Acid In Acetonitrile Research Articles

Development and Validation of a Robust LC-MS/MS Method for Quantitation of a Novel Kinase Inhibitor, Ritlecitinib, in Rat Plasma: Application in Pharmacokinetic Study

Background Ritlecitinib (RIT) is a novel kinase inhibitor with promising therapeutic applications in the treatment of autoimmune disease alopecia areata. Accurate quantification of RIT in biological matrices is essential for pharmacokinetic studies and drug development. Objectives This study aims to develop and validate a sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantitation of RIT in rat plasma and to apply this method in a pharmacokinetic study. Materials and Methods A sensitive and selective LC-MS/MS method was developed in a AB Sciex QTRAP 5500 coupled with Shimadzu Prominence LC-20AD UFLC (ultra-fast liquid chromatography) System using an C18 column (Xselect HSS T3 2.5 µm, 2.1x150 mm) with a mobile phase consisting of 0.1% formic acid in high-performance liquid chromatography (HPLC) grade water and 0.1% formic acid in acetonitrile (70:30, v:v). RIT ( m/z 286.1 ↓ 105.7) and [ 13 C4]-ritlecitinib (internal standard, IS) ( m/z 290.1 ↓ 109.7) were detected in a positive mode by electrospray ionization (ESI) using multiple reaction monitoring (MRM) and extracted from rat plasma samples through protein precipitation We validated the method according to United States Food and Drug Administration (U.S. FDA) guidelines, assessing parameters such as linearity, accuracy, precision, selectivity, sensitivity, recovery and stability. The validated method was then applied to a pharmacokinetic study in rats following the administration of RIT. Results On rat plasma, the LC-MS/MS method has shown exceptional linearity across the concentration range of 5–100 ng/mL. The FDA’s approved standards of ±15% matched the accuracy and precision measurements. The matrix effect was negligible, and the mean recovery was about 93%, indicating that RIT was fully extracted from plasma. Different experimental conditions found RIT to be stable. RIT is quickly absorbed, with a peak plasma concentration (Cmax) of 43.82 ng/mL occurring 0.5–0.75 hours after the dose. It then drops in concentration in a way that is biexponential, with a half-life at the end of 1.5 hours. Conclusion A highly sensitive and specific LC-MS/MS method for quantitation of RIT in rat plasma was successfully developed and validated. The method was effectively applied to a pharmacokinetic study, providing critical data on the absorption and elimination characteristics of RIT in rats. This established approach may support additional preclinical pharmacokinetic investigations of RIT.

Determination of eight phthalate metabolites in urine of pregnant women and evaluation of neonatal birth outcomes based on solid-phase extraction-high performance liquid chromatography-tandem mass spectrometry

Phthalates (PAEs) are endocrine-disrupting chemicals that are widely present in everyday life and enter the human body through various pathways. The release of PAEs into the environment through pathways that include leaching, evaporation, abrasion, and the use of personal care products exposes humans to PAEs via ingestion, inhalation, and dermal absorption. Pregnant women, as a particularly vulnerable population, risk adverse newborn growth and development when exposed to PAEs. While the concentrations of PAEs in urine reflect recent exposure levels in humans, urinary levels of phthalate metabolites (mPAEs) are commonly used as biomarkers of internal exposure owing to the relatively short biological half-lives of PAEs (<24 h). In this study, we developed a solid-phase extraction-high performance liquid chromatography-tandem mass spectrometry (SPE-HPLC-MS/MS) method for simultaneously detecting eight mPAEs in the urine of pregnant women. Urine samples were enzymatically hydrolyzed with β-glucosidase and then purified using the Bond Elut Plexa SPE column, with subsequent elution, concentration, and redissolved performed prior to HPLC-MS/MS. Separation was achieved using an Agilent Eclipse Plus C18 column (100 mm×3 mm, 3.5 μm), with gradient elution performed using 0.1% acetic acid aqueous solution and 0.1% acetic acid acetonitrile as mobile phases. Multiple reaction monitoring (MRM) mode was used for detection, with quantification performed using the internal-standard method. Good linearities were obtained in the range of 0.1-200 ng/mL for the eight mPAEs, with limits of detection (LODs) and quantification (LOQs) of 0.015-0.048 and 0.050-0.160 ng/mL, respectively. The eight mPAEs exhibited recoveries of 80.2%-99.7% at three spiked levels (1, 10, and 50 ng/mL). This method was subsequently used to analyze the eight mPAEs levels in urine samples of 497 pregnant women from the Ezhou Maternity and Child Health Care Hospital. The participants exhibited widespread exposure to PAEs, with monobutyl phthalate (MBP) showing the highest median level of 104.46 ng/mL, and monobenzyl phthalate (MBzP) showing the lowest (0.22 ng/mL). In addition, this study assessed neonatal birth outcomes. Linear regression modeling revealed that gestational age decreased by 0.11 weeks (95% confidence interval (CI): -0.18--0.03) for every natural-log (ln) increase in the level of monoethyl phthalate (MEP) in urine samples of pregnant woman. Moreover, the birth weight decreased by 39.28 g (95% CI: -76.48--2.09) and 39.62 g (95% CI: -73.73--5.52), for every ln increase in mono(2-ethyl-5-oxohexyl) phthalate (MEOHP) and mono(2-ethylhexyl) phthalate (MECPP) levels, respectively. The developed method is characterized by its simplicity, low LODs, high accuracy, and precision. This study provides clear evidence that PAE exposure during pregnancy negatively affects newborn growth and development by measuring the levels of eight mPAEs in the urine of pregnant women and linking these findings to neonatal outcomes. Further large-scale cohort studies are needed to validate these findings, along with mechanistic studies using animal models or in-vitro systems that elucidate the biological pathways through which mPAEs contribute to adverse birth outcomes.

Construction of a two-dimensional magnetic solid phase extraction microfluidic chip/tandem mass spectrometry platform for the determination of cytarabine in plasma

A novel concept of two-dimensional magnetic solid phase extraction microfluidic chip (2D-MSPE-chip) was proposed, in which two or even more different separation mechanisms of solid phase extraction units were designed and constructed on microfluidic chips in series. The synthesized Fe3O4-UiO-66-C18 was used in the first dimension to retain protein and phospholipid through hydrophobic effect and ZrOP coordination, which could almost eliminate the interference of the matrix effect on the mass spectrum. The second dimensional material utilized Fe3O4-UiO-66-NH2 to enable cytarabine enrichment via π-π and hydrogen bonding. The parameters of 2D MSPE-Chip/tandem MS method such as desorption solvent (5 % acetic acid acetonitrile), loading solvent (20 % acetonitrile), and flow rate for extraction and desorption (10 μL/min) were select. The LOD of the established method for cytarabine in plasma was 0.13 ng/mL, and the linear range was between 2.0 ng/mL and 1000 ng/mL with correlation coefficient (R2 = 0.9991). The matrix effect was −5.0 %. The recovery rate of real samples ranged from 95.5 % to 104.8 %, with RSD ≤ 5.2 %. The chip design was simple and efficient and could be reused, thereby decreasing analysis expenses. It held considerable potential for application in rapid clinical diagnostics and precision medicine.

Development of UHPLC‐MS/MS Method for Evaluation of the Acute Oral Toxicity and Pharmacokinetics of Ursodeoxycholic Acid and Silymarin Combination in Rats

ABSTRACTThe combination of ursodeoxycholic acid (UDCA) and silymarin has been shown to alleviate various hepatic conditions. This study aimed to evaluate the acute oral toxicity and in vivo pharmacokinetics of the combination in female Sprague Dawley rats. The acute oral toxicity and the pharmacokinetic studies were conducted using 17 rats divided into five groups and 30 rats assigned into four control groups and one treatment group, respectively. A new UHPLC/MS‐MS method was applied to determine silibinin (SIL) and UDCA in plasma, urine, and feces. This method used the BEH C8 column (1.7 µm, 100 × 2.1 mm) with a gradient elution of 0.01% formic acid in water and 0.01% formic acid in acetonitrile. The acute oral toxicity study revealed that administering the combination at 5000 mg/kg preserves a wide safety margin. In the pharmacokinetic study, the combination resulted in a significant enhancement of the absorption of both drugs and a reduction of the renal elimination of silymarin. The oral LD50 of the combination was higher than 5000 mg/kg, so according to the OECD guidelines, it was classified in Category 5 GHS. The presented data serve as scientifically defensible safety data with a defined absorption and elimination profile for the combination.

A Validated Liquid Chromatography-Tandem Mass Spectrometry Method for the Determination of Methylophiopogonanone A in Rat Plasma and Its Application to Pharmacokinetic Study.

Methylophiopogonanone A (MOA) is one of the homoisoflavonoids isolated from Ophiopogon japonicus, which has been demonstrated to have extensive pharmacological activities. The aim of this study was to develop and validate a liquid chromatography-tandem mass spectrometry method for the measurement of MOA in rat plasma. Methylophiopogonanone B (MOB) was used as internal standard. After precipitation with acetonitrile, the samples were separated using a Waters ACQUITY HSS T3 column with 0.1% formic acid solution and acetonitrile as mobile phase. Mass detection was monitored using multiple reactions monitoring mode with precursor-to-product ion transition of m/z 343.2 > 135.1 for MOA and m/z 329.2 > 121.1 for internal standard. The assay showed good linearity over the concentration range of 1-1000 ng/mL, with correlation coefficient > 0.997. The lower limit of quantification (LLOQ) was 1 ng/mL. Acetonitrile-mediated precipitation showed high extraction efficiency (> 80%). The accuracy and precision were within the acceptable limits. MOA was demonstrated to be stable in rat plasma under the tested storage conditions. After validation, the proposed method was applied to the pharmacokinetic study of MOA in rat plasma, and the data revealed that MOA showed rapid absorption and elimination from rat plasma. The oral bioavailability of MOA in rat was 24.5%.

Development and validation of HPLC-UV and LC-MS/MS methods for the quantitative determination of a novel aminothiazole in preclinical samples

Aminothiazoles are the important class of chemical groups which have proven their broad range of biological activities. A novel aminothiazole (21MAT) was quantified in analytical solutions using a high-performance liquid chromatography (HPLC) approach that was developed and partially validated for the analysis of in vitro experimental samples. An isocratic elution on reverse phase Phenomenex® Luna C18 (50 mm × 4.6 mm, 5 μm) column with 55% 0.1% v/v orthophosphoric acid in water and 45% of orthophosphoric acid in acetonitrile at a flow rate of 1 mL/min was used. The analyte was detected at 272 nm. Similar to this, a robust bioanalytical technique, LC-mass spectrometry (LC-MS/MS) was created and verified to measure 21MAT in rat plasma for use in in vitro screening study samples and early-stage pharmacokinetic research. The protein precipitation method was used to extract 21MAT from plasma. The mixture of 95: 5% v/v methanol: acetonitrile and 0.1% v/v formic acid, along with 15% of 5 mM ammonium formate solution, was used to separate the mixture on a reverse phase Waters Xterra RP® C18 (150 mm × 4.6 mm, 5 μm) column at a flow rate of 1 mL/min. Using electro spray ionisation mode in multiple reaction monitoring mode, the analyte and internal standard (a structural analogue) were both identified. According to current criteria, all validation parameters (specificity, selectivity, accuracy, precision, recovery, matrix factor, hemolysis effect, and stability) were evaluated in rat plasma. The area response of 21MAT was found to be linear over the concentration range of 1.25–1250 ng/mL in rat plasma. Both techniques are suitable for use in any format of preclinical research and were sufficiently reliable to measure 21MAT precisely in various matrices. In silico prediction helped in understanding absorption, distribution, metabolism, excretion, and toxicity (ADMET) behaviour of the molecule. Both developed LC-MS/MS and HPLC-UV methods were successfully used to quantify the analyte in in vitro screening study samples.

A novel, sensitive, and fast ultra-high-performance liquid chromatography tandem mass spectrometry method for TNG908 determination in dog plasma and pharmacokinetic study.

TNG908 is a potent and selective protein arginase methyltransferase 5 (PRMT5) inhibitor that is currently going through phase I/II clinical development for the treatment of non-small cell lung cancer. To facilitate pharmacokinetic and toxicokinetic studies of TNG908, here, we reported an ultra-high-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the detection of TNG908 in dogs. The dog plasma samples were precipitated by acetonitrile and analyzed using a Waters ACQUITY BEH C18 column combined with a Thermo triple quadrupole mass spectrometer. The mobile phase consisted of 0.1% formic acid solution and acetonitrile, at a flow rate of 0.3mL/min. TNG908 and internal standard were monitored by selective reaction monitoring (SRM) with m/z 410.2 > 150.1 and m/z 394.2 > 278.1, respectively. The method demonstrated excellent linearity over the concentration range of 1-1000 ng/mL, with a correlation coefficient greater than 0.995. Acetonitrile-mediated protein precipitation showed high extraction efficiency and a recovery above 80%. The validated assay was further applied to measure TNG908 in dog plasma after oral and intravenous administration and achieved success. The obtained pharmacokinetic parameters indicated low clearance of TNG908 (3.7 ± 0.8mL/min/kg) and moderate oral bioavailability (>36.4%).

Study on the Mechanism of Raspberry (Rubi fructus)in Treating Type 2 Diabetes Based on UPLC-Q-Exactive Orbitrap MS, Network Pharmacology, and Experimental Validation.

The aim of this study is to analyze the chemical composition of raspberry using liquid chromatography-mass spectrometry (LC-MS) technology, predict the potential effects of raspberry in treating type 2 diabetes through network pharmacology, and conduct preliminary validation through invitro experiments. A Waters CORTECS C18 column (3.0 mm × 100 mm, 2.7 μm) was used; mobile phase A consisted of 0.1% formic acid in water and mobile phase B consisted of 0.1% formic acid in acetonitrile. Gradient elution was performed with full-scan mode in both positive and negative ion modes, covering a mass range of m/z 100-1500. The chemical components of raspberry were analyzed and identified based on secondary spectra from databases and relevant literature. The disease targets related to type 2 diabetes were searched, and protein-protein interaction network analysis as well as gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were conducted on the intersecting targets of the active components of raspberry and the disease. HepG2 cells were used for experimental validation, with high glucose-induced insulin resistance models established. The CCK-8 method was employed to assess the effects of raspberry on cell proliferation, while Western blotting was used to measure the expression of proteins related to the AGE/RAGE signaling pathway. A total of 47 components were identified, including 10 organic acids, 15 flavonoids, 12 phenols, 2 alkaloids, 4 terpenoids, 1 miscellaneous compound, 1 stilbene, 1 steroid and its derivatives, and 1 diterpenoid. Through database screening, seven active components were identified: kaempferol, epicatechin, ellagic acid, crocetin, stigmasterol, fisetin, and isorhamnetin. KEGG and GO results indicated that the therapeutic effects of raspberry on type 2 diabetes may be related to the advanced glycation end product (AGE)- receptor for advanced glycation end product (RAGE) signaling pathway. Establishment of an insulin resistance model in HepG2 cells demonstrated that, compared to the control group, the raspberry treatment group upregulated p53 protein expression while downregulating the expression of RAGE, Akt1, and Caspase-3 proteins. This study preliminarily elucidates that the therapeutic effects of raspberry in treating type 2 diabetes may be mediated through the inhibition of the AGE-RAGE signaling pathway, providing important references for the study of the pharmacological basis and clinical application of raspberry.

Determination of 42 antimicrobials in disinfection products by dispersive solid phase extraction-high performance liquid chromatography-tandem mass spectrometry

Antimicrobials inhibit the growth and reproduction of microorganisms thereby alleviating skin and other problems caused by microorganisms. Antimicrobials are classified into different categories, including antibacterials, antifungals, and antivirals, among others, and include azoles, sulfonamides, tetracyclines, quinolones, and many other classes of synthetic and natural compound. The inappropriate or excessive use of antimicrobials can damage skin and other human organs and increase antimicrobial resistance. Relevant regulations and standards clearly state that antimicrobials are prohibited for use as ingredients in disinfection products. However, since antimicrobials enhance the disinfection or antibacterial effect of a product, with a significant short-term effect, antimicrobials are occasionally illegally added to disinfectant products, including those intended for human use. Therefore, establishing testing methods that provide technical support for enforcing regulations is an urgent objective. Herein, a method was established for the analysis of 42 antimicrobials in disinfection products, that is applicable to common types of disinfection-product matrix, including creams, gels, and aqueous solutions, using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) combined with dispersive solid phase extraction. The 42 antimicrobials comprise antibacterials, antifungals, and antivirals, and include seven sulfonamides, ten quinolones, three lincosamides, five tetracyclines, three macrolides, eight azoles, three purine nucleoside analogs, one furan, one nonpolyene antifungal, and one steroid. Briefly, 0.2 g of a sample was first dispersed in 2 mL of water and then extracted with 10 mL of 0.5% formic acid in acetonitrile, with 3 g of anhydrous Na2SO4 added to remove water. After centrifugation, 5 mL of the supernatant was cleaned using dispersive solid phase extraction with EMR-Lipid as the adsorbent. Lipids, waxes, surfactants, and moisturizing lubricants are commonly used as cream and gel matrices. Matrix substances containing long carbon chains dissolved in acetonitrile were removed using the EMR-Lipid adsorbent. Target analytes were separated on a Poroshell 120 EC-C18 analytical chromatography column (150 mm×3.0 mm, 2.7 μm), with 0.1% formic acid in acetonitrile and 0.1% formic acid aqueous solution used as mobile phases under gradient-elution conditions. The target analytes in the test solution were detected in positive ionization (ESI+) and multi-reaction-monitoring (MRM) modes. Analytes were characterized in terms of their retention times and selected ions, and quantified using the external-standard method. The main factors affecting method response, recovery, and sensitivity, such as the extraction method and solvent, purification method and adsorbent, mobile phase, and MS conditions, were examined during sample pretreatment and instrumental analysis. The 42 antimicrobials were effectively separated under the optimized experimental conditions; the target compounds exhibited linear working curves in the 0.25-5.0 mg/kg concentration range, with correlation coefficients (r) greater than 0.99. Limits of detection (LODs) for the 42 antimicrobials were determined from the signal-to-noise ratios (S/N) of their chromatographic peaks. LODs of 0.03-0.10 mg/kg were determined for the three matrices using 0.2-g samples and 10-mL test solution. Recoveries of 80.3-109.8%, with relative standard deviations (RSDs) of less than 9.8%, were obtained by determining three levels of each target analyte added to the three blank matrices; this process was repeated for six parallel samples. The developed method was used to analyze antimicrobials in commercially available disinfection products, with two sample batches testing positive. The established method is simple, accurate, precise, and suitable for the rapid screening and quantification of antimicrobials in disinfection products. This study provides powerful technical support for regulating the illegal addition of related antimicrobials to disinfection products.

Simultaneous determination of 17 perfluoroalkyl substances in beef by EMR-Lipid dispersive solid-phase extraction and ultra performance liquid chromatography-tandem mass spectrometry

The ability to accurately analyze perfluoroalkyl substance (PFAS) levels in beef is imperative in order to effectively assess food-safety risks and ensure consumer safety because PFASs are harmful and prevalent in beef. In this study, we developed a rapid and accurate method for the simultaneously determination of the 17 PFASs in beef using dispersive solid-phase extraction (d-SPE) and ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), and optimized the mobile phase system, extraction solvent, and d-SPE materials. Samples were finally extracted using 0.1% (v/v) formic acid in acetonitrile, cleaned using d-SPE with PSA, C18, GCB, and EMR-Lipid, separated using an Acquity Premier BEH C18 column (100 mm×2.1 mm, 1.7 μm) with 0.5 mmol/L ammonium fluoride aqueous solution and methanol as the mobile phases at a flow rate of 0.3 mL/min. Analytes were detected in negative ion switching mode (ESI-) with multiple reaction monitoring (MRM) scanning, and quantitatively analyzed using the internal standard method. The 17 PFASs exhibited linearity in the 0.2-20.0 μg/L range under the optimal experimental conditions, with correlation coefficients of 0.9915-0.9999. The method delivered limits of detection (LODs) of 0.003-0.007 μg/kg and limits of quantification (LOQs) of 0.01-0.02 μg/kg. The 17 PFASs exhibited recoveries of 71.1%-127.4% with RSDs of 0.6%-14.4% when spiked at three levels (0.05, 0.5, and 1.8 μg/kg). We optimized the mobile phase system, which revealed that, compared with 2.0, 5.0, and 10.0 mmol/L ammonium formate or ammonium acetate in aqueous methanol, 0.5 mmol/L ammonium fluoride in aqueous methanol exhibited higher sensitivities for all the 17 PFASs, with PFASs bearing long-chain carboxylic acids (C10-C18) showing 1-2 fold increases in sensitivity. PFASs do not dissociate in acidic environments, favoring their entry into the organic phase. Therefore, we investigated the effect of extractant acidity, which revealed that the 17 PFASs were better extracted using 0.1% (v/v) formic acid in acetonitrile. The beef matrix has a complex composition; consequently, d-SPE adsorbents were required to purify samples and reduce matrix effects. The purification effects of four adsorbents (PSA, C18, GCB, and EMR-Lipid) toward the 17 PFASs and the amount of EMR-Lipid used were investigated, which revealed that 100 mg PSA+80 mg C18+40 mg GCB+150 mg EMR-Lipid exhibited superior matrix-purification behavior. We also investigated the effects of various injection solutions and types of syringe filter, with pure methanol selected for reconstitution and high-speed supernatant centrifugation applied prior to injection. The developed method is simple, rapid, sensitive, and reproducible, and can be used to simultaneously, rapidly, and accurately determine various perfluoroalkyl compounds in beef.

A simple, robust and high-throughput LC-MS/MS method for the therapeutic drug monitoring of polymyxin B1, polymyxin B2, polymyxin B3, isoleucine-polymyxin B1, polymyxin E1 and polymyxin E2 in human plasma.

To facilitate clinical therapeutic drug monitoring (TDM) of polymyxin B (PB) and polymyxin E (PE), we developed and validated a simple LC-MS/MS method for simultaneous determination of PB (including polymyxin B1 (PB1), polymyxin B2 (PB2), polymyxin B3 (PB3) and isoleucine-polymyxin B1 (ile-PB1)) and PE (including polymyxin E1 (PE1) and polymyxin E2 (PE2)) in human plasma. PB or PE was extracted from 20.0μL plasma using a 5% (v/v) formic acid acetonitrile solution and separated on a BEH-C18 column (2.1 × 100 mm, 1.7μm) with a mobile phase consisting of 0.8% formic acid aqueous solution and 0.2% formic acid acetonitrile solution. Gradient elution was performed over 5.5min at a flow rate of 0.250 mL/min. Quantitative analysis was conducted in positive ion scanning mode by electrospray ionization and multiple reaction monitoring. The method validation was conducted based on bioanalytical method validation guidance, including specificity, calibration curve, precision, accuracy, recovery, matrix effect, stability and dilution integrity and all of the results satisfied the requirements. The method was simple, robust and high-throughput and is currently being used to provide a TDM service to enhancing therapeutic efficacy and safety use of the PB and PE.

A quality by design paradigm, novel, and fast LC-MS/MS method to quantify vortioxetine in rat brain homogenate and its assessment of greenness, whiteness, and blueness tools

Vortioxetine (VOR) is an antidepressant drug with multimodal mechanism of action. In this work a sensitive and simple bioanalytical LC-MS/MS method for analysis of VOR in spiked rat brain tissue was developed and validated. Chromatographic analyses were performed in isocratic mode, and Citalopram (CT) was found as a suitable internal standard. Supelco Ascentis® Express Phenyl-Hexyl column was used as stationary phase, and the mobile phase was 0.15 % formic acid in acetonitrile:water (55:45, v/v). A Box-Behnken Design − based method optimization was applied to explore the impact of analytical conditions on chromatographic separation of VOR and CT. Detection was realized via a triple quadrupole LC-MS/MS system; the mass transition ion-pair has been followed as m/z 299.05 → 149.90 for VOR, m/z 325.20 → 109.05 for CT. Analytical method validation was performed according to International Council for Harmonisation (ICH) guideline M10 on bioanalytical method validation. The calibration curve was within the range of 30 to 450 ng/mL. LOD and LLOQ were calculated at 10 and 30 ng/mL, respectively. Recovery was 90 % for the lower limit of quantification and between 98 % to 105 % for the other concentration levels. Total run time was 5 min, retention times of citalopram and vortioxetine were 2.4 and 3.1 min, respectively, and a good chromatographic separation was obtained under optimal conditions. The solvents used to make the method greener were evaluated with the Green Solvent Selection Tool and Green Index Spider chart. The AGREE score for the greenness of the developed method was calculated as 0.61 and was also evaluated with the ComplexGAPI pictogram. The BAGI score for blueness was calculated as 72.5, and the RGBfast score for whiteness was calculated as 50. The method was determined to be most green, white, and blue.

A novel quantitative method for the determination of 10B-carrier boronophenylalanine in rat plasma by UHPLC-MS/MS and comparison with ICP-MS

L-Boronophenylalanine (BPA), a widely used 10B carrier for clinical boron neutron capture therapy (BNCT), was quantified in rat plasma through a simple, effective and stable ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method. Chromatographic separation was performed on an ACQUITY UPLC HSS T3 (100 mm × 2.1 mm, 1.8 μm) column with the mobile phase of 0.5 % formic acid aqueous solution and acetonitrile. For the detector, the m/z ion pairs used for quantification were 209.1→120.1 for BPA and 210.1→120.1 for internal standard in a positive mode by electrospray ionization (ESI) using multiple reaction monitoring (MRM). The method is specific and robust with rare affection by endogenous substances in the matrix. A good linear relationship was observed over 80–80000 ng/mL (r2 = 0.9993). The values of inter- and intra-day accuracy and precision were within the acceptance criteria of ±15 %. BPA was found to be stable under different experimental conditions. This developed method was successfully applied on a pharmacokinetic experiment on Sprague-Dawley rats (intravenous injection, 125 mg/kg) and a comparation between UHPLC-MS/MS and ICP-MS for BPA was carried out.

Simultaneous analysis of certain tree-nut allergens in cookies by LC-MS/MS

In this study, four tree-nut allergens were identified using liquid chromatography-mass spectrometry (LC-MS/MS) with a reverse-phase XSelect HSS C18 SB column (100 mm x 2.1 mm, 3.5 &amp;micro;m) and corresponding guard column. The gradient solvent program employed 0.1% formic acid in water and 0.1% formic acid in acetonitrile, with a flow rate of 0.5 mL/min, a positive ion electrospray ionization (ESI+) source, and a multiple reaction monitoring (MRM) mode. The method was validated following the guidelines of AOAC International. Results showed that the method had good specificity, with calibration curves constructed in the concentration ranges of 30.5 - 1220 &amp;micro;g/g for almonds, 102 - 4080 &amp;micro;g/g for cashews, 128 - 5120 &amp;micro;g/g for hazelnuts, and 122 - 4880 &amp;micro;g/g for pistachios. Limits of detection were 9 &amp;micro;g/g for almonds, 30.6 &amp;micro;g/g for cashews, 38.4 &amp;micro;g/g for hazelnuts, and 36.6 &amp;micro;g/g for pistachios, while limits of quantification were 30.5 &amp;micro;g/g for almonds, 102 &amp;micro;g/g for cashews, 128 &amp;micro;g/g for hazelnuts, and 122 &amp;micro;g/g for pistachios. The repeatability and accuracy of the method met AOAC requirements. The method was used to analyze the content of four groups of nut allergens (almond, cashew, hazelnut, and pistachio) in ten cookie samples from the Hanoi area. Results indicated that three samples contained undeclared allergens on the food labels.

Determination of QLNC-3A6 in canine plasma by UHPLC-MS/MS and its application in pharmacokinetic studies

Multi-targeted tyrosine kinase inhibitor QLNC-3A6 Di-maleate, a structurally novel small molecule compound, has therapeutic efficacy for the treatment of canine cutaneous mast cell tumor (CMCT) caused by mutations in the c-Kit gene. Since pharmacokinetic (PK) information plays an important role in the development and application of new drugs, etc., a rapid, highly sensitive and selective UHPLC-MS/MS analytical method was developed and validated for the first time in this study for the quantitative detection of QLNC-3A6 in canine plasma. 100 µL of plasma was precipitated using 350 µL of acetonitrile, and Chromatographic separation was performed on a Phenomenex Kinetex C18 column (50 × 2.1 mm, 2.6 µm) at a flow rate of 0.4 mL/min, the mobile phases were set to 0.1% formic acid aqueous solution (A) and 0.1% formic acid acetonitrile (B). The calibration curve linear range was 0.5–100 ng/mL (R 2>0.99). The intraday and interday precision values (relative standard deviation, RSD) were 2.06–13.57% and 6.90–9.14%. Intraday and interday accuracies were −10.73 to 9.54% and −3.86 to 0.70% respectively. The dilution integrity RSD value and stability RSD value were less than 3.77 and 7.45%, respectively. Subsequently, the pharmacokinetics were investigated in canine after oral administration of QLNC-3A6 Di-maleate tablets at a dose of 3 mg/kg BW using this method. The results showed that QLNC-3A6 showed fast absorption rate, rapid distribution and slow metabolic elimination in canine plasma. The results of the main PK parameters including λz, T 1/2λz, C max, T max and AUC last were 0.07 ± 0.01/h, 11.00 ± 2.57 h, 50.88 ± 31.94 ng/mL, 9.08 ± 11.57 h and 836.48 ± 230.53 ng h/mL, respectively.

Evaluating quackery formulations: LC-MS/MS based method for detecting glucocorticoid content

Glucocorticoids are widely used as highly effective drugs for treating inflammatory diseases. In this study, a method was developed and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to simultaneously determine four glucocorticoids, including betamethasone, dexamethasone, hydrocortisone, and prednisolone in unauthorized or unregulated medicinal powders often associated with quackery formulations. Commercially available standards were used for method development and glucocorticoid detection. Glucocorticoids were extracted from the samples with methanol, which were then chromatographically separated using two mobile phases (0.1 % formic acid in water and 0.1 % formic acid in acetonitrile) in an isocratic flow on an Agilent Poroshel 120 C18 column (2.1 mm x 75 mm x 2.7 m). The validated analytical measuring range (AMR) of betamethasone and dexamethasone was 7.8–500 ng/mL, whereas, for hydrocortisone and prednisolone, AMR was 7.8–1000 ng/mL. The method showed an excellent coefficient of determination (r2) >0.990 for betamethasone, hydrocortisone, and prednisolone, while for dexamethasone 0.986. Accuracy and precision (intra/inter days) of these glucocorticoids showed a bias of 6–15 % (<20 %) and a coefficient of variation (CV) of <15 %. For each dilution factor, the integrity of samples was maintained after dilution. The developed method is sensitive and valuable for detecting, quantifying, and confirming the selected glucocorticoids in various quackery formulation powders commonly used in Pakistani setups.

Quantitation of the DNA-dependent protein kinase inhibitor peposertib (M3814) and metabolite in human plasma by LC-MS/MS.

The DNA-dependent protein kinase (DNA-PK) is an abundant nuclear protein that mediates DNA double-strand break repair by nonhomologous end joining (NHEJ). As such, DNA-PK is critical for V(D)J recombination in lymphocytes and for survival in cells exposed to ionizing radiation and clastogens. Peposertib (M3814) is a small molecule DNA-PK inhibitor currently in preclinical and clinical development for cancer treatment. We have developed a high-performance liquid chromatography-mass spectrometry method for quantitating peposertib and its metabolite in 0.1 mL human plasma. After MTBE liquid-liquid extraction, chromatographic separation was achieved with a Phenomenex Synergi polar reverse phase (4μm, 2× 50 mm) column and a gradient of 0.1% formic acid in acetonitrile and water over an 8min run time. Mass spectrometric detection was performed on an ABI SCIEX 4000 with electrospray, positive-mode ionization. The assay was linear from 10 to 3000 ng/mL for peposertib and 1-300 ng/mL for the metabolite and proved to be both accurate (97.3%-103.7%) and precise (<8.9%CV) fulfilling criteria from the Food and Drug Administration (FDA) guidance on bioanalytical method validation. This liquid chromatography-tandem mass spectroscopy (LC-MS/MS) assay will support several ongoing clinical studies by defining peposertib pharmacokinetics.

B-147 Enhancing the Detection of Vitamin D Metabolites by LC-MS/MS Using a Novel Derivatization Kit

Abstract Background Vitamin D is crucial for various biological functions and exists in various tissues, including bones, intestines, and kidneys. 25-hydroxyvitamin D serves as a key marker for assessing vitamin D status in the body. Currently, LC-MS/MS is the gold standard for measuring vitamin D levels. The JeoQuant Kit for LC/MS Analysis of Vitamin D Metabolites (JEOL, Tokyo, Japan), which is used for the determination of vitamin D metabolites in serum based on the DAP-PA (Seki et al, 2019), enhances the ionization efficiency in the LC-MS/MS system for four vitamin D metabolites (25(OH)D3, 25(OH)D2, 24,25(OH)2D3 and 3-epi-25(OH)D3), enabling accurate fractionation and quantification. DAP-PA was developed as a caged Cookson-type reagent, demonstrating significantly enhanced stability compared to previous non-caged Cookson-type reagents. Our research aimed to assess whether the kit works practically. Methods The JeoQuant Kit contains internal standards of stable isotope-labelled compounds at appropriate concentrations, derivatization reagents, quality control materials derived from human serum and calibration materials. An Acquity H-Class UPLC coupled with an Acquity TQD mass spectrometer (Waters, MA, US) was used for analysis. CAPCELL CORE C18 2.1mm*100mm (Osaka-soda, Tokyo, Japan) was used as the analytical column. For the pretreatment, an Isolute SLE+ column (Biotage, Tokyo, Japan) was used. Eluent of LC-MS/MS was used 0.1% formic acid aqueous solution and 0.1% formic acid acetonitrile, with binary gradient. Results Using attached control L/H, reproducibility was shown by intra- and inter-assay CVs which were below 10% and 15%, respectively. Storage tests of derivatization agents at -20°C, 4°C, and room temperature for one week showed no significant difference on peak areas (one-way ANOVA). Through this derivatization, it was possible to achieve more than a 20-fold increase in sensitivity compared to measurements without derivatization. Notably, low concentrations of 3-epi-25(OH)D3 were distinctly separated and detected. Conclusions This study showed that the JeoQuant Kit effectively increases the sensitivity of vitamin D metabolites through derivatization. It also emphasized the reagent's notable stability. Furthermore, it enabled the detection of 3-epi-25(OH)D3 with less sensitive LC-MS/MS models, offering clear separation from 25(OH)D3. The kit, which is complete with calibrants, internal standards, and quality control samples, simplifies analytical procedures for vitamin D metabolite quantification, potentially aiding in developing Vitamin D- related biomarkers. Efforts to compare analytical performance of this kit with that of other methods are underway to establish its benefits.

Accurate determination of three new antihypertensive drugs illegally added to food using ultra-high performance liquid chromatography-tandem mass spectrometry

Effective strategies are required to address food safety issues related to the illegal addition of antihypertensive drugs to food and claims of antihypertensive function. In this study, a novel ultra-high performance liquid chromatography-triple-quadrupole mass spectrometry (UHPLC-MS/MS) method was developed for the simultaneous determination of three antihypertensive drugs (azilsartan, candesartan cilexetil, and lacidipine) in 12 food matrices (pressed candies, solid beverages, alternative teas, tea drinks, biscuits, jellies, mixed liquors, oral liquids, medicinal teas, tablets, hard capsules, and soft capsules). Initially, mass spectrometry parameters, such as the collision energies of the three antihypertensive drugs, were optimized. Subsequently, the response intensities and chromatographic separation conditions of the three drugs in different mobile phases were compared. In addition, to enhance the recoveries, various extraction solvents and purification methods, including solid-phase extraction (SPE) columns and the QuEChERS technique, were investigated. In the developed method, sample determination involved three steps. First, the sample was extracted using 0.2% (v/v) formic acid in acetonitrile and then filtered using high-speed centrifugation, in addition, the extracted solution of alternative tea and medicinal tea was purified using the QuEChERS technique. Second, the supernatant was diluted with water, and filtered through a 0.22 μm polytetrafluoroethylene (PTFE) membrane. Finally, the analytes were separated on an Agilent Eclipse Plus RRHD C18 column (50 mm×2.1 mm, 1.8 μm) using a 5 mmol/L ammonium formate aqueous solution and acetonitrile as the mobile phases under gradient elution conditions and then detected using UHPLC-MS/MS with positive electrospray ionization (ESI) in the multiple reaction monitoring (MRM) mode. Quantitative analysis was performed using a matrix-matched external standard method. Methodological validation showed good linear relationships for all three antihypertensive drugs in the investigated concentration ranges, with correlation coefficients (r2) greater than 0.996. The limit of detection (LOD) and limit of quantification (LOQ) of lacidipine were 0.02 mg/kg and 0.04 mg/kg, respectively, whereas those of the other two drugs were 0.01 mg/kg and 0.02 mg/kg, respectively. In the 12 food matrices, the average recoveries of the drugs ranged from 86.6% to 107.5% with relative standard deviations (RSDs) of 1.1%-10.9% (n=6) at low, medium, and high spiked levels. Furthermore, this method was successfully applied to the analysis of real food samples. Overall, the newly developed method is simple, rapid, sensitive, accurate, and suitable for the qualitative and quantitative determination of antihypertensive drugs in different food matrices. This work could provide technical support for food safety agencies in implementing measures against the illegal addition of antihypertensive drugs to food.

Development and Validation of an HPLC Method for Niraparib Analysis: A Comprehensive Approach.

Background: Niraparib is a significant PARP inhibitor utilized in cancer therapy. This study aims to develop and validate a high-performance liquid chromatography (HPLC) method for the quantification of Niraparib in pharmaceutical formulations and biological samples. Results: A Phenomenex Kinetex XB-C8 column (150 x 4.6 mm, 5 µ) was employed with a mobile phase of 0.1% formic acid in acetonitrile (60:40, % v/v). The analysis was performed at an oven temperature of 30℃ with a flow rate of 0.5 mL/min, and detection was achieved at 240 nm. The method demonstrated excellent linearity (R² = 0.9998) across concentrations from 40 to 60 μg/mL. Validation studies confirmed high accuracy, with a mean recovery of 99.96%. Conclusion: The validated HPLC method is robust and reliable for quantifying Niraparib in various formulations, aiding in the stability assessment and ensuring the efficacy and safety of cancer treatment protocols. This method facilitates informed decisions in pharmaceutical development.