Characterization of the in vitro metabolic profile of nazartinib in HLMs using UPLC-MS/MS method: In silico metabolic lability and DEREK structural alerts screening using StarDrop software.

The orally given, irreversible, third-generation inhibitor of the epidermal growth factor receptor (EGFR), known as Nazartinib (EGF816), is now undergoing investigation in Phase II clinical trials conducted by Novartis for Non-Small Cell Lung Cancer. The primary aim of the current research was to establish a rapid, specific, environmentally friendly, and highly versatile UPLC-MS/MS methodology for the determination of nazartinib (NZT) levels in human liver microsomes (HLMs). Subsequently, same approach was used to examine the metabolic stability of NZT. The UPLC-MS/MS method employed in HLMs was validated as stated in the bioanalytical method validation criteria outlined by the US- FDA. The evaluation of the metabolic stability of NZT and the identification of potentially structural alarms were performed using the StarDrop software package that includes the P450 and DEREK software. The calibration curve for NZT showed a linearity in the range from 1 to 3000 ng/mL. The inter-day accuracy and precision exhibited a range of values between -4.33 % and 4.43 %, whereas the intra-day accuracy and precision shown a range of values between -2.78 % and 7.10 %. The sensitivity of the developed approach was verified through the determination of a LLOQ of 0.39 ng/mL. The intrinsic clearance and in vitro half-life of NZT were assessed to be 46.48 mL/min/kg and 17.44 min, respectively. In our preceding inquiry, we have effectively discerned the bioactivation center, denoted by the carbon atom between the unsaturated conjugated system and aliphatic linear tertiary amine. In the context of computational software, making minor adjustments or substituting the dimethylamino-butenoyl moiety throughout the drug design process may increase the metabolic stability and safety properties of new synthesized derivatives. The efficiency of utilizing different in silico software approaches to conserve resources and reduce effort was proved by the outcomes attained from in vitro incubation experiments and the use of NZT in silico software.

Determination of doxorubicin in plasma and tissues of mice by UPLC-MS/MS and its application to pharmacokinetic study.

A rapid and sensitive ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was established for the simultaneous determination of doxorubicin (DOX) in mouse plasma and tissues, including the heart, liver, spleen, lung, kidney and tumor, and to investigate the pharmacokinetics and distribution in mice. In this study, daunorubicin (DNR) was used as an internal standard, and the mobile phase consisted of ammonium formate 2 mM containing 0.1 % formic acid (A) and acetonitrile (B), the chromatographic column was ACQUITY UPLC BEHTM C18 with a gradient elution at a flow rate of 0.2 mL/min. Electrospray ionization (ESI) in positive ion pattern was utilized for the ion separation of DOX, with the ions used for quantitative analysis being DOX m/z 544.28 â†’ 397.10 and DNR m/z 528.35 â†’ 321.08, respectively. The results showed that a good linear relationship in the calibration curve range of 1-800 ng/mL in mouse plasma and 1-2500 ng/g in tissues (R2 > 0.99) with the limits of quantification of 1 ng/mL in plasma and tissues. The method exhibited good matrix effect and extraction recovery, with the intra-day and inter-day precision of plasma and tissue were less than 10.3 % and 15.4 %, and the relative error (RE) were both less than ±14.8 % and ±18.9 %, respectively. The stability results under different conditions were found to be accurate. It also revealed the distribution of DOX in various tissues of mice, with the concentration ranking as liver > heart > kidney > spleen > lung > tumor. This method was successfully used to the study for the pharmacokinetics in plasma and drug distribution in tissues of BALB/c mice.

Development of a UPLC-MS/MS method for quantifying KPT-335 (Verdinexor) in feline plasma for a study of PK.

KPT-335 (Verdinexor) is a novel SINE that potently inhibits the nucleoprotein Exportin 1 (XPO1/CRM1) of tumor cell lines and reduces the replication level of the influenza virus. KPT-335 is mainly used for the treatment of canine tumors. Drugs for the effective treatment of feline tumors are currently unavailable in China. KPT-335 may have potential in the treatment of cat tumors. However, the effects of KPT-335 in cats are unreported, and no relevant methodology has been established for pharmacokinetic studies. In this study, a UPLC-MS/MS method was developed to determine KPT-335 concentrations in cat plasma, followed by pharmacokinetic studies. Briefly, plasma proteins are precipitated with acetonitrile, and the supernatant was collected for detection after centrifugation. The linearity for KPT-335 in cat plasma was in the range of 5-1,000 ng/mL. Satisfactory accuracy and precision were obtained. The intra-day accuracy was between -4.10% and 10.48%, the precision was ≤4.65%; the inter-day accuracy was between -0.11% and 8.09%, and the precision was ≤5.85%. Intra-day and inter-day accuracy and precision were within regulatory limits. The results of preliminary pharmacokinetic studies were as follows: Tmax was 1.46 ± 0.51 h; Cmax was 239.54 ± 190.60 ng·mL-1; T1/2 was 5.16 ± 2.30 h; AUC0-t was 1439.85 ± 964.64 ng·mL-1·h. The AUC0-∞ was 1589.82 ± 1003.75 ng·mL-1·h. The purpose of this study was to develop a rapid and simple UPLC-MS/MS method to detect KPT-335 concentration in cat plasma and to conduct preliminary pharmacokinetic studies to support the future application of KPT-335 in felines.

Chemical profiling and quality evaluation of raw and vinegar-processing frankincense by multiple UPLC-MS/MS techniques.

INTRODUCTION: Frankincense is used for analgesic, tumor-suppressive, and anti-inflammatory treatments in Traditional Chinese Medicine but poses toxicological concerns. Vinegar processing is a common technique used to reduce the toxicity of frankincense. OBJECTIVE: This study aimed to investigate the chemical composition and quality evaluation of raw and vinegar-processing frankincense by multiple UPLC-MS/MS techniques. Additionally, we purposed refining the vinegar processing technique and identifying potentially harmful ingredients in the raw frankincense. METHODOLOGY: Sub-chronic oral toxicity studies were conducted on raw and vinegar-processing frankincense in rats. The composition of frankincense was identified by UPLC-Q-TOF-MS/MS. Chemometrics were used to differentiate between raw and vinegar-processing frankincense. Potential chemical markers were identified by selecting differential components, which were further exactly determined by UPLC-QQQ-MS/MS. Moreover, the viability of the HepG2 cells of those components with reduced contents after vinegar processing was assessed. RESULTS: The toxicity of raw frankincense is attenuated by vinegar processing, among which vinegar-processing frankincense (R40) (herb weight: rice vinegar weight = 40:1) exhibited the lowest toxicity. A total of 83 components were identified from frankincense, including 40 triterpenoids, 37 diterpenoids, and 6 other types. The contents of six components decreased after vinegar-processing, with the lowest levels in R40. Three components, specifically 3α-acetoxy-11-keto-ß-boswellic acid (AKBA), 3α-acetoxy-α-boswellic acid (α-ABA), and 3α-acetoxy-ß-boswellic acid (ß-ABA), inhibited the viability of HepG2 cells. The processing of frankincense with vinegar at a ratio of 40:1 could be an effective method of reducing the toxicity in raw frankincense. CONCLUSION: Our research improves understanding of the toxic substance basis and facilitates future assessments of frankincense quality.

Development of UPLC-MS/MS method for studying the pharmacokinetic interactions of fuzuloparib with curcumin in rats.

Fuzuloparib is a novel orally bioactive poly-ADP-ribose polymerase inhibitor (PARPi), which was approved by the Chinese Regulatory Agency (CRA) in 2020 for the treatment of platinum-sensitive recurrent ovarian, fallopian tube, and primary peritoneal cancers. This study firstly presents a rapid and accurate ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for analyzing the levels of fuzuloparib and its major metabolite (SHR165202), and to investigate drug-drug interaction between fuzuloparib and curcumin in vitro and in vivo studies. After protein precipitation with acetonitrile, mobile phase consisted of acetonitrile and 0.1 % formic acid with a gradient elution was used to successfully separate fuzuloparib, SHR165202 and talazoparib (internal standard, IS). The results indicated that fuzuloparib and SHR165202 had good linearity over the calibration range of 2-50 ng/mL and 1-20 ng/mL, respectively. The precision, accuracy, stability, matrix effect, and extraction recovery required for methodological validation all complied with the requirements of the Bioanalytical Method Validation Guidelines. In vitro microsome incubation experiments, curcumin exhibited inhibitory effect on fuzuloparib in both rat liver microsomes (RLM) and human liver microsomes (HLM) with half-maximal inhibitory concentration (IC50) value of 10.54 µM and 47.64 µM, respectively, and the corresponding mechanism was non-competitive. Furthermore, the inhibitory mechanism of curcumin on fuzuloparib was validated through molecular docking. In pharmacokinetic experiments in rats, curcumin significantly altered the plasma exposure of fuzuloparib, resulting in significant increases in AUC(0-t) and Cmax of fuzuloparib and a significant decrease in CLz/F. Moreover, the metabolite SHR165202 showed significant increases in AUC(0-t), AUC(0-∞), Tmax and Cmax and a significant decrease in CLz/F. This further supports the notion that curcumin could inhibit the metabolism of fuzuloparib. Therefore, when co-administering fuzuloparib and curcumin in clinic, it is recommended to monitor plasma levels of fuzuloparib and pay close attention to adverse effects. If necessary, the dose of fuzuloparib needs to be reduced.

Ultra-fast UPLC-MS/MS approach for estimating X-376 in human liver microsomes: Evaluation of metabolic stability via in silico software and in vitro analysis.

X-376 is a novel anaplastic lymphoma kinase (ALK) inhibitor that is capable of penetrating the blood brain barrier. This makes it suitable for use in patients with ALK-positive non-small cell lung cancer (NSCLC) who have metastases in the central nervous system. This study developed a highly sensitive, fast, eco-friendly, and reliable UPLC-MS/MS approach to quantify X-376 in human liver microsomes (HLMs). This approach was used to evaluate X-376's metabolic stability in HLMs in vitro. The UPLC-MS/MS analytical technique validation followed US-FDA bio-analytical method validation guidelines. StarDrop software, containing P450 metabolic and DEREK modules, was utilized to scan X-376's chemical structure for metabolic lability and hazardous warnings. X-376 and Encorafenib (ENF as internal standard) were resoluted on the Eclipse Plus C18 column utilizing an isocratic mobile phase method. The X-376 calibration curve was linear from 1 to 3000 ng/mL. The precision and accuracy of this study's UPLC-MS/MS approach were tested for intra- and inter-day measurements. Inter-day accuracy was -1.32% to 9.36% while intra-day accuracy was -1.5% to 10.00%. The intrinsic clearance (Clint) and in vitro half-life (t1/2) of X-376 were 59.77 mL/min/kg and 13.56 min. The high extraction ratio of X-376 supports the 50 mg twice-daily dose for ALK-positive NSCLC and CNS metastases patients. In silico software suggests that simple structural changes to the piperazine ring or group substitution in drug design may improve metabolic stability and safety compared to X-376.

UPLC-MS/MS determination of 71 neuropsychotropic drugs in human serum.

In this study, a UPLC-MS/MS method was developed for the rapid detection of 71 neuropsychotropic drugs in human serum for drug concentration monitoring and toxicity screening. The analytes were separated from the biological matrix by protein precipitation using a methanol-acetonitrile solvent mixture. The chromatographic separation was performed on a Kromasil ClassicShell C18 column (2.1*50 mm, 2.5 µ m) with gradient elution using acetonitrile-0.2 % acetic acid and 10 mM ammonium acetate as the mobile phases (flow rate 0.4 mL/min, column temperature 40 °C, injection volume 5 µL). An electrospray ion source in both positive and negative ion modes with multiple ion monitoring was used. The total run time was 6 min. All compounds were quantified using the isotope internal standard method. Totally, 71 drugs were detected within their linear ranges with correlation coefficients greater than 0.990. The intra- and inter-batch precision relative standard deviations (RSDs) for the low, medium, and high concentration points were less than 15 %, with an accuracy of 90%-110 %. All compounds except Moclobemide N-oxindole are stabilised within 7 days. The relative matrix effect results for each analyte were within ±20 % of the requirements. The method is validated according to Clinical and Laboratory Standards Institute guidelines, easy to use, and has a low cost.

Development of UPLC-MS/MS method for the simultaneous quantification of valproic acid and phenytoin in human plasma and application to study pharmacokinetic interaction in epilepsy patients.

Valproic acid and phenytoin are two prevalent antiepileptic medications known for their narrow indices and propensity for cardiovascular and respiratory system toxicity. Therefore, therapeutic drug monitoring (TDM) of valproic acid (VAL) and phenytoin (PHE) concentrations in patient plasma is extremely beneficial for improving clinical choices, avoiding adverse reactions, and optimizing treatment for individual patients. In this study, a rapid and sensitive ultra-performance liquid chromatographic tandem mass spectrometer (UPLC-MS/MS) method was developed and validated for the simultaneous quantitative determination of valproic acid (VAL) and phenytoin (PHE) in human plasma. Negative electron spray ionization (ESI-) mode with selective ion recording (SIR) was employed to determine the transitions of m/z 142.98 and m/z 250.93 for VAL and PHE, respectively. The internal standard (IS) betamethasone (BETA) was ionized using positive electron spray ionization (ESI+) and detected by multi-reaction monitoring (MRM) mode to obtain precursor ions and specific fragment ions for quantification, and the MRM transition was chosen to be m/z 393.17 â†’ 355.16. The separation was performed using a Phenomenex Synergi Hydro-RP (4 µm, 250 × 4.6 mm, I.D.) with an isocratic mobile phase consisting of acetonitrile - water (75:25, v/v) at a flow rate of 0.8 mL/min. The column temperature was maintained at 25 °C. The lower limit of quantification of VAL and PHE was 3.6 µg/mL and 0.72 µg/mL, respectively, which resulted in a recovery of more than 85 % for most analytes. According to US-FDA bioanalytical technique validation, the specificity, intra- and inter-day precision and accuracy, matrix effect, carryover, dilution, and stability of all analytes were within acceptable ranges. This analytical method was successful in evaluating the levels of valproic acid and phenytoin in human plasma from epileptic patients.

A robust ultra-performance liquid chromatography-tandem mass spectrometry method for simultaneous determination of 10 components in glutathione cycle.

Glutathione (GSH) is an important antioxidant that is generated and degraded via the GSH cycle. Quantification of the main components in the GSH cycle is necessary to evaluate the process of GSH. In this study, a robust ultra-performance liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of 10 components (GSH; γ-glutamylcysteine; cysteinyl-glycine; n-acetylcysteine; homocysteine; cysteine; cystine; methionine; glutamate; pyroglutamic acid) in GSH cycle was developed. The approach was optimized in terms of derivative, chromatographic, and spectrometric conditions as well as sample preparation. The unstable thiol groups of GSH, γ-glutamylcysteine, cysteinyl-glycine, n-acetylcysteine, cysteine, and homocysteine were derivatized by n-ethylmaleimide. The derivatized and underivatized analytes were separated on an amino column with gradient elution. The method was further validated in terms of selectivity (no interference), linearity (R2 > 0.99), precision (% relative standard deviation [RSD%] range from 0.57 to 10.33), accuracy (% relative error [RE%] range from -3.42 to 10.92), stability (RSD% < 5.68, RE% range from -2.54 to 4.40), recovery (RSD% range from 1.87 to 7.87) and matrix effect (RSD% < 5.42). The validated method was applied to compare the components in the GSH cycle between normal and oxidative stress cells, which would be helpful in clarifying the effect of oxidative stress on the GSH cycle.

Effects of the antitumor drugs adagrasib and asciminib on apixaban metabolism in vitro and in vivo.

Apixaban is an oral anticoagulant that directly inhibits the target Factor Xa (FXa). In this study, we focused on the in vivo and in vitro effects of adagrasib and asciminib on apixaban metabolism, to discover potential drug-drug interactions (DDI) and explore their inhibitory mechanisms. The levels of apixaban and its metabolite, O-desmethyl-apixaban (M2), were determined by ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). In vitro evaluation, the maximum half inhibitory concentration (IC50) of adagrasib in rat liver microsomes (RLM) and human liver microsomes (HLM) against apixaban was 7.99 µM and 117.40 µM, respectively. The IC50 value of asciminib against apixaban in RLM and HLM was 4.28 µM and 18.42 µM, respectively. The results of the analysis on inhibition mechanisms showed that adagrasib inhibited the metabolism of apixaban through a non-competitive mechanism, while asciminib inhibited the metabolism of apixaban through a mixed mechanism. Moreover, the interaction of apixaban with adagrasib and asciminib in Sprague-Dawley (SD) rats was also investigated. It was found that the pharmacokinetic characteristics of apixaban were significantly changed when combined with these two antitumor drugs, where AUC(0-t), AUC(0-∞), t1/2, Tmax, and Cmax were increased, while CLz/F was significantly decreased. But both drugs did not appear to affect the metabolism of M2 in a significant way. Consistent results from in vitro and in vivo demonstrated that both adagrasib and asciminib inhibited the metabolism of apixaban. It provided reference data for the future clinical individualization of apixaban.

A UPLC-MS/MS method for quantification of metabolites in the ethylene biosynthesis pathway and its biological validation in Arabidopsis.

The plant hormone ethylene is of vital importance in the regulation of plant development and stress responses. Recent studies revealed that 1-aminocyclopropane-1-carboxylic acid (ACC) plays a role beyond its function as an ethylene precursor. However, the absence of reliable methods to quantify ACC and its conjugates malonyl-ACC (MACC), glutamyl-ACC (GACC), and jasmonyl-ACC (JA-ACC) hinders related research. Combining synthetic and analytical chemistry, we present the first, validated methodology to rapidly extract and quantify ACC and its conjugates using ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). Its relevance was confirmed by application to Arabidopsis mutants with altered ACC metabolism and wild-type plants under stress. Pharmacological and genetic suppression of ACC synthesis resulted in decreased ACC and MACC content, whereas induction led to elevated levels. Salt, wounding, and submergence stress enhanced ACC and MACC production. GACC and JA-ACC were undetectable in vivo; however, GACC was identified in vitro, underscoring the broad applicability of the method. This method provides an efficient tool to study individual functions of ACC and its conjugates, paving the road toward exploration of novel avenues in ACC and ethylene metabolism, and revisiting ethylene literature in view of the recent discovery of an ethylene-independent role of ACC.

Network pharmacology study on fermented soybeans for the prevention of Alzheimer's disease in older individuals.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the disruption of synaptic communication among millions of neurons. Recent research has highlighted the potential therapeutic effectiveness of natural polyphenolic compounds in addressing AD. Soybeans are abundant in polyphenols, and their polyphenolic composition undergoes significant alteration through fermentation by Eurotium cristatum. Through comprehensive database searches, we identified active components within fermented soybean polyphenols and genes associated with AD. Subsequently, we utilized Venn diagrams to analyze the overlap between AD-related genes and these components. Furthermore, we visualized the network between intersecting targets and proteins using Cytoscape software. The anti-AD effects of soybeans were further explored through comprehensive analysis, including protein-protein interaction analysis, pathway enrichment analysis, and molecular docking studies. Our investigation unveiled 6-hydroxydaidzein as a major component of fermented soybean polyphenols, shedding light on its potential therapeutic significance in combating AD. The intersection between target proteins of fermented soybeans and disease-related targets in AD comprised 34 genes. Protein-protein interaction analysis highlighted key potential targets, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glycogen synthase kinase 3 beta (GSK3B), amyloid precursor protein (APP), cyclin-dependent kinase 5 (CDK5), and beta-site APP cleaving enzyme 1 (BACE1). Molecular docking results demonstrated a robust binding effect between major components from fermented soybeans and the aforesaid key targets implicated in AD treatment. These findings suggest that fermented soybeans demonstrate a degree of efficacy and present promising prospects in the prevention of AD.

Method optimization for a simultaneous determination of neonicotinoid, carbamate/thiocarbamate, triazole, organophosphate and pyrethroid pesticides and their metabolites in urine using UPLC-MS/MS.

An accurate and sensitive method for the determination of a total of 23 pesticides and their metabolites in human urine has been optimised. The methodology is based on a previously published method based on solid-phase extraction with methanol and acetone followed by ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) in the selected reaction mode (SRM) with both positive and negative electrospray ionization (ESI+/-). The detection settings of the previous method, which allowed to determine the metabolites from 6 organophosphate and 2 pyrethroid pesticides, were optimised in order to include further pesticide groups, such as 11 neonicotinoids, 3 carbamates/thiocarbamates and 2 triazoles. The 5-windows method enduring 22 min was optimized with acceptable results in relation to accuracy (recoveries >75 %), precision (coefficients of variation <26 %) and linearity (R2> 0.9915). The limits of detection ranged between 0.012 ng/mL and 0.058 ng/mL. Samples from the German External Quality Assessment Scheme (G-EQUAS) encompassing 2 pyrethroids, 2 organophosphate and one neonicotinoid (6-chloronicotinic acid, a common metabolite of imidacloprid and acetamiprid) were analysed, and the latter, included in this newest optimization, provided good reference results. The method is optimal as a human biomonitoring tool for health risk assessment in large population surveys.

Quantification and analyses of seven tyrosine kinase inhibitors targeting hepatocellular carcinoma in human plasma by QuEChERS and UPLC-MS/MS.

Tyrosine kinase inhibitors (TKIs) are commonly used to treat various cancers. Literature suggests that the blood concentration of TKIs strongly correlates with their efficacy and adverse effects. Therefore, establishing a Therapeutic Drug Monitoring (TDM) methodology for TKI drugs is crucial to improving their clinical efficacy and minimizing the treatment-related adverse effects. However, quantifying their concentrations in the plasma using existing methods to avoid potential toxicity is challenging. Herein, seven TKIs, namely sorafenib tosylate, axitinib, erlotinib, cediranib, brivanib, linifanib, and golvatinib, were successfully analyzed in human plasma by following a quick, easy, cheap, effective, rugged, and safe (QuEChERS) pretreatment method combined with ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Briefly, biological samples were extracted using 1 mL of methanol, followed by the sequential addition of 250 mg of anhydrous magnesium sulfate and 25 mg of N-propylethylenediamine (PSA) for salinization and purification by adsorption, respectively. In this study, dovitinib was used as the internal standard. The seven TKIs were detected by the gradient elution method for 4 min in the positive ion electrospray mode. The mobile phase comprised methanol (phase A) and 0.1 % aqueous formic acid solution (phase B) on the Agilent Zorbax RRHD Stablebond Aq, (2.1 × 50 mm; 1.8 µm). Brivanib, linifanib, axitinib, sorafenib tosylate, and golvatinib exhibited good linearity in the range of 5-500 ng/mL, and erlotinib and cediranib exhibited good linearity in the range of 10-1000 ng/mL, with linear correlation coefficients (R2) ≥ 0.99. The limits of detection and quantification were 0.60-0.18 ng/mL and 5-10 ng/mL, respectively. The intraday and interday accuracy values ranged from -6.12 % to 7.31 %, with a precision (RSD) of ≤ 10.57 %. The method was rapid, accurate, specific, simple, reproducible, and suitable for the quantitative determination of the seven TKIs in human plasma.

Comprehensive analysis of the dynamic changes of volatile and non-volatile metabolites in boletus edulis during processing by HS-SPME-GC-MS and UPLC-MS/MS analysis.

In order to investigate the dynamic changes of flavor compounds, Ultra Performance Liquid Chromatography Tandem Mass Spectrometry (UPLC-MS/MS) combined with Headspace Solid Phase Microextraction Gas Chromatography Mass Spectrometry (HS-SPME-GC-MS) was used to detect the metabolites in different drying processes. A total of 80 volatile compounds and 1319 non-volatile compounds were identified. The trend in the changes of C-8 compounds and sulfur-containing compounds were generally consistent with the trend of key enzyme activities. 479 differential metabolites were identified and revealed that metabolic profiles of compounds in Boletus edulis were altered with increased organic acids and derivatives and lipids and lipid-like molecules. Fatty acids and amino acids were transformed into volatile compounds under the action of enzymes, which played a significant role in the formation of the distinctive flavor of Boletus edulis. Our study provided a theoretical support for fully comprehending the formation mechanism of flavor from Boletus edulis during drying processes.

Simultaneous determination of multiple components in rat plasma by UPLC-MS/MS for pharmacokinetic studies after oral administration of Pogostemon cablin extract.

Introduction: Pogostemon cablin (PC) is used in traditional Chinese medicine and food, as it exerts pharmacological effects, such as immune-modulatory, antibacterial, antioxidant, antitumor, and antiviral. Currently, the pharmacokinetics (PK) studies of PC mainly focus on individual components. However, research on these individual components cannot reflect the actual PK characteristics of PC after administration. Therefore, the simultaneous determination of multiple components in rat plasma using UPLC-MS/MS was used for the pharmacokinetic study after oral administration of PC extract in this study, providing reference value for the clinical application of PC. Methods: In the present study, a reliable and sensitive ultra-high performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the simultaneous determination of 15 prototype components (vanillic acid, vitexin, verbascoside, isoacteoside, hyperoside, cosmosiin, apigenin, ß-rhamnocitrin, acacetin, ombuin, pogostone, pachypodol, vicenin-2, retusin, and diosmetin-7-O-ß-D-glucopyranoside) in rat plasma after oral administration of the PC extract. Plasma samples were prepared via protein precipitation using acetonitrile, and icariin was used as the internal standard (IS). Results: The intra-day and inter-day accuracies ranged from -12.0 to 14.3%, and the precision of the analytes was less than 11.3%. The extraction recovery rate of the analytes ranged from 70.6-104.5%, and the matrix effects ranged from 67.4-104.8%. Stability studies proved that the analytes were stable under the tested conditions, with a relative standard deviation lower than 14.1%. Conclusion: The developed method can be applied to evaluate the PK of 15 prototype components in PC extracts of rats after oral administration using UPLC-MS/MS, providing valuable information for the development and clinical safe, effective, and rational use of PC.

Repurposing Castanea sativa Spiny Burr By-Products Extract as a Potentially Effective Anti-Inflammatory Agent for Novel Future Biotechnological Applications.

The concept of a "circular bioeconomy" holds great promise for the health, cosmetic, and nutrition sectors by re-using Castanea sativa (Mill.) by-products. This sustainable resource is rich in bioactive secondary metabolites with antioxidant and anti-inflammatory properties. By transforming these by-products into high-value products for human health, we can promote sustainable economic growth and reduce the environmental impact of traditional waste disposal, adding value to previously underutilized resources. In the present study, we investigated the antioxidant capacity, phytochemical composition, and in vitro antioxidant and anti-inflammatory activity of C. sativa burr (CSB) aqueous extract. The spectrophotometric study revealed high total phenolic content (TPC) values with significant antioxidant and anti-radical properties. Using UPLC-MS/MS techniques, the phytochemical investigation identified 56 metabolites, confirming the presence of phenolic compounds in CSBs. In addition, CSBs significantly downregulated pro-inflammatory mediators in LPS-stimulated RAW 264.7 macrophage cells without significant cell toxicity. Lastly, in silico studies pinpointed three kinases from RAW 264.7 cells as binding partners with ellagic acid, the predominant compound found in our extract. These findings strongly advocate for the recycling and valorization of C. sativa by-products, challenging their conventional classification as mere "waste".

Determination of vancomycin and meropenem in serum and synovial fluid of patients with prosthetic joint infections using UPLC-MS/MS.

Numerous studies have suggested that intra-articular administration of antibiotics following primary revision surgery may be one of the methods for treating prosthetic joint infection (PJI). Vancomycin and meropenem are the two most commonly used antibiotics for local application. Determining the concentrations of vancomycin and meropenem in the serum and synovial fluid of patients with PJI plays a significant role in further optimizing local medication schemes and effectively eradicating biofilm infections. This study aimed to establish a rapid, sensitive, and accurate ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for determining the concentrations of vancomycin and meropenem in human serum and synovial fluid. Serum samples were processed using acetonitrile precipitation of proteins and dichloromethane extraction, while synovial fluid samples were diluted before analysis. Chromatographic separation was achieved in 6 min on a Waters Acquity UPLC BEH C18 column, with the mobile phase consisting of 0.1% formic acid in water (solvent A) and acetonitrile (solvent B). Quantification was carried out using a Waters XEVO TQD triple quadrupole mass spectrometer with an electrospray ionization (ESI) source in positive ion mode. The multiple reaction monitoring (MRM) mode was employed to detect the following quantifier ion transitions: 717.95-99.97 (norvancomycin), 725.90-100.04 (vancomycin), 384.16-67.99 (meropenem). The method validation conformed to the guidelines of the FDA and the Chinese Pharmacopoeia. The method demonstrated good linearity within the range of 0.5-50 µg/ml for serum and 0.5-100 µg/ml for synovial fluid. Selectivity, intra-day and inter-day precision and accuracy, extraction recovery, matrix effect, and stability validation results all met the required standards. This method has been successfully applied in the pharmacokinetic/pharmacodynamic (PK/PD) studies of patients with PJI.

Metabolic Composition of Methanolic Extract of the Balkan Endemic Species Micromeria frivaldszkyana (Degen) Velen and Its Anti-Inflammatory Effect on Male Wistar Rats.

Extracts from medicinal plants are widely used in the treatment and prevention of different diseases. Micromeria frivaldszkyana is a Balkan endemic species with reported antioxidant and antimicrobial characteristics; however, its phytochemical composition is not well defined. Here, we examined the metabolome of M. frivaldszkyana by chromatography-mass spectrometry (GC-MS), ultra-performance liquid chromatography-mass spectrometry (UPLC-MS-MS), and inductively coupled plasma mass spectrometry (ICP-MS). Amino acids, organic acids, sugars, and sugar alcohols were the primary metabolites with the highest levels in the plant extract. Detailed analysis of the sugar content identified high levels of sucrose, glucose, mannose, and fructose. Lipids are primary plant metabolites, and the analysis revealed triacylglycerols as the most abundant lipid group. Potassium (K), magnesium (Mg), zinc (Zn), and calcium (Ca) were the elements with the highest content. The results showed linarin, 3-caffeoil-quinic acid, and rosmarinic acid, as well as a number of polyphenols, as the most abundant secondary metabolites. Among the flavonoids and polyphenols with a high presence were eupatorin, kaempferol, and apigenin-compounds widely known for their bioactive properties. Further, the acute toxicity and potential anti-inflammatory activity of the methanolic extract were evaluated in Wistar rats. No toxic effects were registered after a single oral application of the extract in doses of between 200 and 5000 mg/kg bw. A fourteen-day pre-treatment with methanolic extract of M. frivaldszkyana in doses of 250, 400, and 500 mg/kg bw induced anti-inflammatory activity in the 1st, 2nd, and 3rd hours after carrageenan injection in a model of rat paw edema. This effect was also present in the 4th hour only in the group treated with a dose of 500 mg/kg. In conclusion, M. frivaldszkyana extract is particularly rich in linarin, rosmarinic acid, and flavonoids (eupatorin, kaempferol, and apigenin). Its methanolic extract induced no toxicity in male Wistar rats after oral application in doses of up to 5000 mg/kg bw. Additionally, treatment with the methanolic extract for 14 days revealed anti-inflammatory potential in a model of rat paw edema on the 1st, 2nd, and 3rd hours after the carrageenan injection. These results show the anti-inflammatory potential of the plant, which might be considered for further exploration and eventual application as a phytotherapeutic agent.

[Simultaneous determination of 31 banned veterinary drugs during egg-laying period in poultry eggs by ultra performance liquid chromatography-tandem mass spectrometry].

The consumption of poultry eggs has increased in recent years owing to the abundance of production and improvements in living standards. Thus, the safety requirements of poultry eggs have gradually increased. At present, few reports on analytical methods to determine banned veterinary drugs during egg-laying period in poultry eggs have been published. Therefore, establishing high-throughput and efficient screening methods to monitor banned veterinary drugs during egg-laying period is imperative. In this study, an analytical method based on ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) combined with QuEChERS-based techniques was developed for the simultaneous determination of 31 banned veterinary drugs encompassing nine drug classes (macrolides, antipyretic and analgesic drugs, sulfonamides, antibacterial synergists, anticoccidials, antinematodes, quinolones, tetracyclines, amphenicols) in different types of poultry eggs. The main factors affecting the response, recovery, and sensitivity of the method, such as the extraction solvent, purification adsorbent, LC separation conditions, and MS/MS parameters, were optimized during sample pretreatment and instrumental analysis. The 31 veterinary drug residues in 2.00 g eggs were extracted with 2 mL of 0.1 mol/L ethylene diamine tetraacetic acid disodium solution and 8 mL 3% acetic acid acetonitrile solution, and salted out with 2 g of sodium chloride. After centrifugation, 5 mL of the supernatant was cleaned-up using the QuEChERS method with 100 mg of octadecylsilane-bonded silica gel (C18), 50 mg of N-propylethylenediamine (PSA), and 50 mg of NH2-based sorbents. After nitrogen blowing and redissolution, the 31 target analytes were separated on a Waters CORTECS UPLC C18 analytical chromatographic column (150 mm×2.1 mm, 1.8 µm) at a flow rate, column temperature, and injection volume of 0.4 mL/min, 30 ℃, and 5 µL, respectively. Among these analytes, 26 analytes were acquired in dynamic multiple reaction monitoring (MRM) mode under positive electrospray ionization (ESI+) conditions using (A) 5 mmol/L ammonium acetate (pH 4.5) and (B) acetonitrile as mobile phases. The gradient elution program was as follows: 0-2.0 min, 12%B-30%B; 2.0-7.5 min, 30%B-50%B; 7.5-10.0 min, 50%B; 10.0-10.1 min, 50%B-100%B; 10.1-12.0 min, 100%B; 12.0-12.1 min, 100%B-12%B; The five other target analytes were acquired in MRM mode under negative electrospray ionization (ESI-) conditions using (A) H2O and (B) acetonitrile as mobile phases. The gradient elution program was as follows: 0-2.0 min, 12%B-40%B; 2.0-6.0 min, 40%B-80%B; 6.0-6.1 min, 80%B-100%B; 6.1-8.0 min, 100%B; 8.0-8.1 min, 100%B-12%B. Matrix-matched external standard calibration was used for quantification. The results showed that all the compounds had good linear relationships within their respective ranges, with correlation coefficients of >0.99. The limits of detection (LODs) and quantitation (LOQs) were 0.3-3.0 µg/kg and 1.0-10.0 µg/kg, respectively. The average recoveries of the 31 banned veterinary drugs spiked at three levels (LOQ, maximum residue limit (MRL), and 2MRL) in poultry eggs ranged from 61.2% to 105.7%, and the relative standard deviations (RSDs) ranged from 1.8% to 17.6%. The developed method was used to detect and analyze banned veterinary drugs in 30 commercial poultry egg samples, including 20 eggs, 5 duck eggs, and 5 goose eggs. Enrofloxacin was detected in one egg with a content of 12.3 µg/kg. The proposed method is simple, economical, practical, and capable of the simultaneous determination of multiple classes of banned veterinary drugs in poultry eggs.