Ultra-high Performance Liquid Chromatography-tandem Research Articles

Functionalized magnetic metal organic framework nanocomposites for high throughput automation extraction and sensitive detection of antipsychotic drugs in serum samples

Due to the complexity of biological sample matrix, the automated and high-throughput pretreatment technology is urgently needed for monitoring the antipsychotic drugs for mental patients. In this study, functionalized magnetic zirconium-based organic framework nanocomposites (Fe3O4@SiO2@Zr-MOFs) were successfully designed and synthesized by the layer-by-layer growth. Among them, Fe3O4@SiO2@UiO-67-COOH showed the best adsorption performance, and at the same time it exhibited excellent water dispersibility, high thermal stability, chemical stability and high hydrophobicity. Results of adsorption kinetics, isotherm and FT-IR showed that the adsorption process was dominated by chemical adsorption (hydrogen bond, electrostatic interaction, π-π interaction) and monolayer adsorption. Moreover, the smaller pore size improved the protein exclusion rate which reached 98.9–99.8%. Based on the above result, the synthesized magnetic nanoparticles were introduced to 96-well automatic extractor, antipsychotic drugs in 96 serum samples were automatically extracted within 9 min, which most greatly saved the time and labor costs and avoided artificial errors. By further integrating with ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), antipsychotic drugs can be detected in the range of 0.2–3.0 ng mL−1 with a quantitative limit of 0.06–0.9 ng mL−1. The recoveries of antipsychotic drugs and their metabolites in serum ranged from 95.7% to 112.3% within 1.4–6.5% of RSD. These features indicate that the proposed method is promising for high throughput and sensitively monitoring of drugs and other hazardous substances.

Non-targeted metabolomics study for discovery of hepatocellular carcinoma serum diagnostic biomarker

Hepatocellular carcinoma (HCC) is one of the most prevalent malignant cancers worldwide. Due to the asymptomatic features of HCC at early stages, patients are often diagnosed at advanced stages and missed effective treatment. Thus, there is an urgent need to identify sensitive and specific biomarkers for HCC early diagnosis. In the present study, an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) approach was used to profile serum metabolites from HCC patients, liver cirrhosis (LC) patients, and normal controls (NC). Univariate and multivariate statistical analyses were performed to obtain the metabolomic differences of the three groups and select significantly changed metabolites that can be used as diagnostic biomarkers. In total, 757 differential metabolites were quantified among the three groups, and pathway enrichment analysis of these metabolites indicated that glycerophospholipid metabolism, pentose and glucuronate interconversions, phenylalanine, tyrosine and tryptophan biosynthesis, and linoleic acid metabolism were the most altered pathways involved in HCC development. Receiver operating characteristic (ROC) curve analysis was performed to select and evaluate the diagnostic biomarker performance. Seven metabolites were identified as potential biomarkers that can differentiate HCC from LC and NC, and LC from NC with the good diagnostic performance of area under the curve (AUC) from 0.890 to 0.990. In summary, our findings provide highly effective biomarker candidates to differentiate HCC from LC and NC, LC, and NC, which shed insight into HCC pathological mechanisms and will be helpful in better understanding and managing HCC.

Abstract B072: Reproducibility of plasma metabolome over 1 year in a population-based study of Black breast cancer survivors

Abstract Background: African American/Black women have higher rates of breast cancer (BrCa) mortality and morbidity than White women. The metabolomics approach using blood samples from epidemiological studies has the potential to elucidate pathways or uncover biomarkers for adverse BrCa outcomes. Therefore, understanding the within-person reproducibility of the blood metabolome of BrCa survivors and factors that influence metabolite levels over time is crucial. However, the stability of blood metabolome among Black BrCa survivors is unknown. Objective: This study aimed to estimate the within-person reproducibility of metabolites over 1 year via global untargeted metabolomic profiling of plasma samples collected in the Women’s Circle of Health Follow-Up Study (WCHFS), a population-based longitudinal study of Black BrCa survivors in New Jersey. The study also aims to identify factors associated with reproducibility. Methods: We estimated the metabolite reproducibility among 61 Black women with BrCa diagnosed between 2012 and 2017 in WCHFS. Plasma samples were collected once for each participant at ~2 and 3 years post-diagnosis, respectively, and analyzed at Metabolon via ultrahigh performance liquid chromatography-tandem mass spectrometry. Using a mixed-effects model, we calculated the intraclass correlation coefficients (ICC) for each metabolite by dividing the between-person variance by the total variance (i.e., within- and between-person variances). ICCs were compared by age, obesity status, estrogen receptor (ER) status, and seasons, fasting status and time interval between two visits when blood samples were collected. Statistical significance was determined using the Wilcoxon test. Results: Of the 857 named metabolites identified after excluding those undetectable in ≥80% of samples or with a coefficient of variance≥0.25, the median ICC was 0.58 (interquartile range [IQR]: 0.44-0.70). 16.6% had high within-person reproducibility (ICC≥0.75), spanning across all metabolite classes, while 65.6% had an ICC within 0.4-0.75, and the remaining 17.9% had an ICC<0.4. Significant differences in ICCs were noted by participants’ age (median ICC [IQR; same below]: 0.56 [0.40 - 0.68] for younger women, i.e. ≤ 55.6 years vs. 0.61 [0.44 - 0.73] for older women), obesity status (0.55 [0.40 - 0.68] for non-obese vs. 0.62 [0.46 - 0.75] for obese), and ER status (0.56 [0.41 - 0.68] for ER+ vs. 0.62 [0.45 - 0.79] for ER-). The reproducibility also differed by fasting status (0.63 [0.45 - 0.77] for fasting vs. 0.53 [0.39 - 0.67] for non-fasting), but not the seasons or time interval between blood collections. Conclusion: The within-person reproducibility of plasma metabolites over 1 year among Black breast cancer survivors was generally reasonable, suggesting a single-time-point measurement could be useful in evaluating associations between metabolites and BrCa outcomes. However, the reproducibility varied across metabolites and was influenced by participant characteristics and certain sample collection conditions, which need consideration in future analysis or design. Citation Format: Bo Qin, Madhir Vyas, Steven C. Moore, Xiaoyang Su, Eileen White, Christine B. Ambrosone, Kitaw Demissie, Chi-Chen Hong, Elisa V. Bandera. Reproducibility of plasma metabolome over 1 year in a population-based study of Black breast cancer survivors [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr B072.

Simultaneous determination of three curative flavonoids and neurotoxic anisatin in star anise fruits by ultra-high performance liquid chromatography-tandem mass spectrometry.

Chinese star anise is an anise-scented star-shaped fruit of the evergreen Illicium verum tree. It is generally used as a spice and folk medicine for treating digestive illness. Chinese star anise may be adulterated with Japanese star anise (fruit of Illicium anisatum tree with similar morphology to the former), containing high content of toxic anisatin leading to severe physical disorders. In this work, a UHPLC-MS/MS method has been developed to determine three quality markers (quercetin, kaempferol, and luteolin) and toxicity marker (anisatin) of star anise using quercetin-d3, an exogenous heavy isotopic compound, as internal standard. Chromatographic separation of quality and toxicity marker compounds was achieved using a T3 column within two minutes. The method was successfully developed, validated, and used to quantify the multiple marker compounds in both botanical reference materials and star anise samples. This method can be used for quality control and assessment of Chinese star anise products.

An ultrahigh-performance liquid chromatography-tandem mass spectrometry method for quantification of methotrexate and 7-hydroxy-methotrexate and application for therapeutic drug monitoring in patients with central nervous system lymphoma.

A method using ultrahigh-performance liquid chromatography-tandem mass spectrometry was developed, validated, and applied to simultaneously determine plasma methotrexate (MTX) and 7-hydroxy-methotrexate (7-OH-MTX) in 117 patients with central nervous system (CNS) lymphoma. The ion transitions utilized were m/z 455.2 > 308.2 for MTX and m/z 471.2 > 324.1 for 7-OH-MTX. Samples were prepared through protein precipitation using methanol. Chromatographic separation was achieved within 3.0 min on a CMS9030 column (Ruixi, 2.1 × 50 mm, 3 μm) through a gradient elution of methanol and a 10% ammonium acetate solution at a flow rate of 0.4 mL/min. The method demonstrated linearity in the concentration range of 0.05-10 μM for MTX and 0.25-50 μM for 7-OH-MTX. The intra- and inter-day inaccuracy ranged from -7.38% to 7.83%, and the imprecision was less than 6.00% for both analytes. The recovery and matrix effect normalized by the internal standard (MTX-D3 ) remained consistent. Both analytes remained stable under nine different storage conditions. In patients with CNS lymphoma, MTX levels at 12 h and 7-OH-MTX levels at 12, 36, and 60 h after dosing in individuals with impaired renal function were significantly higher compared with those with normal renal function. 7-OH-MTX could potentially serve as a superior indicator for nephrotoxicity compared with MTX.

Determination of Triazole Fungicides by Ultra-High Performance Liquid Chromatography–Tandem Mass Spectrometry (UHPLC–MS/MS) with a Lipophilicity-Matched Separation Strategy for Reducing Matrix Effects

Matrix effects are a major concern in liquid chromatography coupled to tandem mass spectrometry for the determination of pesticide residues. In this work, a novel and efficient lipophilicity-matched chromatographic separation strategy was developed to improve the separation efficiency on an alkyl bonded phase chromatographic column and reduce matrix effects. An ultra-high performance liquid chromatography-tandem mass spectrometry method based on this strategy was established to minimize matrix effects for the determination of triazole fungicides. The samples were cleaned-up with dispersive solid phase extraction and separated on a chromatographic column with similar lipophilicity to the fungicides. The results showed that the proposed method effectively minimized the matrix effects and improved the separation. The matrix effects of 21 fungicides ranged from −8.3% to 4.7%. Moreover, the average recoveries were between 91.4% and 108.1% using solvent calibration for quantification, and the limits of quantitation were between 0.5 and 3.5 μg/kg. These results demonstrate the effectiveness of the proposed strategy in improving the chromatographic efficiency. This work provides a practical strategy and reference for the liquid chromatography-tandem mass spectrometry determination of pesticide residues.

Rapid profiling of cytokinins using supercritical fluid chromatography coupled with tandem mass spectrometry

BackgroundThe determination of plant hormones is still a very challenging analytical discipline, mainly due to their low concentration in complex plant matrices. Therefore, the involvement of very sensitive high-throughput techniques is required. Cytokinins (CKs) are semi-polar basic plant hormones regulating plant growth and development. Modern methods for CK determination are currently based on ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), which enables the separation of CK isomeric forms occurring endogenously in plants. Here, ultra-high performance supercritical fluid chromatography coupled with tandem mass spectrometry (UHPSFC-MS/MS) was used for the simultaneous determination of 37 CK metabolites. ResultsThe chromatographic conditions were tested on three different columns with various retention mechanisms. Hybrid silica modified with 2-picolylamine was selected as the stationary phase. Several parameters such as column temperature, back pressure regulation, mobile phase composition and make-up solvent were investigated to achieve efficient separation of CK isomers and reasonable sensitivity. Compared to UHPLC-MS/MS, a 9-min chromatographic analysis using a mobile phase of supercritical CO2 and 5 mM ammonia in methanol represents a three-fold acceleration of total run time. The quantification limit of UHPSFC-MS/MS method was in the range of 0.03–0.19 fmol per injection and the method validation showed high accuracy and precision (below 15 % for most analytes). The method was finally applied to the complex plant matrix of the model plant Arabidopsis thaliana and the obtained profiles of CK metabolites were compared with the results from the conventional UHPLC-MS/MS method. SignificanceThe presented work offers a novel approach for quantification of endogenous CKs in plants. Compared to the conventional UHPLC-MS/MS, the total run time is shorter and the matrix effect lower for the key CK metabolites. This approach opens the opportunity to utilize UHPSFC-MS/MS instrumentation for targeted plant hormonomics including other plant hormone families.

High-throughput detection and dietary exposure risk assessment of 44 mycotoxins in Mango, Litchi, Longan, and their products in South China

Mycotoxins exposure from food can trigger serious health hazards. This study aimed to establish an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous detection of 44 mycotoxins in fruits and their products, followed by dietary exposure risk assessment. The optimized UPLC-MS/MS method exhibited a good linear relationship with correlation coefficients ≥ 0.99041. The limits of detection (LOD) and the limits of quantification (LOQ) were within the range of 0.003 ∼ 0.700 μg/kg and 0.01 ∼ 2.00 μg/kg, respectively. The three fruits and their corresponding value-added products, with a total sampling size of 42, were subjected to analysis and detected with mycotoxins. Further dietary exposure risk assessment revealed that the hazard quotient (HQ) and hazard index (HI) of mycotoxins were 1.213 ∼ 60.032 % and 5.573 ∼ 93.750 %, indicating a low risk for Chinese consumers. However, we still need be cautious about 15-acetyl-deoxynivalenol (15-ADON), as it had 78.6 % occurrence among all samples. This work provides an accurate analysis strategy for 44 mycotoxins and contributes to mycotoxins supervision.

Flower-like calix[6]arene-based covalent organic framework for membrane extraction of sulfonamides in animal-derived food through host-guest interaction prior to determination with ultra-high performance liquid chromatography-tandem mass spectrometry

Supramolecular macrocycle-based covalent organic frameworks (COFs) are promising adsorbents for adsorption of hazards due to their host-guest recognition property. However, most supramolecular macrocycles are conformationally flexible, making them challenging to introduce into COFs. In this work, a calix[6]arene-based COF (CX6-BD COF) was fabricated with a unique flower-like morphology and high crystallinity. Especially, the cavity of CX6 exhibited host-guest inclusion interaction for sulfonamides (SAs), which was verified by quantum chemistry calculation. The integration of the porosity of COFs with the recognition cavity of CX6 made CX6-BD COF display excellent enrichment performance for SAs, with good enrichment factors (EFs) between 77 and 96. The material was employed as an adsorbent for COF membrane filter extraction, coupled with ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) to simultaneously enrich and determine seven SAs in animal-derived food. The analytical method showed a wide linear range (0.01–100 µg/L and 0.05–100 µg/L) and low detection limits (3–10 ng/L). The established method was successfully applied to sensitively determine SAs in chicken, pork and beef samples, which achieved satisfactory recoveries (73.8–113%). These results demonstrated CX6-BD COF has good application potential in determination of trace and ultra-trace SAs in complex food matrices as an adsorbent.

Competitive adsorption and bioaccumulation of sulfamethoxazole and roxithromycin by sediment and zebrafish (Danio rerio) during individual and combined exposure in water

Antibiotics are extensively used for health protection and food production, causing antibiotic pollution in the aquatic environment. This study aims to determine the bioavailability and bioaccumulation of typical antibiotics sulfamethoxazole (SMX) and roxithromycin (RTM) in zebrafish under environmentally realistic conditions. Four different microcosms, i.e. water, water-sediment, water-zebrafish, and water-sediment-zebrafish were constructed, with three replicates in parallel. The concentrations of SMX and RTM in water, sediment and zebrafish were extracted and analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) to assess their kinetic behavior and bioavailability. In the water-sediment system, the dissolved concentration of both SMX and RTM decreased with time following the first-order kinetic while their adsorption by sediment increased with time. In the water-zebrafish system, SMX and RTM bioaccumulation was increasing with time following the pseudo second-order kinetics. RTM bioaccumulation in zebrafish (up to 16.4 ng/g) was an order of magnitude higher than SMX (up to 5.2 ng/g), likely due to RTM being more hydrophobic than SMX. In addition, the bioaccumulation factor (BAF) value of SMX in zebrafish was greater than its sediment partition coefficient, while the opposite trend was observed for RTM, demonstrating the importance of antibiotics properties in affecting their bioavailability. Furthermore, increasing dissolved organic carbon concentration in water reduced SMX bioaccumulation, but increased RTM bioaccumulation at the same time. The findings are important in future studies of environmental fate and bioavailability of toxic chemicals with different pollution sources and physicochemical properties.

Plasma metabolomics profiles in Black and White participants of the Adventist Health Study-2 cohort

BackgroundBlack Americans suffer disparities in risk for cardiometabolic and other chronic diseases. Findings from the Adventist Health Study-2 (AHS-2) cohort have shown associations of plant-based dietary patterns and healthy lifestyle factors with prevention of such diseases. Hence, it is likely that racial differences in metabolic profiles correlating with disparities in chronic diseases are explained largely by diet and lifestyle, besides social determinants of health.MethodsUntargeted plasma metabolomics screening was performed on plasma samples from 350 participants of the AHS-2, including 171 Black and 179 White participants, using ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and a global platform of 892 metabolites. Differences in metabolites or biochemical subclasses by race were analyzed using linear regression, considering various models adjusted for known confounders, dietary and/or other lifestyle behaviors, social vulnerability, and psychosocial stress. The Storey permutation approach was used to adjust for false discovery at FDR < 0.05.ResultsLinear regression revealed differential abundance of over 40% of individual metabolites or biochemical subclasses when comparing Black with White participants after adjustment for false discovery (FDR < 0.05), with the vast majority showing lower abundance in Blacks. Associations were not appreciably altered with adjustment for dietary patterns and socioeconomic or psychosocial stress. Metabolite subclasses showing consistently lower abundance in Black participants included various lipids, such as lysophospholipids, phosphatidylethanolamines, monoacylglycerols, diacylglycerols, and long-chain monounsaturated fatty acids, among other subclasses or lipid categories. Among all biochemical subclasses, creatine metabolism exclusively showed higher abundance in Black participants, although among metabolites within this subclass, only creatine showed differential abundance after adjustment for glomerular filtration rate. Notable metabolites in higher abundance in Black participants included methyl and propyl paraben sulfates, piperine metabolites, and a considerable proportion of acetylated amino acids, including many previously found associated with glomerular filtration rate.ConclusionsDifferences in metabolic profiles were evident when comparing Black and White participants of the AHS-2 cohort. These differences are likely attributed in part to dietary behaviors not adequately explained by dietary pattern covariates, besides other environmental or genetic factors. Alterations in these metabolites and associated subclasses may have implications for the prevention of chronic diseases in Black Americans.

Untargeted metabolomic profiling in children identifies novel pathways in asthma and atopy

BackgroundAsthma and other atopic disorders can present with varying clinical phenotypes marked by differential metabolomic manifestations and enriched biological pathways. ObjectiveTo identify these unique metabolomic profiles in atopy and asthma. MethodsWe analyzed baseline non-fasted plasma samples from a large multi-site pediatric population of 470 children < 13 years from three different sites in the US and France. Atopy (At+) was defined as skin prick test ≥ 3 mm and/or specific IgE ≥ 0.35 kUi/L, and/or total IgE ≥ 173 IU/mL. Asthma (As+) was based on physician’s diagnosis. The cohort was divided into four groups of varying combinations of As and At and six pairwise analyses were conducted to best assess differential metabolomic profiles between groups. Results210 children were classified as At-As-, 42 as At+As-, 74 as At-As+ and 144 as At+As+. Untargeted global metabolomic profiles were generated through Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectroscopy. We applied two independent machine learning classifiers and shortlisted 362 metabolites as discriminant features. Our analysis showed the most diverse metabolomic profile in the At+As+ / At-As- comparison, followed by the At-As+ / At-As- comparison, indicating that asthma is the most discriminant condition associated with metabolomic changes. At+As+ metabolomic profiles were characterized by higher levels of bile acids, sphingolipids, and phospholipids, and lower levels of polyamine, tryptophan, and gamma-glutamyl amino-acids. ConclusionAt+As+ phenotype displays a distinct metabolomic profile suggesting underlying mechanisms such as modulation of host-pathogen and gut microbiota interactions, epigenetic changes in T-cell differentiation, and lower antioxidant properties of the airway epithelium.

Plasma metabolomics and proteomics reveal novel molecular insights and biomarker panel for cholelithiasis

BackgroundCholelithiasis is a gastrointestinal disease that is associated with the highest socioeconomic cost. A diagnosis of cholelithiasis based on clinical features is significantly limited, and direct molecular insights into cholelithiasis and the relationship between cholelithiasis and clinical biochemical parameters are unclear. ObjectivesUncovering direct molecular insights into cholelithiasis and the relationship between cholelithiasis and clinical biochemical parameters. Identifying sensitive and specific biomarkers for this disease. MethodsParallel metabolomic and proteomic analyses of plasma from cholelithiasis patients (CPs) and healthy control individuals (HCs) without cholelithiasis were performed using ultrahigh-performance liquid chromatography-tandem mass spectrometry. A multimodule coexpression network analysis and integrated machine learning methods, including least absolute shrinkage and selection operator, random forest, and support vector machine, were used for bioinformatic analyses. An independent cohort and the cross-validation of the combination of two cohorts were used to evaluate the diagnostic performance of the panel. ResultsArachidonic acid metabolism was significantly different between the CP and HC groups. Glyceraldehyde-3-phosphate dehydrogenase, actin beta, phosphoglycerate mutase 1, Enolase 1, Myeloperoxidase, and actin alpha 1 were identified as potential proteins related to cholelithiasis. The correlation between the merged modules and clinical biochemical tests was calculated. A diagnostic panel composed of four candidate biomarkers, including 3-oxotetradecanoic acid, 12-hydroxydodecanoic acid, hemoglobin subunit delta (HBD), and fibrinogen beta chain (FGB), was proposed based on three modules that were significantly associated with cholelithiasis. The classification according to the diagnostic panel detected CPs with an area under the curve (AUC) of 0.955. External validation in an independent cohort resulted in similar accuracy (AUC=0.995). ConclusionsThis study provided some direct molecular insights into cholelithiasis by showing the differences in plasma metabolic and protein profiles between CPs and HCs and presented a potential biomarker panel with two metabolites (3-oxotetradecanoic acid, 12-hydroxydodecanoic acid) and two proteins (HBD, FGB) for predicting cholelithiasis. We also explored the potential correlation of clinical biochemical parameters with combined modules. These findings may provide some reference for the diagnosis of cholelithiasis in clinical practice.

An improved ultra-high performance liquid chromatography-tandem mass spectrometry method determining hispidulin and homoplantaginin in rat plasma and associated pharmacokinetic studies

AbstractFlavonoids are the most abundant components in Salvia plebeia, with significant pharmacological antioxidant and hepatoprotective properties. Hispidulin and homoplantaginin are the main flavonoid components in S. Plebeia. In this study, we established an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to determine hispidulin and homoplantaginin in rat plasma samples, which were precipitated using acetonitrile-methanol (9:1, v/v). We used a UPLC HSS T3 (100 mm × 2.1 mm, 1.7 μm diameter) chromatographic column, an acetonitrile-water (containing 0.1% formic acid) mobile phase, and a gradient elution flow rate of 0.4 mL min−1 in an elution time of 4 min. We used electrospray ionization (ESI) detection in positive ion mode, and multiple reaction monitoring mode (MRM) for quantitative analysis: m/z 301 → 286 for hispidulin, m/z 463 → 301 for homoplantaginin, and m/z 465 → 303 for internal standard (IS). In pharmacokinetic studies, 24 rats were orally administered hispidulin and homoplantaginin (5 mg kg−1) and received sublingual intravenous injections (1 mg kg−1) at two different doses, four groups with six rats/group. Differences in hispidulin and homoplantaginin pharmacokinetics in rat plasma were evaluated. The calibration curve showed good linearity in the 0.5–1,000 ng mL−1 range, with r &gt; 0.99. Precision, accuracy, recovery, matrix effects, and stability results all met standard biological sample detection requirements. Our pharmacokinetic studies showed hispidulin bioavailability was much higher than homoplantaginin at 17.8% and 0.1%, respectively.

Simultaneous determination of seven bile acids to study the effect of ivermectin on their plasma levels in rat by UHPLC–MS/MS

Bile acids (BAs) are considered to be important diagnostic biomarkers to understand the pathophysiology of hepatobiliary and metabolic diseases. BAs regulate lipid and glucose metabolism by binding to farnesoid X receptor (FXR). To date, there were no reports on the effect of an exogenous FXR modulator, ivermectin (IVM), on the plasma BA profiles in rats. To explore the effect of IVM on plasma BA levels in rat, an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC–MS/MS) method was developed and validated for simultaneous determination of seven major BAs in rat plasma. The developed method was selective, specific, accurate and precise for the quantification of plasma BAs. Sprague–Dawley rats were orally administered with IVM at a dose of 5 mg/kg once a day for 14 days and the plasma BAs were determined before and after IVM exposure using developed UHPLC–MS/MS method. Once-daily administration of IVM for 14 days resulted in significant reduction in cholic acid and deoxycholic acid levels while glycodeoxycholic acid and taurodeoxycholic acid levels were not affected. Interestingly, tauro-α-muricholic acid and tauro-β-muricholic acid levels were significantly increased. This study revealed that IVM has an important effect on plasma BA profiles in rats. This report provides an analytical methodology that can be applied to investigate the effect of drugs or pathophysiological factors on plasma BA levels.

Metabolic pathway of tebuconazole by soil fungus Cunninghamella elegans ATCC36112.

Tebuconazole is the most widely used fungicide in agriculture. Due to its long half-life, tebuconazole residues can be found in the environment media such as in soil and water bodies. Here, the metabolic pathway of tebuconazole was studied in Cunninghamella elegans (C. elegans). Approximately 98% of tebuconazole was degraded within 7days, accompanied by the accumulation of five metabolites. The structures of the metabolites were completely or tentatively identified by gas chromatography-mass spectrometry (GC-MS) and ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). To identify representative oxidative enzymes that may be involved in the metabolic process, treatment with piperonyl butoxide (PB) and methimazole (MZ) was performed. PB had a strong inhibitory effect on the metabolic reactions, while MZ had a weak inhibitory effect. The results suggest that cytochrome P450 (CYP) and flavin-dependent monooxygenase are involved in the metabolism of tebuconazole. Based on the results, we propose a metabolic pathway for the fungal metabolism of tebuconazole. Data are of interest to gain insight into the toxicological effects of tebuconazole and for tebuconazole bioremediation.

Development and validation of an ultra-high performance liquid chromatography-tandem mass spectrometry method for quantifying lenacapavir plasma concentrations: Application to therapeutic monitoring

Although current antiretroviral therapy (ART) effectively suppresses HIV in the blood, regimens may fail due to suboptimal treatment history and non-adherence to ART. In these scenarios, accumulation of viral resistance mutations to ART drug classes may occur. For these treatment-experienced people living with HIV (PLWH), activity against resistant viral strains is required; lack of therapeutic efficacy will result in continued viral replication and progression to acquired immunodeficiency syndrome. New treatment options have emerged. Lenacapavir is a first-in-class long-acting HIV-1 capsid inhibitor approved for the treatment of HIV in treatment-experienced patients. Lenacapavir is approved with an initiation regimen of oral and subcutaneous injection dosing followed by subcutaneous self-injection every 6 months. With infrequent dosing, therapeutic drug monitoring may be necessary to ensure adequate concentrations are consistently achieved in the plasma to assure treatment adherence and prevent further HIV resistance formation. To this end, we developed and validated a highly selective ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method to quantify lenacapavir concentrations in human plasma. A simple protein precipitation with acetonitrile followed by supernatant dilution was performed. Lenacapavir and its stable labeled internal standard were separated at 1.90 min using a multi-step UPLC gradient. The assay for lenacapavir quantification was extensively validated according to the United States Food and Drug Administration Bioanalytical Guidelines over a clinically relevant range of 0.1 to 500 ng/mL with excellent linearity (R2 ≥ 0.9960). This analytical method achieves acceptable performance of trueness (89.7–104.1 %), repeatability, and precision (CV < 15 %). We applied this method to quantify a clinical sample and to determine the percent protein-unbound. This method can be utilized for the therapeutic monitoring of lenacapavir in human plasma for monitoring HIV treatment efficacy.

Determination of restricted dyes in textile raw material solid wastes by ultra-high performance liquid chromatography-tandem mass spectrometry

A rapid and highly sensitive method for the quantification of 34 restricted dyes (including acid, basic, disperse, direct, and azo dyes) in solid textile raw material wastes was developed by employing ultrasonic extraction coupled with ultra-high performance liquid chromatography-tandem mass spectrometry(UHPLC-MS/MS). More specifically, the proposed method employed methanol as the extraction solvent, while the mobile phases consisted of acetonitrile and 10 mmol/L ammonium acetate + 0.05% ammonia. A good linearity was achieved over the concentration range of 0.01–200 ng/mL with correlation coefficients (R) between 0.991–0.999, limits of detection (LODs) of 0.25–40.0 µg/kg (S/N = 3) and limits of quantification (LOQs) of 0.84–133.4 µg/kg (S/N = 10). 34 dyes were recovered at three levels ranging from 84.5 to 106.9% with relative standard deviation (RSDs) ranging from 0.59% to 10.61%. Further, the method was applied for the accurate analysis of 32 counts of cotton yarn, waste cotton, and printed fabrics within 15 min. The dyestuffs accurately quantified by this rapid chromatographic procedure covered a wide range of carcinogenic and allergenic dyestuffs listed in the Oeko-Tex Standard 100 (version 02.2023) colourants. The ultrasound technique combined with the ultra-high performance liquid chromatography-tandem mass spectrometry method proposed in this work is thus suitable for the rapid screening, confirmation, and quantitative detection of industrial synthetic dyes within solid waste originating from textile raw materials.

Determination of Nε-(1-Carboxymethyl)-L-Lysine in brewing soy sauce by ultrahigh performance liquid chromatography coupled with tandem mass spectrometry

A method for the determination of Nε-(1-Carboxymethyl)-L-Lysine (CML) in brewing soy sauce was developed using ultrahigh performance liquid chromatography-tandem mass spectrometry. A mixed cation exchange solid phase extraction cartridge was used for purification, while gradient elution of a hydrophilic interaction chromatography column was performed with a 0.1% formic acid aqueous solution-acetonitrile. The adding standard recovery rate of this method ranged from 86.94 to 92.61%, and the relative standard deviation (RSD) was between 3.69 and 6.18%. The limit of detection (LOD) was 0.038 ng/mL, and the limit of quantification was 0.127 ng/mL. The RSD values of the repeatability test and the parallel test were 3.44 and 6.56%, respectively. Using this method, the CML contents determined in eight brewing soy sauce samples were 3.16 ± 0.18 – 15.10 ± 1.19 µg/mL. This method is simple, accurate, sensitive, and precise, and it is an effective method for monitoring the CML content in brewing soy sauce.

Linderae Radix water extract treats diarrhea-predominant irritable bowel syndrome in rats: a serum metabolomics study

This study aims to investigate the mechanism of Linderae Radix water extract(LRWE) in the prevention and treatment of diarrhea-predominant irritable bowel syndrome(IBS-D) based on serum metabolomics. Eighteen 2-week-old male SD rats were randomized into control, IBS-D model, and LRWE groups. The rats in other groups except the control group received gavage of senna concentrate combined with restraint stress for the modeling of IBS-D. The rats in the LRWE group were administrated with LRWE(5.4 g·kg~(-1)) by gavage, and those in the control and IBS-D model groups with an equal volume of distilled water for a total of 14 days. The visceral sensitivity was evaluated by the abdominal withdrawal reflex(AWR) score, and the degree of diarrhea was assessed by the fecal water content(FWC). The morphological changes of the colon and the morphology and number of goblet cells were observed by hematoxylin-eosin(HE) and periodic acid-schiff(PAS) staining, respectively. Ultra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS) was used for the screening of the potential biomarkers in the rat serum and their related metabolic pathways. The results showed that LRWE reduced the AWR score, decreased FWC, and alleviated visceral sensitivity and diarrhea symptoms in IBS-D rats. HE and PAS staining showed that LRWE mitigated low-grade intestinal inflammation and increased the number of mature secretory goblet cells in the colonic epithelium of IBS-D rats. A total of 25 potential biomarkers of LRWE in treating IBS-D were screened out in this study, which were mainly involved in riboflavin, tryptophan, glycine, serine and threonine metabolism, glyoxylate and dicarboxylate metabolism, and cysteine and methionine metabolism. The regulatory effects were the most significant on the riboflavin and tryptophan metabolism pathways. LRWE may alleviate the visceral hypersensitivity by promoting energy metabolism and amino acid metabolism, enhancing intestinal barrier function, and improving intestinal immune function in IBS-D rats.