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The global surge in multi-drug resistant bacteria, including extended-spectrum β-lactamase (ESBL)-producing Escherichia coli has led to a growing need for new antibacterial compounds. Despite being promising, the potential of fish-derived antimicrobial peptides (AMPs) in combating ESBL-producing E. coli is largely unexplored. In this study, native African catfish antimicrobial peptides (NACAPs) were extracted from the skin mucus of farmed African catfish, Clarias gariepinus, using a combination of 10% acetic acid solvent hydrolysis, 5 kDa ultrafiltration, and C18 hydrophobic interaction chromatography. Peptides were then sequenced using Orbitrap Fusion Lumos Tribrid Mass Spectrometry. The identified peptides were screened for potential antibacterial activity using Random Forest and AdaBoost machine learning algorithms. The most promising peptide was chemically synthesized and evaluated in vitro for safety on rabbit red blood cells and activity against ESBL-producing E. coli (ATCC 35218) utilizing spot-on-lawn and broth dilution methods. Eight peptides ranging from 13 to 22 amino acids with molecular weights between 968.42 and 2434.11 Da were identified. Peptide NACAP-II was non-hemolytic to rabbit erythrocytes (p > 0.05) with a zone of inhibition (ZOI) of 22.7 ± 0.9 mm and a minimum inhibitory concentration (MIC) of 91.3 ± 1.2 μg/mL. The peptide is thus a candidate antibacterial compound with enormous potential applications in the pharmaceutical industry. However, further studies are still required to establish an upscale production strategy and optimize its activity and safety in vivo.

The effects of the simultaneous consumption of amphetamine or amphetamine derivatives and alcohol have not yet been adequately clarified, particularly concerning potential condensation products resulting from the endogenous reaction between these substances and their metabolites (e.g., acetaldehyde, a metabolite of ethanol). In this study, we developed an LC–MS/MS method employing liquid–liquid extraction for the qualitative detection of some relevant condensation products belonging to the class of tetrahydroisoquinolines and their derivatives in human blood, brain, and liver samples. This includes the analysis of the substrates amphetamine, methamphetamine, methylenedioxymethamphetamine, methylenedioxyamphetamine, as well as the condensation products 1,3-dimethyl-1,2,3,4-tetrahydroisoquinoline, N-methyl-1,3-dimethyl-1,2,3,4-tetrahydroisoquinoline, 1,3-dimethyl-7,8-methylenedioxy-1,2,3,4-tetrahydroisoquinoline, and N-methyl-1,3-dimethyl-7,8-methylenedioxy-1,2,3,4-tetrahydroisoquinoline. Therefore, the reference standards of the mentioned tetrahydroisoquinolines were synthesized in advance and the method was validated with regard to the question of the qualitative detection of these compounds. The validation parameters included selectivity, specificity, limit of detection, lower limit of quantification, recovery, matrix effects, and stability for blood, brain, and liver samples. Following the analysis of human blood and post-mortem tissue samples, evidence of the condensation product 1,3-dimethyl-1,2,3,4-tetrahydroisoquinoline originating from the interaction between amphetamine and acetaldehyde was identified in two liver samples. On the contrary, no evidence of this or other tetrahydroisoquinolines was found in the remaining tissue and serum samples. [ABSTRACT FROM AUTHOR]

Per- and polyfluoroalkyl substances (PFAS) are widely used in industry, residential, and consumer products. Studies have shown associations between high PFAS exposure and adverse health effects. In 2022, the National Academies of Science, Engineering, and Medicine (NASEM) published Guidance on PFAS Exposure, Testing, and Clinical Follow-up providing laboratory and clinical direction. The Guidance suggests nine PFAS should be measured in serum or plasma specimens and summed to provide a total PFAS concentration using a NASEM-recommended method. Follow-up clinical recommendations are based on the calculated PFAS NASEM summation. We developed and validated a liquid chromatography-tandem mass spectrometry (LC–MS/MS) method in accordance with NASEM recommendations but distinguished by the ability to separate closely related structural isomers. As part of our validation, PFAS prevalence was evaluated in a population survey comprised of clinical donor and remnant specimens (n = 1023 in total). In this study, 82.2% of the specimens had PFAS NASEM summations of 2 to < 20 ng/mL and 2.5% had a summation ≥ 20 ng/mL. The median PFAS NASEM summation was 4.65 ng/mL in this study, lower than the 7.74 ng/mL median observed in the 2017–2020 Centers for Disease Control and Prevention, National Health and Nutrition Examination Survey (n = 3072). This lower median PFAS NASEM summation may reflect a decline in PFAS population levels over time or sample population exposure differences. [ABSTRACT FROM AUTHOR]

Per- and polyfluoroalkyl substances (PFAS) are persistent environmental contaminants with bioaccumulation potential, particularly affecting aquatic ecosystems and human health also via fish consumption. There is therefore a need for reliable extraction methods and studies to accurately assess PFAS levels in fish, crucial for understanding bioaccumulation and potential toxicological effects on both fish and humans through consumption. This study investigated PFAS levels in freshwater fish from Swiss lakes, focusing on six common species: Coregonus wartmanni, Cyprinus carpio, Oncorhynchus mykiss, Perca fluviatilis, Salmo trutta, and Squalius cephalus. Utilizing an optimized QuEChERS extraction method, 15 PFAS were analyzed in 218 fish fillet samples using liquid chromatography-mass spectrometry (LC–MS/MS). The results were compared to EU regulations and EFSA guidelines for tolerable weekly intake (TWI), with a specific focus on correlations between fish size and PFAS concentration. Our findings reveal significant PFAS contamination, particularly in Perca fluviatilis with perfluorooctane sulfonic acid (PFOS) and perfluorohexane sulfonic acid (PFHxS) levels often exceeding EU safety limits. TWI, calculated for a person of 70 kg body weight and an intake of 200 g of fish fillet, is exceeded in 95% of Coregonus wartmanni, 100% of Squalius cephalus, and in 55%, 50%, and 36% of the specimens Oncorhynchus mykiss, Salmo trutta, and Perca fluviatilis respectively. Correlation analysis between PFAS concentration and fish size in 121 Salmo trutta specimens revealed significant positive correlations for perfluorobutane sulfonic acid (PFBS), perfluorodecanoic acid (PFDA), and perfluorohexane sulfonic acid (PFHxS), and a negative correlation for perfluoropentanoic acid (PFPeA). These results underscore the critical need for continuous monitoring and regulatory efforts to mitigate PFAS exposure risks to both ecosystems and human health. [ABSTRACT FROM AUTHOR]

AbstractMiniaturized procedures for chemical analysis give the possibility of a significant reduction in quantities of organic solvents used during isolation and determination processes. Liquid-liquid microextraction (LLME) using butanol as an extractant was investigated to selectively isolate oxytetracycline (OTC) antibiotic residues from dairy products. The optimal conditions of LLME were established after deproteinization of the samples. The isolation procedure for OTC was performed using 700 µL of butanol. The extracts were analysed by HPLC-UV and LC-MS/MS. A satisfactory analyte recovery was achieved in the range 97.6%–98.9%. The concentration of the linearity range of the studied antibiotic should allow its determination at the level of MRL values. The limit of detection during HPLC-UV and LC-MS/MS determination of OTC in milk samples was 9.9·10−8 mol L−1 (47.85 µg kg−1) and 5.7·10−9 mol L−1 (2.75 µg kg−1), respectively. The precision (1.7%–8.6%) of the methods using LLME isolation of OTC varied depending on the kind of sample and the chromatographic technique used. The developed methods were applied to the analysis of milk and cottage cheese to estimate the presence and detection of OTC content. [ABSTRACT FROM AUTHOR]

Background: Heat stress has deleterious effects on physiological and performance traits in livestock. Within this context, using tropically adapted cattle breeds in pure herds or terminal crossbreeding schemes to explore heterosis is attractive for increasing animal production in warmer climate regions. This study aimed to identify biological processes, pathways, and potential biomarkers related to thermotolerance in Caracu, a tropically adapted beef cattle breed, by proteomic analysis of blood plasma. To achieve this goal, 61 bulls had their thermotolerance evaluated through a heat tolerance index. A subset of 14 extreme animals, including the seven most thermotolerant (HIGH group) and the seven least thermotolerant (LOW group), had their blood plasma samples used for proteomic analysis by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The differentially regulated proteins detected between HIGH and LOW groups were used to perform functional enrichment analysis and a protein-protein interaction network analysis. Results: A total of 217 proteins were detected only in the HIGH thermotolerant group and 51 only in the LOW thermotolerant group. In addition, 81 and 87 proteins had significantly higher and lower abundancies in the HIGH group, respectively. Regarding proteins with the highest absolute log-fold change values, we highlighted those encoded by DUSP5, IGFALS, ROCK2, RTN4, IRAG1, and NNT genes based on their functions. The functional enrichment analysis detected several biological processes, molecular functions, and pathways related to cellular responses to stress, immune system, complement system, and hemostasis in both HIGH and LOW groups, in addition to terms and pathways related to lipids and calcium only in the HIGH group. Protein-protein interaction (PPI) network revealed as important nodes many proteins with roles in response to stress, hemostasis, immune system, inflammation, and homeostasis. Additionally, proteins with high absolute log-fold change values and proteins detected as essential nodes by PPI analysis highlighted herein are potential biomarkers for thermotolerance, such as ADRA1A, APOA1, APOB, APOC3, C4BPA, CAT, CFB, CFH, CLU, CXADR, DNAJB1, DNAJC13, DUSP5, FGA, FGB, FGG, HBA, HBB, HP, HSPD1, IGFALS, IRAG1, KNG1, NNT, OSGIN1, PROC, PROS1, ROCK2, RTN4, RYR1, TGFB2, VLDLR, VTN, and VWF. Conclusions: Identifying potential biomarkers, molecular mechanisms and pathways that act in response to heat stress in tropically adapted beef cattle contributes to developing strategies to improve performance and welfare traits in livestock under tropical climates. [ABSTRACT FROM AUTHOR]

Background: Diabetes mellitus affects millions globally, necessitating effective management strategies. Glenmark Pharmaceutical Limited introduced a fixed-dose combination of teneligliptin and dapagliflozin in 2022 to address this need. However, existing methods for their simultaneous detection are limited, lacking forced degradation studies essential for assessing drug stability. Results: A stability-indicating RP-HPLC method was developed for the simultaneous determination of dapagliflozin and teneligliptin in pharmaceutical formulations. The separation was efficiently achieved employing a Zorbax Eclipse Plus C18 column (150 mm × 4.6 mm, 5 µm). The mobile phase comprised a mixture of 10 mM ammonium acetate buffer in water, methanol, and acetonitrile in a suitable proportion and delivered at a flow rate of 0.6 mL/min. Detection of the isocratic eluents was performed at 224 nm using a photodiode array (PDA) detector. Validation against various stress conditions, as per ICH guidelines, revealed significant degradation of teneligliptin primarily under acidic, basic, and oxidative stress. Moreover, liquid chromatography tandem mass spectrometry (LC-MS/MS)-based characterization was conducted for the primary degradation products of teneligliptin generated under acidic, basic, and oxidative conditions. Conclusions: The developed RP-HPLC method with a stability-indicating approach provides an efficient means for the simultaneous determination of dapagliflozin and teneligliptin in pharmaceutical formulations. The significant degradation was observed under various stress conditions and also successfully separated the degradation products by this method. The proposed method directly applied for the characterization of degradation products and based on LC-MS/MS data the structure of degradation products was generated and also degradation pathways under various stress conditions were predicted. The proposed method ensured accurate and precise results in quality control process in pharmaceutical industry, and adherence to ICH validation guidelines affirms its reliability in assessing the stability of these compounds. [ABSTRACT FROM AUTHOR]

Queuosine (Q) is a hypermodified 7-deaza-guanosine nucleoside exclusively synthesized by bacteria. This micronutrient and its respective nucleobase form queuine (q) are salvaged by humans either from gut microflora or digested food. Depletion of Q-tRNA in human or mouse cells causes protein misfolding that triggers endoplasmic reticular stress and the activation of the unfolded protein responses. In vivo, this reduces the neuronal architecture of the mouse brain affecting learning and memory. Herein, a sensitive method for quantifying free q and Q in human blood was developed, optimised and validated. After evaluating q/Q extraction efficiency in several different solid-phase sorbents, Bond Elut PBA (phenylboronic acid) cartridges were found to have the highest extraction recovery for q (82%) and Q (71%) from pooled human plasma. PBS with 4% BSA was used as surrogate matrix for method development and validation. An LC–MS/MS method was validated across the concentration range of 0.0003–1 µM for both q and Q, showing excellent linearity (r2 = 0.997 (q) and r2 = 0.998 (Q)), limit of quantification (0.0003 µM), accuracy (100.39–125.71%) and precision (CV% < 15.68%). In a sampling of healthy volunteers (n = 44), there was no significant difference in q levels between male (n = 14; mean = 0.0068 µM) and female (n = 30; mean = 0.0080 µM) participants (p = 0.50). Q was not detected in human plasma. This validated method can now be used to further substantiate the role of q/Q in nutrition, physiology and pathology. [ABSTRACT FROM AUTHOR]

The analysis of gangliosides and glycosphingolipids is crucial for understanding cellular membrane structure and function as well as to accurately diagnose certain inborn errors of metabolism. GM2-gangliosidosis represents a rare and fatal group of lysosomal storage disorders characterized by accumulation of GM2 gangliosides in various tissues and organs. These disorders arise due to deficiency or functional impairment of the β-hexosaminidase A or B enzymes, which are responsible for degradation of GM2 ganglioside. Deficient enzyme activity primarily leads to the accumulation of GM2 gangliosides within the lysosomes of cells. Accurate and rapid diagnostic methods that detect increased levels of GM2 gangliosides in patients with GM2-gangliosidosis can play a significant role in early diagnosis and appropriate treatment of this condition. To address this need, we developed a multiplexed liquid chromatography-tandem mass spectrometry method targeting 84 species of gangliosides and other glycosphingolipids involved in ganglioside metabolism. Reproducibility, linearity, extraction efficiency, and sample stability were evaluated and proof-of-concept data obtained from analysis of serum samples from confirmed cases of GM2-gangliosidosis. This method has the potential to simultaneously monitor the biosynthesis of gangliosides and the lysosomal catabolic pathway serving as a valuable tool for screening and diagnosing an important group of lysosomal storage disorders. [ABSTRACT FROM AUTHOR]

Certain population in rural zones are still getting assistance from the traditional and folkloric medical systems. Before they cease to exist, this practice and knowledge need to be documented. Acorus calamus is a well-known aromatic herb with a rhizome that has been exhibited various therapeutic applications like gastroenteritis, antibacterial, antioxidant, antifungal, neuroprotective, antidiabetic, anti-inflammatory, anticancer, and anti-asthmatic activity. In Siddha system, the thermally treated dried rhizome of A. calamus is generally used to cure gastrointestinal and neurological ailments occur especially in infants by mixing the powder of rhizome with milk, ghee, and water. In this study, we evaluate the effect of thermal treatment on screening of bioactive compounds from A. calamus rhizome. The dried rhizome was thermally treated at different temperatures and incubation periods followed by ethanol extraction for 12 h to obtain the bioactive compounds profile. The temperature was optimized to 500 °C for 100 s of incubation based on the number of identified compounds through GC–MS analysis. The optimized samples were further analyzed by using LC–MS/MS, FTIR, micro-Raman, DLS-zeta potential, and XRD. A number of sesquiterpenoids, alkaloids, fatty acids, amino acid derivatives, sterols, and glucosides were detected in the aforementioned methods. The difference was observed in the size and charge of thermally treated and control dried rhizome. This indicates the short time exposure of thermally treated rhizome to be enough to retrieve the major bioactive compounds and also has tremendous application to revert the ailments, over to dried control rhizome. [ABSTRACT FROM AUTHOR]

The purpose of this paper was to evaluate the phytochemical profile of Acacia cyclops trunk bark methanol extract using LC-MS/MS, as well as to assess its antioxidant and anti-tyrosinase activities. Thus, total phenolic and flavonoid contents of the studied extract were established and 19 compounds were detected and quantified. In addition of their antioxidant potential against DPPH and ABTS assays, in vitro and in silico studies were adopted to evaluate tyrosinase inhibitory property of A. cyclops extract. Methanol trunk bark extract showed significant total phenolic content, antioxidant potential in terms of free radical scavenging, as well as an interesting tyrosinase inhibitory action (IC50= 05.12 ± 0.41 μg/mL). The molecular docking analysis and the drug-likeness prediction of the major selected compounds supported the significant anti-tyrosinase activity of the studied extract. The obtained results suggest that A. cyclops extract could be a promising candidate in the treatment of skin hyperpigmentation disorders. [ABSTRACT FROM AUTHOR]

The cardioprotective drug cyclocreatine phosphate has been awarded Food and Drug Administration-orphan drug designation for the prevention of ischemic injury to enhance cardiac graft recovery and survival in heart transplantation. Cyclocreatine phosphate is the water-soluble derivative of cyclocreatine. Estimating the levels of Cyclocreatine phosphate, Adenosine triphosphate, Creatine Phosphate, Creatine and Cyclocreatine helps us in understanding the energy state as well as evaluating the heart cells' function. The quantification of endogenous compounds imposes a challenging task for analysts because of the absence of a true blank matrix, whose use is required according to international guidelines. Recently, the International Council for Harmonization issued a new guideline that contains guidance on the validation of methods used to quantify endogenous components, such as the background subtraction approach that was employed in our current study. Specifically, we developed and validated a sensitive, reliable and accurate liquid chromatography-tandem mass spectrometry assay to determine simultaneously the levels of mentioned endogenous compounds in rat heart tissue. Tissue samples were prepared by protein precipitation extraction using water: methanol (1:1). Using Ultra Performance Liquid Chromatography, Chromatographic separation was achieved with ZORBAX Eclipse Plus C18 4.6 × 100 mm,3.5 μm column and conditions as following: ammonium acetate (pH 8.5): acetonitrile, 70:30 mobile phase, 0.7 mL/min flow rate and 25 °C temperature. Electrospray ionization mass detector with Multiple reaction monitoring mode was then employed, using both positive and negative modes, Analysis was carried out using 5.00–2000.00 ng/mL linear concentration range within 2 min for each analyte. According to Food and Drug Administration guidelines for bioanalytical methods, validation was carried out. We investigated the matrix effect, recovery efficiency and process efficiency for the analyte in neat solvent, postextraction matrix and tissue. The results stated mean percentage recoveries higher than 99%, accuracy 93.32–111.99%, and Relative Standard Deviation (RSD) below 15% within the concentration range of our study which indicated that target analytes' stability in their real matrix is sufficient under the employed experimental conditions. Highlights: Development of a validated LC‒MS/MS method to quantify levels of CCrP, ATP, CrP, Crt, and CCr, simultaneously. LC‒MS/MS method is sensitive, simple and suitable for preclinical trials for CCrP quantitation in different biological fluids. Validated method with good selectivity, linearity, and stability for CCrP in the rat heart. Efficient CCrP quantitation to improve outcomes for heart transplant patients. [ABSTRACT FROM AUTHOR]

Isosorbide dinitrate and hydralazine hydrochloride tablets treat heart failure in addition to conventional therapy, prolong hospitalization for heart failure, and enhance patient-reported functional status. LC-MS/MS methods developed and validated separately for the quantification of hydrazine mono-lactose adduct and hydrazine di-lactose adduct impurities in the isosorbide and hydralazine hydrochloride tablets. Separation of hydrazine mono-lactose adduct impurity achieved on ZIC-HILIC (100 × 4.6 mm, 5 µm) column with 0.1% formic acid in water as mobile phase A and acetonitrile as mobile phase B. The HPLC method gradient elution is; Tmin/% of B: 0/90, 15/40, 15.1/90, and 20/90. The flow rate is 1.0 mL per minute, and the injection volume is 20 µL. Separation of hydrazine di-lactose adduct impurity achieved on Inertsil HILIC (150 × 4.6 mm, 5 µm) column with 10 Mm ammonium acetate in water as mobile phase A and acetonitrile as mobile phase B. The HPLC method gradient elution is; Tmin/% of B: 0/90, 15/40, 15.1/90, and 18/90. The flow rate is 1.0 mL per minute, and the injection volume is 40 µL. Method validation has demonstrated both methods are specific, sensitive, linear, precise, accurate, stable and robust. [ABSTRACT FROM AUTHOR]

Background Type 2 diabetes mellitus (T2DM) intricately involves disrupted lipid metabolism. Exosomes emerge as carriers of biomarkers for early diagnosis and monitoring. This study aims to identify lipid metabolites in serum exosomes for T2DM diagnosis. Methods Serum samples were collected from newly diagnosed T2DM patients and age and body mass index−matched healthy controls. Exosomes were isolated using exosome isolation reagent, and untargeted/targeted liquid chromatography−tandem mass spectrometry (LC-MS/MS) was used to identify and validate altered lipid metabolites. Receiver operating characteristic curve analysis was used to evaluate the diagnostic value of candidate lipid metabolites. Results Serum exosomes were successfully isolated from both groups, with untargeted LC-MS/MS revealing distinct lipid metabolite alterations. Notably, phosphatidylethanolamine (PE) (22:2(13Z,16Z)/14:0) showed stable elevation in T2DM-serum exosomes. Targeted LC-MS/MS confirmed significant increase of PE (22:2(13Z,16Z)/14:0) in T2DM exosomes but not in serum. PE (22:2(13Z,16Z)/14:0) levels not only positively correlated with hemoglobin A1C levels and blood glucose levels, but also effectively distinguished T2DM patients from healthy individuals (area under the curve = 0.9141). Conclusion Our research sheds light on the importance of serum exosome lipid metabolites in diagnosing T2DM, providing valuable insights into the complex lipid metabolism of diabetes. [ABSTRACT FROM AUTHOR]

Total testosterone (TT) and free testosterone (FT) are important biochemical markers for anabolism of the human body, and can also serve as early screening indicators for overtraining syndrome (OTS). Presently, there is no fast and reliable serum TT and FT determination method in the field of sport science that can meet the requirements of sports research. Thus, a rapid and accurate determination method for serum TT and FT to fill the gap is needed urgently in sports training. Herein, a simple and reliable liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of TT and FT in serum was developed and fully validated, followed by the application of professional athletes in training monitoring. Efficient pretreatments based on only one-step liquid–liquid extraction (LLE) for TT and one-step LLE after a 20 min ultrafiltration for FT were adopted in this study, and the isotope internal standard of testosterone-13C3 was used to ensure the reliability of the whole procedure. A linear range of four orders of magnitude with 0.02–100 ng/mL can meet the concentration range requirement between a higher limit for male TT and a lower limit for female FT. The accuracy, precision, stability, and matrix effect were all within the limits of the guidelines. The serum TT and FT levels of 200 professional athletes (98 male athletes and 102 female athletes) were investigated by this method. Serum TT, FT, and FT/TT levels of professional athletes were significantly higher than the general population, and serum TT levels were significantly higher by LC-MS/MS than by a chemiluminescence immunoassay. In conclusion, the LC-MS/MS method for TT and FT measurement developed in this study is time-saving and easy to operate, which can be used as a reliable method for the determination of serum TT and FT in sports training, offering valuable information for monitoring anabolism of athletes and screening OTS in the early stage. [ABSTRACT FROM AUTHOR]

Erectile dysfunction is a common complication of type 2 diabetes. The combination of saxagliptin and avanafil can effectively manage both conditions in individuals with type 2 diabetes. However, evaluating the potential pharmacokinetic interactions for co‐administration of saxagliptin and avanafil is crucial. Here, we developed a sensitive bioanalytical method for simultaneously quantifying saxagliptin and avanafil in rat plasma using liquid chromatography hyphenated to triple quadrupole mass spectrometry. The chromatographic separation of saxagliptin, avanafil, and irbesartan (internal standard) was achieved on an Aquity BEH C18 column (100 × 2.1 mm, 1.7 µm), using 0.1% formic acid in water and acetonitrile as mobile phase with the gradient elution at a flow rate of 0.2 mL/min. Mass detection was carried out using selective reaction monitoring mode, with the product ions for saxagliptin, avanafil, and irbesartan being 180.2, 375.1, and 207.1 m/z, respectively. The developed liquid chromatography coupled to tandem mass spectrometry method was validated as per the US Food and Drug Administration guidelines in rat plasma. The validation findings confirmed the method's robustness and suitability for accurate quantification of saxagliptin and avanafil in rat plasma with the limit of quantification of 1 ng/mL for both the drugs and were linear in the range of 1–2000 ng/mL. The study found no significant pharmacokinetic interaction between saxagliptin and avanafil, suggesting they can be safely co‐administered to patients with type 2 diabetes. [ABSTRACT FROM AUTHOR]

An enhanced quick, easy, cheap, effective, rugged, and safe (QuEChERS) purification method utilizing multi‐walled carbon nanotubes (MWCNTs) has been developed and coupled with liquid chromatography‐tandem mass spectrometry for the determination of eight quinolone drug residues in quail eggs. The sample was extracted using ethylenediamine tetraacetic acid‐McIlvaine buffer (pH = 4.0) and 2% formic acid in acetonitrile, followed by salting out with MgSO4, and purification with primary secondary amine (PSA), MWCNT, and Octadecylsilane (C18). The method demonstrated linear relationships for the target compounds within the range of 2–100 µg/L with correlation coefficients (r) exceeding 0.99. The limits of detection and quantification were determined to be 0.4 and 0.8 µg/kg, respectively. Validation through a three‐level spiking test (low, medium, and high) in quail eggs resulted in an average recovery rate ranging from 86.8% to 113.7%, with relative standard deviations between 2.6% and 10.2% (n = 6). This method offers rapid operation, excellent purification efficiency, and high sensitivity, making it suitable for the qualitative and quantitative analysis of quinolone drug residues in eggs. Application of this method to a batch of 20 quail eggs revealed the presence of enrofloxacin and ciprofloxacin, suggesting a potential risk of non‐compliant quinolones use in quail egg production. [ABSTRACT FROM AUTHOR]

Heat shock factor 2 (HSF2) is a versatile transcription factor that regulates gene expression under stress conditions, during development, and in disease. Despite recent advances in characterizing HSF2‐dependent target genes, little is known about the protein networks associated with this transcription factor. In this study, we performed co‐immunoprecipitation coupled with mass spectrometry analysis to identify the HSF2 interactome in mouse testes, where HSF2 is required for normal sperm development. Endogenous HSF2 was discovered to form a complex with several adhesion‐associated proteins, a finding substantiated by mass spectrometry analysis conducted in human prostate carcinoma PC‐3 cells. Notably, this group of proteins included the focal adhesion adapter protein talin‐1 (TLN1). Through co‐immunoprecipitation and proximity ligation assays, we demonstrate the conservation of the HSF2‐TLN1 interaction from mouse to human. Additionally, employing sequence alignment analyses, we uncovered a TLN1‐binding motif in the HSF2 C terminus that binds directly to multiple regions of TLN1 in vitro. We provide evidence that the 25 C‐terminal amino acids of HSF2, fused to EGFP, are sufficient to establish a protein complex with TLN1 and modify cell–cell adhesion in human cells. Importantly, this TLN1‐binding motif is absent in the C‐terminus of a closely related HSF family member, HSF1, which does not form a complex with TLN1. These results highlight the unique molecular characteristics of HSF2 in comparison to HSF1. Taken together, our data unveil the protein partners associated with HSF2 in a physiologically relevant context and identifies TLN1 as the first adhesion‐related HSF2‐interacting partner. [ABSTRACT FROM AUTHOR]

• The content of Melilotus officinalis was revealed by LC-MS/MS analysis. • A novel potentiometric sensor detected antioxidant activity levels in extracts. • Active sites of the most abundant molecules were shown by molecular docking. Conducting content analysis is very important to elucidate and expand the different usage areas of plant components. Today's content analysis methods provide very reliable results. However, the high cost of these methods and the need for trained personnel bring other challenges. Therefore, simple, fast, and low-cost content analysis methods are a great need. In this study, the phenolic content of Melilotus officinalis flower extracts was investigated using LC-MS/MS analysis, and its antioxidant activity was determined with a novel method employing a PVC membrane sensor. In the method, for the first time, the antioxidant activities of M. officinalis flowers were determined in an economical, sensitive, fast, and stable manner using a PVC potentiometric sensor. Sensor measurements were performed using DPPH and FCR molecules, resulting in 25.71 mg.g-1 activity values and 117.524 mg.g-1, respectively. LC-MS/MS analysis was conducted to investigate the presence of 45 standard phenolic compounds in M. officinalis. The results revealed that coumarin and isoquercitrin were the major phenolic compounds in the M. officinalis flower extract. The findings of LC-MS/MS analyses of the extracts show moderate phenolic content presence in the M. officinalis flower extracts. The total phenolic content determined by LC-MS/MS analysis was consistent with the PVC membrane sensor analysis results. The studies of molecular docking aimed to identify the most effective active sites, and the Moldock scores (-57.13, -105.93) and binding energies (4.5 kcal.mol-1, -7.9 kcal.mol-1) of coumarin and isoquercitrin molecules. The results of molecular docking studies suggest that isoquercitrin exhibits a stronger inhibitory effect on DD-peptidase enzymes compared to coumarin. [ABSTRACT FROM AUTHOR]

The contents and distribution of different collagen types in bones, skins, and guts of salmon and cod were assessed by chemical and mass spectroscopic methods. The highest levels of collagen were found in the skin (89–98%), followed by the bones (79–89%), and the guts (25–36%). A variety of different collagen α-chain proteins were detected in the assessed tissues, with proteins from 12 to 6 different collagen types quantified in salmon and cod, respectively. The less well-known collagen IV was found in significant levels in all tissues, and the most diverse collagen composition was found in the bones. [ABSTRACT FROM AUTHOR]

The natural environment of Geobacter sulfurreducens is oligotrophic and described as limiting in both electron donors and terminal electron acceptors (TEA). In previous studies we examined the effects of long-term TEA (fumarate) limitation, and in this study we examine long-term batch cultures under limiting electron donor. The microorganism survived under long-term electron donor (acetate) starvation, maintaining a stable population of ∼1–2 × 108 cells mL−1 for >650 days. Proteins that varied in abundance with a high level of statistical significance (p < 0.05) for stages between mid-log to survival phase (acetate starved) were identified using iTRAQ based mass spectroscopy. The most highly represented proteins that significantly increased in level in the survival phase cells are generally membrane-associated and are involved in energy metabolism and protein fate. These results document that changes in the outer and cytoplasmic membranes may help G. sulfurreducens survive during starvation through detection and transport of nutrients into the cell. A sizeable portion of the identified proteins with unknown or hypothetical function further suggest that much of the biological process involved in survival have yet to be fully understood. Geobacter sulfurreducens was also able to survive under long-term TEA-starvation conditions with ferric citrate as TEA and maintained a stable population of 1.5–3 × 107 cells mL−1 for >650 days. We also found that survival phase cells from fumarate-limiting conditions were able to quickly resuscitate and reduce metal such as ferric iron as compared to the mid-log phase cells. [ABSTRACT FROM AUTHOR]

Long‐term sample stability of five atypical antipsychoticdrugs risperidone, paliperidone, clozapine, quetiapine and olanzapine and the antidepressant drug mirtazapine in serum was studied by use of a newly developed and validated analytical method based on solid‐phase extraction and liquid chromatography–tandem mass spectrometry. Ascorbic acid was used as an antioxidative agent to stabilize olanzapine during storage and sample preparation. We assessed analyte stability on long‐term storage in serum samples at 25°C, 5°C, −20°C and −80°C, and during five freeze–thaw cycles. Analytes were stable for 23 days at room temperature except for olanzapine and mirtazapine (17 days). All analytes were stable for at least 30 days at 5°C. All analytes were stable for 270 days at −20°C, except for paliperidone and mirtazapine with 60 days and 180 days, respectively. All analytes were stable for 270 days at −80°C. Furthermore, all analytes were stable for five freeze–thaw cycles. We recommend storage at −80°C when samples drawn for analysis of antipsychotic drugs are stored for more than 60 days, whereas a temperature of −20°C is sufficient for storage less than 60 days. [ABSTRACT FROM AUTHOR]

The sesquiterpene lactones (SL) of Inula helenium (I. helenium) roots were extracted using high-pressure homogenization (HPH) and compared against those derived from maceration. The effect of process conditions on the extraction efficiency of bioactive compounds (alantolactone, isoalantolactone, total phenols, and flavonoids); the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical-scavenging activity, ferric-reducing antioxidant power (FRAP), and antimicrobial activity of the extract; and the inhibition of advanced glycated end product (AGE) formation were assessed. The HPH of 90 MPa for 4 passes demonstrated the highest alantolactone (38.1 ± 0.7 mg/g) and isoalantolactone yields (34.4 ± 0.2 mg/g), respectively, which were significantly higher than the rates obtained using maceration (20.0 ± 0.2 mg/g and 18.9 ± 0.7 mg/g). Under optimal extraction conditions of 90 MPa for 4 passes, inhibition of AGE formation in the extract reached a rate of 97.16 ± 1.86%, with the highest DPPH and FRAP of 231.63 ± 2.96 µg/mL and 949.43 ± 1.86 µmol TE/100 mL, respectively. HPH exhibited lethal activity against all tested bacteria and fungi strains (Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Aspergillus niger, Fusarium oxysporum, and Penicillum expansum) at a concentration of 15 µL. SEM results proved that HPH severely damaged the cell structures of roots, increased solvent permeability, and improved the extraction rate of bioactive compounds. Additionally, this study also demonstrated that HPH yields high recovery with a specific energy consumption of 0.22 kWh/kg, which is close to the recommended minimum energy demand for an extraction procedure within industrial production processes. Therefore, these findings showed that HPH can be used as an efficient extraction technique for the production of SL in related industries. [ABSTRACT FROM AUTHOR]

Steroid hormone levels of patients may be monitored via dried blood spot (DBS) sampling at home. Stability of steroid hormones in DBS samples, however, needs to be established. DBS samples from healthy volunteers were collected and stored at various temperatures. Steroid hormone concentrations in DBS were measured directly, at day 2, day 7 and day 14 following storage at 37 °C and after 7 days, 14 days, 3 months and 6 months following storage at −20 °C, 4 °C and room temperature (RT). Cortisol, cortisone, corticosterone, testosterone, androstenedione, and 17-hydroxyprogesterone (17-OHP) were assessed using LC-MS/MS. All steroids were stable (±15 %) up to 14 days when stored at 37 °C, except for cortisone (only stable until 2 days). All steroids were stable up to 6 months when stored at −20 °C, 4 °C and RT. However, there were some exceptions, for androstenedione at RT (only stable until 7 days), for 17-OHP when stored at −20 °C (only stable until 3 months), for cortisone at RT and 4 °C (only stable until 14 days), and cortisol at RT (only stable until 3 months). Overall, we demonstrated stability of steroid hormone concentrations in DBS under various conditions which may be encountered during shipping to the diagnostic laboratory and during long-term storage before analysis. [ABSTRACT FROM AUTHOR]

Field trials were conducted in two consecutive years to evaluate the terminal residue levels, dissipation trend of cupric nonyl phenolsulfonate in citrus and soil. Analyses were carried out using QuEChERS (Quick, Easy, Cheap, Effective, Rugged, Safe) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine the concentration of cupric nonyl phenolsulfonate in citrus and soil. The results showed a good linear relationship within the range of 0.01 to 5.0 mg·L−1 (R2 = 0.9999). Average recoveries were 77.70%-105.25% for cupric nonyl phenolsulfonate at the spiked levels of 0.01, 0.1,1, and 5 mg·kg−1, the relative standard deviations (RSDs) were 2.23–7.22%. Half-lives (t1/2) of cupric nonyl phenolsulfonate were 2.76–5.87 and 1.27–3.22 days in citrus and soil, respectively. The results showed that the final contents of cupric nonyl phenolsulfonate in citrus peel were the highest while that of citrus pulp were the lowest. As for soil, the final contents range from < 0.010 to 4.081 mg·kg−1. The dietary risk assessment of citrus was evaluated with risk quotients. The risk quotient values were found to be significantly < 100%, indicating that using recommended doses of cupric nonyl phenolsulfonate in citrus fruits poses risks to human health within acceptable risk levels. The results of this study contribute to the guidance of safe and reasonable use of cupric nonyl phenolsulfonate. [ABSTRACT FROM AUTHOR]

Major public health concern is raised by the evidence that common drugs like heroin are now frequently laced or replaced with highly potent novel synthetic opioids (NSOs). The objective of this study was to explore the prevalence and patterns of NSOs in a cohort of Swiss opioid users by hair analysis. Hair analysis is considered an ideal tool for retrospective consumption monitoring. Hair samples from 439 opioid users in Zurich were analyzed. Study inclusion required a previous positive hair test result for heroin metabolites, oxycodone, fentanyl, methadone, or tramadol. The samples were extracted with a two‐step extraction procedure, followed by a targeted LC–MS/MS (QTRAP® 6500+) analysis in multiple reaction monitoring mode for a total of 25 NSOs. The method underwent full validation and demonstrated good selectivity and sensitivity with limits of detection (LOD) as low as 0.1 pg/mg. The analyzed sample cohort demonstrated a positivity rate for NSOs of 2.5%, including the following NSOs: butyrylfentanyl, acrylfentanyl, furanylfentanyl, methoxyacetylfentanyl, ocfentanil, U‐47700, isobutyrylfentanyl and benzylfentanyl. Furthermore, we were able to identify specific consumption patterns among drug users. The results indicate that hair analysis is a valuable tool for investigating the prevalence of NSOs in drug‐using populations, which seems to be low in the case of Swiss opioid users. Nevertheless, the results highlight the need for sensitive analytical detection methods in forensic toxicology to identify and monitor substance distribution in different populations. [ABSTRACT FROM AUTHOR]

Chlortalidone (CLT) is a thiazide‐type diuretic with high affinity for the erythrocyte carbonic anhydrase. Therapeutically, it is mostly used to treat edema and hypertension due to liver cirrhosis, heart insufficiency, or renal dysfunction. Although diuretics and masking agents are prohibited by the World Anti‐Doping Agency (WADA) at all times in sports, substances belonging to this category are constantly detected in athlete samples, according to WADA's annual testing figures. Within this group of structurally diverse compounds, a threshold of 20 ng/mL has been introduced for six substances solely due to their presence as contaminants in other permitted drugs because of pharmaceutical production processes. In a recent presumptive doping case with a low urinary CLT concentration, the question of unintentional doping, for example, by contaminated non‐steroidal anti‐inflammatory drug intake, arose. To examine this potential scenario, a co‐elimination of low‐dose CLT and hydrochlorothiazide (HCTA; 20 × 50 μg, 0.2 mg/day each) was conducted on five consecutive days in two volunteers. Urine samples were subjected to liquid chromatography‐tandem mass spectrometry (LC–MS/MS). Moreover, we examined the incorporation of CLT in scalp hair. HCTA is rapidly excreted renally in comparatively high concentrations. In contrast, the elimination of CLT is considerably slower (terminal elimination half‐life extended by a factor of 12) and, consequently, much less concentrated in corresponding urine samples (45 and 53 ng/mL, respectively). Conversely, a higher hair incorporation of chlorthalidone was observed with simultaneous dosing of both. The results suggest that an unintentional intake of sub‐therapeutic CLT doses due to contamination might result in an adverse analytical finding. [ABSTRACT FROM AUTHOR]

Five active compounds, daidzein, luteolin, alpinumisoflavone (AI), 6,8-diprenylgenistein (DG), and warangalone (WA), were identified from the fruits of Maclura tricuspidata via LC-Q/TOF-MS. WA and DG were shown to reverse the high glucose (HG)-induced injury in human umbilical vein endothelial cells (HUVECs), indicating their potential protective effects in alleviating diabetic symptoms. Network pharmacology was conducted to reveal the potential mechanisms of action of the compounds, and Hsp90α (degree: 47), Src (degree: 49), Akt (degree: 69) and p53 (degree: 60) were shown as the core targets related to antidiabetic properties. Further experimental verification suggested that the compounds could enhance phosphorylation of Src and Akt, increase p53 expression act as Hsp90 inhibitors, and protect against HG induced endothelial dysfunction. Our findings will provide a comprehensive understanding of the active substances of M. tricuspidata, which will be helpful for their utilisation. [ABSTRACT FROM AUTHOR]

Rice straw is used as livestock feed and compost. Ferimzone and tricyclazole, common fungicides for rice blast control, can be found in high concentrations in rice straw after unmanned aerial vehicle (UAV) spraying, potentially affecting livestock and human health through pesticide residues. In this study, an optimized method for the analysis of the two fungicides in rice straw was developed using the improved QuEChERS method. After the optimization of water and solvent volume, extraction conditions including ethyl acetate (EtOAc), acetonitrile (MeCN), a mixed solvent, and MeCN containing 1% acetic acid were compared. Different salts, including unbuffered sodium chloride, citrate, and acetate buffer salts, were compared for partitioning. Among the preparation methods, the MeCN/EtOAc mixture with unbuffered salts showed the highest recovery rates (88.1–97.9%, RSD ≤ 5.1%). To address the severe matrix effect (%ME) of rice straw, which is characterized by low moisture content and cellulose-based complex matrices, samples were purified using 25 mg each of primary–secondary amine (PSA) and octadecylsilane (C18), without pesticide loss. The developed method was validated with a limit of quantification (LOQ) of 0.005 mg/kg for target pesticides, and recovery rates at levels of 0.01, 0.1, and 2 mg/kg met the permissible range (82.3–98.9%, RSD ≤ 8.3%). The %ME ranged from −17.6% to −0.3%, indicating a negligible effect. This optimized method was subsequently applied to residue studies following multi-rotor spraying. Fungicides from all fields and treatment groups during harvest season did not exceed the maximum residue limits (MRLs) for livestock feed. This confirms that UAV spraying can be safely managed without causing excessive residues. [ABSTRACT FROM AUTHOR]

This study investigates the combination of FGFR inhibitor futibatinib (FTB) and MEK inhibitor binimetinib (BTB) for KRASmt NSCLC therapy. An analytical method was developed and validated for measuring FTB and BTB concentrations in rat plasma, adhering to USFDA guidelines. Using liquid–liquid extraction on 45‐μL plasma samples, a 6.5‐min run time was achieved. The linear calibration curve ranged from 2 to 100 ng/mL. Intra‐day and inter‐day accuracy ranged between 92.06% and 100.08%. Four blank injections post high‐concentration samples resolved significant carryover. Extraction recoveries averaged 92.06% to 102.37% across concentrations. No significant endogenous interference was detected in blank plasma. The LLOQ for both drugs was 2.0 ng/mL. Selectivity, matrix effects, stability, and dilution integrity met the acceptance criteria. The method assessed FTB and BTB interaction potential in combination therapy at 5 mg/kg. The findings provide essential pharmacokinetics insights for future clinical trials. [ABSTRACT FROM AUTHOR]

Given the limitations of untargeted metabolomics in precise metabolite quantification, our current research employed a novel approach by integrating untargeted and targeted metabolomics utilizing ultra‐high‐performance liquid chromatography quadrupole time‐of‐flight tandem mass spectrometry (UHPLC‐QTOF‐MS/MS) to analyze the metabolic profile and potential biomarkers for tuberculosis (TB). A cohort of 36 TB patients and 36 healthy controls (HC) was enlisted to obtain serum samples. Multivariate pattern recognition and univariate statistical analysis were employed to screen and elucidate the differential metabolites, whereas dot plots and receiver operating characteristic (ROC) curves were established for the identification of potential biomarkers of TB. The results indicated a distinct differentiation between the two groups, identifying 99 metabolites associated with five primary metabolic pathways in relation to TB. Of these, 19 metabolites exhibited high levels of sensitivity and specificity, as evidenced by the area under curve values approaching 1. Following targeted quantitative analysis, three potential metabolites, namely, L‐asparagine, L‐glutamic acid, and arachidonic acid, were demonstrated excellent discriminatory ability as evidenced by the results of the ROC curve, dot plots, and random forest model. Particularly noteworthy was the enhanced diagnostic efficacy of the combination of these three metabolites compared to singular biomarkers, suggesting their potential utility as serum biomarkers for TB diagnosis. [ABSTRACT FROM AUTHOR]

This research paper introduces novel strategies to address the stability issues arising with vildagliptin, marking the first attempt to tackle this challenge comprehensively. The study incorporates malic acid into the human plasma, a crucial step in stabilizing vildagliptin and preventing its degradation. Additionally, optimization of the elution process on a C18 Asentis Express column, fine‐tuned with a combination of acetonitrile and ammonium trifluoroacetate 5mM, ensures optimal chromatographic conditions. For detection and quantification, electrospray ionization (ESI) is employed, monitoring multiple reactions for vildagliptin (304.2 → 154.2) and vildagliptin D7 (311.1 → 161.2). Meticulous validation of the method demonstrates high accuracy (97.30%–104.15%) and precision [(0.32%–3.09% coefficient of variance (CV)] for vildagliptin calibration curve standards (CC STD), establishing its sensitivity and reliability in measuring vildagliptin levels. This refined methodology offers numerous advantages, including the elimination of stability concerns, reduced human plasma sample volume (100 μL), exceptional reproducibility, shortened run time (~2.2 min), and a wide concentration range (1.00 to 851.81 ng/mL). These attributes make it exceptionally well‐suited for diverse research applications, spanning from extensive sampling in therapeutic drug monitoring units to bioequivalence and bioavailability studies, as well as pharmacokinetic investigations of vildagliptin. [ABSTRACT FROM AUTHOR]

A liquid chromatography electrospray ionization tandem mass spectrometry method with amoxicillin‐d4 as the stable isotope‐labeled internal standard for simultaneous quick detection of amoxicillin and clavulanic acid in human plasma was developed and validated. Chromatographic separations were performed on a Hedera ODS‐2 column (2.1 × 150 mm, 5 μm). The mobile phases for gradient elution were aqueous solution containing 0.2% acetic acid (AA) (mobile phase A) together with organic phase solution (acetonitrile and methanol mixed solution, mobile phase B). Mass spectrometry was performed using negative electrospray ionization in multiple reaction monitoring mode. The target fragment ion pairs of amoxicillin, clavulanic acid and amoxicillin‐d4 were m/z 364.1 → 223.1, 198.1 → 135.9 and 368.1 → 227.1, respectively. The linear ranges of this method were 40–5,000 ng/ml for amoxicillin and 30–2,500 ng/ml for clavulanic acid, with coefficient of determination > 0.9900. This method validation included selectivity, standard curve, lower limit of quantitation, accuracy, precision, recovery, matrix effect (hemolytic matrix and hyperlipidemic matrix), carryover, stability, dilution reliability and incurred sample reanalysis study. A successful application of this method was realized in a pharmacokinetic study after administration of amoxicillin–clavulanic acid potassium granules. [ABSTRACT FROM AUTHOR]

Betel nut (Areca nut, AN) products are extensively consumed across East and South Asian countries, with Binglang being the predominant chewable AN product in China. Given the increased risk of carcinogenicity associated with alkaloid nitrogen oxides compared to the primary alkaloids in AN, this study aimed to establish a precise and sensitive method for quantitative analysis of the arecoline N-oxide (ACNO) and arecaidine N-oxide (ADNO) at three different processing stages of Binglang using liquid chromatography-tandem mass spectrometry (LC–MS/MS). The developed method underwent comprehensive validation, evaluating key analytical parameters including selectivity, linearity, sensitivity, accuracy, precision, and stability. Notably, the method demonstrated excellent sensitivity, with the limits of detection (LOD) ranging from 0.029 to 0.135 ng/mL, while the limits of quantification (LOQ) ranging from 0.095 to 0.450 ng/mL. Subsequently, the established analytical technique was applied to analyze 15 Binglang samples at three different processing stages, providing the first evidence of the content and variability of alkaloid nitrogen oxides throughout these stages. ACNO and ADNO were detected in all samples, and their total contents ranged from 4.31 to 32.82 μg/g. The changes observed across different processing stages indicated that the roasting process at 80 ℃ resulted in the promotion of alkaloid nitrogen oxides. This analytical approach constitutes a valuable tool for the quality control and risk assessment of Binglang and can be extended to other AN products, thereby assisting in addressing health concerns associated with these products. [ABSTRACT FROM AUTHOR]

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Environmental factors influence fungal growth and mycotoxin production in stored grains. However, the concentrations of free mycotoxins and their conjugates and how they are impacted by different interacting environment conditions have not been previously examined. The objectives of this study were to examine the impact of storage conditions (0.93–0.98 aw) and temperature (20–25 °C) on (a) the concentrations of deoxynivalenol and zearalenone and their respective glucosides/conjugates and (b) the concentrations of emerging mycotoxins in both naturally contaminated and irradiated wheat grains inoculated with Fusarium graminearum. Contaminated samples were analysed for multiple mycotoxins using Liquid Chromatography Tandem Mass Spectrometry (LC–MS/MS). Method validation was performed according to the acceptable performance criteria set and updated by the European Commission regulations No. 2021/808/EC. As an important conjugate of deoxynivalenol, the concentrations of deoxynivalenol-3-glucoside were significantly different from its precursor deoxynivalenol at 0.93 aw (22% moisture content- MC) at 25 °C in the naturally contaminated wheat with a ratio proportion of 56:44% respectively. The high concentrations of deoxynivalenol-3-glucoside could be influenced by the wheat's variety and/or harvested season/fungal strain type/location. Zeralenone-14-sulfate concentrations were surprisingly three times higher than Zearalenone in the naturally contaminated wheat at 0.98 aw (26% MC) at both temperatures. Emerging mycotoxins such as moniliformin increased with temperature rise with the highest concentrations at 0.95 aw and 25 °C. These findings highlight the influence and importance of storage aw x temperature conditions on the relative presence of free vs conjugated mycotoxins which can have implications for food safety. [ABSTRACT FROM AUTHOR]

Introduction: CEP-37440 was synthesized and supplied by the research and development division of Teva Branded Pharmaceutical Products (West Chester, PA, United States). CEP-37440 represents a newly developed compound that exhibits selectivity inhibition of Focal Adhesion Kinase and Anaplastic Lymphoma Kinase FAK/ALK receptors, demonstrating novel characteristics as an orally active inhibitor. The simultaneous inhibition of ALK and FAK can effectively address resistance and enhance the therapeutic efficacy against tumors through a synergistic mechanism. Methods: The objective of this research was to create an LC-MS/MS method that is precise, efficient, environmentally friendly, and possesses a high level of sensitivity for the quantification of CEP-37440 in human liver microsomes (HLMs). The aforementioned approach was subsequently employed to evaluate the metabolic stability of CEP-37440 in HLMs in an in vitro setting. The validation procedures for the LC-MS/MS analytical method in the HLMs were performed following the bio-analytical method validation guidelines set out by the US-FDA. The AGREE program was utilized to assess the ecological impacts of the current LC-MS/MS methodology. Results and Discussion: The calibration curve linearity was seen in the range of 1–3000 ng/mL. The inter-day accuracy (% RE) exhibited a range of −2.33% to 3.22%, whilst the intra-day accuracy demonstrated a range of −4.33% to 1.39%. The inter-day precision (% RSD) exhibited a range of 0.38% to 3.60%, whilst the intra-day precision demonstrated a range of 0.16% to 6.28%. The determination of the in vitro half-life (t1/2) and moderate intrinsic clearance (Clint) of CEP-37440 yielded values of 23.24 min and 34.74 mL/min/kg, respectively. The current manuscript is considered the first analytical study for CEP-37440 quantification with the application to metabolic stability assessment. These results suggest that CEP-37440 can be categorized as a pharmaceutical agent with a moderate extraction ratio. Consequently, it is postulated that the administration of CEP-37440 to patients may not lead to the accrual of dosages within the human organs. According to in silico P450 metabolic and DEREK software, minor structural alterations to the ethanolamine moiety or substitution of the group in drug design have the potential to enhance the metabolic stability and safety profile of novel derivatives in comparison to CEP-37440. [ABSTRACT FROM AUTHOR]

Propolis is a sticky substance produced by bees because of the reaction of beeswax, pollen, and bee enzymes. Particularly, their biological activity and chemical content attract attention. Thus, in this study, the total amount of phenolic and flavonoid substances, Fe3+‐Fe2+, Cu2+ (cupric ions reducing activity [CUPRAC]), and Fe3+‐TPTZ (ferric ions reducing antioxidant power [FRAP]) reducing, and DPPH• and ABTS•+ scavenging assays in vitro antioxidant properties of propolis samples obtained from four different provinces of Türkiye were determined. In addition, the chemical content of propolis samples was quantitatively determined by LC–MS/MS, and the antiglaucoma property was revealed by hCAII enzyme inhibition. Propolis samples from Ordu presented the highest amounts of total phenolic and flavonoid content (492.3 ± 5.8 and 96.1 ± 2.1, respectively) and also highest antioxidant activity (DPPH• and ABTS•+ IC50 [μg/mL]: 8.884 ± 0.84 and 4.589 ± 0.80, respectively; Fe+3, CUPRAC, and FRAP: 1.051 ± 0.012, 1.021 ± 0.008, and 0.957 ± 0.007 μg/mL, respectively). hCAII enzyme inhibition was highest in Muş propolis (IC50 [μg/mL]: 8.6) as determined. By LC–MS/MS, 53 different components were screened and 35 bioactive components were determined. According to the results, propolis was found to be a raw material because it contains high concentrations of acacetin, chrysin, caffeic acid, and quinic acid (123.824, 24.759, 47.779, and 16.32 mg analyte/g extract, respectively). [ABSTRACT FROM AUTHOR]

Plant-pathogenic bacteria are one of the major constraints on agricultural yield. In order to selectively treat these bacteria, it is essential to understand the molecular structure of their cell membrane. Previous studies have focused on analyzing hydrolyzed fatty acids (FA) due to the complexity of bacterial membrane lipids. These studies have highlighted the occurrence of branched-chain fatty acids (BCFA) alongside normal-chain fatty acids (NCFA) in many bacteria. As several FA are bound in the intact phospholipids of the bacterial membrane, the presence of isomeric FA complicates lipid analysis. Furthermore, commercially available reference standards do not fully cover potential lipid isomers. To address this issue, we have developed a reversed-phase high-performance liquid chromatography (RP-HPLC) method with tandem mass spectrometry (MS/MS) to analyze the phospholipids of various plant-pathogenic bacteria with a focus on BCFA containing phospholipids. The study revealed the separation of three isomeric phosphatidylethanolamines (PE) depending on the number of bound BCFA to NCFA. The validation of the retention order was based on available reference standards in combination with the analysis of hydrolyzed fatty acids through gas chromatography with mass spectrometry (GC/MS) after fractionation. Additionally, the transferability of the retention order to other major lipid classes, such as phosphatidylglycerols (PG) and cardiolipins (CL), was thoroughly examined. Using the information regarding the retention behavior, the phospholipid profile of six plant-pathogenic bacteria was structurally elucidated. Furthermore, the developed LC–MS/MS method was used to classify the plant-pathogenic bacteria based on the number of bound BCFA in the phospholipidome. [ABSTRACT FROM AUTHOR]

In recent years, instrumental improvements have enabled the spread of mass spectrometry–based lipidomics platforms in biomedical research. In mass spectrometry, the reliability of generated data varies for each compound, contingent on, among other factors, the availability of labeled internal standards. It is challenging to evaluate the data for lipids without specific labeled internal standards, especially when dozens to hundreds of lipids are measured simultaneously. Thus, evaluation of the performance of these platforms at the individual lipid level in interlaboratory studies is generally not feasible in a time-effective manner. Herein, using a focused subset of sphingolipids, we present an in-house validation methodology for individual lipid reliability assessment, tailored to the statistical analysis to be applied. Moreover, this approach enables the evaluation of various methodological aspects, including discerning coelutions sharing identical selected reaction monitoring transitions, pinpointing optimal labeled internal standards and their concentrations, and evaluating different extraction techniques. While the full validation according to analytical guidelines for all lipids included in a lipidomics method is currently not possible, this process shows areas to focus on for subsequent method development iterations as well as the robustness of data generated across diverse methodologies. [ABSTRACT FROM AUTHOR]

Background: Cyclosporine A (CsA) is used as a steroid-sparing or alternative immunosuppressing agent in cats with various immune-mediated diseases such as immune-mediated hemolytic anemia. Daily treatment dosages of 5–20  mg/ kg have been described. Interindividual variations in CsA blood levels are known to occur. To determine when steady-state conditions are reached and thus the earliest advisable time for monitoring CsA blood levels during the course of treatment, a prospective experimental study was conducted in six healthy adult Domestic Shorthair cats. Materials and methods: Cats were treated with an oral dosage of 7  mg/kg CsA q 12  h for 10  days. On days 1, 2, 3, 5, 7, and 10 after the start of CsA administration (i.e., after 1, 3, 5, 9, 13, and 19 CsA administrations), EDTA blood was collected to measure the CsA level 12  h after the CsA administration (trough values) using high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS/MS). Results: Statistical analysis revealed a significant increase in mean CsA blood levels up to day 5 (2,050  ±  964.2  ng/mL [mean  ±  SD], 832–3,203  ng/mL [minimum–maximum]; repeated-measures ANOVA: p  =  0.0021), while values on days 5 and 7 did not differ significantly from CsA concentrations on day 10. CsA concentrations showed markedly interindividual variability. Conclusion: Cyclosporine A blood levels reached a steady state on day 5 of high dosages of CsA q 12  h (i.e., after nine CsA administrations), indicating that this time point is suitable for monitoring blood levels in clinical patients. Results confirmed the well-known remarkable interindividual variability of CsA, indicating the need for treatment monitoring. The assessed treatment regime resulted in significantly higher mean CsA trough levels than the target range for immunosuppressive therapy (200–600  ng/mL). [ABSTRACT FROM AUTHOR]

Abstract\nHIGHLIGHTSThis study aims to develop and validate a method for the simultaneous quantification of JWH-073, JWH-018, and their two main metabolites 3-OH JWH-073 and 4-OH JWH-018 in human blood and urine samples by using liquid chromatography combined with a mass spectrometer (LC-MS/MS). The method was validated according to current FDA and EMA guidelines and was tested on 10 urine samples from suspected synthetic cannabinoid abusers and 10 plasma samples from drug-free volunteers. Experimental data showed that the limits of detection (LOD) for the analytes were very low at 0.01 ng/mL in plasma samples and 0.003 ng/mL in urine samples. The limits of quantification (LLOQ) were 0.03 ng/mL in plasma samples and 0.01 ng/mL in urine samples. The linear correlation coefficients (R2) for these analytes ranged from 0.9914 to 0.9929 in plasma samples and 0.9820 to 0.9892 in urine samples. Among the 10 urine samples from suspected synthetic cannabinoid abusers, the parent JWH-073 and JWH-018 analytes were detected in six and three samples, with concentrations of 0.01 − 8.14 ng/mL and 7.16 − 17.50 ng/mL, respectively. At the same time, their two main metabolites, 3-OH JWH-073 and 4-OH JWH-018, were also found in the samples containing parent drugs, with concentrations of 32.13 − 84.24 ng/mL and 14.48–32.97 ng/mL, respectively. Three samples (T3, T5, and T6) contained only 4-OH JWH-018 metabolite (with concentrations of 25.69 − 51.87 ng/mL) and one sample (T10) contained only 3-OH JWH-073 metabolite (with concentration of 56.94 ng/mL) without the presence of their parent drugs. The plasma samples from drug-free volunteers were all negative for the analytes.Simultaneous quantification of JWH-073 and JWH-018 and their two main metabolites (3-OH JWH-073 and 4-OH JWH-018) in plasma samples (LOD: 0.01 ng/mL; LLOQ: 0.03 ng/mL) and in urine samples (LOD: 0.003 ng/mL; LLOQ: 0.01 ng/mL).The parent JWH-073 and JWH-018 drugs were detected in six and three samples, with concentrations of 0.01 − 8.14 ng/mL and 7.16 − 17.50 ng/mL, respectively. Their main metabolites also were detected in these samples: 3-OH JWH-073 (concentrations of 32.13 − 84.24 ng/mL) and 4-OH JWH-018 (concentrations of 14.48–32.97 ng/mL).Three samples (T3, T5, and T6) only had the metabolite 4-OH JWH-018 present (with concentrations of 25.69 − 51.87 ng/mL) and one sample (T10) only had the metabolite 3-OH JWH-073 present (concentration of 56.94 ng/mL) without the presence of the parent drugs.Plasma samples from drug-free volunteers were all negative for the analytes.Simultaneous quantification of JWH-073 and JWH-018 and their two main metabolites (3-OH JWH-073 and 4-OH JWH-018) in plasma samples (LOD: 0.01 ng/mL; LLOQ: 0.03 ng/mL) and in urine samples (LOD: 0.003 ng/mL; LLOQ: 0.01 ng/mL).The parent JWH-073 and JWH-018 drugs were detected in six and three samples, with concentrations of 0.01 − 8.14 ng/mL and 7.16 − 17.50 ng/mL, respectively. Their main metabolites also were detected in these samples: 3-OH JWH-073 (concentrations of 32.13 − 84.24 ng/mL) and 4-OH JWH-018 (concentrations of 14.48–32.97 ng/mL).Three samples (T3, T5, and T6) only had the metabolite 4-OH JWH-018 present (with concentrations of 25.69 − 51.87 ng/mL) and one sample (T10) only had the metabolite 3-OH JWH-073 present (concentration of 56.94 ng/mL) without the presence of the parent drugs.Plasma samples from drug-free volunteers were all negative for the analytes. [ABSTRACT FROM AUTHOR]

This study aimed to determine the effectiveness of different green extraction techniques (GETs) on targeted bioactive compounds from artichoke leaf by-products using deep eutectic solvent extraction (DESE), supercritical CO2 extraction (SCO2E), subcritical water extraction (SWE), and ultrasound-assisted extraction (UAE). Moreover, (HR) LC-ESI-QTOF MS/MS and HPLC-PDA analyses were used to perform qualitative–quantitative analysis on the extracts, enabling the detection of several bioactive compounds, including luteolin, luteolin 7-O-glucoside, luteolin 7-O-rutinoside, apigenin rutinoside, chlorogenic acid, and cynaropicrin as the most representative ones. SWE showed better results than the other GETs (TPC: 23.39 ± 1.87 mg/g of dry plant, dp) and appeared to be the best choice. Regarding UAE, the highest total phenols content (TPC) was obtained with 50:50% v/v ethanol: water (7.22 ± 0.58 mg/g dp). The DES obtained with choline chloride:levulinic acid showed the highest TPC (9.69 ± 0.87 mg/g dp). Meanwhile, SCO2E was a selective technique for the recovery of cynaropicrin (48.33 ± 2.42 mg/g dp). Furthermore, the study examined the antioxidant activity (1.10–8.82 mmol Fe2+/g dp and 3.37–31.12 mmol TEAC/g dp for DPPH• and FRAP, respectively) and total phenols content via Folin–Ciocalteu's assay (198.32–1433.32 mg GAE/g dp), of which the highest values were detected in the SWE extracts. The relationship among the GETs, antioxidant assays, and compounds detected was evaluated using Principal Component Analysis (PCA). PCA confirmed the strong antioxidant activity of SWE and showed comparable extraction yields for the antioxidant compounds between UAE and DESE. Consequently, GETs selection and extraction parameters optimization can be employed to enrich artichoke leaf by-products' extracts with targeted bioactive compounds. [ABSTRACT FROM AUTHOR]

Aim & Objective: This study evaluates the potential of combining paclitaxel (PTX) and bortezomib (BTZ) for breast cancer therapy. Materials & Methods: The nanoformulation was optimized via Box-Behnken Design (BBD), with method validation adhering to US-FDA guidelines. Results: Multiple reaction monitoring transitions for PTX, BTZ and internal standard were m/z 855.80→286.60, 366.80→226.00 and 179.80→110.00, respectively. Elution done on C18 Luna column with 0.1% FA in MeOH:10 mM ammonium acetate. The size of nanoformulation was 133.9 ± 1.97 nm, PDI 0.19 ± 0.01 and zeta potential -19.20 ± 1.36 mV. Pharmacokinetics showed higher Cmax for PTX-BTZ-NE (313.75 ± 10.71 ng/ml PTX, 11.92 ± 0.53 ng/ml BTZ) versus free PTX-BTZ (104 ± 13.06 ng/ml PTX, 1.9 ± 0.08 ng/ml BTZ). Conclusion: Future findings will contribute to the treatment of breast cancer using PTX and BTZ. Graphical Abstract Article highlights The importance of paclitaxel (PTX) and bortezomib (BTZ) in combination with chemotherapy is demonstrated by recently published studies. An obstacle to measuring the results of translational research is the lack of an analytical or bioanalytical approach for simultaneously estimating PTX and BTZ. A gap in the scientific literature has been filled by the development and validation of an LC-MS/MS method for the co-estimation or simultaneous measurement of PTX and BTZ. Both moieties were resolved in a Phenomenex Luna (C18), (7.5 × 4.6 mm, 3μ) column with a 0.1% FA in MeOH:10 mM ammonium acetate, 80:20 (% v/v) mobile phase at 0.6 ml/min flow rate. According to the US-FDA, the developed method was validated in female Sprague–Dawley rats plasma for different characteristics, such as linearity, precision and accuracy, LLOQ and stability studies. The oral P.K profile of PTX and BTZ co-loaded in a nanoformulation has been investigated in young female Sprague–Dawley rats to support the applicability of the established LC-MS/MS methodology. With an LLOQ of 1.0 ng/ml for each drug and a run time of 6.0 min, the calibration curve was found linear for the 1–600 ng/ml concentration range. The proposed LC-MS/MS method was confirmed to be repeatable, precise and accurate. The developed LC-MS/MS method was successfully illustrated for the co-estimation or simultaneous estimation of both drugs in rat plasma to generate an oral P.K profile. US-FDA Guidance of Industry and Bioanalytical Method Validation criteria was followed for the co-estimation of PTX and BTZ and was applied to nanoformulation development and bioanalytical investigations. [ABSTRACT FROM AUTHOR]

This study aims to investigate the influence of yeast fermentation and inoculation methods on the quality of coffee across 3 different coffee cultivars. Fermenting Sacchoromyces cerevisiae SafAle s-33 on Coffea arabica cv. Caltimor via bucket inoculation outperforms spray inoculation across all aspects including microbial cell adhesion, the chemical profile, reducing sugar content and sensory analysis conducted by a Q-grader. The PCA analysis revealed distinct profiles of organic acids and volatile compounds in roasted beans in yeast fermentation and natural fermentation of all 3 coffee varieties. Particularly, volatile compounds such 1-(4-Methoxyphenyl)octan-1-one, 8-Oxabicyclo[3.2.1]oct-6-en-3-one and 5,6-Dimethoxy-2-(2-hydroxyethyl-1-thio)-3 trimethylsilyl methyl-1,4-benzoquinone and, furfuryl ethyl ether and 5-methyl furfural were detected only in yeast fermented bean. The malic acid also contained a higher amount in roasted coffee beans from the yeast fermentation of all 3 coffee varieties than the natural fermentation. Those compounds present a notably positive influence on the sensory aspects of the coffee i.e. sweet caramel, dry fruit, vanilla and hazelnut-like flavors. In addition, roasted beans from the yeast fermentation of C. arabica cv. Caltimor and C. canephora cv. Robusta displayed higher sensory scores when compared to those from natural fermentation, conversely in C. arabica cv. Bourbon, beans from both yeast fermentation and natural fermentation displayed similar quality levels. Our study concluded that the commercial yeast strain within the beer and wine industry proved to be an alternative source for coffee fermentation. Nevertheless, the selection of the coffee variety plays a crucial role when utilizing S. cerevisiae SafAle s-33 as a starter. [ABSTRACT FROM AUTHOR]

Pemphigus vulgaris (PV) stands as a rare autoimmune bullous disease, while the precise underlying mechanism remains incompletely elucidated. High‐throughput proteomic methodologies, such as LC‐MS/MS, have facilitated the quantification and characterisation of proteomes from clinical skin samples, enhancing our comprehension of PV pathogenesis. The objective of this study is to elucidate the signalling mechanisms underlying PV through proteomic analysis. Proteins and cell suspension were extracted from skin biopsies obtained from both PV patients and healthy volunteers and subsequently analysed using LC‐MS/MS and scRNA‐seq. Cultured keratinocytes were treated with PV serum, followed by an assessment of protein expression levels using immunofluorescence and western blotting. A total of 880, 605, and 586 differentially expressed proteins (DEPs) were identified between the lesion vs. control, non‐lesion vs. control, and lesion vs. non‐lesion groups, respectively. The oxidative phosphorylation (OXPHOS) pathway showed activation in PV. Keratinocytes are the major cell population in the epidermis and highly expressed ATP5PF, ATP6V1G1, COX6B1, COX6A1, and NDUFA9. In the cellular model, there was a notable increase in the expression levels of OXPHOS‐related proteins (V‐ATP5A, III‐UQCRC2, II‐SDHB, I‐NDUFB8), along with STAT1, p‐STAT1, and p‐JAK1. Furthermore, both the OXPHOS inhibitor metformin and the JAK1 inhibitor tofacitinib demonstrated therapeutic effects on PV serum‐induced cell separation, attenuating cell detachment. Metformin notably reduced the expression of V‐ATP5A, III‐UQCRC2, II‐SDHB, I‐NDUFB8, p‐STAT1, p‐JAK1, whereas tofacitinib decreased the expression of p‐STAT1 and p‐JAK1, with minimal impact on the expression of V‐ATP5A, III‐UQCRC2, II‐SDHB, and I‐NDUFB8. Our results indicate a potential involvement of the OXPHOS and JAK‐STAT1 pathways in the pathogenesis of PV. [ABSTRACT FROM AUTHOR]

Empagliflozin (EFZ), a sodium-glucose co-transporter 2 (SGLT2) inhibitor used to treat type 2 diabetes, faces challenges with poor solubility and bioavailability. This study aimed to evaluate a Self-Nanoemulsifying Drug Delivery System (SNEDDS), specifically SN1C, for enhancing EFZ's dissolution rate and pharmacokinetics compared to pure EFZ powder and a marketed tablet (MKT). We assessed dissolution profiles in vitro and conducted pharmacokinetic studies in rats following oral administration of the three formulations. Plasma samples were analyzed using LC-MS/MS, and pharmacokinetic parameters were determined via non-compartmental analysis. SN1C demonstrated the highest dissolution rate in vitro. In vivo, SN1C achieved a significantly higher maximum plasma concentration (Cmax) and area under the curve (AUC(inf)) than EFZ and MKT, with approximately 1.5-fold higher Cmax and a significantly greater AUC(inf) (p < 0.05). Tmax and half-life (t1/2) were similar across all formulations, indicating that SNEDDS did not negatively impact these parameters. The results suggest that SN1C substantially improves the bioavailability of EFZ, highlighting the efficacy of SNEDDS technology in enhancing the oral delivery and therapeutic effectiveness of poorly soluble drugs like EFZ. [ABSTRACT FROM AUTHOR]

Linear alkyl benzene sulfonates (LASs) with alkyl chains from C10 to C14 are the most commonly used anionic surfactants in household laundry. However, LAS toxicity toward agricultural soil further could be formed at a risk for fruits and vegetables grown on the land, so that to develop a sensitive and precise analytical technique for determination of trace residual sulfonates in vegetables is very important for health. This study developed a QuEChERS extraction coupled to liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for analyzing LASs in vegetables and fruits. The best results were obtained using acetonitrile as the extraction solvent combined with 25 mg primary secondary amine and 25 mg graphitized carbon black as sorbents. The linearity ranged between 1 and 250 ng/mL as well as limit of quantification (LOQ) was obtained in the range of 0.01–0.23 ng/mL of the proposed analytical method. The proposed method was utilized to detect the real spinach samples and water spinach planted with LASs‐spiked water, and the results showed the trace LASs were detected with concentration between 1.8 and 29.1 ng/g. In this study, a thorough optimization and validation of different amount of sorbent cleanup efficiency approaches was performed for the first time for the analysis of LAS. [ABSTRACT FROM AUTHOR]

The aim of this study was to establish a simple, fast, and sensitive method with liquid chromatography–tandem mass spectrometry (LC–MS/MS) for simultaneously determining ibuprofen enantiomers using mouse blood in very small volumes. LC–MS/MS equipped with an electrospray ionization (ESI) source was used in negative ion mode and multiple‐reaction monitoring mode. Enantiomer chromatographic separation was carried out on a Lux® 5 μm Cellulose‐3 (250 × 4.6 mm, 5 μm) column at a flow rate of 0.6 mL/min. Samples were pretreated by extracting only 5 μL of blood with 40 μL of acetonitrile (containing 1.3% formic acid) so that a concentration‐time profile could be completed using a single mouse. 2‐(4‐Propylphenyl) propanoic acid was used as an internal standard. Standard curves for each enantiomer were linear from 0.04 to 80.00 μg/mL, demonstrating a lower limit of quantitation (LLOQ) than all previously reported methods. This method was completely validated and successfully executed to investigate the pharmacokinetics of ibuprofen enantiomers after intravenous administration of racemic ibuprofen, (S)‐(+)‐ibuprofen, and (R)‐(−)‐ibuprofen in Kunming mice, respectively. The results showed that the pharmacokinetic profiles of the (R)‐(−)‐ibuprofen and (S)‐(+)‐ibuprofen were significantly different, indicating the unidirectional inversion of R‐(−)‐ibuprofen to (S)‐(+)‐ibuprofen. [ABSTRACT FROM AUTHOR]

Bacterial keratitis (BK) is an infection that causes inflammation of the cornea and, if severe, can result in blindness. Topical fluoroquinolones combined with corticosteroids have been shown to be useful in the treatment of BK. A rapid, selective, and sensitive bioanalytical method for simultaneous quantification of Gatifloxacin (GAT) and Dexamethasone (DEX) has been developed and validated using tandem mass spectrometry (LC–MS/MS). Optimal separation was accomplished in under 5 min using an Agilent Zorbax C18 column (100 mm × 4.6 mm, 3.5 μm). The mobile phase was composed of a blend of 0.2% formic acid in triple distilled water and methanol with a flow rate of 0.65 mL/min in isocratic mode. GAT and DEX were detected in positive electrospray ionization multiple reaction monitoring mode (MRM), and the retention time was found to be at 1.64 and 2.93 min, respectively. The linearity of GAT and DEX was found to be in the range of 1.56–400 ng mL−1 with good precision and accuracy. The method was validated according to USFDA regulatory guidelines. The validated method was effectively utilized for preclinical pharmacokinetic analysis of GAT and DEX in rabbit tear fluid following the topical application of a commercial formulation. [ABSTRACT FROM AUTHOR]