total thiol compounds in serum from 10 healthy volunteers at normal and stress states.

Simultaneous determination of DL-cysteine, DL-homocysteine, and glutathione in saliva and urine by UHPLC-Q-Orbitrap HRMS: Application to studies of oxidative stress.

A high-sensitivity and -selectivity mass spectrometry derivatization reagent, (R)-(5-(3-isothiocyanatopyrrolidin-1-yl)-5-oxopentyl) triphenylphosphonium (NCS-OTPP), was developed for the enantiomeric separation of chiral thiol compounds as prospectively important diagnostic markers for oxidative stress-related diseases. Complete separation of GSH, DL-Cys, and DL-Hcy was achieved. The parent ions of all derivatives had a fragment of m/z 473.18 and a structure of m/z 75.95 (R-S = C-S-R'), conducive to qualitative and quantitative analysis. Good linear relationships were obtained for all analytes (R2≥ 0.9995). The intra-day and inter-day precision were 0.82-5.16 % and 1.02-4.18 % in saliva, and 0.81-3.45 % and 0.99-6.47 % in urine, with mean recoveries of 83.31-105.66 % and 84.09-101.11 %, respectively. The limit of detection (S/N = 3) was 19.20-57.60 nM. Free and total GSH, DL-Cys, and DL-Hcy were detected simultaneously in saliva and urine from 10 volunteers in the normal, stressed, and stable states by UHPLC-Q-Orbitrap HRMS. The thiol compounds were quantitatively related to oxidative stress state changes.

A convenient sampling and noninvasive dried spot method of uric acid in human saliva: Comparison of serum uric acid value and salivary uric acid in healthy volunteers and hyperuricemia patients.

Concentration of uric acid (UA) in serum is one of the markers used to diagnose gout and hyperuricemia. However, serum treatment and storage are cumbersome, and wounds are susceptible to infection. Therefore, a new sampling and analysis method using noninvasive biological samples has been developed, called the dried spot method of UA in human saliva (DSM-UHS). Saliva (5 µL) was dropped on filter paper (a spot with a diameter of 5 mm) containing hypoxanthine (IS) (5 µL) and dried at room temperature for 30 min. The filter paper was immersed in 200 µL of lithium carbonate solution and shaken in a block bath shaker for 5 min at 30 °C. Afterward, the extraction was concentrated and reconstituted with 100 µL of lithium carbonate solution analyzed by HPLC-UV. When comparing the concentration of UA in the human saliva of hyperuricemia patients (HPs) and with that of healthy volunteers (HVs), we observed the concentration of UA was higher in the HPs than in the HVs (p < 0.0001). In addition, the results showed a significant linear relationship between the content of UA in saliva and the content of UA in the serum (r = 0.6243). The content of UA in human saliva could indirectly reflect the content of UA in human serum. Then DSM-UHS could be used to determine the content of UA in the saliva of HVs and HPs. This study provides a new research method and strategy for the determination of human UA content and the clinical prewiring of hyperuricemia.