Biomonitoring urinary organophosphorus flame retardant metabolites by liquid-liquid extraction and ultra-high performance liquid chromatography-tandem mass spectrometry and their association with oxidative stress.

Organophosphate flame retardants (OPFRs) are widely used as substitutes for traditional brominated flame retardants, necessitating a reliable and sensitive method for biomonitoring their urinary metabolites to assess human exposure. This study conducted biomonitoring of 10 metabolites of OPFRs in 152 adults and assessed their association with oxidative stress biomarkers 8-hydroxydeoxyguanosine and 8-hydroxyguanosine. Urinary metabolites of OPFRs were released via enzymatic deconjugation. The addition of sodium chloride to the urine samples increases the ionic strength, inducing a salting-out effect that reduces the solubility of these compounds, thereby facilitating their extraction with a mixture of ethyl acetate and acetonitrile. Then, the metabolites of OPFRs were quantified by ultra-high performance liquid chromatography-tandem mass spectrometry, and we validated the method for linear range, precision, matrix effect, and method detection limit. The detection limit of the metabolites of OPFRs ranged from 0.01 to 0.2 µg/L, and these metabolites were detected with high frequencies ranging from 25.0 to 98.68% in the urine samples. The concentration of bis (2-chloroethyl) phosphate was significantly higher in males than in females, with the geometric mean concentration of 0.88 µg/L for males and 0.53 µg/L for females, respectively. Spearman correlation analysis revealed weak but statistically significant positive correlations among the urinary metabolites. Bayesian kernel machine regression analysis showed a significant positive association between elevated urinary concentrations of metabolites of OPFRs and increased oxidative stress levels. Di-n-butyl phosphate was identified as the metabolite that significantly contributed to the elevated level of 8-hydroxyguanosine.

Analysis of 14 ß-agonists in pork using an automated wooden tip-based solid-phase microextraction device and ultra-high-performance liquid chromatography-tandem mass spectrometry.

This study introduces a cost-effective, automated ultra-high-performance liquid chromatography-tandem mass spectrometry method for the detection of 14 ß-agonists in pork using a novel solid-phase microextraction probe composed of polyacrylonitrile and molecularly imprinted polymer. Integrated into an automated extraction device, the probe optimizes extraction prior to analysis while reducing expenses and time compared to traditional solid-phase extraction procedures. The method validation followed the Chinese National Standard (GB/T 27404-2008) and examined limits of detection, limits of quantification, matrix effects, linearity, intraday, and interday precision. Average recovery rates ranged from 71.6% to 82.2%, with relative standard deviations less than 15%. Limits of detection and limits of quantification ranged from 0.09 to 0.39 and 0.27 to 0.99 µg/kg, respectively. The new method identified positive samples more accurately than the current National Standard GB/T 31658.22-2022 and demonstrated its potential for routine assessment and regulatory compliance in the detection of ß-agonists in pork.

Comparative pharmacokinetics of 11 major bioactive components between two dosage forms of Qixue Shuangbu Prescription in rats by ultra-high-performance liquid chromatography-tandem mass spectrometry.

Although Qixue Shuangbu Prescription (QSP) is a classic Chinese medicine prescription for treating chronic heart failure. Low bioavailability due to the insolubility and poor biofilm permeability of the main bioactive ingredients of QSP is still a key factor limiting its efficacy. In this study, a novel self-microemulsifying drug delivery system was proposed to effectively improve the bioavailability of QSP. The qualified ultra-high-performance liquid chromatography-tandem mass spectrometry methodology was established to investigate the pharmacokinetics characteristics of the QSP self-microemulsifying drug delivery system. Our results showed that 11 components in the self-microemulsifying drug delivery system group had prolonged T1/2 and MRT0-t values compared with QSP extract. The Cmax of calycosin-7-glucoside (CG), vanillic acid and paeoniflorin increased 2.5 times, 2.4 times and 2.3 times, respectively. The relative bioavailability values of CG, paeoniflorin and ononin were most significantly affected, increasing by 383.2%, 336.5% and 307.1%, respectively. This study promoted the development of new dosage forms of QSP and provided a useful reference for improving dosage forms to solve the problem of low bioavailability of traditional Chinese medicine.

Follicular fluid lipidomics analysis reveals altered lipid signatures in patients with polycystic ovary syndrome.

BACKGROUND: This research investigates the metabolic profiles of follicular fluid (FF) samples from patients with polycystic ovary syndrome (PCOS) undergoing in vitro fertilisation and aims to identify diagnostic and therapeutic biomarkers for PCOS through lipidomic analysis. METHODS: We performed non-targeted lipid analysis of FF samples from women with PCOS (n = 6) and normal controls (n = 6) using ultra-high-performance liquid chromatography-tandem mass spectrometry. Differential lipids between the two groups were screened using multidimensional statistical analysis, followed by fold change analysis and t-tests to identify potential PCOS biomarkers. RESULTS: Multivariate statistical analysis revealed significant differences in FF lipid levels between the PCOS and control groups. Five different lipids were selected as standards, with p < .05. Phosphatidylcholine (PC), the main differentially expressed lipid, was significantly increased in the FF of the POCS group and was closely related to other lipids. CONCLUSIONS: Using ultra-high-performance liquid chromatography-tandem mass spectrometry, we investigated lipid biomarkers based on FF lipidomics to provide useful information for the discovery of diagnostic markers for PCOS. Our study identified five distinct lipids as potential markers of PCOS, with PC being the primary aberrant lipid found in the FF of patients with PCOS.

Follicular fluid (FF) is a complex microenvironment involved in oocyte growth, follicular maturation and germ cell­somatic cell communication. All metabolites during oocyte growth are collected from the FF. This study used lipidomic analysis to identify differences in FF lipids between normal women and those diagnosed with polycystic ovary syndrome (PCOS). The pathogenesis of PCOS is associated with abnormal metabolism of glyceroglycolipids and sphingomyelin. Here, we found that phosphatidylcholine is the main abnormal lipid in FF in patients with PCOS. Our study informs the future research into the development of diagnostic markers for PCOS to be used in clinical practice.

[Determination of six bisphenols in human urine by ultra-high performance liquid chromatography-tandem mass spectrometry].

OBJECTIVE: To develop and validate a solid phase extraction-ultra-high performance liquid chromatography-tandem mass spectrometry method for the determination of six bisphenols(bisphenol S, bisphenol F, bisphenol A, 2, 2'-methylenediphenol, bisphenol AF, bisphenol AP) in urine. METHODS: After enzymolysis of urine sample, the target substances were quickly purified and extracted by WAX solid phase extraction column. On ACQUITY BEH C_(18) column(2.1 mm×100 mm, 1.7 µm), the mobile phase of water and methanol was used to separate. Finally, multi-reaction detection was carried out under electrospray negative ion scanning, and quantification was carried out by internal standard method. RESULTS: The correlation coefficients(r) of the target compounds were all more than 0.998 in the range of 0.1-50.0 ng/mL, the linearity was good, and the detection limits were all lower than 0.1 ng/mL. The recoveries of the three standard concentrations(0.5, 5.0 and 50.0 ng/mL) were all between 80% and 120%, and the relative standard deviation was less than 20%(n=5). The standard reference material was detected and the concentration was within the reference range. CONCLUSION: This method can be used to detect six bisphenols in urine quickly and accurately, is suitable for the trace analysis of bisphenol compounds in human urine.

Simultaneous determination and pharmacokinetics study of three triterpenoid saponins in rat plasma by ultra-high-performance liquid chromatography tandem mass-spectrometry after oral administration of Astragalus Membranaceus leaf extract.

A selective and sensitive ultra-high-performance liquid chromatography-tandem mass spectrometry method was developed and validated for the determination of three triterpenoid saponins isolated from Astragalus membranaceus leaf extract. In this article, a method for simultaneous determination of Huangqiyenin A, Huangqiyenin E, and Huangqiyenin K was established for the first time. The method was successfully applied to the pharmacokinetic study of Astragalus membranaceus leaf extract after oral administration. Liquid-liquid extraction was applied to plasma sample preparation. Multiple reaction monitoring mode with an electrospray ion source in positive electrospray ionization was chosen to quantify the analytes. Chromatographic separation was performed on a Waters HSS T3 column, using gradient elution with a mobile phase composed of acetonitrile and 5 mM ammonium acetate/water. The pharmacokinetic results showed that all three compounds had the characteristics of rapid absorption-slow metabolism trend. The time of maximum plasma concentration of Huangqiyenin A is higher than Huangqiyenin E and Huangqiyenin K. And the maximum plasma concentration of Huangqiyenin A is higher as well. The pharmacokinetic results revealed the pharmacokinetic characteristics of the three analytes in rat plasma, which could provide a helpful reference for the further study of Astragalus membranaceus leaf extract.

Development of UHPLC-MS/MS method for simultaneous determination of tacrolimus and sirolimus in human whole blood and comparisons with two immunoassays.

Tacrolimus (TAC) and sirolimus (SIR) antirejection medications are widely used in organ transplantation. We aimed to develop an ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) assay for quantifying TAC and SIR simultaneously and evaluating agreement with chemiluminescence microparticle immunoassay (CMIA) and electrochemiluminescence immunoassay (ECLIA). Whole blood samples collected from 209 TAC and 208 SIR patients were assessed by UHPLC-MS/MS, CMIA and ECLIA. The agreement of the three techniques was assessed using the Bland-Altman plot. The UHPLC-MS/MS assay had a calibration range of 1-100 ng/ml for TAC and SIR. The accuracy and precision were -2.73-4.32% and <4.71% for TAC, respectively, and 0.07-4.84% and <6.5% for SIR, respectively. The three methods had good correlation. In comparison with UHPLC-MS/MS, two immunoassays showed a slight deviation in proportion. An UHPLC-MS/MS method for simultaneously detecting TAC and SIR in human whole blood was developed, validated and comparatively analyzed with CMIA and ECLIA. For determining TAC and SIR, immunoassays displayed acceptable analytical performances in terms of precision and correlation compared with UHPLC-MS/MS. However, further investigation is warranted to examine the novel method.

Simultaneous determination of serum tryptophan metabolites in an older Chinese population.

Tryptophan (TRP) and its metabolites exhibit significant biological effects and are strongly associated with age-related disease and mortality. However, reports on quantitatively analyzing these metabolites in older individuals are not available. We used ultra-high-performance liquid chromatography-tandem mass spectrometry to optimize and validate a method for isotope dilution analysis of TRP metabolites in older individuals. The targeted analytes are TRP, serotonin or 5-hydroxytryptamine, kynurenine, kynurenic acid, xanthurenic acid, indole-3-acetic acid, indole-3-propionic acid, and tryptamine. The serum sample was purified using solid-phase extraction and was separated on a Waters HSS T3 column (100 mm × 2.1 mm, 1.8 µm). The analytes were detected in the multiple reaction monitoring mode under positive ionization. TRP was confirmed and measured after being diluted 100 times. This method exhibited satisfactory linearity (r > 0.99). The intrabatch and interbatch accuracies (85.7-114%) and precisions (<15%) were acceptable. The standard-normalized matrix effects ranged from 51.6 to 145%. This method was successfully applied to a cohort of 1021 older Chinese individuals, and this study may enable further understanding of the metabolic phenotypes associated with TRP in other populations.

Qualitative Analysis of Visible Foreign Solids in Armillarisin A Injection Formulations Using Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry.

During the trial production of Armillarisin A for injection (AA-I), unidentified needle-like yellow-brown crystals were occasionally observed. Here, we report an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS) method for determining the source of the visible foreign bodies in the formulations of Armillarisin A active pharmaceutical ingredient (AA-API). AA-API, photolyzed samples, the intermediate polymer, and the excipient analyzed determined after the separation on a Waters Symmetry C18 (3.5 µm, 4.6 × 75 mm) column with a mobile phase consisting of a methanol/acetic acid (0.1 mol/L) aqueous solution (50:50). Furthermore, the crystal type of the visible foreign bodies, the intermediate polymer and AA-API were investigated by X-ray powder diffraction (XRD). The results revealed that the characteristics of the visible foreign solids were the same as those of AA-API as regards UPLC peak position (368 nm) and MS spectrum in negative ion detection mode. The visible foreign solids were thus identified as unpolymerized crystals of AA-API and were attributed to AA-API itself. The results showed that the production process could be improved by changing the stirring method and frequency as well as by optimizing the polymerization temperature to ensure the safety, stability, and control of the product quality in the stage of batch production.

An Automated Solid-Phase Extraction-UPLC-MS/MS Method for Simultaneous Determination of Sulfonamide Antimicrobials in Environmental Water.

The large-scale use of sulfonamide antimicrobials in human and veterinary medicine has seriously endangered the ecological environment and human health. The objective of this study was to develop and validate a simple and robust method for the simultaneous determination of seventeen sulfonamides in water using ultra-high performance liquid chromatography-tandem mass spectrometry coupled with fully automated solid-phase extraction. Seventeen isotope-labeled internal standards for sulfonamides were used to correct matrix effects. Several parameters affecting extraction efficiency were systematically optimized, and the enrichment factors were up to 982-1033 and only requiring about 60 min per six samples. Under the optimized conditions, this method manifested good linearity (0.05-100 µg/L), high sensitivity (detection limits: 0.01-0.05 ng/L), and satisfactory recoveries (79-118%) with acceptable relative standard deviations (0.3-14.5%, n = 5). The developed method can be successfully utilized for the determination of 17 sulfonamides in pure water, tap water, river water, and seawater. In total, six and seven sulfonamides were detected in river water and seawater, respectively, with a total concentration of 8.157-29.676 ng/L and 1.683-36.955 ng/L, respectively, and sulfamethoxazole was the predominant congener.

An Untargeted Metabolomics Approach to Study the Variation between Wild and Cultivated Soybeans.

The differential metabolite profiles of four wild and ten cultivated soybeans genotypes were explored using an untargeted metabolomics approach. Ground soybean seed samples were extracted with methanol and water, and metabolic features were obtained using ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS) in both positive and negative ion modes. The UHPLC-HRMS analysis of the two different extracts resulted in the putative identification of 98 metabolites belonging to several classes of phytochemicals, including isoflavones, organic acids, lipids, sugars, amino acids, saponins, and other compounds. The metabolic profile was significantly impacted by the polarity of the extraction solvent. Multivariate analysis showed a clear difference between wild and cultivated soybean cultivars. Unsupervised and supervised learning algorithms were applied to mine the generated data and to pinpoint metabolites differentiating wild and cultivated soybeans. The key identified metabolites differentiating wild and cultivated soybeans were isoflavonoids, free amino acids, and fatty acids. Catechin analogs, cynaroside, hydroxylated unsaturated fatty acid derivatives, amino acid, and uridine diphosphate-N-acetylglucosamine were upregulated in the methanol extract of wild soybeans. In contrast, isoflavonoids and other minor compounds were downregulated in the same soybean extract. This metabolic information will benefit breeders and biotechnology professionals to develop value-added soybeans with improved quality traits.

[Determination of amantadine, rimantadine and dimethylamantadine residues in poultry by ultra-performance liquid chromatography-tandem mass spectrometry].

OBJECTIVE: To establish a method for determination of amantadine, rimantadine and dimethylamantadine residues in poultry matrix by ultra-performance liquid chromatography-tandem mass spectrometry. METHODS: Poultry samples were extracted with acid acetonitrile, salting out, and then the organic phase was cleaned up by C_(18) and PSA. A Waters ACQUITYTM UPLC HSS T3 column(100 mm×2.1 mm, 1.7 mm)was used for liquid chromatography separation, ESI positive ion scan was used with multiple reaction monitoring(MRM) mode and quantified by matrix-matched external standard method. RESULTS: At the spiked level of 0.5, 1.0 and 5.0 µg/kg, the recoveries of each compound were in the range of 81.3%-91.1% with the relative standard deviations of 6.5%-11.3%. The qualitative limits of detections were 0.06-0.2 µg/kg and the quantitative limits were 0.2-0.5 µg/kg for the 3 target compounds. The established method was applied to the detection of the 3 target compounds in 30 poultry samples, and none of the target compounds exceeded the residue limits. CONCLUSION: The method is simple, rapid, high sensitivity and good stability, with a wide variety and a certain development. It can be used for the daily monitoring of the veterinary drug residues in poultry.

Rapid Determination of Bucinnazine in Blood by UPLC-MS/MS.

OBJECTIVES: To establish a rapid method for the analysis of bucinnazine in blood by UPLC-MS/MS and to apply the method to the practical case. METHODS: After the internal standard was added to blood, the protein was precipitated with 900 µL mixed solution (Vacetonitrile∶Vwater=8∶2). After vortex and centrifugation, the protein was measured through 0.22 µm filter membrane. The separation was performed on C18 chromatography column, with acetonitrile and 5 mmol/L ammonium acetate containing 0.1% formic acid aqueous as mobile phase gradient elution at the flow rate of 0.4 mL/min. Multiple reaction monitoring scan was performed in electrospray positive ion mode, quantitative measurement was performed by internal standard method, and methodological verification was carried out. RESULTS: The linear relationship of bucinnazine in blood was good in the range of 0.5-200 µg/L, the correlation coefficient (r) was 0.999 7, the limit of detection was 0.1 µg/L, the limit of quantitation was 0.5 µg/L, and the recovery was 78.3%-83.8% at 1, 10 and 100 µg/L mass concentration levels. The matrix effect was 69.4%-73.8%, the intra-day precision was 1.9%-2.8%, and the inter-day precision was 2.8%-3.2%, the accuracy was 3.1%-3.5%. The stability test results of 1 and 100 µg/L mass concentrations at -25 ℃ showed that the accuracy (bias) of 10 d was less than 4.5%. CONCLUSIONS: This method has the advantages of simple pre-treatment process, fast sample processing speed, high sensitivity of instrument analysis, good stability of content determination and reliable identification results, and can meet the needs of case identification.

UPLC-MS/MS Method for Detection of Etomidate and Its Metabolite Etomidate Acid Quantity in Blood.

OBJECTIVES: To establish a method for the simultaneous quantitative analysis of etomidate and its metabolite etomidate acid in blood, and to discuss its application value in actual cases. METHODS: Acetonitrile precipitate protein method was used, and C18 column was selected. Gradient elution was performed with acetonitrile and 5 mmol/L ammonium acetate within 6 min. Electrospray ionization source in positive ion mode was used. The internal standard etomidate acid-d5 was obtained by etomidate-d5 alkaline hydrolysis reaction. Ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used for quantitative analysis. The methodological verification was conducted. RESULTS: Etomidate and etomidate acid in blood showed good linear relationship in the quantitative linear range (r>0.999), with the lower limit of quantification was 2.5 ng/mL and 7.5 ng/mL, respectively. The accuracy, precision, recovery rate, and matrix effect of the method met the professional verification standards. The practical application results showed that etomidate and etomidate acid could be detected in the blood of the abusers, and their mass concentrations ranged from 17.24 to 379.93 ng/mL. CONCLUSIONS: The method established in this study can simultaneously quantify etomidate and etomidate acid in blood, which is simple and convenient to operate with accuracy. It can meet the detection needs of actual cases and provide technical support for law enforcement to crack down on etomidate abuse.

[Determination of phenylglyoxylic acid and mandelic acid in urine by ultra high performance liquid chromatography tandem mass spectrometry].

Objective: To develop a method for the analysis of phenylglyoxylic acid (PGA) and mandelic acid (MA) in urine by ultra-high performance liquid chromatography tandem mass spectrometry. Methods: The study was conducted in April 2022. Urine samples were directly diluted with the initial mobile phase, separated by Waters HSS T3 column after passing through the membrane, and analyzed under negative ionization mode (ESI(-)) and multiple reaction monitoring (MRM) conditions, the contents of PGA and MA in human urine were quantitatively determined by external standard method. Results: The determination of PGA and MA showed a good linear relationship within the range of 10-1000 ng/ml, with a correlation coefficient of 0.9999. The linear regression equation of PGA was y=1141.4x+2157.3, the detection limit and lower limit of quantification of the method were 0.081 ng/ml and 0.269 ng/ml, and the recovery rate was 90.47%-99.83%. The linear regression equation of MA was y=62.8x+140.3, the detection limit and lower limit of quantification of the method were 0.551 ng/ml and 1.836 ng/ml, and the recovery rate was 92.75%-101.09%. The intra and inter batch precision of PGA and MA were both<5%. Conclusion: An ultra-high performance liquid chromatography tandem mass spectrometry method for the analysis of PGA and MA in urine was established.The sample pretreatment operation is simple, and the accuracy and precision of the method meet the standard requirements. It can be used for monitoring and evaluating PGA and MA in urine of the general population and occupational contact population.

Metabolic signatures of hepatolithiasis using ultra-high performance liquid chromatography-tandem mass spectrometry.

BACKGROUND & AIMS: A metabolomic study of hepatolithiasis has yet to be performed. The purpose of the present study was to characterize the metabolite profile and identify potential biomarkers of hepatolithiasis using a metabolomic approach. METHODS: We comprehensively analyzed the serum metabolites from 30 patients with hepatolithiasis and 20 healthy individuals using ultra-high performance liquid chromatography-tandem mass spectrometry operated in negative and positive ionization modes. Statistical analyses were performed using univariate (Student's t-test) and multivariate (orthogonal partial least-squares discriminant analysis) statistics and R language. Receiver operator characteristic (ROC) curve analysis was performed to identify potential predictors of hepatolithiasis. RESULTS: We identified 277 metabolites that were significantly different between hepatolithiasis serum group and healthy control serum group. These metabolites were principally lipids and lipid-like molecules and amino acid metabolites. The steroid hormone biosynthesis pathway was enriched in hepatolithiasis serum group. In all specific metabolites, 75 metabolites were over-expressed in hepatolithiasis serum group. The AUC values for 60 metabolites exceeded 0.70, 4 metabolites including 18-ß-Glycyrrhetinic acid, FMH, Rifampicin and PC (4:0/16:2) exceeded 0.90. CONCLUSIONS: We have identified serum metabolites that are associated with hepatolithiasis for the first time. 60 potential metabolic biomarkers were identified, 18-ß-Glycyrrhetinic acid, FMH, Rifampicin and PC (4:0/16:2) may have the potential clinical utility in hepatolithiasis.

Simultaneous determination of penthiopyrad enantiomers and its metabolite in vegetables, fruits, and cereals using ultra-high performance liquid chromatography-tandem mass spectrometry.

Penthiopyrad is a novel succinate dehydrogenase inhibitor that has one chiral center and exists a metabolite, 1-methyl-3-trifluoromethyl-1H-pyrazole-4-carboxamide in its residue definition. An efficient analytical method for the simultaneous determination of penthiopyrad enantiomers and its metabolite in eight matrices were developed using modified quick, easy, cheap, effective, rugged, safe method, coupled with chiral stationary phase and ultra-high performance liquid chromatography-tandem mass spectrometry. The absolute configuration of penthiopyrad enantiomers was confirmed by polarimetry and electronic circular dichroism. Eight polysaccharide-based chiral stationary phases were evaluated in terms of the enantioseparation of penthiopyrad and separation-related factors (the mobile phase, flow rate and the column temperature) were optimized. To obtain an optimal purification, different sorbent combinations were assessed. The linearities of this method were acceptable in the range of 0.005 to 1 mg/L with R2  > 0.998, while the limits of detection and quantification were 0.0015 mg/kg and 0.01 mg/kg for two enantiomers and its metabolite. The average recoveries of R-(-)-penthiopyrad, S-(+)-penthiopyrad and the metabolite ranged from 75.4 to 109.1, 69.5 to 112.8, and 70.0 to 108.5%, respectively. The intra-day and inter-day relative standard deviations were less than 18.8%. The analytical method was accurate and convenient, which can support their further research on stereoselective degradation, residual monitoring and risk assessment.

Comprehensive pseudotargeted metabolomics analysis based on two-phase liquid extraction-UHPLC-MS/MS for the investigation of depressive rats.

Pseudotargeted analysis combines the advantages of untargeted and targeted metabolomics methods. This study proposed a comprehensive pseudotargeted metabolomics method based on two-phase liquid extraction using ultra-high-performance liquid chromatography-tandem mass spectrometry. Two-phase liquid extraction, composed of both aqueous and organic phases, extracted a wide range of metabolites from polar to nonpolar in plasma samples. Besides, the two phases were combined and detected in a single injection to save analytical time. A total of 486 potential metabolites were detected by the developed approach. Compared with the conventional methanol-based protein precipitation method, the two-phase liquid extraction method significantly increased the metabolite coverage by 20.29%. Besides, the proposed pseudotargeted metabolomics method exhibited higher sensitivity and better repeatability than the untargeted method. Finally, we applied the established pseudotargeted method to the metabolomics study of depressive rats and screened 53 differential variables. Sixteen determined differential metabolites were mainly in four metabolic pathways including glycerophospholipid, arachidonic acid, sphingolipid metabolisms, pentose and glucuronate interconversions. The results indicated that the pseudotargeted method based on two-phase liquid extraction broadened the metabolite coverage with good sensitivity and repeatability, exhibiting significant potential for discovering differential metabolites in metabolomics studies.

Development and Comprehensive SPE-UHPLC-MS/MS Analysis Optimization, Comparison, and Evaluation of 2,4-Epibrassinolide in Different Plant Tissues.

A determination method for trace 24-epibrassinolide (EBL) in plant tissues was developed using ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The plant tissue samples were extracted using a methanol-formic acid solution, and the corresponding supernatant was purified with ODS C18 solid-phase extraction column. The extracts were separated using a Zorbax Eclipse Plus C18 (2.1 mm × 50 mm, 1.8 µm) column with methanol and 0.1% formic acid as the mobile phase. The ion source for the mass spectrometry was an electrospray ionization source with positive ion mode detection. The linear range of the target compound was 0.7~104 µg/kg, the limit of detection (LOD) was 0.11~0.37 µg/kg, the limit of quantification (LOQ) was 0.36~1.22 µg/kg, the recovery rate was 84.0~116.3%, and the relative standard deviation (RSD%) was 0.8~10.5. The samples of maize plumule, brassica rapeseed flower, and marigold leaf were detected using the external standard method. The optimization of the extraction method and detection method of EBL improved the detection sensitivity, laid a foundation for the artificial synthesis of EBL, improved the extraction rate of EBL, and provided a theoretical basis for the study of EBL in many plants.

[Analytical method of organophosphorus flame retardants in fish by ultra-high performance liquid chromatography-tandem mass spectrometry based on EMR-Lipid purification].

OBJECTIVE: A method for simultaneous determination of 11 kinds of organophosphorus flame retardants in fish was established by using the EMR-Lipid cleaning agent and ultra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS). METHODS: The samples were extracted by ultrasonic with 0.5% formic acid acetonitrile solution. The lipid removal product EMR-lipid was used for lipid purification. The co-extracts were further removed by magnesium sulfate, N-propyl ethylene amine(PSA) and graphitized carbon black(GCB) purification agent. The target compounds were separated on an ACQUITY UPLC® BEH C_(18) column(100 mm×2.1 mm, 1.7 µm), detected by electrospray ionization(ESI) and positive ion multiple reaction monitoring mode, the internal standard method and external standard method are quantitatively evaluated, and external standard method was adopted. RESULTS: The 11 kinds of organophosphorus flame retardants had a good linear relationship in the range of 0.5-50 µg/L(tris(2-ethylhexyl) phosphate 0.05-5 µg/L) with r>0.999. The limits of detection were 0.004-1.029 µg/kg and the limits of quantitation were 0.012-3.094 µg/kg. The average recoveries at three spiked levels(low, medium and high) were 80.0%-111.2% with the relative standard deviations all less than 10%(n=6). CONCLUSION: The method could be used for the determination of trace organophosphorus flame retardants in freshwater fish with accurate and reliable result.