Metabolomics reveals the importance of metabolites in Mussaenda pubescens for antioxidant properties and quality traits.

Mussaenda pubescens (Mp) is a valuable medicinal plant that has traditionally been used for medicinal purposes or as a tea substitute. However, there are few studies on the comprehensive and dynamic evaluation of Mp metabolites. This study used an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) approach and biochemical analysis to investigate substance changes in leaves at three different stages and elucidate the relationship between metabolites and antioxidant capacity. The findings showed that Mp leaves contained 957 metabolites, the majority of which were phenolic acids, lipids, and terpenoids. The metabolite profiling of Mp leaves was significantly influenced by their growth and development at different stages. A total of 317 differentially accumulated metabolites (DAMs) were screened, including 150 primary metabolites and 167 secondary metabolites, with 202 DAMs found in bud leaf vs. tender leaf, 54 DAMs in tender leaf vs. mature leaf, and 254 DAMs in bud leaf vs. mature leaf. Total phenolics, flavonoids, and anthocyanin concentrations decreased as Mp leaves grew and developed, whereas terpenoids increased significantly. The secondary metabolites also demonstrated a positive correlation with antioxidant activity. Phenolics, flavonoids, terpenoids, and anthocyanins were the primary factors influencing the antioxidant activity of leaves. These findings provide new insights into the metabolite formation mechanism, as well as the development and utilization of Mp tea.

Integrated metabolomic and transcriptomic analyses provide insights into regulation mechanisms during bulbous stem development in the Chinese medicinal herb plant, Stephania kwangsiensis.

BACKGROUND: Stephania kwangsiensis Lo (Menispermaceae) is a well-known Chinese herbal medicine, and its bulbous stems are used medicinally. The storage stem of S. kwangsiensis originated from the hypocotyls. To date, there are no reports on the growth and development of S. kwangsiensis storage stems. RESULTS: The bulbous stem of S. kwangsiensis, the starch diameter was larger at the stable expanding stage (S3T) than at the unexpanded stage (S1T) or the rapidly expanding stage (S2T) at the three different time points. We used ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and Illumina sequencing to identify key genes involved in bulbous stem development. A large number of differentially accumulated metabolites (DAMs) and differentially expressed genes (DEGs) were identified. Based on the differential expression profiles of the metabolites, alkaloids, lipids, and phenolic acids were the top three differentially expressed classes. Compared with S2T, significant changes in plant signal transduction and isoquinoline alkaloid biosynthesis pathways occurred at both the transcriptional and metabolic levels in S1T. In S2T compared with S3T, several metabolites involved in tyrosine metabolism were decreased. Temporal analysis of S1T to S3T indicated the downregulation of phenylpropanoid biosynthesis, including lignin biosynthesis. The annotation of key pathways showed an up-down trend for genes and metabolites involved in isoquinoline alkaloid biosynthesis, whereas phenylpropanoid biosynthesis was not completely consistent. CONCLUSIONS: Downregulation of the phenylpropanoid biosynthesis pathway may be the result of carbon flow into alkaloid synthesis and storage of lipids and starch during the development of S. kwangsiensis bulbous stems. A decrease in the number of metabolites involved in tyrosine metabolism may also lead to a decrease in the upstream substrates of phenylpropane biosynthesis. Downregulation of lignin synthesis during phenylpropanoid biosynthesis may loosen restrictions on bulbous stem expansion. This study provides the first comprehensive analysis of the metabolome and transcriptome profiles of S. kwangsiensis bulbous stems. These data provide guidance for the cultivation, breeding, and harvesting of S. kwangsiensis.

Simultaneous quantification of six proteins related to liver injury using nano liquid chromatography-tandem mass spectrometry.

RATIONALE: In clinical diagnosis of liver injury, which is an important health concern, serum aminotransferase assays have been the go-to method used worldwide. However, the measurement of serum enzyme activity has limitations, including inadequate disease specificity and enzyme specificity. METHODS: With the high selectivity and specificity provided by nano liquid chromatography-tandem mass spectrometry (LC/MS/MS), this work describes a method for the simultaneous determination of six proteins in liver that can be potentially used as biomarkers for liver injury: glutamic-pyruvic transaminase 1 (GPT1), glutamic oxaloacetic transaminase 1 (GOT1), methionine adenosyl transferase 1A (MAT1A), glutathione peroxidase 1 (GPX1), cytokeratin 18 (KRT18) and apolipoprotein E (APOE). RESULTS: In validation, the method was shown to have good selectivity and sensitivity (limits of detection at pg/mL level). The analytical method revealed that, compared with normal mice, in carbon tetrachloride-induced acute liver injury mice, liver MAT1A and GPX1 were significantly lower (p < 0.01 and p < 0.05, respectively), KRT18 was significantly higher (p < 0.05) and APOE and GPT1 were marginally significantly lower (p between 0.05 and 0.1). This is the first work reporting the absolute contents of GPT1, GOT1, MAT1A, GPX1 and KRT18 proteins based on LC/MS. CONCLUSIONS: The proposed method provides a basis for establishing more specific diagnostic indicators of liver injury.

LC-MS Characterization and Stability Assessment Elucidate Correlation Between Charge Variant Composition and Degradation of Monoclonal Antibody Therapeutics.

Aggregation stability of monoclonal antibody (mAb) therapeutics is influenced by many critical quality attributes (CQA) such as charge and hydrophobic variants in addition to environmental factors. In this study, correlation between charge heterogeneity and stability of mAbs for bevacizumab and trastuzumab has been investigated under a variety of stresses including thermal stress at 40 °C, thermal stress at 55 °C, shaking (mechanical), and low pH. Size- and charge-based heterogeneities were monitored using analytical size exclusion chromatography (SEC) and cation exchange chromatography (CEX), respectively, while dynamic light scattering was used to assess changes in hydrodynamic size. CEX analysis revealed an increase in cumulative acidic content for all variants of both mAbs post-stress treatment attributed to increased deamidation. Higher charge heterogeneity was observed in variants eluting close to the main peak than the ones eluting further away (25-fold and 42-fold increase in acidic content for main and B1 of bevacizumab and 19-fold for main of trastuzumab, respectively, under thermal stress; 50-fold increase in acidic for main and B1 of bevacizumab and 10% rise in basic content of main of trastuzumab under pH stress). Conversely, variants eluting far away from main exhibit greater aggregation as compared to close-eluting ones. Aggregation kinetics of variants followed different order for the different stresses for both mAbs (2nd order for thermal and pH stresses and 0th order for shaking stress). Half-life of terminal charge variants of both mAbs was 2- to 8-fold less than main indicating increased degradation propensity.

Determination of trace chelating carboxylic acids in rice by green extraction combined with liquid chromatography-mass spectrometry analysis and its application in the evaluation of old and new rice.

RATIONALE: Accurate identification of old rice samples from new ones benefits their market circulation and consumers. However, the current detection methods are still not satisfactory because of their insufficient accuracy or (and) time-consuming process. METHODS: Chelating carboxylic acids (CCAs) were selectively extracted from rice, by stirring with chelating resin and a dilute Na2CO3 solution. The green analytical chemistry guidelines for sample preparation were investigated by using the green chemistry calculator AGREE prep. The extractant was determined by liquid chromatography-mass spectrometry (LC/MS), and statistical analysis of the analytical data was carried out to evaluate the significance of the difference by ChiPlot. RESULTS: The limit of quantitation for the CCAs is in the range of 1 to 50 ng/mL, with a reasonable reproducibility. The CCAs in 23 rice samples were determined within a wide concentration range from 0.03 to 1174 µg/g. Intriguingly, the content of citric acid, malonic acid, α-ketoglutaric acid and cis-aconite acid in new rice was each found to be distinctively higher than that in old rice by several times. Even mixtures of old and new rice were found to show much difference in the concentration of citric acid and malic acid. CONCLUSION: A green analytical method has been developed for the simultaneous determination of CCAs by LC/MS analysis, and the identification of old rice samples from new ones was easily carried out according to their CCA content for the first time. The results indicated that the described method has powerful potential for the accurate identification of old rice samples from new ones.

Circulating ceramide levels and ratios in Emirati youth under 18 years: associations with cardiometabolic risk factors.

BACKGROUND: Circulating ceramide (Cer) drives various pathological processes associated with cardiovascular diseases, liver illness, and diabetes mellitus. Although recognized as predictors of cardiometabolic diseases (CMD) in research and clinical settings, their potential for predicting CMD risk in individuals under 18 remains unexplored. OBJECTIVES: This study was designed to utilize Liquid Chromatography-Mass Spectrometry (LC-MS/MS) methodology to determine the biological reference ranges for Cer in plasma samples of Emirati children and develop a risk assessment score (CERT-1) based on Cer concentrations. METHODS: Using LC-MS/MS, we developed a method to measure five Cer species in plasma samples of 582 Emirati participants aged 5-17. We used the circulating concentrations of these Cer to determine their reference intervals in this population. We employed traditional statistical analyses to develop a risk score (CERT-1) and assess the association between Cer levels and conventional biomarkers of CMD. RESULTS: We validated a high-throughput methodology using LC-MS/MS to quantify five Cer species in human plasma. Reference values for this population (n = 582) were quantified: CerC16:0 (0.12-0.29 µmol/L), CerC18:0 (0.019-0.067 µmol/L), CerC22:0 (0.102-0.525 µmol/L), CerC24:0 (0.65-1.54 µmol/L) and CerC24:1 (0.212-0.945 µmol/L). We devised a risk assessment score (CERT-1) based on plasma Cer content in the study participants, showing that 72.5% have low to moderate risk and 9.3% are at a higher risk of developing CMD. Our analyses also revealed a significant correlation (P < 0.05) between this score and the conventional risk factors linked to CMD, indicating its potential clinical implication. CONCLUSION: This study presents a clinical-scaled LC-MS/MS methodology for assessing clinically relevant Cer, setting reference ranges, and developing a risk score (CERT-1) for young Emirati individuals. Our findings can enhance primary risk prediction and inform the management and follow-up of CMD from an early age.

Analytical method development, identification, and characterization of stress degradation products of idelalisib by ultrahigh-performance liquid chromatography with photodiode array and ultrahigh-performance liquid chromatography with electrospray ionization quadrupole time-of-flight mass spectrometry studies.

RATIONALE: As per International Council for Harmonization (ICH) drug stability test guideline Q1A(R2), inherent stability characteristics of a drug should be studied. This work was designed to investigate inherent degradation characteristics of the drug idelalisib under ICH prescribed stress conditions, identify its degradation products, and postulate their corresponding degradation pathways. METHODS: Idelalisib was subjected to the ICH prescribed conditions of hydrolytic (neutral, acidic, and alkaline), photolytic, oxidative, and thermal stress according to ICH guideline Q1A(R2). An ultrahigh-performance liquid chromatography with photodiode array (UHPLC-PDA) method was developed to adequately resolve the drug from its degradation products, validated as per the ICH guidelines, and subsequently extended to UHPLC with electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QTOFMS) studies to identify the degradation products. RESULTS: Significant degradation was noted under conditions of acidic/alkaline hydrolysis, acid photolysis, and oxidative stress. The UHPLC/ESI-QTOFMS studies revealed the generation of four degradation products (I-IV), which were satisfactorily resolved from the drug by UHPLC on a Kinetex® C18 (100 × 4.6 mm; 2.6 µm) column by the developed isocratic elution method. Detection wavelength was selected as 270 nm. All the degradation products (I-IV) could be identified and characterized from their mass spectral data. The degradation pathways for the generation of various products from the drug were postulated. CONCLUSIONS: A UHPLC-PDA method was developed and validated for idelalisib. Four degradation products of idelalisib were revealed through UHPLC/ESI-QTOFMS studies, and corresponding degradation pathways were postulated for the same.

Biomonitoring of benzophenones in guano samples of wild bats in Poland.

Benzophenones (BPs) are substances used in the production of sunscreens, cosmetics, and personal care products. However, there is a lack of knowledge of BPs in wild animals. Therefore, the study aimed to assess the concentration of selected BPs commonly used in the cosmetic industry in guano samples collected from 4 colonies of greater mouse-eared bats (Myotis myotis). Liquid chromatography with tandem mass spectrometry (LC-MS/MS) was used to determine guano concentrations of benzophenone 1 (BP-1), benzophenone 2 (BP-2), benzophenone 3 (BP-3) and benzophenone 8 (BP-8). BP-1 levels above the method quantification limit (MQL) were noted in 97.5% of samples and fluctuated from <0.1 ng/g to 259 ng/g (mean 41.50 ng/g, median 34.8). The second most common was BP-3, which fluctuated from <0.1 ng/g to 19 ng/g (mean 6.67 ng/g, median 5.05), and its levels higher than MQL were observed in 40% of samples. BP-2 and BP-8 concentrations did not exceed the method detection limit (0.04 ng/g) in any analyzed sample. There were visible differences in the BP-1 and BP-3 levels among the studied bat colonies. Mean BP-1 concentration fluctuated from 11.23±13.13 ng/g to 76.71±65.51 ng/g and differed significantly between the colonies. Mean BP-3 concentration fluctuated from 5.03±6.03 ng/g to 9.18±7.65 mg/g, but it did not differ significantly between the colonies. The results show that guano is a suitable matrix for the assessment of wildlife exposure to BPs. This could be particularly advantageous in protected species, where not disturbing and stressing the animals are crucial.

Optimization, validation, and application of a liquid chromatography-tandem mass spectrometry method for the determination of 47 banned drug and related chemical residues in livestock urine using graphitized carboxyl multi-walled carbon nanotubes-based QuEChERS extraction.

The establishment of an efficient method for the analysis of drug residues in animal urine facilitates the real-time monitoring of drugs used in the production of animal-derived food. A modified QuEChERS extraction-liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was established for the determination of 47 banned drug and related chemical residues in livestock urine. The sample was extracted with acetonitrile by converting the acid-base environment. The sample cleanup effects of seven solid phase extraction cartridges and two EMR-Lipid products were compared, and three materials, including graphitized carboxyl multi-walled carbon nanotubes (MWCNTs), PSA, and C18, were selected as QuEChERS adsorbents from 24 materials. All analytes showed good linearity, with correlation coefficients (R2) greater than 0.9936. Low limits of quantification could be obtained, ranging from 0.2 to 5.5 ng/mL. The average recoveries at low, medium, and high spiked levels were in the range of 70.8-114.9 %, with intra-day precision ranging from 2.4 % to 11.2 % and inter-day precision ranging from 4.5 % to 16.1 %. Swine urine and bovine urine samples collected from different farms were effectively analyzed using the developed method, and metronidazole was detected in three swine urine samples.

In-silico method development and optimization of on-line comprehensive two-dimensional liquid chromatography via a shortcut model.

Comprehensive two-dimensional liquid chromatography (LCxLC) represents a valuable alternative to conventional single column, or one-dimensional, liquid chromatography (1D-LC) for resolving multiple components in a complex mixture in a short time. However, developing LCxLC methods with trial-and-error experiments is challenging and time-consuming, which is why the technique is not dominant despite its significant potential. This work presents a novel shortcut model to in-silico predicting retention time and peak width within an RPLCxRPLC separation system (i.e., LCxLC systems that use reversed-phase columns (RPLC) in both separation dimensions). Our computationally effective model uses the hydrophobic-subtraction model (HSM) to predict retention and considers limitations due to the sample volume, undersampling and the maximum pressure drop. The shortcut model is used in a two-step strategy for sample-dependent optimization of RPLCxRPLC separation systems. In the first step, the Kendall's correlation coefficient of all possible combinations of available columns is evaluated, and the best column pair is selected accordingly. In the second step, the optimal values of design variables, flow rate, pH and sample loop volume, are obtained via multi-objective stochastic optimization. The strategy is applied to method development for the separation of 8, 12 and 16 component mixtures. It is shown that the proposed strategy provides an easy way to accelerate method development for full-comprehensive 2D-LC systems as it does not require any experimental campaign and an entire optimization run can take less than two minutes.

Profiling Intact Glycosphingolipids with Automated Structural Annotation and Quantitation from Human Samples with Nanoflow Liquid Chromatography Mass Spectrometry.

Sphingolipids are an essential subset of bioactive lipids found in most eukaryotic cells that contribute to membrane biophysical properties and are involved in cellular differentiation, recognition, and mediating interactions. The described nanoHPLC-ESI-Q/ToF methodology utilizes known biosynthetic pathways, accurate mass detection, optimized collision-induced disassociation, and a robust nanoflow chromatographic separation for the analysis of intact sphingolipids found in human tissue, cells, and serum. The methodology was developed and validated with an emphasis on addressing the common issues experienced in profiling these amphipathic lipids, which are part of the glycocalyx and lipidome. The high sensitivity obtained using nanorange flow rates with robust chromatographic reproducibility over a wide range of concentrations and injection volumes results in confident identifications for profiling these low-abundant biomolecules.

Screening for Forensically Relevant Drugs Using Data-Independent High-Resolution Mass Spectrometry.

Forensic and clinical laboratories are expected to provide a rapid screening of samples for a wide range of analytes; however, the ever-changing landscape of illicit substances makes analysis complicated. There is a great need for untargeted methods that can aid these laboratories in broad-scope drug screening. Liquid chromatography hyphenated with high-resolution mass spectrometry (LC-HRMS) has become a popular technique for untargeted screening and presumptive identification of drugs of abuse due to its superior sensitivity and detection capabilities in complex matrices. An untargeted extraction and data acquisition method was evaluated for the broad screening of high-priority drugs of abuse in whole blood. A total of 35 forensically relevant target analytes were identified and extracted at biologically relevant low and high (10× low) concentrations from whole blood using supported liquid extraction. Data-independent acquisition was accomplished using ultraperformance liquid chromatography and a quadrupole time-of-flight mass spectrometry. Results were acceptable for screening assays, with limits of detection at or below the recommended low-concentration cutoffs for most analytes. Analyte ionization varied from 30.1 to 267.6% (average: 110.5%) at low concentrations and from 8.6 to 383.5% (average: 93.6%) at high concentrations. Extraction recovery ranged from 8.5 to 330.5% (average: 105.3%) at low concentrations and from 9.4 to 127.5% (average: 82.7%) at high concentrations. This variability was also captured as precision, ranging from 4.7 to 135.2% (average: 36.5%) at low concentrations and from 0.9 to 59.0% (average: 21.7%) at high concentrations. The method described in this work is efficient and effective for qualitative forensic toxicology screening, as demonstrated by analysis of 166 authentic suspected impaired driver and postmortem specimens. That said, it is critical that laboratories establishing untargeted LC-HRMS screening assays be aware of the strengths and limitations across diverse drug categories and chemical structures.

Concomitant investigation of crustacean amphipods lipidome and metabolome during the molting cycle by Zeno SWATH data-independent acquisition coupled with electron activated dissociation and machine learning.

BACKGROUND: DIA (Data-Independent Acquisition) is a powerful technique in Liquid Chromatography coupled with high-resolution tandem Mass Spectrometry (LC-MS/MS) initially developed for proteomics studies and recently emerging in metabolomics and lipidomics. It provides a comprehensive and unbiased coverage of molecules with improved reproducibility and quantitative accuracy compared to Data-Dependent Acquisition (DDA). Combined with the Zeno trap and Electron-Activated Dissociation (EAD), DIA enhances data quality and structural elucidation compared to conventional fragmentation under CID. These tools were applied to study the lipidome and metabolome of the freshwater amphipod Gammarus fossarum, successfully discriminating stages and highlighting significant biological features. Despite being underused, DIA, along with the Zeno trap and EAD, holds great potential for advancing research in the omics field. RESULTS: DIA combined with the Zeno trap enhances detection reproducibility compared to conventional DDA, improving fragmentation spectra quality and putative identifications. LC coupled with Zeno-SWATH-DIA methods were used to characterize molecular changes in reproductive cycle of female gammarids. Multivariate data analysis including Principal Component Analysis and Partial Least Square Discriminant Analysis successfully identified significant features. EAD fragmentation helped to identify unknown features and to confirm their molecular structure using fragmentation spectra database annotation or machine learning. EAD database matching accurately annotated five glycerophospholipids, including the position of double bonds on fatty acid chain moieties. SIRIUS database predicted structures of unknown features based on experimental fragmentation spectra to compensate for database incompleteness. SIGNIFICANCE: Reproducible detection of features and confident identification of putative compounds are pivotal stages within analytical pipelines. The DIA approach combined with Zeno pulsing enhances detection sensitivity and targeted fragmentation with EAD in positive polarity provides orthogonal fragmentation information. In our study, Zeno-DIA and EAD thereby facilitated a comprehensive and insightful exploration of pertinent biological molecules associated with the reproductive cycle of gammarids. The developed methodology holds great promises for identifying informative biomarkers on the health status of an environmental sentinel species.

Simultaneous determination of folic acid photolysis products and oxidized guanine derivatives in plasma by liquid chromatography-tandem mass spectrometry.

Folic acid (FA) is easily photodegraded to yield 6-formylpterin and pterin-6-carboxylic acid, which can generate reactive oxygen species and result in the formation of oxidized guanine derivatives such as 8-hydroxy-2'-deoxyguanosine and 8-hydroxy-guanosine. In this study, we developed a simple, rapid, and sensitive liquid chromatography-tandem mass spectrometry strategy for the simultaneous determination of FA photolysis products and oxidized guanine derivatives in plasma samples. Chromatographic separation was performed on a Waters HSS T3 column (2.1 × 100 mm, 5.0 µm) with gradient elution at a flow rate of 0.25 mL/min. Plasma samples were first pretreated with 1% formic acid, followed by protein precipitation with methanol. The developed method showed good linear relationships between 1 and 2000 ng/mL (r2 > 0.99). The intra- and inter-day precisions ranged from 2.6% to 7.5% and from 2.5% to 6.5%, respectively. Recoveries of the analytes were between 75.4% and 112.4% with the relative standard deviation < 9.1%. Finally, the method was applied to quantify FA photolysis products and oxidized guanine derivatives in rats with light and non-light conditions.

Preparation of Monolith for Online Extraction and LC-MS Analysis of ß-Estradiol in Serum Via a Simple Multicomponent Reaction.

Multicomponent reactions offer efficient and environmentally friendly strategies for preparing monoliths suitable for applications in analytical chemistry. In the described study, a multicomponent reaction was utilized for the one-pot miniaturized preparation of a poly(propargyl amine) polymer inside commercial silica-lined PEEK tubing. The reaction involved only small amounts of reagents and was characterized by atom economy. The resulting monolithic column was incorporated into an autosampler system for the online extraction and cleanup of ß-estradiol from human serum. Sample pretreatment was simplified to a simple dilution with methanol and centrifugation to remove proteins. The resulting platform included LC-MS analysis in multiple reaction monitoring for quantitative analysis of ß-estradiol. The method was validated in serum, demonstrating practical applicability for the monitoring of fertile women. Recoveries were above 94%, and LOD and LOQ values at 0.008 and 0.18 ng mL-1, respectively. The developed platform proved to be competitive with previous methods for solid-phase microextraction of ß-estradiol in serum, with comparable recovery and sensitivity but with the advantage of nearly complete automation. The environmental impact of the process was evaluated as acceptable due to the miniaturization of the monolith synthesis and the automation of extraction. The drawback associated with the LC-MS technique can be reduced by the inclusion of additional analytes in a single investigation. The work demonstrates that multicomponent reactions are versatile, economical, and possibly a green methodology for producing reversed-phase and mixed-mode sorbents, enabling miniaturization of the entire analytical procedure from the preparation of extraction sorbents to analysis.

Simplified processing and rapid quantification of buprenorphine, norbuprenorphine, and their conjugated metabolites in human plasma using UPLC-MS/MS: Assessment of buprenorphine exposure during opioid use disorder treatment.

Opioid use disorder (OUD) is a chronic neurobehavioral ailment and is prevalent in pregnancy. OUD is commonly treated with methadone or buprenorphine (BUP). Pregnancy is known to alter the pharmacokinetics of drugs and may lead to changes in drug exposure and response. A simple, specific, and sensitive analytical method for measuring the parent drug and its metabolites is valuable for assessing the impact of pregnancy on drug exposure. A new liquid chromatography-tandem mass spectrometric method that utilized a simple protein precipitation procedure for sample preparation and four deuterated internal standards for quantification was developed and validated for BUP and its major metabolites (norbuprenorphine [NBUP], buprenorphine-glucuronide [BUP-G], and norbuprenorphine-glucuronide [NBUP-G]) in human plasma. The standard curve was linear over the concentration range of 0.05-100 ng/mL for BUP and NBUP, and 0.1-200 ng/mL for BUP-G and NBUP-G. Intra- and inter-day bias and precision were within ±15% of nominal values for all the analytes. Quality controls assessed at four levels showed high recovery consistently for all the analytes with minimal matrix effect. Adequate analyte stability was observed at various laboratory conditions tested. Overall, the developed method is simple, sensitive, accurate and reproducible, and was successfully applied for the quantification of BUP and its metabolites in plasma samples collected from pregnant women in a clinical study assessing BUP exposure during OUD treatment.

Study of Bitespiramycin Distribution in Rats and Cerebrospinal Fluid of Patients by a Sensitive LC-MS/MS Method with Rapid Sample Preparation.

Bitespiramycin, has been shown to have a therapeutic effect against respiratory tract inflammation, including a potential effect against COVID-19. A current clinical trial in China showed that bitespiramycin was an effective treatment for severe pneumonia and intracranial infection. However, there is lack of an analytical method to elucidate the distribution of bitespiramycin. In this study, a highly sensitive, rapid and reliable UPLC-MS/MS method was developed to comprehensively characterize the bitespiramycin distribution in various bio-samples, which is significantly improved upon the published work. A rapid sample preparation method was developed by using n-butanol as the solvent to extract bitespiramycin from different bio-samples. The extract was then directly analyzed by UPLC-MS/MS coupled with an alkaline-resistant column after centrifugation which avoids the time-consuming concentration process under nitrogen and redissolution. The method was employed to accurately quantify bitespiramycin and its metabolites in rat plasma, tissues, and human cerebrospinal fluid. Notably, the presence of bitespiramycin and its metabolites was identified for the first time in various rat organs including brain, testis, bladder and prostate as well as in human cerebrospinal fluid. This newly developed approach shows great promise for drug distribution assays including other antibiotics and can help elucidate the ADME of bitespiramycin.

LC-MS-Based Metabolomics Reveals the Mechanism of Protection of Berberine against Indomethacin-Induced Gastric Injury in Rats.

Berberine is a natural isoquinoline alkaloid with low toxicity, which exists in a wide variety of medicinal plants. Berberine has been demonstrated to exhibit potent prevention of indomethacin-induced gastric injury (GI) but the related mechanism remains unclear. In the present study, liquid chromatography-mass spectrometry (LC-MS)-based metabolomics was applied for the first time to investigate the alteration of serum metabolites in the protection of berberine against indomethacin-induced gastric injury in rats. Subsequently, bioinformatics was utilized to analyze the potential metabolic pathway of the anti-GI effect of berberine. The pharmacodynamic data indicated that berberine could ameliorate gastric pathological damage, inhibit the level of proinflammatory factors in serum, and increase the level of antioxidant factors in serum. The LC-MS-based metabolomics analysis conducted in this study demonstrated the presence of 57 differential metabolites in the serum of rats with induced GI caused by indomethacin, which was associated with 29 metabolic pathways. Moreover, the study revealed that berberine showed a significant impact on the differential metabolites, with 45 differential metabolites being reported between the model group and the group treated with berberine. The differential metabolites were associated with 24 metabolic pathways, and berberine administration regulated 14 of the 57 differential metabolites, affecting 14 of the 29 metabolic pathways. The primary metabolic pathways affected were glutathione metabolism and arachidonic acid metabolism. Based on the results, it can be concluded that berberine has a gastroprotective effect on the GI. This study is particularly significant since it is the first to elucidate the mechanism of berberine's action on GI. The results suggest that berberine's action may be related to energy metabolism, oxidative stress, and inflammation regulation. These findings may pave the way for the development of new therapeutic interventions for the prevention and management of NSAID-induced GI disorders.

Liquid Chromatography with Tandem Mass Spectrometry Analysis of Carboxymethyl Lysine in Indonesian Foods.

There is little data on directly measured carboxymethyl lysine (CML) content in Indonesian foods. This study aimed to generate a database of CML values in foods commonly consumed in West Java and West Sumatra. The results were to be used to update our previous estimated CML values. CML values in food samples were measured using high-pressure liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). Food protein content was analyzed by Kjeldahl's method or inferred from the nutrition facts' label. A total of 210 food samples were examined, with the food groups of meat and poultry (1.06 mg CML/100 g edible food), and starchy foods (0.21 mg/100 g edible food) having the highest and lowest mean CML levels, respectively. We found that the foods with the top three highest CML content were fried starch dough (cimol), fried fish crackers, and chicken gulai. The mean of the estimated values (0.80 mg CML/100 g edible food) was higher than the directly measured values (0.66 mg CML/100 g edible food), [p < 0.035]. Conclusion: This database provides information on CML values in Indonesian foods, and can be further used to make a guide policy for the selection of foods to reduce non-communicable diseases. Further measurements are needed on Indonesian dishes to complete the database.

An Integrated Hepatocyte Stability Assay for Simultaneous Metabolic Stability Assessment and Metabolite Profiling.

The determination of metabolic stability is critical for drug discovery programs, allowing for the optimization of chemical entities and compound prioritization. As such, it is common to perform high-volume in vitro metabolic stability experiments early in the lead optimization process to understand metabolic liabilities. Additional metabolite identification experiments are subsequently performed for a more comprehensive understanding of the metabolic clearance routes to aid medicinal chemists in the structural design of compounds. Collectively, these experiments require extensive sample preparation and a substantial amount of time and resources. To overcome the challenges, a high-throughput integrated assay for simultaneous hepatocyte metabolic stability assessment and metabolite profiling was developed. This assay platform consists of four parts: 1) an automated liquid-handling system for sample preparation and incubation, 2) a liquid chromatography and high-resolution mass spectrometry-based system to simultaneously monitor the parent compound depletion and metabolite formation, 3) an automated data analysis and report system for hepatic clearance assessment; and 4) streamlined autobatch processing for software-based metabolite profiling. The assay platform was evaluated using eight control compounds with various metabolic rates and biotransformation routes in hepatocytes across three species. Multiple sample preparation and data analysis steps were evaluated and validated for accuracy, repeatability, and metabolite coverage. The combined utility of an automated liquid-handling instrument, a high-resolution mass spectrometer, and multiple streamlined data processing software improves the process of these highly demanding screening assays and allows for simultaneous determination of metabolic stability and metabolite profiles for more efficient lead optimization during early drug discovery. SIGNIFICANCE STATEMENT: Metabolic stability assessment and metabolite profiling are pivotal in drug discovery to fully comprehend metabolic liabilities for chemical entity optimization and lead selection. Process of these assays can be repetitive and resource demanding. Here, we developed an integrated hepatocyte stability assay that combines automation, high-resolution mass spectrometers, and batch-processing software to improve and combine the workflow of these assays. The integrated approach allows simultaneous metabolic stability assessment and metabolite profiling, significantly accelerating screening and lead optimization in a resource-effective manner.