Search

Aims: Postpartum haemorrhage (PPH) is the leading cause of maternal mortality worldwide. To prevent PPH, the WHO recommends administration of oxytocin (OT) immediately after birth, i.e. during the third stage of labour (TSL). Previous studies demonstrate that methods to quantify OT in biological matrices, e.g. enzyme‐linked immunosorbent assays (ELISA), radioimmunoassays (RIA) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) lack the specificity and/or sensitivity to accurately quantify OT in plasma from women administered OT during TSL. This is due to increased metabolic clearance of OT in late‐stage pregnancy and at the time of childbirth, resulting in extremely low OT plasma concentrations. This study describes the development of an ultra‐sensitive bioanalytical method that overcomes the issues previously reported and enables accurate pharmacokinetic analyses of exogenously administered OT in TSL. Methods: A selective and sensitive assay to quantify OT in TSL plasma was developed. Immunoprecipitation (IP) was applied to selectively extract OT from the TSL plasma, thereby generating clean extracts compatible with nanoflow LC (nLC). nLC–MS/MS was chosen for its high sensitivity and ability to differentiate between OT and potentially co‐captured OT‐like immunoreactive products. Results: The presented methodology is accurate and precise, with a good linear fit between 100–10 000 fg mL−1 OT. TSL plasma samples from a clinical phase 1 study (NCT02999100) were analysed successfully, enabling OT quantification down to 100 fg mL−1. Conclusions: The presented IP–nLC–MS/MS method succeeded in overcoming the sensitivity challenge related to the assay of OT in TSL plasma and thereby revealing the PK profiles of OT in TSL plasma clinical study samples. [ABSTRACT FROM AUTHOR]

Thyroid hormones (THs), including triiodothyronine (T3), thyroxine (T4), and their metabolites, are essential for regulating development, growth, and energy metabolism. Thyroglobulin (Tg) produced by thyroid follicular cells acts as an essential substrate for TH synthesis. The combination of THs with Tg is a widely used serological laboratory test for thyroid function assessment. Early detection and timely intervention are significant for preventing and managing thyroid disease. In recent years, liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) has emerged as a powerful tool for the precise detection of small molecular analytes and steroid hormones in clinical practice as a result of its high sensitivity and specificity. While LC‐MS/MS has been increasingly used for detecting THs and Tg recently, its application in clinical practice is still in its early stages. Recent advances in the assessment of thyroid metabolism using LC‐MS/MS in clinical samples published during 2004–2023 were reviewed, with a special focus on the use of this technique for quantifying molecules involved in thyroid diseases. [ABSTRACT FROM AUTHOR]

Ceftriaxone (CTRX) is a commonly used cephalosporin antibiotic. It is suggested that monitoring plasma/serum concentrations is helpful for its safe use. This study aimed to develop and validate an analytical method for measuring CTRX concentrations in human serum according to International Conference on Harmonization guideline M10. Ten microliters of serum sample was purified using a salting‐out assisted liquid–liquid extraction procedure with magnesium sulfate. The upper layer was then diluted threefold and analyzed using a liquid chromatography–tandem mass spectrometry‐based method with a total run time of 12 min. The linear calibration curve was obtained over the concentration range 5–500 μg/ml. The within‐run accuracy varied from 0.2 to 6.5%, and the precision was ≤8.0%. The between‐run accuracy and precision ranged from 0.7% to 5.6% and ≤6.4%, respectively. Significant carryover was resolved by injecting four blanks after high‐concentration CTRX samples. The recovery rates from spiked serum at low and high concentrations were 44.4 and 43.4%, respectively. Other factors, including selectivity, matrix effects, stability, dilution integrity and reinjection reproducibility also met the acceptance criteria. Serum concentrations in 14 samples obtained from two participants receiving 2 g/day of CTRX were successfully determined using this method. [ABSTRACT FROM AUTHOR]

Immunoglobulin nephropathy (IgAN) stands as the most prevalent primary glomerular nephropathy globally, typically diagnosed through an invasive renal biopsy. Emerging research suggests the significant involvement of chiral amino acids in kidney disease progression. This study introduces a nonderivative LC–tandem mass spectrometry approach, offering efficient separation outcomes within 15 min for identifying chiral amino acids in human urine samples. Subsequently, using this method, the analysis of l‐ and d‐amino acids in the urine of both patients with IgAN and healthy individuals was conducted. Fourteen d‐amino acids and 20 l‐amino acids were identified in the urine samples obtained from 17 patients with IgAN and 21 healthy individuals. The results indicated notable variances in the concentrations of both l‐ and d‐amino acids between the IgAN and healthy control groups. In contrast to the healthy group, the IgAN group exhibited higher mean urine concentrations of most l‐amino acids and lower concentrations of d‐amino acids. Furthermore, correlations between amino acids and clinical markers were investigated. These results propose a novel method for monitoring trace amino acids in urine samples and introduce a new concept for potential markers of IgAN. [ABSTRACT FROM AUTHOR]

The contents of organic acids (OAs) in tea beverage and their relationship with taste intensity have not been fully understood. In this work, a rapid (10 min for a single run) and sensitive (limits of quantification: 0.0044–0.4486 µg/mL) method was developed and validated for the simultaneous determination of 17 OAs in four types of tea, based on liquid chromatography‐tandem mass spectrometry with multiple reaction monitoring mode. The contents of 17 OAs in 96 tea samples were measured at levels between 0.01 and 11.80 g/kg (dried weight). Quinic acid, citric acid, and malic acid were determined as the major OAs in green, black, and raw pu‐erh teas, while oxalic acid and tartaric acid exhibited the highest contents in ripe pu‐erh tea. Taking the OAs composition as input features, a partial least squares regression model was proposed to predict the sourness intensity of tea beverages. The model achieved a root‐mean‐square error of 0.58 and a coefficient of determination of 0.84 for the testing set. The proposed model provides a theoretical way to evaluate the sensory quality of tea infusion based on its chemical composition. [ABSTRACT FROM AUTHOR]

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

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

The physicochemical diversity of the structurally related aromatic probe drugs, used together in a drug cocktail to assess metabolic and transport phenotypes, require optimized analytical procedures for simultaneous quantification. The analytical conditions can greatly influence the analyte selectivity, retention, stability, and ultimately the robustness of the method. The aim of this study was to assess the selectivity of the structurally related ionizable analytes between the commonly used C18 column chemistry and an alternative biphenyl column chemistry as well as the influence of changes in the analytical conditions on method robustness using liquid chromatography‐tandem mass spectrometry. A repeated measure two‐factor analysis of variance with Geisser‐Greenhouse correction was used to determine statistical significance. The results showed that a biphenyl stationary phase in combination with methanol as the organic eluent, could provide improved resolution and analyte selectivity. Changes in analytical conditions caused statistically significant variation in the retention behavior, selectivity, column efficiency, and sensitivity of the analytes of interest The robustness experiment confirmed the importance of controlling analytical conditions to ensure the reproducibility and reliability of the quantitative method. [ABSTRACT FROM AUTHOR]

Regorafenib is a small‐molecule tyrosine kinase inhibitor with severe hepatotoxicity. It undergoes metabolism mainly by CYP3A4 to generate active metabolites regorafenib‐N‐oxide (M2) and N‐desmethyl‐regorafenib‐N‐oxide (M5). Wuzhi capsule (WZC) is an herbal preparation derived from Schisandra sphenanthera and is potentially used to prevent regorafenib‐induced hepatotoxicity. This study aims to explore the effect of WZC on the pharmacokinetics of regorafenib in rats. An efficient and sensitive liquid chromatography‐tandem mass spectrometry method was developed to quantitatively determine regorafenib and its main metabolites in rat plasma. The proposed method was applied to the pharmacokinetic study of regorafenib in rats, with or without WZC. Coadministration of regorafenib with WZC resulted in a prolonged mean residence time (MRT) of the parent drug but had no statistically significant difference in other pharmacokinetic parameters. While for the main metabolites of regorafenib, WZC decreased the area under the curve and maximum concentration (Cmax), delayed the time to reach Cmax, and prolonged the MRT of M2 and M5. These results indicate that WZC delayed and inhibited the metabolism of regorafenib to M2 and M5 by suppressing CYP3A4. Our study provides implications for the rational use of the WZC‐regorafenib combination in clinical practice. [ABSTRACT FROM AUTHOR]

Introduction: Treatment strategies for depression based on interventions for glucose and lipid metabolism disorders are receiving increasing attention. Investigating the mechanism of their antidepressant effect and exploring new diagnostic and therapeutic biomarkers have attracted increasing attention. Dulaglutide, a long‐acting GLP‐1 receptor agonist, has been reported to alleviate cognitive deficits and neuronal damage. However, the antidepressant effect of dulaglutide and, especially, the underlying mechanism are still poorly understood. In this study, we aimed to explore the underlying biomarkers of depression and potential modulatory targets of dulaglutide in chronic mild stress (CMS) mice. Methods: Sixty mice were randomly divided into a control group (CON group), a CMS+Vehicle group (CMS+Veh group), a CMS+0.3 mg/kg dulaglutide group (Low Dula group), and a CMS+0.6 mg/kg dulaglutide group (High Dula group). Numerous behavioral tests, mainly the open field test, forced swimming test, and tail suspension test, were applied to evaluate the potential effect of dulaglutide treatment on anxiety‐ and depression‐like behaviors in mice exposed to chronic stress. Furthermore, a liquid chromatography–tandem mass spectrometry‐based metabolomics approach was utilized to investigate the associated mechanisms of dulaglutide treatment. Results: Three weeks of dulaglutide treatment significantly reversed depressive‐like but not anxiety‐like behaviors in mice exposed to chronic stress for 4 weeks. The results from the metabolomics analysis showed that a total of 20 differentially expressed metabolites were identified between the CON and CMS+Veh groups, and 46 metabolites were selected between the CMS+Veh and High Dula groups in the hippocampus of the mice. Comprehensive analysis indicated that lipid metabolism, amino acid metabolism, energy metabolism, and tryptophan metabolism were disrupted in model mice that experienced depression and underwent dulaglutide therapy. Conclusion: The antidepressant effects of dulaglutide in a CMS depression model were confirmed. We identified 64 different metabolites and four major pathways associated with metabolic pathophysiological processes. These primary data provide a new perspective for understanding the antidepressant‐like effects of dulaglutide and may facilitate the use of dulaglutide as a potential therapeutic strategy for depression. [ABSTRACT FROM AUTHOR]

A good and accessible biomarker is of great clinical value in neuroendocrine tumor (NET) patients, especially considering its frequently indolent nature and long‐term follow‐up. Plasma chromogranin A (CgA) and 5‐hydroxyindoleacetic acid (5‐HIAA) are currently used as biomarkers in NET, but their sensitivity and specificity are restricted. 5‐HIAA is the main metabolite of serotonin, an important neurotransmitter of the tryptophan pathway. The aim of this study is to estabish a sensitive and accurate method for the quantification of tryptophan pathway metabolites in plasma. We further aimed to evaluate its utility as a clinical tool in NET disease. We obtained plasma samples from NET patients and healthy controls recruited from the University Hospital of North Norway, Tromsø. Samples were analyzed by liquid chromatography‐tandem mass spectrometry (LC–MS/MS), and eight metabolites of the tryptophan pathway were quantified. We included 130 NET patients (72/130 small intestinal [SI] NET, 35/130 pancreatic NET, 23/130 other origin) and 20 healthy controls. In the SI‐NET group, 26/72 patients presented with symptoms of carcinoid syndrome (CS). We found that combining tryptophan metabolites into a serotonin/kynurenine pathway ratio improved diagnostic sensitivity (92.3%) and specificity (100%) in detecting CS patients from healthy controls compared with plasma 5‐HIAA alone (sensitivity 84.6%/specificity 100%). Further, a clinical marker based on the combination of plasma serotonin, 5‐HIAA, and 5OH‐tryptophan, increased diagnostic capacity identifying NET patients with metastasized disease from healthy controls compared with singular plasma 5‐HIAA, serotonin, or CgA. In addition, this marker was positive in 61% of curatively operated SI‐NET patients compared with only 10% of healthy controls (p <.001). Our results indicate that simultaneous quantification of several tryptophan metabolites in plasma, using LC–MS/MS, may represent a clinically useful diagnostic tool in NET disease. [ABSTRACT FROM AUTHOR]

Although filtration is one of the most common steps in sample preparation for chemical analysis, filter membrane materials can leach contaminants and/or retain some analytes in the filtered solutions. In multiclass, multiresidue analysis of veterinary drugs, it is challenging to find one type of filter membrane that does not retain at least some of the analytes before injection in ultrahigh‐performance liquid chromatography‐tandem mass spectrometry (UHPLC‐MS/MS). In this study, different filter membranes were tested for use in UHPLC‐MS/MS analysis of 183 diverse drugs in bovine muscle, kidney, and liver tissues. Membranes evaluated consisted of polytetrafluoroethylene (PTFE), polyvinylidene difluoride (PVDF), polyethersulfone, nylon, and regenerated cellulose. Drug classes represented among the analytes included β‐agonists, β‐lactams, anthelmintics, macrolides, tetracyclines, sulfonamides, tranquilizers, (fluoro)quinolones, anti‐inflammatories, nitroimidazoles, coccidiostats, phenicols, and others. Although the presence of a matrix helped reduce the binding of analytes on surface active sites, all of the filter types partially retained at least some of the drugs in the final extracts. In testing by flow‐injection analysis, all of the membrane filters were also observed to leach interfering components. Ultimately, filtration was avoided altogether in the final sample preparation approach known as the quick, easy, cheap, effective, rugged, safe, efficient, and robust (QuEChERSER) mega‐method, and ultracentrifugation was chosen as an alternative. [ABSTRACT FROM AUTHOR]

Hemp‐based materials have gained interest as alternative feed ingredients for livestock. However, safety concerns arise regarding the transfer of cannabinoids from the plant to the animals. Addressing these concerns requires the use of methods capable of detecting and quantifying cannabinoids in livestock. In this study, a fast and sensitive method was developed for quantification of cannabinoids and cannabinoid metabolites in cattle plasma using liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). The extraction of cannabinoids from the plasma matrix was achieved by combining the Captiva Enhanced Matrix Removal‐Lipid clean‐up and salting‐out assisted liquid–liquid extraction procedure. The developed method underwent validation using various analytical parameters, and the results demonstrated good accuracy, precision, specificity, and high sensitivity. The method was applied to real plasma samples obtained from cattle fed hemp for 2 weeks, and successfully detected various cannabinoids, including delta‐9‐tetrahydrocannabinol. Furthermore, the study revealed that 7‐carboxy cannabidiol, a metabolite of cannabidiol, was the predominant cannabinoid present in the cattle plasma throughout the feeding period, which could remain detectable for weeks after the hemp feeding had ended. [ABSTRACT FROM AUTHOR]

During bioanalytical assay development and validation, maintaining the stability of the parent drug and metabolites of interest is critical. While stability of the parent drug has been thoroughly investigated, the stability of unanalyzed metabolites is often overlooked. When an unstable metabolite is known or suspected to interfere with measurement of the parent drug or other metabolites of interest through back‐conversion or other routes, additional tests with these unstable metabolites should be conducted. Here, the development and validation of two assays for quantification of rosuvastatin, one in human plasma and one in human urine, was reported. To this end, additional sets of quality control samples were added during assay validation to ensure the reliability of the assays. Acid treatment of samples is shown to be necessary for rosuvastatin quantification. In this regard, stability issues caused by the metabolite, rosuvastatin lactone, may have been overlooked if assay development and validation had only considered the parent drug, rosuvastatin. These assays represent a case study for how to develop and validate assays with unstable metabolites. Taken together, unstable metabolites should be included in all applicable stability tests. [ABSTRACT FROM AUTHOR]

Despite aggressive treatment approaches, the overall survival of glioblastoma (GBM) patients remained poor with a strong need for more effective chemotherapeutic agents. A previous study has shown that ARN14988 is more cytotoxic to GBM cells compared to US Food and Drug Administration‐approved temozolomide. This finding makes ARN14988 a desirable candidate for further pharmacological assessment. Therefore, an efficient analytical method is needed to quantify ARN14988. Herein, we have developed and validated sample preparation and LC–MS/MS triple quadrupole (QQQ) method for quantification of ARN14988 in mouse plasma. In this method, the liquid–liquid extraction of ARN14988 from mouse plasma was performed using 5% ethyl acetate in hexane. The chromatographic separation was achieved using a C18‐column with mobile phases of 10 mm ammonium acetate (pH 5) and 0.1% formic acid in methanol, within a runtime of 10 min. The monitored transitions were m/z 391.20 → m/z 147.00 for ARN14988, and m/z 455.30 → m/z 165.00 for verapamil (internal standard) in positive electrospray ionization. The developed method for ARN14988 showed linearity over the range of 10–5,000 ng/ml (r2 > 0.99). The selectivity, sensitivity, matrix effect, recovery, stability, inter‐day and intraday accuracy and precision were determined using four quality control samples. This validated method was successfully applied to the pharmacokinetic study of ARN14988 in mice. [ABSTRACT FROM AUTHOR]

Although the antibiotics inhibit or kill pathogens, the abuse leads to the resistance formation and even "Super Bacteria." Therefore, it is urgent to explore the natural and safe alternatives such as bacteriocin. In this study, an uncharacterized bacteriocin gene cluster for Lysinibacillus boronitolerans was first predicted by genome sequencing and bioinformatics analysis, of which including two biosynthetic genes, a regulatory gene, a transport‐related gene, and six other genes. Subsequently, the 10.24‐kb gene cluster was expressed in Escherichia coli BL21, and the lysate effectively inhibited the growths of pathogenic bacteria containing Bacillus pumilus, Bacillus velezensis, Pseudomonas syringae pv. tomato DC3000, and Xanthomonas axonopodis pv. manihotis. The antibacterial substance was purified by 70% ammonium sulfate precipitation and further identified by liquid chromatography‐tandem mass spectrometry. The results showed that the antibacterial substance consisted of 44 amino acids and had 24.1% sequence identity with the cyanobacterin Piricyclamide 7005 E4 PirE4, a bacteriocin analogue. The minimal set of genes required for the biosynthesis of the antibacterial substance was determined by site‐directed mutagenesis, suggesting both a transcriptional repressor and a phosphohydroxythreonine transaminase were essential. Subsequently, the evolution and conservation of the two proteins were analyzed among 22 Lysinibacillus species. Among them, the residues responsible for functions were identified. Collectively, our results set a solid foundation for investigation of the biosynthesis and application of bacteriocin. [ABSTRACT FROM AUTHOR]

Sucrose esters (SEs) are crucial tobacco smoke flavor precursors and play a significant role in tobacco's functionality. Due to their structural complexity, the separation and analysis of SEs in tobacco remain a major challenge, and massive structures of SEs have not yet been fully identified. In this study, the fractions enriched in SEs were obtained from oriental and flue‐cured tobacco through a series of pretreatments, and two types of SEs (Types I and II) were distinguished by liquid chromatography‐tandem mass spectrometry (LC‐MSn) analysis, with Type II SEs newly characterized in tobacco. Five groups of main SEs were further purified using preparative high‐performance LC (HPLC) coupled to an evaporative light scattering detector, and their structures were characterized by nuclear magnetic resonance spectrometry techniques including 1H, 13C, correlation spectroscopy, heteronuclear single quantum correlation, and heteronuclear multiple bond correlation. By combining LC‐MSn and nuclear magnetic resonance spectrometry, the structures of eight SE isomers were finally proposed, of which four were newly identified. These findings further enhance the understanding of the structural diversity of SEs in tobacco, serving as a valuable reference for future research on the elucidation, synthesis, and metabolism of SEs. [ABSTRACT FROM AUTHOR]

Ribociclib is a cyclin‐dependent kinase (CDK4/6) inhibitor and is a standard of care for treating metastatic breast cancer. The drug has moderate oral bioavailability and exhibits permeability‐controlled absorption. Novel formulations to enhance ribociclib pharmacokinetics are being developed and tested in rats. This requires developing analytical assays for quantifying ribociclib monitoring in rat plasma. We present a fully validated, sensitive, and simple LC‐MS/MS method for measuring ribociclib in rat plasma. Ribociclib‐D6 was utilized as an internal standard, and a simple protein precipitation procedure with acetonitrile was used in sample preparation. Excellent assay linearity was observed over a standard curve concentration of 1.008–1027.624 ng/mL. Acceptable intra‐ and inter‐day accuracy and reproductivity were demonstrated for ribociclib quality controls (bias and CV% within ±15%). Complete extraction recovery of ribociclib was achieved, and a negligible matrix effect of analyte to internal standard ratio was observed. Ribociclib was stable at various conditions, including bench‐top, freeze–thaw, and short‐term stability. Overall, the presented method is simple, sensitive, accurate, and precise and was successfully applied to quantify ribociclib in plasma samples from a pharmacokinetic study of ribociclib suspension and nanoparticle formulation in rats. [ABSTRACT FROM AUTHOR]

The opioid epidemic has affected the United States (US) for decades with fentanyl and its analogs accounting for a recent surge in morbidity and mortality. Currently, there is a relative lack of information characterizing fentanyl‐related fatalities specifically in the Southern US. A retrospective study was conducted to examine all postmortem fentanyl‐related drug toxicities in Travis County, Texas, encompassing Austin (one of the fastest‐growing cities in the US), from 2020 to 2022. Fentanyl contributed to 2.6% and 12.2% of deaths submitted for toxicology between 2020 and 2022, respectively, representing a 375% increase in fentanyl‐related deaths over this 3‐year period (n = 517). Fentanyl‐related fatalities primarily occurred in males in their mid‐30s. Fentanyl and norfentanyl concentrations ranged from 0.58 to 320 ng/mL and 0.53 to 140 ng/mL with mean (median) concentrations of 17.2 ± 25.0 (11.0) and 5.6 ± 10.9 (2.9) ng/mL, respectively. Polydrug use was present in 88% of cases, with methamphetamine (or other amphetamines) (25%), benzodiazepines (21%), and cocaine (17%) representing the most frequently identified concurrent substances. Co‐positivity rates of various drugs and drug classes widely varied over time. Scene investigations reported illicit powder(s) (n = 141) and/or illicit pill(s) (n = 154) in 48% (n = 247) of fentanyl‐related deaths. Illicit oxycodone (44%, n = 67) and illicit "Xanax" (38%, n = 59) pills were frequently reported on scene; however, toxicology only identified oxycodone and alprazolam in 2 and 24 of these cases, respectively. The results of this study provide a better understanding of the fentanyl epidemic in this region creating an opportunity to promote increased awareness, shift focus to harm reduction, and aid in minimizing public health risks. [ABSTRACT FROM AUTHOR]

This research aims to synthesize a specific and efficient sorbent to use in the extraction of apixaban from human plasma samples and its determination by high‐performance liquid chromatography‐tandem mass spectrometry. High specific surface area of metal‐organic framework, magnetic property of iron oxide nanoparticles, selectively of molecular imprinted polymer toward the analyte, and the combination of dispersive solid‐phase extraction method with a sensitive analysis system provided an efficient analytical method. In this study, first, a molecularly imprinted polymer combined with magnetic metal organic framework nanocomposite was prepared and then characterized using different techniques. Then the sorbent particles were used for selective extraction of the analyte from plasma samples. The efficiency of the method was improved by optimizing effective parameters. According to the validation results, wide linear range (1.02–200 ng mL−1), acceptable coefficient of determination (0.9938), low limit of detection (0.32 ng mL−1) and limit of quantification (1.02 ng mL−1), high extraction recovery (78%), and good precision (relative standard deviations ≤ 2.9% for intra‐ (n = 6) and interday (n = 6) precisions) were obtainable using the proposed method. These outcomes showed the high potential of the proposed method for screening apixaban in the human plasma samples. [ABSTRACT FROM AUTHOR]

Cannabis is the world's most used illegal drug. The main psychoactive component of cannabis is Δ9‐tetrahydrocannabinol (THC). To aid the identification of cannabis‐impaired individuals, a simple but effective workflow for reliable quantification of THC and its metabolites in oral fluid samples collected with the Greiner Bio‐One Saliva Collection System is presented. Sampling involves rinsing the oral cavity with an extraction solution containing a citrate buffer stimulating salivary flow. Sample processing targeted the cannabinoid fraction interacting with proteins and other insoluble constituents that can be separated by centrifugation. Approximately 50% of the total amount of cannabinoids included in the oral fluid was recovered from the obtained pellet by extraction with acetonitrile. Liquid chromatography–tandem mass spectrometry was used for cannabinoid quantification. Fitness of the developed workflow for application in forensic and clinical cannabis testing was demonstrated by evaluating multiple performance parameters, including selectivity, linearity, limits of quantification (LOQs), accuracy, precision, matrix effects, extraction recoveries, process efficiencies and stability. Furthermore, sensitivity and specificity of the developed oral fluid−based cannabis test was demonstrated by analysing 195 samples collected either from opioid addicts or persons suspected of driving under the influence of drugs. The accuracy of identifying a person with the presence of THC in blood was found to be 97.9%. [ABSTRACT FROM AUTHOR]

Fatty acids have multitudinous biological functions and play a crucial role in many biological processes, but due to poor ionization efficiency and lack of appropriate internal standards, the comprehensive quantification of fatty acids by liquid chromatography‐tandem mass spectrometry is still challenging. In this study, a new, accurate, and reliable method for quantifying 30 fatty acids in serum using dual derivatization was proposed. Indole‐3‐acetic acid hydrazide derivants of fatty acids were used as the internal standard and indole‐3‐carboxylic acid hydrazide derivants of them were used to quantify. The derivatization conditions were systematically optimized and the method validation results showed good linearity with R2 > 0.9942, low detection limit (0.03–0.6 nM), precision (1.6%–9.8% for intra‐day and 4.6%–14.1% for inter‐day), recovery (88.2%–107.2% with relative standard deviation < 10.5%), matrix effect (88.3%–105.2% with the relative standard deviation < 9.9%) and stability (3.4%–13.8% for fatty acids derivants in 24 h at 4°C and 4.2%–13.8% for three freeze‐thaw cycles). Finally, this method was successfully applied to quantify fatty acids in serum samples of Alzheimer's patients. In contrast to the healthy control group, nine fatty acids showed a significant increase in the Alzheimer's disease group. [ABSTRACT FROM AUTHOR]

In this exploratory study from a randomized double‐blinded crossover trial including 70 patients with coronary heart disease and self‐perceived muscular side effects of statins, we aimed to determine the relationship between low‐density lipoprotein cholesterol (LDL‐C) reduction and atorvastatin metabolite plasma concentrations. All patients underwent a 7 weeks treatment period with atorvastatin 40 mg/day and a 7 weeks placebo period in random order. Nonlinear regression with a three‐parameter equation explored the relationship between percentage LDL‐C reduction (statin vs. placebo) and the pharmacokinetic variables. Mean LDL‐C reduction was 49% (range 12% to 71%). The sum of 4‐OH‐atorvastatin acid and lactone correlated moderately with the LDL‐C response (Spearman ρ 0.27, 95% confidence interval [CI]: 0.03 to 0.48). Accordingly, nonlinear regression showed R2 of 0.14 (95% CI: 0.03 to 0.37, R2 adjusted equaled 0.11). Even a perfect underlying correlation of 1.0 showed R2 = 0.32 by simulation, using historical intra‐individual LDL‐C variation (8.5%). The 90% inhibitory concentration was 2.1 nmol/L, and the 4‐OH‐metabolite sum exceeded this threshold in 34% of the patients. In conclusion, trough plasma concentrations of 4‐OH‐atorvastatin metabolites correlated moderately to the LDL‐C reduction. A plateau LDL‐C response was observed above a pharmacokinetic threshold, below which the response was highly variable. The usefulness of monitoring concentrations of atorvastatin metabolites to optimize the individual dosage have limitations, but its supportive potential may be pursued in relevant patient subsets to achieve adequate efficacy at the lowest possible dose. The results add knowledge to the overall understanding of the variable LDL‐C response mediated by atorvastatin. [ABSTRACT FROM AUTHOR]

N,N–dimethylacetamide is an excipient used in intravenous busulfan formulations, a drug used in hematopoietic stem cell transplantation conditioning. The aim of this study was to develop and validate a liquid chromatography‐tandem mass spectrometry method for simultaneous quantification of N,N‐dimethylacetamide, and its metabolite N‐monomethylacetamide in plasma from children receiving busulfan. A 4 μl aliquot of patient plasma was extracted using 196 μl 50% methanol solution and quantified against calibrators prepared in the extraction solvent given negligible matrix effects across three concentrations. 9[H2]‐N,N‐dimethylacetamide was used as an internal standard. Separation of N,N‐dimethylacetamide and N‐monomethylacetamide was achieved using a Kinetex EVO C18 stationary phase (100 mm × 2.1 mm × 2.6 μm) running an isocratic mobile phase of 30% methanol containing 0.1% formic acid at a flow of 0.2 ml/min over 3.0 min. The injection volume was 1 μl. Calibration curves for N,N‐dimethylacetamide and N‐monomethylacetamide were linear up to 1200 and 200 μg/L, respectively, with a lower limit of quantification 1 μg/L for both analytes. Calibrator accuracy and precision were within ± 10% of the test parameters across four concentration levels. Analytes were stable over 14 days at three different storage conditions. This method was successfully applied to measure N,N‐dimethylacetamide and N‐monomethylacetamide concentrations in a total of 1265 plasma samples from 77 children. [ABSTRACT FROM AUTHOR]

Societal Impact Statement: Evidence has emerged that the antioxidant ergothioneine may be important in preventing many inflammatory diseases in humans. However, ergothioneine is not produced by humans or plants and is only made by fungi and some bacteria in soils. As such, humans get ergothioneine from eating fungi (mushrooms) or plants that take it up from the soil. In this study, we found that growing plants with beneficial fungi called arbuscular mycorrhizal fungi increased the amount of ergothioneine in plant tissues. This suggests that promoting agricultural practices that maintain healthy populations of beneficial soil fungi may improve the nutritional quality of crops. Summary: The amino acid ergothioneine (ERGO) has recently gained attention as an antioxidant that benefits human health. ERGO is produced by fungi and mycobacteria in soils and is acquired only from diet. The mechanism by which ERGO is transferred from soil to plant is unknown. Recent work has shown that tillage reduces the amount of ERGO in crops. As tillage also reduces arbuscular mycorrhizal fungi (AMF) populations, we examined the relationship between AMF and plant ERGO uptake.We grew asparagus, black beans, wheat, and oats with a variety of single and mixed species AMF inocula and compared ERGO levels of these plants to plants that were uninoculated.Mycorrhizal inoculation enhanced ERGO content across all plants. There was a positive correlation between AMF colonization level and plant ERGO content.AMF appear to be important mediators of plant ERGO uptake. Future research is needed to identify the mechanism that leads to higher ERGO in plants colonized by AMF in order to promote farming practices that enhance AMF populations and increase crop ERGO concentration in field settings. [ABSTRACT FROM AUTHOR]

The aim of this study was to develop a validated liquid chromatography–tandem mass spectrometry method to quantify nine important transporter proteins, so as to support the quantification of transporter proteins abundance in different human tissues and organs. Quantification was performed by targeted proteomics in positive multiple reaction monitoring mode with liquid chromatography–tandem mass spectrometry. The method was validated with respect to specificity, linearity, accuracy, precision, and stability. All the correlation coefficients (r) were larger than 0.99 and the validation ranges of multidrug‐resistant transporter 1, breast cancer resistance protein, organic cation transporter 2, and organic anion transporter 1 were 0.036–3.636 nM, organic anion transporter 3 was 0.018–1.818 nM, organic anion transporting polypeptide transporter 1B1 and multidrug and toxin extrusion transporter 1 were 0.091–9.091 nM, organic anion transporting polypeptide transporter 1B3 and multidrug and toxin extrusion transporter 2K were 0.182–18.182 nM. The results of intra‐day and inter‐day accuracy and precision were all in line with the quantification requirements of biological samples and the stability results indicated that the samples were stable under current processing and storage conditions. The method is sensitive and efficient for the quantification of nine transporters in human tissues. [ABSTRACT FROM AUTHOR]

Cancer cells are known to survive in a hypoxic microenvironment by altering their lipid metabolism as well as their energy metabolism. In this study, Caco‐2 cells derived from human colon cancer, were found to have elevated intracellular levels of phosphatidic acid and its lysoform, lysophosphatidic acid (LPA), under hypoxic conditions. Our results suggested that the elevation of LPA in Caco‐2 cells was mainly due to the combined increases in cellular levels of lysophosphatidylcholine and lysophosphatidylethanolamine by phospholipase A2 and subsequent hydrolysis to LPA by lysophospholipase D. We detected the Ca2+‐stimulated choline‐producing activities toward exogenous lysophosphatidylcholines in whole Caco‐2 cell homogenates, indicating their involvement in the LPA production in intact Caco‐2 cells. [ABSTRACT FROM AUTHOR]

Quick, easy, cheap, effective, rugged, and safe extraction strategies are becoming increasingly adopted in various analytical fields to determine drugs in biological specimens. In the present study, we developed two fully automated quick, easy, cheap, effective, rugged, and safe extraction methods based on acetonitrile salting‐out assisted liquid‐liquid extraction (method 1) and acetonitrile salting‐out assisted liquid‐liquid extraction followed by dispersive solid‐phase extraction (method 2) using a commercially available automated liquid‐liquid extraction system. We applied these methods to the extraction of 14 psychotropic drugs (11 benzodiazepines and carbamazepine, quetiapine, and zolpidem) from whole blood samples. Both methods prior to liquid chromatography–tandem mass spectrometry analysis exhibited high linearity of calibration curves (correlation coefficients, > 0.9997), ppt level detection sensitivities, and satisfactory precisions (< 8.6% relative standard deviation), accuracies (within ± 16% relative error), and matrix effects (81–111%). Method 1 provided higher recovery rates (80–91%) than method 2 (72–86%), whereas method 2 provided higher detection sensitivities (limits of detection, 0.003–0.094 ng/mL) than method 1 (0.025–0.47 ng/mL) owing to the effectiveness of its dispersive solid‐phase extraction cleanup step. These fully automated extraction methods realize reliable, labor‐saving, user‐friendly, and hygienic extraction of target analytes from whole blood samples. [ABSTRACT FROM AUTHOR]

A new multifactor analysis assessment strategy was developed for evaluating, optimizing, and comparing analytical techniques for acrylamide in frying oils. Based on five indices (absolute recovery, absolute matrix effect, the intensity of the full ion scan, and the precursor ion scan to m/z 184 and m/z 241), the proposed strategy was performed with radar analysis, relative contribution analysis, and the entropy‐weighted technique for order performance by similarity to ideal solution analysis. Two novel methods based on quick, easy, cheap, effective, rugged, and safe extraction methodology and gel permeation chromatography–liquid–liquid extraction followed by liquid chromatography–tandem mass spectrometry have been developed for the analysis of acrylamide in frying oils. Two methods were suitable for rapid and sensitive analysis of acrylamide in oils in different laboratories, with a limit of quantitation at 2 μg/kg, and the average recovery ranging from 92.5% to 107.8%, with relative standard deviations below 10%. When considering automation efficiency and matrix effects, gel permeation chromatography is the most efficient method, whereas the other method has an advantage when analyzing large samples. The developed methods were used in a pilot study to analyze frying oils with acrylamide content below 9.82 μg/kg, showing that the repeated frying process did not produce significant content of acrylamide in oils. [ABSTRACT FROM AUTHOR]

Dispersive liquid–liquid microextraction was used in conjunction with liquid chromatography–atmospheric pressure chemical ionization–tandem mass spectrometry to quantitate vitamins K1 and K2 in vitamin‐fortified emulsions, and vital microextraction parameters were optimized using response surface methodology coupled with Box–Behnken design. Under optimal microextraction conditions, highly linear (R2 >.999) calibration curves were obtained for both vitamins in a broad concentration range (1–1000 μg/L), and vitamin recoveries exceeded 90%. The detection and quantitation limits equaled 1.89 and 5.72 μg/L for vitamin K1, respectively, and 5.00 and 15.15 μg/L for vitamin K2, respectively. When applied to vitamin‐K‐loaded nanoemulsions and solid lipid nanoparticles, the developed method achieved excellent results, outperforming the currently employed Korean Food Code method, and therefore holding great promise for the quantitation of vitamin K in vitamin‐fortified food products. [ABSTRACT FROM AUTHOR]

To assess the bioequivalence of a generic safinamide tablet (test) vs a brand‐name safinamide tablet (reference) and effects of food on the pharmacokinetics of safinamide in healthy Chinese subjects, a single‐center, single‐dose, randomized, open‐label, 2‐preparation, 2‐period study with a 15‐day washout period was undertaken. A total of 56 healthy subjects were recruited in this study (fasting, n = 28; fed, n = 28). A single dose of a 100‐mg test or reference safinamide tablet was administered to each subject in a randomized sequence. Blood samples were obtained at 5 minutes before drug administration and during the 120 hours after dosing. The safinamide concentration in plasma was determined by high‐performance liquid chromatography–tandem mass spectrometry. Pharmacokinetic parameters were analyzed with noncompartmental methods. Safety was also monitored. The major pharmacokinetic parameters including maximum plasma drug concentration, area under plasma concentration‐time curve (AUC) from zero to time t (AUC0‐t), and AUC from time 0 to infinity (AUC0‐∞) were similar between the test and reference tablets under fasting and fed conditions. The 90% CIs of the test/reference ratios of log‐transformed maximum plasma drug concentration, AUC from zero to time t, and AUC from time 0 to infinity all fell within the equivalence interval (80.0%‐125%) whether under fasting condition or fed condition. In conclusion, the 2 formulations of safinamide tablets were bioequivalent and well tolerated under both fasting and fed conditions in healthy Chinese volunteers. High‐fat food delayed the absorption of safinamide but did not affect the final bioavailability. [ABSTRACT FROM AUTHOR]

An active substance of pyrano[3,2‐a]phenazine, also called CPUL1, is a synthesized phenazine derivative and displays broad‐spectrum anticancer activities. Quantitative assessment of CPUL1 in biological samples has not been well established, hindering pharmaceutical development and application. According to international guidelines, a sensitive and selective liquid chromatography‐tandem mass spectrometry method in negative ion mode was developed and validated for quantification of CPUL1 in human plasma, colorectal cancer cell lines, and rat plasma, whereby linearity and accuracy were demonstrated for the range of 1–1000 ng/ml. The validated liquid chromatography‐tandem mass spectrometry method was successfully employed in pharmacokinetic studies of CPUL1 in vitro and in vivo. Notably, the cellular pharmacokinetic behavior of CPUL1 varies in colorectal cancer cell lines. Regarding the pharmacokinetic processes in vivo, oral absorption was less effective than an injection, with a bioavailability of 23.66%. CPUL1 was linearly eliminated after a single administration; however, it could accumulate in tissues (heart, liver, spleen, lung, and kidney) after multiple injections. In summary, this study established a capable bioanalytical method for CPUL1 and provided exploratory pharmacokinetic data, paving the way for use of this promising derivative in disease models. [ABSTRACT FROM AUTHOR]

Sperm cryopreservation technology has laid the foundation for promoting the popularity of artificial insemination in donkey reproduction, but the freeze–thaw process can cause sperm damage, and the viability of frozen sperm is greatly reduced, resulting in low insemination ability. Sperm metabolites play an important role in the freezing process of spermatozoa and have a major influence on the freezability of spermatozoa. The aim of this study was to explore the differential metabolites in donkey spermatozoa before and after cryopreservation by liquid chromatography–tandem mass spectrometry (LC–MS/MS). We analysed ejaculate samples from male donkeys obtained before and after freezing and identified 1323 metabolites. Compared with fresh sperm (F), the metabolites of cryopreserved sperm (CRY) were significantly changed, and 570 metabolites were significantly different between the two groups (p <.05). Among them, 277 metabolites were higher in frozen sperm, while the opposite was true for 293 metabolites. These metabolites mainly include phospholipids, lysophospholipids and amino acids., most of which are associated with oxidative stress and sperm capacitation. We describe significantly different metabolites before and after freezing that are significantly associated with decreased sperm motility post‐freezing and can be used as biomarkers of decreased sperm motility post‐freezing. [ABSTRACT FROM AUTHOR]

Antimicrobial agents are essential to protect human and animal health. During the coronavirus disease 2019 pandemic, antimicrobials such as cephalosporins were widely used as prophylactics and to prevent bacterial co‐infection. Undoubtedly, the prevalence of antibiotics in the aquatic environment will ultimately affect the degree of resistance against these bacteria in animals and the environmental systems. In order to monitor 16 cephalosporins in the aquatic environment, we developed a new liquid chromatography‐tandem mass spectrometry method that functioned simultaneously under positive and negative electrospray ionization switching modes. The chromatographic separation has been implemented using a pentafluorophenyl propyl column kept at 40°C. The limits of detection and quantitation for the studied cephalosporins ranged from (8 × 10−4) to (7.11 × 10−2) ng/ml and from (2.61 × 10−3) to (2.37 × 10−1) ng/ml, respectively. The percent extraction efficiency (apparent recovery) and relative standard deviations for the analyzed cephalosporins ranged from 61.69% to 167.67% and 2.45% to 13.48%, respectively. The overall findings showed that the effluent from the wastewater treatment plants that receive wastewater from pharmaceutical factories had a higher detected amount of cephalosporins than that of domestic sewage. Moreover, seven cephalosporins, including cefuroxime, ceftazidime, cefradine, cefprozil, cefixime, cefalexin, and cefadroxil (0.68–105.45 ng/L) were determined in the aquatic environment. [ABSTRACT FROM AUTHOR]

The study evaluated the pharmacokinetic features of azithromycin (AZM) in 15 freshwater crocodiles (Crocodylus siamensis) in Thailand. The crocodiles were administered a single intramuscular (i.m.) injection of AZM at three different dosages of 2.5, 5, and 10 mg/kg body weight (b.w.). Blood samples were collected at pre‐assigned times up to 168 h. The plasma concentrations of AZM were measured using a validated liquid chromatography–tandem mass spectrometry method. The plasma concentration of AZM were quantifiable for up to 168 h after i.m. administration at the three different dosages. A non‐compartmental model was used to fit the plasma concentration of AZM versus the time curve for each crocodile. The elimination half‐life values of AZM were 33.70, 38.11, and 34.80 h following i.m. injection after dosages of 2.5, 5, and 10 mg/kg b.w., respectively. There were no significant differences among groups. The results indicated that the overall rate of elimination of AZM in freshwater crocodiles was relatively slow. The maximum concentration and area under the curve from zero to the last values of AZM increased in a dose‐dependent fashion. The average binding percentage of AZM to plasma protein was 48.66%. Based on the pharmacokinetic data, the susceptibility break‐point and the surrogate PK‐PD index (T > MIC), the intramuscular administration of AZM at a dose of 10 mg/kg b.w. might be appropriate for the treatment of susceptible bacterial infections (MIC < 4 μg/ml) in freshwater crocodiles. [ABSTRACT FROM AUTHOR]

The illegal use of clenbuterol seriously endangers food safety and human health. Accurate monitoring of the illegal use of clenbuterol in livestock can efficiently prevent the clenbuterol residue pork products from entering the consumer market. Thus, in this study, a simple, rapid, and sensitive method for the determination of clenbuterol in swine urine was developed using electromembrane extraction combined with liquid chromatography‐tandem mass spectrometry. It should be noted that the electromembrane extraction method presented many advantages of simple operation, fast mass transfer rate, good sample clean‐up capability, and less organic solvent consumption. The effect of important factors on the extraction efficiency of clenbuterol was investigated. Under the optimal conditions, good linearity was achieved for clenbuterol over the range of 1–1000 ng/ml (linear correlation [R2] = 0.9996). The recoveries of clenbuterol in swine urine at three spiked levels ranged from 83.7% to 110.0% with relative standard deviation values lower than 9.7% (n = 4). The limits of detection and quantification for clenbuterol were 0.07 and 0.25 ng/ml, respectively. These results suggested that the proposed method has great potential for the extraction and determination of trace analyte in a complex sample matrix for monitoring illegal use in livestock. [ABSTRACT FROM AUTHOR]

A fast, uncomplicated, sensitive and fully validated high‐performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method has been developed for estimating l‐amino acids in the plasma of schizophrenic patients. The gradient‐elution chromatographic method was implemented with the Luna® PFP column (50 × 2.0 mm, 5 μm), and a mobile phase of 0.1% formic acid in water and methanol was used. The intra‐ and interday variability of the l‐amino acids was <13.11%, their accuracy ranged from 85.14 to 116.75% at the quality control levels and the lower limit of quantification ranged from 2.5 to 15 nm. The extraction efficiency (apparent recovery) of amino acids from healthy plasma was employed by spiking the plasma with standard amino acids at the quality control levels. Their percentage recoveries ranged from 80.4 to 119.94%. Our method has a short run time and fast sample preparation compared with existing methods, which suffer from long preparative steps and/or time‐consuming analysis, restricted reagents and the suboptimal performance characteristics of presently available technologies. Therefore, the proposed HPLC–MS/MS method was effectively applied for monitoring the l‐amino acids in the plasma of schizophrenic patients and healthy volunteers. [ABSTRACT FROM AUTHOR]

Lysophosphatidylethanolamine (LPE) is a major lysophospholipid produced by phospholipids and binds to human serum albumin (HSA). LPEs may play various roles in vivo depending on the differences in their acyl chains. However, only few reports have been published on the biological functions of LPEs. Hence, we determined the exact relative abundance of the major LPEs in the serum of healthy participants (n = 8) using liquid chromatography–tandem mass spectrometry. Consequently, LPE 18:2 (24.1 ± 5.2%) was found to be the most abundant in serum. To understand the distribution of LPEs, the serum separated via gel‐filtration high‐performance liquid chromatography was subjected to quantitative measurement. LPEs were more observed in the albumin fraction than the lipoprotein fraction. We also performed a fluorescence displacement assay and an in silico molecular docking experiment using AutoDock to confirm the affinity and binding sites of the LPEs on HSA. The binding affinities of the LPEs for drug sites 1 and 2 on HSA were relatively low, with Ki values of approximately 11 and 3.8 μM, respectively. AutoDock analysis revealed the conformation of the LPEs bound to drug sites and the possibility of LPEs binding to other HSA sites. These findings could help to elucidate the biological and pathological functions of LPEs. [ABSTRACT FROM AUTHOR]

Shuanghuanglian oral liquid is a common traditional Chinese medicine used to treat respiratory tract infections. Its major components are baicalin, chlorogenic acid, and forsythin. In this study, the main drug‐related components in human plasma after oral administration of Shuanghuanglian were initially identified using ultra‐performance liquid chromatography‐ultraviolet detector/quadrupole time‐of‐flight mass spectrometry. Thirteen components from baicalin were identified, including the parent drug baicalin and aglycone baicalein. Only one metabolite related to chlorogenic acid, a sulfate conjugate formed after hydrolysis, and one metabolite related to forsythin, a sulfate conjugate of forsythin aglycone, were detected. Subsequently, a liquid chromatography‐tandem mass spectrometry method was established and validated to simultaneously determine baicalin and baicalein, the primary active components. After simple protein precipitation, the analytes were separated on a BEH C18 column using a 5 min‐gradient elution to avoid interference from baicalin isomers and their in‐source dissociation. Excellent linearity was observed over the concentration ranges of 5.00–2000 ng/ml for baicalin and 1.00–100 ng/ml for baicalein. The validated method was successfully applied to a pharmacokinetic study of an oral administration of 60 ml Shuanghuanglian in healthy subjects. This study provided a foundation to investigate the clinical efficacy and safety of Shuanghuanglian further. [ABSTRACT FROM AUTHOR]

Phenytoin is a powerful antiseizure drug with complex pharmacokinetic properties, making it an interesting model drug to use in preclinical in vivo investigations, especially with regards to formulations aiming to improve drug delivery to the brain. Moreover, it has a major metabolite, 5‐(4‐hydroxyphenyl)‐5‐phenylhydantoin, which can be simultaneously studied to achieve a better assessment of its behaviour in the body. Here, we describe the development and validation of a sensitive LCMS/MS method for quantification of phenytoin and 5‐(4‐hydroxyphenyl)‐5‐phenylhydantoin in rat plasma and brain which can be used in such preclinical studies. Calibration curves produced covered a range of 7.81 to 250 ng/mL (plasma) and 23.4 to 750 ng/g (brain tissue) for both analytes. The method was validated for specificity, sensitivity, accuracy, and precision and found to be within the acceptable limits of ±15% over this range in both tissue types. The method when applied in two in vivo investigations: validation of a seizure model and to study the behaviour of a solution of intranasally administered phenytoin as a foundation for future studies into direct nose‐to‐brain delivery of phenytoin using specifically developed particulate systems, was highly sensitive for detecting phenytoin and 5‐(4‐hydroxyphenyl)‐5‐phenylhydantoin in rat plasma and brain. [ABSTRACT FROM AUTHOR]

A liquid chromatography‐tandem mass spectrometry method was developed and validated for the simultaneous quantification of vincristine and tariquidar in 10 μL of mouse whole blood using volumetric absorptive microsampling devices. Samples were extracted from the devices and quantified against calibrators prepared in a human blood plasma matrix. Separation of vincristine and tariquidar was achieved using a Shimpack XR ODS III C18 stationary phase and H2O and methanol mobile phase solvents containing 0.1% formic acid, running a gradient elution at a flow rate of 0.2 mL/min over 6.0 min. The method was linear up to 1200 ng/mL (R2 > 0.99 for both analytes), with calibrator accuracy within ± 15% of the nominal concentrations and analyte coefficient of variance <15% for both vincristine and tariquidar. Pharmacokinetic assessment of both analytes was successfully applied in mice as both single‐agent therapy and combination therapy over a 24‐h period, and a 2.3‐fold increase in vincristine drug exposure was observed in combination with tariquidar. This study validates the use of this approach for longitudinal analysis of drug exposure in animal studies. [ABSTRACT FROM AUTHOR]

The expression of spidroins in the major ampullate, minor ampullate, flagelliform, and tubuliform silk glands of Trichonephila clavipes spiders was analyzed using proteomics analysis techniques. Spidroin peptides were identified and assigned to different gene products based on sequence concurrence when compared with the whole genome of the spider. It was found that only a relatively low proportion of the spidroin genes are expressed as proteins in any of the studied glands. In addition, the expression of spidroin genes in different glands presents a wide range of patterns, with some spidroins being found in a single gland exclusively, while others appear in the content of several glands. The combination of precise genomics, proteomics, microstructural, and mechanical data provides new insights both on the design principles of these materials and how these principles might be translated for the production of high‐performance bioinspired artificial fibers. Research highlights: ‐Proteomics was used to analyse the spidroin content of MA, mi, Flag and Tub glands.‐Peptides were compared with spidroin genes.‐Only a fraction of the genes is expressed in the glands.‐Two proteins were identified in the four glands. [ABSTRACT FROM AUTHOR]

Thiamylal is an ultrashort‐acting barbiturate used for intravenous administration or general anesthesia induction. However, some cases of poisoning and suicide with thiamylal administration have been reported. Additionally, there are few reports on its analysis in the organs and adipose tissue, which requires purification by column chromatography and evaporation. A rapid and sensitive method was developed for quantifying thiamylal and its metabolite, secobarbital, in the adipose tissue, serum, and liver using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Samples were prepared using modified QuEChERS extraction. For adipose tissue samples, an acetonitrile–hexane partitioning step was added to the extraction. This method was applied to investigate a suspected self‐poisoning autopsy case. The quantitation accuracy for thiamylal added to porcine pericardial fat (0.18 µg/g), human serum (0.015 µg/mL), and porcine liver (0.18 µg/g) was 103%, 113%, and 95.3%, respectively. The quantitation limits calculated for porcine pericardial fat, human serum, and porcine liver at a signal‐to‐noise ratio of 10 were 0.06 µg/g, 0.005 µg/mL, and 0.06 µg/g, respectively. In addition, the thiamylal and secobarbital levels in the forensic autopsy case were 140 and 1.5 µg/g, respectively, in myocardial fat; 3.5–4.9 and 0.12–0.20 µg/mL, respectively, in serum; and 6.2–42 and 0.58–1.1 µg/g, respectively, in liver tissue. Thiamylal is especially distributed in the adipose tissue. The thiamylal‐to‐fat ratio may help estimate the time from administration to death. The developed modified QuEChERS extraction method with acetonitrile–hexane partitioning is suitable for analyzing hydrophobic compounds, such as thiamylal, in the adipose tissue. [ABSTRACT FROM AUTHOR]

We present a liquid chromatography tandem mass spectrometry method for the simultaneous analysis of 16 endogenous steroids (androgens, estrogens, glucocorticoids and progestogens) in human serum. Samples (250 μl of matrix) were extracted with t‐butylmethyl ether prior to LC–MS/MS analysis. The chromatographic separation was achieved on a reversed‐phase column using a methanol–water gradient. The HPLC was coupled to a triple quadrupole mass spectrometer equipped with an electrospray ionization source with acquisition in multiple reaction monitoring mode. The method was validated using surrogate matrices and human serum samples. The specificity of the method was confirmed for all of the considered steroids; linearity was also assessed (R2 > 0.99, lack‐of‐fit test) in the ranges of concentrations investigated. The lower limits of quantification were in the range 10–400 pg/ml depending on the target steroid. Accuracy was in the range 85–115% for all target steroids except for the lower limit of quantitation levels where it was 80–120%. The extraction recovery was always >65%. No significant matrix effects were observed. To test the reliability of the method, the analysis of serum samples collected from 10 healthy subjects (5 M/5F) was performed. The present method can be used to identify the trajectories of deviation from the concentration normality ranges applied to disorders of the gonadal and adrenal axes. [ABSTRACT FROM AUTHOR]

Oral fluid is an alternative matrix that has proven to be useful for the detection of drugs. Oral fluid is easy to collect, noninvasive, and may indicate recent drug use. There are limited methods available that analyze cognitive stimulants in oral fluid. Cognitive stimulants are used to treat attention‐deficit/hyperactivity disorder (ADHD), a neurological disorder that emerges from lack of dopamine in the brain. To combat this disorder, medications inhibit dopamine and norepinephrine reuptake by blocking transporters in the brain. Though commonly diagnosed in children, ADHD may extend beyond adolescence and abuse of medications in college students is not uncommon. The goal of this study was to develop and validate a quantitative method for methylphenidate, ethylphenidate, lisdexamfetamine, and amphetamine in oral fluid using liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). Analytes were isolated by solid‐phase extraction and analyzed on an Agilent 1290 Infinity II Liquid Chromatograph coupled to an Agilent 6470 Triple Quadrupole Mass Spectrometer. The linear range was 0.5–100 ng/ml (except lisdexamfetamine at 5–500 ng/ml). Bias and between‐run precision were acceptable (±11.0% bias and ±12.2%CV). No interferences or carryover were observed and dilution integrity was sustained. This validated method was applied to four authentic oral fluid samples collected with Quantisal® devices from college students. Lisdexamfetamine was quantified in one sample at 5.8 ng/ml while amphetamine was quantified in all four samples at 6.0–78.8 ng/ml. This is the first known quantitative method in oral fluid that includes these analytes using LC‐MS/MS and may give rise to interpretive value in a forensic toxicology setting. [ABSTRACT FROM AUTHOR]

Poisoning from consumption of foraged alternative medicine products is an uncommon yet recognized occurrence. Here, presented is the case of a 40‐year‐old woman who was witnessed to collapse with labored breathing and subsequently died despite emergency medical personnel attendance and resuscitation efforts. Autopsy revealed the presence of plant matter that was visually identified as leaves from Taxus baccata – the English Yew. Isolation of alkaloids from the plant material and subsequent identification of the same alkaloids in the decedent's blood by liquid chromatography‐tandem mass spectrometry indicated a toxicological cause of death. This case illustrates a collaborative team approach among subject matter experts to unexpectedly discover and then confirm the sudden death of this woman from T. baccata toxicity. [ABSTRACT FROM AUTHOR]

Tacrolimus has a narrow therapeutic index and large individual differences in pharmacokinetics. The distribution of tacrolimus in ascitic fluid and its influence on whole‐blood tacrolimus were unclear. In this study, a sensitive ultra‐performance liquid chromatography‐tandem mass spectrometry method was established and validated for the quantification of tacrolimus in the ascitic fluid of liver transplant recipients. Chromatographic separation was achieved on an Agilent ZORBAX Eclipse Plus Phenyl‐Hexyl column (2.1 × 100 mm, 3.5 μm). Mass spectrometry was performed in multiple reaction monitoring conditions of transitions m/z 821.4→768.5 for tacrolimus. The concentrations of tacrolimus in the ascitic fluid range from 0.2 to 3.0 ng/mL, accounting for 1.19–31.87% of whole‐blood tacrolimus concentrations. A linear mixed model showed a statistically significant positive correlation between the steady‐state trough blood concentration of tacrolimus and the corresponding amount of tacrolimus excreted in the ascitic fluid for 24 consecutive hours, especially after normalization by daily dose per unit body weight. These data suggested that the distribution of tacrolimus in the ascitic fluid has great individual differences. The whole‐blood tacrolimus concentration, dose per unit body weight, and other confounding factors may contribute to the excretion of tacrolimus in ascitic fluid, but the influence of tacrolimus excretion in drained ascitic fluid on the whole‐blood tacrolimus concentration is negligible. [ABSTRACT FROM AUTHOR]

Levetiracetam is an antiepileptic drug that is primarily approved by the Food and Drug Administration for the treatment of focal and generalized seizures. This study describes the development and validation of a highly selective and sensitive liquid chromatography‐tandem mass spectrometry method with triple‐stage fragmentation to determine levetiracetam in epileptic patient serum. After simple protein precipitation, the analytes were separated on a short reversed‐phase column (Agilent Poroshell 120 SB‐C18 column, 4.6 × 50 mm, 2.7 μm) using isocratic elution with 25% 0.1% formic acid in water (solvent A) and 75% methanol (solvent B) at a flow rate of 0.8 ml/min. The linear range is 0.5–50 μg/mL (R2 > 0.99). All the validation data, such as lower limit of quantification, linearity, specificity, recoveries, matrix effects, and other parameters, fit the request of biological method validation guidance. Passing–Bablok regression coefficients demonstrated that there is no constant bias and no proportional bias between the liquid chromatography‐tandem mass spectrometry methods with triple‐stage fragmentation and liquid multiple reaction monitoring. Bland–Altman plot showed that the developed liquid chromatography‐tandem mass spectrometry method with triple‐stage fragmentation method is reliable and accurate to determine levetiracetam in human serum. [ABSTRACT FROM AUTHOR]

In this study, well‐defined core‐shell ethylenediamine‐functional magnetic ferroferric oxide polymers were prepared and were fully characterized by transmission electron microscopy, scanning electron microscopy, FTIR spectroscopy, and vibrating sample magnetometry. Then, it was used as a magnetic solid‐phase extraction adsorbent for simultaneous determination of 11 trace quinolone residues in freshwater fish samples coupled to liquid chromatography‐tandem mass spectrometry. The obtained results revealed that the adsorbent showed good extraction efficiency and the adsorption mechanisms referred to hydrogen bond and π‐π stacking interaction. Moreover, the magnetic solid‐phase extraction conditions were also carefully optimized. The limits of quantitation of 11 quinolones were in the range of 0.15–0.36 μg/kg, while spiking recoveries were in the range of 80.2–99.5% for the 11 quinolones in freshwater fish samples at four spiked levels including limits of quantitation, 1.0, 40.0, and 80.0 μg/kg with the relative standard deviations ranging from 0.8 to 9.1%. The proposed method was applied to analyze 45 freshwater fish samples, and enrofloxacin was detected in 91.1% samples with concentrations ranging from 0.659 to 333 μg/kg. It could be concluded that the proposed method is fast, simple, sensitive, and accurate for the routine monitor of freshwater fish. [ABSTRACT FROM AUTHOR]

Herba Lysimachiae inhibits synovial damage in osteoarthritis via regulating two bio labels (integrin alpha 2b/beta 3). However, the relevant active ingredients are still unknown. Here, the active ingredients of herbal medicines were analyzed based on the liquid chromatography–tandem mass spectrometry technology and public bioinformatics platforms. The liquid chromatography–tandem mass spectrometry technology was used for compound analysis, and public databases (PubChem BioAssay and STRING) were applied to establish the links between herbal compounds and both bio labels, and identify which herbal compounds may regulate these bio labels. Subsequently, the osteoarthritis model was used to confirm the results. Totally, ninety compounds in Herba Lysimachiae were identified based on the liquid chromatography–tandem mass spectrometry technology. Bioinformatics analysis showed that five compounds (myricetin, fisetin, esculetin, 7‐hydroxycoumarin‐4‐acetic acid, and caffeic acid) may synergistically regulate bio labels through 11 targets, which may be the active ingredients of Herba Lysimachiae for osteoarthritis treatment. In the verification experiments, five compounds markedly suppressed the overexpression of bio labels in the synovium of the osteoarthritis model. In conclusion, the present study effectively and rapidly analyzed the active ingredients of Herba Lysimachiae for osteoarthritis treatment. [ABSTRACT FROM AUTHOR]