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Abstract We established a method based on ultra performance liquid chromatography tandem mass spectrometry (UPLC‒MS/MS) to quantitatively measure tepotinib, which was validated as acceptable and used in the evaluation of food-drug interactions between tepotinib and naringenin in rats. We used pemigatinib as the internal standard (IS), and acetonitrile and 0.1% formic acid aqueous solution constituted the mobile phase. To extract the target analyte, acetonitrile was used for protein precipitation (PPT). For UPLC‒MS/MS, we performed liquid chromatography using a C18 column, and mass spectrometry was performed in positive multiple reaction monitoring (MRM) mode. Excellent linearity was shown in the range of 0.1–500 ng/mL, and the coefficient of correlation was > 0.99. Notably, the lower limit of quantification (LLOQ) for tepotinib was determined to be 0.1 ng/mL. The intra- and inter-day accuracy of tepotinib ranged from − 1.7 to 7.3%, while the precision was ≤ 8.4%, at three concentrations except LLOQ. The recovery of each substance was ≥ 81.2%, and the matrix effects were within 90.5-98.6%. The stabilities of all analytes under different conditions met all requirements for quantitation in plasma samples. The relevant parameters, such as LLOQ, were evaluated in accordance with the principles of the Food and Drug Administration (FDA) biological verification method. Food-drug interaction study had shown that the plasma concentration of tepotinib could be significantly increased, accompanied by a decrease in clearance rate when administered with 50 mg/kg naringenin. The results showed that naringenin could increase the plasma concentration and decrease the clearance rate of tepotinib when naringenin and tepotinib were administered at the same time.

Abstract Ectoine (ECT) has recently gained considerable interest in the healthcare sector due to its promising therapeutic benefits in a variety of human disorders. This research aimed to quantify the ECT plasma level in rats by creating and optimizing a sensitive and validated UPLC-MS/MS method. Prior to analysis, ECT extraction from the plasma samples was conducted via a protein precipitation procedure, using hydroxyectoine as an internal standard (IS). A 1.7 μm UPLC C8 column (100 mm × 2.1 mm) was selected for the chromatographic separation, using a gradient mobile phase consisting of acetonitrile and 0.05% formic acid. The electrospray ionization mass spectrometry (ESI-MS) was used to detect ECT in the positive ion mode. To determine the specific precursor and the product ions of ECT, multiple reaction monitoring (MRM) methods were carried out. The selected ion pair of ECT was 143.1 > 97 and 159.1 > 113.13 for the IS. The ECT’s linearity range in rat plasma was found to be 1-1000 ng/mL, with a recovery rate of 96.48–97.37%. Consistent with FDA guidelines for bio-analytical method validation, the suggested method was validated. The method was efficiently employed to quantify the studied drug in spiked rat plasma with good accuracy and precision with no significant matrix effects. Furthermore, it was effectively used to investigate the pharmacokinetic behavior of ECT in rats after a single oral dose of 30 mg/kg.

Abstract In this study, we firstly established and verified a method by ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) for the analysis of vilazodone and its metabolite M10 in rat plasma, then this method was used to explore the pharmacokinetics of vilazodone and M10 present or absence of 80 mg/kg bergenin in rats. Protein precipitation with acetonitrile was used to prepare the samples in this research. The mobile phase for liquid chromatography was consisted of 0.1% formic acid aqueous solution and acetonitrile. Brexpiprazole was used as the internal standard (IS), and the multiple reaction monitoring (MRM) mode was used for detection. The verification items required by the US Food and Drug Administration (FDA) guidelines such as selectivity, sensitivity, linearity, stability, recovery and matrix effect of this method were all met the standards. Besides, rats were used to explore the drug-drug interaction between vilazodone and bergenin, which were divided into two groups, and separately gavaged with the same-volume of carboxymethyl cellulose sodium (CMC-Na) solution and 80 mg/kg bergenin, respectively. The results showed that bergenin significantly affected the metabolism of vilazodone. It suggested that there was a potential drug-drug interaction between bergenin and vilazodone in rats. In clinical application, we should pay attention to the dose of vilazodone when in combination with bergenin.

The primary objective of this study was to investigate the effects of baicalein and fangchinoline on the metabolism of abemaciclib. We hypothesized that these two natural compounds could significantly affect the metabolism of abemaciclib by inhibiting the activity of the CYP3A4 enzyme, thus potentially increasing its concentration in the body. In vitro, rat liver microsomes (RLM) and human liver microsomes (HLM) were employed to explore the inhibitory effects and mechanisms of baicalein and fangchinoline on abemaciclib. In vivo, twelve healthy male Sprague-Dawley (SD) rats were randomly assigned to three groups: Group A (control group), Group B (baicalein), and Group C (fangchinoline). The concentrations of abemaciclib and its metabolite N-desethylabemaciclib (M2) were evaluated using ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Finally, molecular docking method was employed to understand the interaction between abemaciclib and baicalein. It was indicated by the in vitro findings that both baicalein and fangchinoline inhibited abemaciclib metabolism in RLM through a mixed mechanism of competitive and non-competitive inhibition pathway. In HLM, baicalein inhibited abemaciclib metabolism by employing a hybrid mechanism of uncompetitive and non-competitive inhibition, while fangchinoline exhibited its inhibition in a competitive manner. In vivo, pharmacokinetic experiments revealed significant increases for AUC(0-t) and AUC(0-∞) of abemaciclib in Group B and Group C when compared to Group A, while the plasma clearance (CLz/F) of abemaciclib exhibited significant reductions. Moreover, molecular docking studies showed that both abemaciclib and baicalein docked to the active pocket of CYP3A4. This study demonstrated that the co-administration of baicalein or fangchinoline significantly affected the metabolism of abemaciclib, providing valuable insights for its clinical application.

Abstract Broad-spectrum histone deacetylase inhibitors (HDACi) have excellent anti-tumor effects, such as abexinostat, which was a novel oral HDACi that was widely used in clinical treatment. The purpose of this study was to establish a rapid and reliable method for the detection of abexinostat concentrations in rat plasma using ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The mobile phase we used was acetonitrile and 0.1% formic acid, and the internal standard (IS) was givinostat. Selective reaction monitoring (SRM) was used for detection with ion transitions at m/z 397.93 → 200.19 for abexinostat and m/z 422.01 → 186.11 for givinostat, respectively. The intra-day and inter-day precision of abexinostat were less than 11.5% and the intra-day and inter-day accuracy ranged from − 10.7% to 9.7% using this method. During the analysis process, the stability of the test sample was reliable. In addition, the recovery and matrix effects of this method were within acceptable limits. Finally, the method presented in this paper enabled accurate and quick determination of abexinostat levels in rat plasma from the pharmacokinetic study following gavage at a dose of 8.0 mg/kg abexinostat.

Millions of individuals throughout the world suffer from lymphatic filariasis (LF), which is a morbid disease caused by Wuchereria bancrofti, Brugia malayi, and Brugia timori. These infections belong to tissue-invading nematodes and are one of the major neglected tropical diseases that often result in permanent and enduring disability among individuals in endemic regions. Due to combination therapy, LF eradication has drastically decreased infections globally. The development of blood micro-sampling techniques allowing precise quantitation of drugs in blood would facilitate pharmacokinetic (PK) studies in remote populations. Therefore, an LC-MS/MS bioanalytical method was utilized to analyze albendazole (ABZ), albendazole sulfone (ABZ-ON), albendazole sulfoxide (ABZ-OX), and probenecid (PR) in plasma and dried blood spots. Solid-phase extraction was utilized to extract the analyte from both plasma and blood-spiked DBS. Analytes of interest were eluted with a gradient mobile system using 0.05% formic acid in water (A) and 0.05% formic acid in methanol (B) and separated using a reversed-phase Acquity ®BEH C18 UPLC column (100 × 2.1 mm, 1.7 µm). Precision and accuracy at each QC level were within the acceptable limit, i.e., ±15% for all analytes in both the matrices. Tests for stability under laboratory and storage conditions indicated that no notable changes were observed for plasma and DBS. The LC-MS/MS method demonstrated its capability to consistently identify all target analytes (ABZ, ABZ-ON, ABZ-OX, and PR) at low concentrations, even at the small specimen volumes obtained from DBS cards. This confirms the efficacy and durability of DBS cards as a micro-sampling technique. Moreover, it enhances collection efforts for therapeutic drug monitoring in remote locations for patients infected with lymphatic filariasis.

Two potential genotoxic impurities were identified (PGTIs)-viz. 4-amino-1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one (PGTI-1), and 1-(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2,4(1H,3H)-one (PGTI-II) in the Molnupiravir (MOPR) synthetic routes. COVID-19 disease was treated with MOPR when mild to moderate symptoms occurred. Two (Q)-SAR methods were used to assess the genotoxicity, and projected results were positive and categorized into Class-3 for both PGTIs. A simple, accurate and highly sensitive ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method was optimized for the simultaneous quantification of the assay, and these impurities in MOPR drug substance and formulation dosage form. The multiple reaction monitoring (MRM) technique was utilized for the quantification. Prior to the validation study, the UPLC-MS method conditions were optimised using fractional factorial design (FrFD). The optimized Critical Method Parameters (CMPs) include the percentage of Acetonitrile in MP B, Concentration of Formic acid in MP A, Cone Voltage, Capillary Voltage, Collision gas flow and Desolvation temperature were determined from the numerical optimization to be 12.50 %, 0.13 %, 13.6 V, 2.6 kV, 850 L/hr and 375 °C, respectively. The optimized chromatographic separation achieved on Waters Acquity HSS T3 C18 column (100 mm × 2.1 mm, 1.8 µm) in a gradient elution mode with 0.13% formic acid in water and acetonitrile as mobile phases, column temperature kept at 35 °C and flow rate at 0.5 mL/min. The method was successfully validated as per ICH guidelines, and demonstrated excellent linearity over the concentration range of 0.5–10 ppm for both PGTIs. The Pearson correlation coefficient of each impurity and MOPR was found to be higher than 0.999, and the recoveries were in between the range of 94.62 to 104.05% for both PGTIs and 99.10 to 100.25% for MOPR. It is also feasible to utilise this rapid method to quantify MOPR accurately in biological samples.

The fruit of Acanthopanax senticosus (Rupr. and Maxim.) has been newly developed for the treatment of blood deficiency syndrome clinically, but the effective constituents are still unclear, restricting its quality control and the new medicinal development based on it. This study elucidated the efficacy of A. senticosus fruit (ASF) for treating blood deficiency syndrome and accurately characterize the constituents. Chinmedomics strategy was used to identify the metabolic biomarkers of the model and the overall effect of ASF was evaluated based on the biomarker when it showed intervention effects for blood deficiency syndrome. ultrahigh performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used to analyze the components in the blood absorbed from A. senticosus fruit, and the components highly relevant to the biomarker are regarded as potential effective constituents for blood deficiency syndrome. Twenty-two of the 28 urine metabolites of blood deficiency syndrome were significantly regulated by A. senticosus fruit, 97 compounds included 20 prototype components, and 77 metabolites were found in vivo under the acting condition. The highly relevant constituents were isofraxidin, eleutheroside B, eleutheroside B1, eleutheroside E, and caffeic acid, which might be the effective constituents of A. senticosus fruit. It is a promising new medicinal resource that can be used for treating blood deficiency syndrome.

N- Demethylsinomenine (NDSM), the in vivo demethylated metabolite of sinomenine, has exhibited antinociceptive efficacy against various pain models and may become a novel drug candidate for pain management. However, no reported analytical method for quantification of N- Demethylsinomenine in a biological matrix is currently available, and the pharmacokinetic properties of N- Demethylsinomenine are unknown. In the present study, an ultra-high performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) method for quantification of N- Demethylsinomenine in rat plasma was developed and utilized to examine the preclinical pharmacokinetic profiles of N- Demethylsinomenine. The liquid-liquid extraction using ethyl acetate as the extractant was selected to treat rat plasma samples. The mixture of 25% aqueous phase (0.35% acetic acid-10 mM ammonium acetate buffer) and 75% organic phase (acetonitrile) was chosen as the mobile phases flowing on a ZORBAX C18 column to perform the chromatographic separation. After a 6-min rapid elution, NDSM and its internal standard (IS), metronidazole, were separated successfully. The ion pairs of 316/239 and 172/128 were captured for detecting N- Demethylsinomenine and IS, respectively, using multiple reaction monitoring (MRM) under a positive electrospray ionization (ESI) mode in this mass spectrometry analysis. The standard curve met linear requirements within the concentration range from 3 to 1000 ng/mL, and the lower limit of quantification (LLOQ) was 3 ng/mL. The method was evaluated regarding precision, accuracy, recovery, matrix effect, and stability, and all the results met the criteria presented in the guidelines for validation of biological analysis method. Then the pharmacokinetic profiles of N- Demethylsinomenine in rat plasma were characterized using this validated UPLC-MS/MS method. N- Demethylsinomenine exhibited the feature of linear pharmacokinetics after intravenous (i.v.) or intragastric (i.g.) administration in rats. After i. v. bolus at three dosage levels (0.5, 1, and 2 mg/kg), N- Demethylsinomenine showed the profiles of rapid elimination with mean half-life (T1/2Z) of 1.55–1.73 h, and extensive tissue distribution with volume of distribution (VZ) of 5.62–8.07 L/kg. After i. g. administration at three dosage levels (10, 20, and 40 mg/kg), N- Demethylsinomenine showed the consistent peak time (Tmax) of 3 h and the mean absolute bioavailability of N- Demethylsinomenine was 30.46%. These pharmacokinetics findings will aid in future drug development decisions of N- Demethylsinomenine as a potential candidate for pain analgesia.

The use of pesticides in nutmeg still leaves an accumulation of pesticide residues that can endanger human health and damage the ecological balance. In addition, nutmeg with high pesticide residue content will be rejected by nutmeg export destination countries. This research was conducted to validate the method of testing pesticide residues of carbaryl, carbofuran, acetate, and dimethoate on nutmeg. This pesticide residue test uses the QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) optimization method and is analyzed using UPLC-MS/MS (Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry). This method meets the validation requirements according to SANTE/12682/2019. The limit of quantification (LOQ) of the analyte is below the Maximum Residue Limit (MRL) set by the European Union through Regulation (EC) No. 396/2005.

Oleandrin and adynerin are the main toxic components of oleander, an evergreen shrub or a small tree of the oleander family, which belongs to the class of cardiac glycosides exhibiting delayed action. The pharmacokinetic differences of oleandrin and adynerin in rats were studied by ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) under two different administration modes: oral (5 mg/kg) and sublingual intravenous injection (1 mg/kg). The chromatographic column was UPLC BEH C18 (50 mm × 2.1 mm, 1.7 μm), and the column temperature was set at 40 °C. The mobile phase was acetonitrile–water (containing 0.1 % formic acid), with gradient elution, the flow rate was 0.4 mL/min, and the elution time was 4 min. Electrospray (ESI) positive ion mode detection with multiple reaction monitoring mode (MRM) was used for quantitative analysis: oleandrin m/z 577 → 145, adynerin m/z 534 → 113, and internal standard m/z 237 → 135. The established UPLC–MS/MS method was successfully applied to the pharmacokinetics in rats after administering oleandrin and adynerin. The bioavailability of oleandrin and adynerin was found to be low, 7.0 % and 93.1 %; respectively.

Diacylglycerols (DAGs) are important lipid mediators in cellular signaling transduction and metabolism. Imbalanced production or consumption of DAGs has a negative impact on the physiological functions of the body. However, comprehensive monitoring of structurally diverse DAGs remains a daunting task due to the rapid metabolism and ion suppression characteristics in biofluids. These bottlenecks call for developing a method that enables sensitive quantification of DAGs in biological sample. In this work, a straightforward charge derivatization strategy was developed to insert a series of structure analogs charge label, i.e., N, N-dimethylglycine (DMG) and N, N-dimethylalanine (DMA), on the free hydroxyl group of the DAGs. Owing to the existence of tertiary amino groups in charge label, the mass spectrometry ionization response of the derivatized DAGs was significantly increased in comparison with traditional metal ion adducts. After charge derivatization, the specific neutral loss diagnostic ions (DMG, 103 Da and DMA, 117 Da) were captured by mass spectrometry. Then, the DMG/DMA-oriented paired multiple reaction monitoring methods based on the characteristic diagnostic ions of the derivatized DAGs have been developed as sensitive methods for the detection (detection limit = 16 aM) and quantification (quantification limit = 62.5 aM) of DAGs in serum. Moreover, the tagged 1,2-DAGs and 1,3-DAGs sn-isomers have been well separated on the reversed-phase column in combination with ultra-performance liquid chromatography. Finally, metabolic characterizations of the tagged DAGs were further explored in L-Arginine-induced acute pancreatitis mice and resveratrol treated model mice. The results indicated that 1,2-DAGs were increased in the serum of model mice relative to normal controls and resveratrol significantly altered this metabolic abnormality. The currently established DMG/DMA-oriented paired charge derivatization strategy is promising for depicting DAGs changes more accurately in metabolic studies of lipid-related diseases and accurately evaluating drug treatment strategies.

Entrectinib is an oral selective inhibitor of the neurotrophic T receptor kinase (NTRK). It is used in the treatment of solid tumors in NTRK gene fusion lung cancer. The study aimed to develop and validate an analytical method for quantifying entrectinib plasma by UPLC-MS/MS using quizartinib as an internal standard. The method involves liquid–liquid extraction of entrectinib from plasma using tert butyl methyl ether. The mass-to-charge transitions were 561.23 → 435.1 for entrectinib and 561.19 → 114.1 for quizartinib. The method was successfully validated according to ICH and FDA guidelines. The method has a low quantification limit of 0.5 ng/mL, and the calibration curves constructed over a wide range of 0.5–1000 ng/mL showed good linearity (≥0.997). This method exhibits a tenfold increase in sensitivity compared with the previous method. The method is also accurate, precise, and reproducible, as evidenced by the inter-day and intra-day accuracy and precision values of 82.24–93.33% and 3.64–14.78%, respectively. Principles of green analytical chemistry were considered during all analytical steps to ensure safety. The greenness of the methods was evaluated using two assessment tools. These tools are the Analytical Eco-Scale and the analytical greenness metric approach (AGREE). The results were satisfactory and compatible with the criteria of these tools for green assessment. This method is green, accurate, precise, and reproducible. The method can be used to quantitate entrectinib in plasma and its pharmacokinetics in preclinical, and therapeutic drug monitoring.

Safinamide is an orally active, selective monoamine oxidase-B inhibitor with dopaminergic and non-dopaminergic properties approved by the European Medicine Agency and US Food and Drug Administration for the treatment of mid- to late-stage fluctuating Parkinson’s disease (PD) used in combination with other PD medications such as levodopa. In this study, an analytical quality by design (AQbD) approach was applied to optimize an LC-MS/MS bioanalytical method to determine safinamide in human plasma. A full 33 factorial design was used to optimize safinamide separation conditions, with a method first screened and optimized using chromatographic responses, including peak area and retention time. The results showed that temperature had a significant indirect effect on retention time and peak area (p < 0.05), while ammonium acetate concentration had an insignificant indirect impact on peak area or retention time. However, the temperature was significantly agonistic to the effect of buffer concentration (p < 0.05). The resultant optimized chromatography conditions utilized 9.0 mM ammonium acetate buffer and acetonitrile (22.0:78.0) as mobile phases at a column temperature of 23.2 °C. The assay was linear from 0.1–1000 ng/mL, met acceptance criteria for inter- and intra-assay precision and accuracies across three quality controls, and was successfully applied to in vitro microsomal metabolic stability. The UPLC/MS/MS method was found to be adequately sensitive and suitable for routine safinamide pharmacokinetic studies.

Sanjoin-tang (SJIT) is an ancient oriental medicine prescription listed in the Jinguiyaolue that is mainly used for the treatment of primary insomnia. This study was conducted to develop and validate an ultra-performance liquid chromatography with tandem mass spectrometry (UPLC–MS/MS) simultaneous analysis method for the quality control of SJIT using 18 target compounds. The 18 analytes were separated on an Acquity UPLC BEH C18 column maintained at 45 °C using a mobile phase composed of distilled water and acetonitrile. The MS system was used to simultaneously detect all analytes using the multiple reaction monitoring (MRM) method of Xevo TQ-XS coupled with an electrospray ionization source. The concentrations of the 18 analytes investigated in the SJIT samples ranged from below the limit of detection to 9.553 mg/g. In conclusion, the validated UPLC–MS/MS MRM analysis method can be used to obtain basic data to establish chemical-nonclinical linkage efficacy and for the clinical research and quality evaluation of SJIT.

Infigratinib (INF) is a fibroblast growth factor receptor (FGFR)-specific tyrosine kinase inhibitor for the therapy of advanced cholangiocarcinoma. However, CYP3A4 polymorphisms and CYP3A4 inducers or inhibitors might affect the pharmacokinetics of INF. Clinical evaluation of drug-drug interactions and adverse effects in these patients was necessary with reference to INF levels in vivo. The presently conducted study optimized a reproducible and rapid ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) analytic technique, which was validated and applied to determine INF concentrations in Sprague-Dawley (SD) rat plasma and pharmacokinetic studies. Protein was precipitated by adding acetonitrile to plasma samples, followed by gradient elution on a Waters Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm) for complete chromatographic separation of the analyte and derazantinib (used as internal standard, IS). A gradient elution of 0.1% formic acid aqueous solution and acetonitrile at a flow rate of 0.30 mL/min was applied as the mobile phase for this analysis. The ion transitions of INF and IS were m/z 599.88 → 313.10 and m/z 468.96 → 382.00 during UPLC-MS/MS detection, respectively, by multiple reaction monitoring (MRM). The methodological validation demonstrated that INF presented high linearity over the concentration of 2–600 ng/mL. The lower limit of quantification (LLOQ) for this experiment was 2 ng/mL, of which the precisions and accuracies were within the permissible levels. Inter-day and intra-day precisions were demonstrated to be within reasonable limits, which were within 15%, and the accuracies were determined to be between 2.2% and 11.4%. Moreover, the values of recovery, stability and matrix effect of INF were within the limits of acceptability. The pharmacokinetics of INF in SD rats was investigated by gavage administration of 10 mg/kg INF, followed by the application of the developed UPLC-MS/MS analytical method to detect the content in plasma and derive the main pharmacokinetic parameters.

Umifenovir is one of the most often prescribed antiviral medications for the prevention and treatment of COVID-19 and other viral infections. Herein, a UPLC-MS/MS method is developed through using ibrutinib as an internal standard (IS) for quantifying umifenovir in plasma samples. Both umifenovir and the IS were analytically separated on an Acquity BEH C18 column with a total run time of only 2.5 min. At a flow rate of 0.3 mLmin−1, acetonitrile:15 mM ammonium acetate (80:20) was employed as the mobile phase composition. Electrospray ionization in positive mode was used for ionization of the samples. Detection and quantification were performed in multiple reaction monitoring mode with parent-to-daughter ionization of 477.05 → 279.02 and 441.16 → 84.4 for umifenovir and the IS, respectively. The method was validated through following international guidelines for bioanalytical method validation, and all parameters were within the acceptable limits. Moreover, the eco-scale method using AGREE software was used for the evaluation of greenness, and results showed that the method is very environmentally friendly. The validated assay was successfully employed in the bioavailability assessment of a newly developed formulation of kneaded ternary umifenovir/β-cyclodextrin with 1% poloxamer 188 (KDB).

5-Fluorouracil (5-FU) is now used in eye drops for the management of conjunctival malignant melanoma, intraepithelial neoplasia, and corneal and conjunctival squamous cell carcinoma. The previously used methods for 5-FU quantification in AqH were time-consuming and less sensitive. Herein, a highly perceptive bioanalytical UPLC–MS/MS method was developed for the quantitative determination of 5-FU in the aqueous humor (AqH) of rabbits using allopurinol as the internal standard (IS). The 5-FU and IS were well separated in an Acquity™ HILIC column. Acetonitrile and 10 mM of ammonium acetate at 95:5 (v/v) were isocratically pumped at a 0.3 mL/min flow rate with a total runtime of 2.5 min. AqH samples were processed with a liquid–liquid extraction method in ethyl acetate. The 5-FU and IS were identified in the negative mode with electrospray ionization. The parent to daughter ion transitions for the 5-FU and IS occurred at m/z 128.92→41.68 and 134.80→64.10, respectively, as quantified using the multiple reaction monitoring mode. The developed method was validated with the ICH-Harmonized Guideline for Bioanalytical Method Validation, and the parameters were within acceptable limits. The calibration curve was linear at the 10.5–2000 ng/mL concentration range, with a correlation coefficient (R2) of 0.9946, and the lower limit of detection was 3.55 ng/mL. The developed and validated method was rapid, sensitive, accurate and robustly able to quantify 5-FU in rabbit AqH. The method was effectively applied to establish the ocular pharmacokinetics of 5-FU following the topical instillation of 5-FU-containing preparations in rabbits.

Fenebrutinib (GDC-0853; FNB) is an oral small molecule that was developed by Roche Pharmaceuticals to slow multiple sclerosis progression. FNB is a reversible bruton tyrosine kinase (BTK) inhibitor, which showed the maximum potency of BTK inhibitors in phase III clinical trials for multiple sclerosis. In the current study, a fast, specific, and sensitive UPLC-MS/MS method for FNB quantification in human liver microsomes (HLMs) was established with application to the evaluation of metabolic stability. The UPLC-MS/MS methodology was verified using the stated USFDA validation guidelines for bioanalytical methodologies that involve selectivity, linearity, accuracy and precision, carryover and extraction recovery, stability, and matrix effect. The FNB calibration curve displayed a linearity in the range from 1 ng/mL to 3000 ng/mL (y = 1.731x + 2.013; R2: 0.9954; RSD < 4.37%) in the HLMs matrix. The limit of quantification was 0.88 ng/mL, which verified the UPLC-MS/MS analytical method sensitivity. The intraday and interday precision and accuracy results of the developed UPLC-MS/MS method were −3.99–14.0% and 0.52–3.83%, respectively. FNB and savolitinib (SVB) (internal standard) were chromatographically separated utilizing an isocratic mobile phase system with a ZORBAX Eclipse plus-C18 (50 mm, 2.1 mm, and 1.8 μm) column. The metabolic stability parameters for FNB, involving high intrinsic clearance (58.21 mL/min/kg) and a short in vitro half-life (13.93 min), revealed the high extraction ratio of FNB. Reviewing the literature revealed that the current UPLC-MS/MS method is the first analytical method for FNB quantification in the HLMs matrix with application to the assessment of FNB metabolic stability.

Gabapentin (GAB) is a cyclohexane acetic acid, structurally related to the neurotransmitter gamma-aminobutyric acid (GABA), and considered the principal inhibitory neurotransmitter in the central nervous system (CNS) of mammals. An ultra-performance liquid chromatography–tandem mass spectrophotometry (UPLC-MS/MS) method for assessing pregabalin (PRE) in human plasma, was developed and validated, via PRE usage as an internal standard. The plasma underwent protein precipitation using methanol, prior to analysis. Chromatographic separation was completed using a mobile phase of methanol: 0.1% formic acid solution, (65:35, v/v), at a flow rate of 0.2 mL/min, with an isocratic approach, on an Agilent Eclipse plus column (50 × 2.1 mm and 1.8 μm), in 1.6 min of running time. An Agilent triple quadrupole was used for mass analysis, to detect the ion transitions for GAB and PER, respectively, at m/z of 172.1 → 154.1 and 160.10 → 142.10. The calibration curve, over the linear range of 0.050–10.0 μg/mL, showed a high correlation coefficient, r = 0.9993. The limits of detection and quantitation were 13.37 ng/mL and 40.52 ng/mL, respectively, based on the standard deviation and slope equation. The results for intra- and inter-day measurement accuracy and precision were in acceptable ranges. The method was extended into the assessment of oral administrations of GAB at different doses, of one 600 mg/tablet and two capsules (each one of them has 300 mg of GAB), to volunteers who were used in pharmacokinetics and bioequivalent studies. The AGREE assessment tool was used to visualize the proposed method’s greenness degree, which revealed a high AGREE rating score, supporting the accepted method’s greenness profile.

Capmatinib (CMB) is an orally bioavailable mesenchymal–epithelial transition (MET) inhibitor approved by the US-FDA to treat metastatic non-small cell lung cancer (NSCLC) patients, with MET exon 14 skipping mutation. The current study aimed to establish a specific, rapid, and sensitive ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) analytical method for quantifying CMB in human liver microsomes (HLMs), with therapeutic implications for assessing metabolic stability. Validation of the UPLC-MS/MS analytical method in the HLMs was performed using selectivity, sensitivity, linearity, accuracy, precision, extraction recovery, stability, and matrix effects according to the guidelines for bio-analytical method validation of the US-FDA. CMB was ionized by positive electrospray ionization (ESI) as the ionization source and analysed using multiple reaction monitoring (MRM) as the mass analyser mode. CMB and pemigatinib (PMT) were resolved on the C18 column, with an isocratic mobile phase. The CMB calibration curve showed linearity in the concentration range of 1–3000 ng/mL. The intra- and inter-day accuracy and precision were −7.67–4.48% and 0.46–6.99%, respectively. The lower limit of quantification (LLOQ) of 0.94 ng/mL confirmed the sensitivity of the UPLC-MS/MS analytical method. The intrinsic clearance (Clint) and in vitro half-life (t1/2) of CMB were 61.85 mL/min/kg and 13.11 min, respectively. CMB showed a high extraction ratio. The present study is the first to develop, establish, and standardize UPLC-MS/MS for the purpose of quantifying and evaluating the metabolic stability of CMB.

Developing effective therapies and medicines to conquer nonalcoholic steatohepatitis (NASH) is of great significance for public health and is faced with a major challenge. The activation of the thyroid hormone receptor agonist THRβ could be regulated by target drugs that has brought huge potential to the treatment of NASH. In this work, pyridazinone compound YWS01125 was synthesized for the first time. In this study, an ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) method for YWS01125 determination was established, and the pharmacokinetics of YWS01125 was evaluated. The half-life values (t1/2)of three different doses of YWS01125 was 189.12 ± 95.27, 152.64 ± 37.98, and 181.95 ± 64.25 min, respectively, and the tissue distribution studies demonstrated that YWS01125 was quickly distributed to various tissues. With successful application in the pharmacokinetics study of YWS01125, the UPLC-MS/MS method has shown characteristics of high sensitivity, rapidity, and good selectivity.

Vortioxetine is currently used as the first-line therapy drug for major depressive disorder (MDD). In the present study, we aimed to develop and fully validate an ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous quantification of vortioxetine and its major metabolite Lu AA34443 in plasma and to investigate the effects of dronedarone and amiodarone on vortioxetine metabolism in rats. After protein precipitation with acetonitrile, the separation of vortioxetine, Lu AA34443 and duloxetine (internal standard, IS) were finished on an Acquity BEH C18 (2.1 mm × 50 mm, 1.7 μm) column and their detections were conducted by a Xevo TQ-S triple quadrupole tandem mass spectrometer in the positive ion mode. The assay displayed excellent linearity in the range of 0.5–50 ng/mL for vortioxetine, and 5–1000 ng/mL for Lu AA34443. The results of this method exhibited that the precision, accuracy, matrix effect, recovery, and stability of vortioxetine and Lu AA34443 met all requirements for the quantitation in plasma samples. The validated assay was further successfully employed to study the effects of dronedarone (80 mg/kg) and amiodarone (60 mg/kg) on vortioxetine metabolism in rats. The results showed that dronedarone and amiodarone could increase the concentration of vortioxetine and have inhibitory effect on vortioxetine metabolism. Thus, vortioxetine dose interruption or reduction may be considered.

The mature pericarp of Citri Reticulatae 'Chachi' (PCRC) is one of the six traditional Chinese medicinal materials that should be used after long storage, and it was regarded that the longer the medicine was stored, the better. However, the aging mechanism of the medicine is not clear. To further investigate the effect of aging on the main active flavonoids of PCRC, ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-MS/MS) and metabolomics analysis were used to analyze the flavonoids of PCRC stored for different periods. In the results, 219 flavonoids were detected. 5,7,3′,4′,5′-pentamethoxy dihydroflavone and 2′-hydroxy-3,4,5,3′4′,6′-hexamethoxychalcone were found from PCRC for the first time. According to the clustering analysis of metabolites, aging times of 0 year, 1 year, and 2 year were clustered into one group, and aging times of 3 year, 4 year, and 29 year were clustered into the other group. Quantitative analysis showed that the former group contained a greater amount of 4 flavonoids than the latter group, while the latter group contained a greater amount of 15 polymethoxyflavonoids. The newly harvested PCRC was compared with the other 5 groups of PCRC (stored for 1, 2, 3, 4, and 29 years). Eight flavonoids, tectochrysin, apigenin, 2′-hydroxyisoflavone, luteolin, 6-hydroxyluteolin, gallocatechin, quercetin -O- acetylhexoside and apigenin −7-O-[β-D-glucuronide (1 → 2) -O-β-D-glucuronide], were used as marker components to discriminate newly harvested PCRC and aging PCRC. In addition, the antioxidant potency composite index (APC) indicated that the PCRC stored for three or four years had stronger antioxidant activity than the PCRC stored for other periods. By means of molecular docking, it was reviewed that the amount of antiviral components against SARS-CoV-2 in freshly harvested PCRC was significantly higher than that in aging PCRC. The results in this study supplied scientific data for quality control, evaluation, and rational utilization of PCRC and basic information for further analysis of the metabolic regulation of the active components of the PCRC.

The main objective of our current study is to develop and validate an accurate and direct ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method to simultaneously detect plasma concentrations of tofacitinib and its metabolite M9, and to study the pharmacokinetic profiles of the two compounds in beagle dogs. After rapid precipitation of protein by adding acetonitrile, the chromatographic separation of tofacitinib was completed, as well as M9 and upadacitinib (internal standard, IS) by using an Acquity BEH C18 (1.7 μm, 2.1 mm × 50 mm) column. A Xevo TQ-S triple quadrupole tandem mass spectrometer was employed to determine their concentrations under the positive ion pattern. Selective reaction monitoring (SRM) was used with ion transitions at m/z 313.12 → 148.97 for tofacitinib, m/z 329.10 → 137.03 for M9, and m/z 380.95 → 255.97 for IS, respectively. This assay demonstrated excellent linearity, and the ranges of calibration curves for both tofacitinib and M9 were 0.5–400 ng/mL. The new UPLC-MS/MS assay can reach the values (0.5 ng/mL) of lower limit of quantification (LLOQ) for both tofacitinib and M9. Both intra-day and inter-day accuracy of all analytes ranged from −12.0% to 14.3%, while the precision was ≤13.2%. The recovery rate of all analytes was >88.5%, and more importantly there was no conspicuous matrix effect. In addition, the stability was consistent with the quantificative requirements of plasma samples under all conditions. Finally, the assay on UPLC-MS/MS is able to be employed to determine the pharmacokinetic characteristics of tofacitinib and its metabolite M9 in the plasma of beagle dogs after taking orally a dose of tofacitinib at 2 mg/kg.

Gongjindan (GJD) is a traditional Korean medicine consisting of four herbal medicines and two animal-derived medicines, and is taken as a tonic in Republic of Korea. In this study, the goal was to develop and validate a simultaneous analytical method to quantify eight compounds in commercially available GJD samples using high-performance liquid chromatography (HPLC), ultra-performance liquid chromatography with tandem mass spectrometry (UPLC–MS/MS), and gas chromatography with tandem mass spectrometry (GC–MS/MS) systems. In HPLC and UPLC–MS/MS, seven components (gallic acid, 5-(hydroxymethyl)furfural, morroniside, loganin, nodakenin, decursin, and decursinol angelate) were separated and quantified using a distilled water–acetonitrile mobile phase system on a Capcell Pak UG80 C18 column and an Acquity UPLC BEH C18 column, respectively. Muscone was quantified using GC–MS/MS. The developed assays were validated by evaluating the linearity, limit of detection, limit of quantitation, recovery, and precision. In the regression equations of all compounds, the coefficient of determination was ≥0.9917, showing good linearity. The recovery was 93.70–108.17%, and the relative standard deviation values in the precision test were all

The prime purpose of this research currently being done was to set up an acceptable and high-operational ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method, which could simultaneously analyze the concentration levels of brexpiprazole, assisted medication for treating depression, and DM-3411, the principal metabolite of brexpiprazole in rats, and to examine the impact of baicalin on the metabolism of brexpiprazole in rats. After quick sample preparation using acetonitrile as a protein precipitating agent, the chromatograms of brexpiprazole, DM-3411 and aripiprazole (internal standard, IS) were successfully separated by Acquity BEH C18 (2.1 mm × 50 mm, 1.7 μm) column. The concentrations of the analytes were detected through a Xevo TQ-S triple quadrupole tandem by the positive ion scanning. The ranges of calibration curves for both brexpiprazole and DM-3411 were 0.5–100 ng/mL, and the approach represented fine linearity in the ranges. The lower limit of quantification (LLOQ) of brexpiprazole and DM-3411 could reach 0.5 ng/mL in the study. The range of intra-day and inter-day accuracy of the two analytes was between −10.6% and 12.3%, while the precision was ≤ 11.5%. The recovery rate of each compound was greater than 81.0%, and no obvious matrix effects were observable. In addition, the plasma sample quantitative detection of the compounds in the study remained stable under all conditions. Then, we used this assay to detect the plasma levels of brexpiprazole and DM-3411 from a herb-drug interaction investigation, in which 200 mg/kg baicalin remarkably increased the plasma concentration of brexpiprazole and changed brexpiprazole pharmacokinetics. This study will contribute to a better understanding of the pharmacokinetic properties of brexpiprazole when concurrent use with baicalin, and to an understanding of the unanticipated clinical risks.

Recently, neoadjuvant treatment has turned out to be a feasible alternative for individuals suffering from locally advanced breast cancer. The neoadjuvant therapy is a type of chemotherapy that is given either before or after surgeries to diminish a tumor and minimize the likelihood of recurrence. This article demonstrates the development of a unique bioanalytical validated sensitive method by means of an ultra high performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) approach for the concurrent estimation of neoadjuvant treatments including 5-Fluorouracil, Doxorubicin, and Capecitabine in rat plasma. Samples were prepared using the fine minor QuEChERS process and analyzed using a Shimadzu-C18 column via an isocratic separation. Acetonitrile:water in the ratio of (30:70) (both containing 0.1 percent formic acid v/v) was the mobile phase employed at a flow rate of 0.20 mL/min. At concentrations of 50.00–500.00 ng/mL for 5-Fluorouracil, 25.00–500.00 ng/mL for Doxorubicin, and 5.00–100.00 ng/mL for Capecitabine, the procedure was shown to be linear. The limit of detection (LOD) was assessed in ng/mL and varied from 1.33 to 13.50. Relative standard deviations for precision were below 2.47 percent over the whole concentration range. For all analytes, the average recovery rate varied from 73.79 to 116.98 percent. A preliminary pharmacokinetic study was successfully performed in real rats to evaluate the procedure efficiency.

The purpose of the experiment was mainly to establish and verify a precise and straightforward ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) approach for simultaneously analyzing the concentration levels of amiodarone, dronedarone, and their metabolites (desethylamiodarone and desbutyldronedarone) in the plasma of Sprague-Dawley (SD) rats and to investigate the pharmacokinetics of all analytes in SD rats. After rapid protein precipitation by using acetonitrile, we accomplished the chromatographic separation of amiodarone, desethylamiodarone, dronedarone, desbutyldronedarone and ivabradine (internal standard, IS) by using Acquity BEH C18 column and detected through a mass spectrometer with Xevo TQ-S triple quadrupole tandem, choosing the positive ion mode. The approach showed wonderful linearity, and the range of calibration curve for amiodarone was 1–200 ng/mL, desethylamiodarone was 0.1–20 ng/mL, dronedarone was 0.5–100 ng/mL, and desbutyldronedarone was 0.25–50 ng/mL, respectively. For lower limit of quantification (LLOQ), the current method of UPLC-MS/MS can achieve values of 1.0 ng/mL for amiodarone, 0.1 ng/mL for desethylamiodarone, 0.5 ng/mL for dronedarone, and 0.25 ng/mL for desbutyldronedarone, respectively. The accuracy of intra-day and inter-day of all analytes was between −14.8% to 10.9%, while the precision was ≤ 13.3%. For each substance, the recovery rate was > 82.1%, besides, obvious matrix effect was not found. In all conditions, the stability of all analytes was comfirmed to the plasma sample quantification. In addition, the method of UPLC-MS/MS we developed could also be applied to measure the pharmacokinetic characteristics including amiodarone, desethylamiodarone, dronedarone, and desbutyldronedarone in the plasma of SD rats.

Elagolix, as a competitive gonadotropin-releasing hormone (GnRH) receptor antagonist, has been recently approved by the US FDA for the management of moderate to severe pain due to endometriosis in women. In this study, we developed and verified an analysis assay to detect the concentration level of elagolix in plasma from rats after sample preparation based on a newly validated ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) technique in this study. The process of sample preparation used acetonitrile for a quick and easy protein precipitation method and diazepam was engaged as the internal standard (IS). Then, gradient elution was used to elute elagolix and IS. The mobile phase used in the present experiment was consisted of solvent A (acetonitrile) and solvent B (water having formic acid with the volume ratio of 0.1%), and the type of the C18 column used was named Acquity UPLC BEH C18 column with the specification of 2.1 mm × 100 mm, 1.7 μm. Multiple reaction monitoring (MRM) in positive ion mode for the experiment was engaged to detect the level of elagolix with electrospray ionization (ESI) source by m/z 632.4 → 529.5 transition for quantification and m/z 632.4 → 177.1 transition for qualification. It was found that the method in the scope of 1–2000 ng/mL indicated excellent linearity (r2 > 0.9983). The precision of this assay for intra-day was between 3.5 and 5.5%, and for inter-day was between 9.4 and 12.7%, respectively; the accuracy was 1.2–13.9% for the intra- and inter-day. The stability, extraction recovery, and matrix effect of the method were all in accordance with the rules of assay validation in biological medium proposed by FDA, whose application was also successfully used to determine the concentration of plasma elagolix from an experiment on pharmacokinetic investigation after oral administration of 15 mg/kg elagolix.

Abstract Comtrex® tablets composed of paracetamol, pseudoephedrine and brompheniramine are widely used for relieving symptoms related to common cold. This study has overcome the challenging dosage form ratio (250:15:1) and proposed chromatographic methods for analyzing the ternary combination were utilized displaying different apparatus, solvents and sensitivity ranges. Three chromatographic methods namely thin layer chromatography (TLC), high performance liquid chromatography with ultra-violet detection (HPLC–UV) and ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) were developed and validated for the simultaneous determination of the three drugs. Concerning the TLC method, aluminum TLC plates pre-coated with silica gel 60F254 were used and methanol:water:ammonia (9:1:0.1, v/v/v) was applied as a mobile phase; scanning of the plates was carried out at 254 nm. For the HPLC–UV method C18 column was used with an isocratic elution mobile phase composed of water:acetonitrile (75:25, v/v; pH 3.2) and the detection was at 210 nm. For the UPLC-MS/MS method; separation was performed on a UPLC-BEH C18 column with methanol: 0.1% ammonium formate (60:40, v/v) as the mobile phase utilizing diphenhydramine as an internal standard and mass spectrometry was used for detection. The methods were simple, sensitive, accurate and precise. Statistical analysis revealed no significant difference from the reported methods in regard to accuracy and precision.

This article introduces the aptamer affinity column (AAC) with nucleic acid aptamer as an affinity ligand for the extraction of four aminoglycoside antibiotics (AGs). The AAC was prepared by loading the aptamer functionalized Sepharose into an extraction column, which was conjugated by covalent binding between NHS-activated Sepharose and amino-modified aptamers with a coupling time of 2 h. After the sample solution flowed through the AAC, the AGs were retained because of the affinity between the AGs and aptamer, then AGs were eluted and analyzed by UPLC-MS/MS. Under the optimized conditions, the maximum adsorption of AGs on the AAC could reach 8.0 μg. Moreover, the proposed AAC could be reused more than 20 times. The resultant AAC that conjugated with the aptamer was successfully applied in the enrichment and purification of four AGs in a milk sample and good recovery results in the range of 83.3–98.8% were obtained (with RSD in the range of 0.6–5.8%). The proposed AAC for recognition of multi-target AGs exhibited good enrichment and purification effects, showing great application potential for targets with their related aptamers.

Erdafitinib is an oral pan-fibroblast growth factor receptor (FGFR) inhibitor and has a potent antitumor activity against FGFR-aberrant malignancies. Erdafitinib has a narrow therapeutic index, and its pharmacokinetics are influenced by genetic variability and interacting medication. Routine therapeutic drug monitoring and dose adjustment are recommended. This study aims at developing a new UPLC-MS/MS method for determination and quantitation of erdafitinib in human plasma using ibrutinib as an internal standard. The sample ionization was performed by using electrospray ionization in positive mode, and multiple reaction monitoring was used for the quantification of target analytes. The chromatographic separation of erdafitinib and IS was achieved by an UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm). Erdafitinib metabolic stability was studied using intrinsic clearance and in vitro half-life. The greenness of the developed method was evaluated using appropriate, analytical Eco-Scale and AGREE software. The linearity of the established UPLC-MS/MS assay ranged from 0.5 to 1000 ng/mL with r > 0.99 with a limit of quantitation of 0.5 ng/mL. The accuracy and precision were within acceptable limits and the average recovery and matrix effects were 86.11% and 90.51%, respectively. Erdafitinib metabolic stability was studied and its in vitro half-life was 7.28 min and intrinsic clearance was 95.11 µL/min/mg. The assessment of the greenness profile of the method indicated that the method is eco-friendly. The proposed method can be utilized for therapeutic drug monitoring and pharmacokinetic studies.

Cydonia oblonga miller (quince) plant serves as a potential folk medicine for treating hypertension and cardiovascular diseases in China. However, to the best of our knowledge, no study has been conducted on the polyphenolic profile and anti-adipogenic effect of quince fruit grown in China. In the current study, we aimed to investigate the quince fruit extract’s major phenolic compounds, evaluate their antioxidant activity, and examine their effect on adipogenesis in 3T3-L1 cells. A rapid and sensitive analytical method was established for the simultaneous determination of major polyphenolic compounds by using ultra-pressure liquid chromatography coupled with a triple quadrupole mass spectrometer (UPLC-MS/MS). Among the 10 compounds, the cryptochlorgenic acid was noticed as the most abundant compound of both purified (242.44 ± 0.73 µg/mg dw) and unpurified extract (3.37 ± 0.01 µg/mg dw) followed by quercetin 3-rutinoside and chlorogenic acid. Alternatively, both extracts possessed a high quantity of phenolic acids (purified extract = 483.10 ± 5.16 µg/mg dw and unpurified extract = 7.89 ± 0.02 µg/mg dw). The purified extract exhibited a strong antioxidant capacity (DPPH: EC50 = 3.316 µg/mL, ABTS: EC50 = 36.38 µg/mL) as compared to the unpurified extract. Additionally, our results also showed that the extract at 100 µg/mL significantly suppressed the preadipocyte differentiation and decreased the lipid droplets up to 69% in mature adipocytes. The present study highlights an accurate and fast detection method for quince fruit extract polyphenolic compounds with its antioxidant and antiadipogenic effects. The study also provides the necessary information for the rational development and utilization of quince fruit extract as a source of phytochemicals.

Avapritinib, an orally consumed, highly selective inhibitor of platelet-derived growth factor receptor alpha (PDGFRA), is approved in the USA for PDGFRA exon 18 (including D842V) mutant gastrointestinal stromal tumour (GIST). The research conducted investigates an advanced reliable and fast UPLC-MS/MS method that could verify and determine avapritinib concentration in plasma of rats. An addition of acetonitrile was incorporated along with the plasma sample in order to precipitate protein with the analyte separated from the matrix by a gradient elution procedure on a Waters Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm). The active stage in mobile phase consists of a mixture of 0.1% formic acid in water and acetonitrile with a 0.40 mL/min flow rate. UPLC-MS/MS detection was done employing a mode of multiple reaction monitoring (MRM), and the ion transitions of avapritinib and imatinib (internal standard, IS) was m/z 499.10 → 482.09, and m/z 494.30 → 394.20, respectively. This method has good linearity within 2–4000 ng/mL of avapritinib calibration range and a lower limit of quantification (LLOQ) of 2 ng/mL verified. Avapritinib precisions in both intra-day and inter-day were below 15% with the determined accuracy of −12.9% to 12.0%. The recoveries, stabilities, and matrix effect of avapritinib and IS were credible. 30 mg/kg avapritinib was administered as a single dose orally to rats. The determination of avapritinib level in pharmacokinetic studies was accomplished by efficiently applying a newly optimized UPLC-MS/MS assay.

5-Aminoisoquinoline (5-AIQ) is a water-soluble, potent and selective Poly (ADP-ribose) polymerase 1 (PARP-1) inhibitor, widely used as a biochemical and pharmacological tool to study the inhibitory effect of PARPs enzyme. In this study, a simple, selective and reliable ultra-performance liquid chromatography-tandem mass spectrometry assay has been developed for the quantitative analysis of 5-AIQ in plasma using pantoprazole as an internal standard (IS). Both 5-AIQ and IS were separated on an Acquity CSH18 (2.1 × 100 mm; 1.7 µm) column after chromatographic elution of mobile phase comprising of 10 mM ammonium acetate and acetonitrile (35:65; v/v) at a flow rate of 0.3 mL/min. Electrospray ionization in positive mode was used for sample ionization and precursor to product ion transitions of 145.0 > 91.0; 145.0 > 117.4 for 5-AIQ and 384.0 > 138.1 for IS were used for detection and quantification in multiple reaction monitoring mode. The assay was linear in the concentration range of 1.0 to 666 ng/mL with correlation coefficient of ≥0.995. The precision and bias were within the acceptable limits of ≤12.68% and −8.6 to 5.9%, respectively, with mean recovery of 79.1% from plasma and negligible matrix effects (92.4%). In silico ADME prediction, 5-AIQ showed to be very soluble in water and high gastrointestinal absorption along with blood–brain barrier (BBB) permeability. The validated assay was successfully applied in a metabolic stability study, and 5-AIQ was moderately metabolized by human liver microsomes with an in vitro half-life of 14.5 min and intrinsic clearance of 47.6 µL/min/mg. The validated method can be utilized for future pharmacokinetic and bio-distribution studies.

Oxidative nucleic acid modifications have attracted increasing attention in recent years since they have been found to be related to a number of diseases including cancer. 8-Hydroxy-2′-deoxyguanosine (8-OHdG) and 8-hydroxyguanosine (8-OHG) are the typical markers of oxidative modification of DNA and RNA, respectively, and they are emerging biomarkers for the early detection of diseases. Urine is a favored biofluid for biomarker discovery due to its noninvasiveness to patients. Accurate quantification of these oxidative nucleic acid modifications still has challenges because their amounts in urine are very low and the interferences in urine samples are complicated. Herein, we developed and validated an accurate and robust solid-phase extraction (SPE) coupled with ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous quantification of these oxidative nucleic acid modifications in human urine. Stable isotope dilution strategy was utilized and the method shows good precision on intraday and interday measurements. Meanwhile, recovery was satisfactory by utilizing the Oasis hydrophilic–lipophilic balance (HLB) cartridge for sample pretreatment at three spiked levels. We successfully quantified urinary 8-OHdG and 8-OHG from 60 gastric cancer patients and 70 healthy controls by using this method. The measured contents of 8-OHdG and 8-OHG in urine from gastric cancer patients are both increased, compared with those in urine from healthy controls, indicating these oxidative nucleic acid modifications could act as potential non-invasive markers for early diagnosis of gastric cancer. Moreover, the present study will stimulate investigations of the effects of oxidative stress and nucleic acid modifications on the initiation and progression of gastric cancer.

Aflatoxin M1(AFM1), a major metabolite of Aflatoxin B1(AFB1), has been identified as a potential contaminant in dairy products. Because of its possible carcinogenicity, the legislation limits as set by Commission Regulation (EC) No. 1881/2006 are very strict, namely 0.050 μg kg−1 in milk and 0.025 μg kg−1 in infant formulas. To meet these requirements, a sensitive and accurate method was developed, employing liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Ιmmunoaffinity columns (R-Biopharm) were used for sample purification and preconcentration of the analyte of interest. The quantification of AFM1 was conducted using fortified milk samples, while Aflatoxin B2 (AFB2) was used as an internal standard (IS). The method was validated in terms of linearity, precision, trueness, limits of detection and quantification and uncertainty. The performance criteria for the method were evaluated based on European Commission Regulation (EC) No. 401/2006 and its most recent amendment, as well as the suggested criteria for revision by the EU Reference Laboratory for Mycotoxins and Plant Toxins. The recovery was in the range of 77.9–81.0% for all fortification levels (0.025–0.050–0.075 μg kg−1), with RSDR values (Relative Standard Deviation of intermediate precision) ranging from 6.1% to 12%. The method’s detection and quantification limits were 0.0027 μg kg−1 and 0.0089 μg kg−1, respectively. The occurrence of AFM1 was investigated in 40 samples of different animal origin (cow, goat and sheep milk) provided by Greek producers.

The PI3K/AKT/MTOR signalling pathway plays an important role in the growth and proliferation of tumour cells. N-((3S,4S)-4-(3,4-Difluorophenyl)piperidin-3-yl)-2-fluoro-4-(1-methyl-1H-pyrazol-5-yl)benzamide (Hu7691) is a new-generation selective AKT inhibitor developed at Zhejiang University. In this study, we developed an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for the measurement of Hu7691 in dog plasma. Plasma was precipitated with acetonitrile and then separated on a trifunctionally bonded alkyl column. Excellent separation efficiency and selectivity were achieved by adjusting the mobile phase ratio, with a total running time of only 5 min. The linear dynamic range of the calibration curve was 5–1000 ng/mL. The method was fully validated, and all performance metrics met the criteria. The validated method was used for the pharmacokinetic monitoring and bioavailability assessment of Hu7691 in dogs. The results showed that the area under the curve and peak plasma concentration of Hu7691 increased with increasing dose (oral 5, 10, 20 mg/kg, intravenous 10 mg/kg), and oral bioavailabilities were 86.7%, 50.8%, and 50.5%, respectively, indicating a high bioavailability of Hu7691 in dogs. This provides a test basis for the clinical application of the compound.

A novel UPLC-MS/MS assay was developed for rapid quantification of delafloxacin (a novel fluoroquinolone antibiotic in plasma samples by one step sample cleanup procedure. Delafloxacin (DFX) and internal standard (losartan) were separated on a UPLC BEH C18 column (50 × 2.1 mm; 1.7 μm) by using gradient programing of a mobile phase containing 0.1% formic acid in acetonitrile and 0.1% formic acid in water. The quantification was performed by a using triple-quadrupole mass detector at an electrospray ionization interface in positive mode. The precursor to the product ion transition of 441.1 → 379.1 for the qualifier and 441.1 → 423.1 for the quantifier was used for DFX monitoring, whereas 423.1 → 207.1 was used for the internal standard. The validation was performed as per guidelines of bioanalytical method validation, and the evaluated parameters were within the acceptable range. The greenness assessment of the method was evaluated by using AGREE software covering all 12 principles of green analytical chemistry. The final score obtained was 0.78, suggesting excellent greenness of the method. Moreover, Deming regression analysis showed an excellent linear relationship between this method and our previously reported method, and it is suitable for high-throughput analysis for routine application. The proposed method was effectively applied in a pharmacokinetic study of novel formulation (self-nanoemulsifying drug delivery systems) of DFX in rats.

Due to the high consumption and incorrect disposal of pharmaceutical active compounds (PhACs), they are recognized as contaminants of emerging concern. Wastewater treatment plants (WWTPs) may be inefficient in removing PhACs, therefore discharging them into surface waters. The removal efficiencies of five WWTPs located in the south of Portugal (Alentejo) were evaluated in 2020. Twenty-six PhACs were analyzed in wastewater influents, effluents, and surface waters, upstream and downstream of the WWTPs by solid-phase extraction (SPE) and ultra-performance liquid chromatography coupled with tandem mass detection (UPLC-MS/MS). The most representative PhACs in influents were acetaminophen, caffeine, naproxen, ibuprofen, and diclofenac with minimum-maximum concentrations of 49–225 µg/L, 26–46 µg/L, 5.9–13 µg/L, 5.2–22 µg/L, and 1.3–2.5 µg/L, respectively. For effluents, it was acetaminophen, caffeine, and diclofenac with minimum-maximum concentrations of 0.054–7.8 µg/L, 0.084–4.8 µg/L, and 0.28–3.3 µg/L, respectively. The highest removal efficiencies were observed for acetaminophen, sulfadiazine, cortisone, testosterone, metoprolol, and propranolol (100%). The lowest removal efficiencies were observed for carbamazepine (2.7%) and diclofenac (−13.2%). The risk quotient of sulfamethoxazole and diclofenac were higher than 1 for receiving waters, indicating they probably pose high risks to aquatic organisms.

An ultra-performance liquid chromatography−tandem mass spectrometry (UPLC−MS/MS) method, in combination with a mixed matrix membrane microextraction method for the quantification of nonsteroidal anti-inflammatory drugs (NSAIDs) in environmental water samples, is reported. The extraction device was prepared by casting well-dispersed polymeric bonded octadecyl (C18) particles in a cellulose triacetate matrix solution onto commercially available 200 μL micropipette tips. The membrane formed contains 25% of the adsorbent loading amount and was firmly attached to the inner wall of the membrane tip. The dynamic extraction was performed by withdrawing and dispensing the sample solution through the tip device for effective analyte adsorption, followed by the analyte desorption process into 40 μL of methanol and acetonitrile (1:1) prior to UPLC−MS/MS analysis. NSAIDs—namely diclofenac, ibuprofen, indoprofen, naproxen and sulindac—were chosen as targeted analytes. Several extraction parameters were comprehensively optimized, including sample pH value, ionic strength, dynamic extraction cycle, desorption solvent and desorption time. The optimized conditions demonstrated a linear range from 0.25 to 500 ng L−1, with correlation coefficients (r2) from 0.9988 to 0.9992 and detection limits ranging from 0.08 to 0.40 ng L−1. The recoveries of the spiked water samples were between 92% and 99% and exhibited excellent precision relative to standard deviations (RSDs ≤ 4.9%), and enrichment factors (EFs) were at 201−249 for the developed approach.

A method was developed for the analysis of 31 pharmaceutical compounds in Lisbon's drinking water system, using solid-phase extraction (SPE) and ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). The method was validated through estimation of the linearity range, method detection and quantification limits, matrix effects, precision and accuracy. The method detection and quantification limit ranges were 0.009-10 and 0.03-33 ng/L, respectively. Analytes were quantified in water samples collected from the EPAL (Empresa Portuguesa das Águas Livres S.A.) supply system. Carbamazepine, atenolol, sulfadiazine, sulfamethazine, sulfapyridine, sulfamethoxazole, acetaminophen, caffeine and erythromycin were quantified in the analysed samples.