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• Measurement of intestinal permeability and absorptive capacity in the domestic cat. • Quantification of sugars in plasma using liquid chromatography-mass spectrometry. • Simultaneous analysis of lactulose, rhamnose, xylose, and 3-O-methylglucose (3-OMG) • Use of stable isotope labelled 13C standards for each target sugar. A novel method for quantifying the concentration of lactulose, rhamnose, xylose, and 3-O-methylglucose (3-OMG) in cat plasma using liquid chromatography-mass spectrometry (LC-MS) was developed. Domestic male cats (n = 13) were orally dosed with a solution containing the four sugars to test the permeability and absorptive capacity of their intestinal barrier. Plasma samples were taken 3 h later and were prepared with acetonitrile (ACN), dried under N 2, and reconstituted in 90 % ACN with 1 mM ammonium formate. Stable isotope labelled 13C standards for each analyte were used as internal standards. Chromatographic separation was conducted using a Phenomenex Luna NH2 column with a gradient elution system of deionized water and 90 % ACN with 1 mM ammonium formate at 300 µL/min for 13 min total analysis time. Recovery trials were conducted in triplicate over three days with RSD values (%) for each day ranging from 1.2 to 1.4 for lactulose, 5.4 – 6.0 for rhamnose, 3.3 – 5.5 for xylose, and 2.6 – 5.6 for 3-OMG. Inter-day variations for each analyte were not different (p > 0.05). Limit of detection and quantification were 0.2 and 0.7 µg/mL for lactulose, 0.8 and 2.4 µg/mL for rhamnose, 0.6 and 1.8 µg/mL for xylose, and 0.3 and 1.1 µg/mL for 3-OMG, respectively. Plasma sugar concentrations recovered from cats were above the limit of quantification and below the highest calibration standard, validating the use of this method to test intestinal permeability and absorptive capacity in cats. [ABSTRACT FROM AUTHOR]

• An LC-MS/MS method was optimized for cardiovascular drugs in dried blood spots (DBS). • Drugs in DBS were simply extracted with organic solvents in two-step protein precipitation. • This method was fully validated for linearity, matrix effect, accuracy, and stability. • Drug distribution within spots was analyzed using punched discs. • Feasibility of accuracy-based analysis for cardiovascular drugs in DBS was proposed. Dried Blood Spots (DBS) revolutionize therapeutic drug monitoring using LC-MS for the precise quantification of cardiovascular drugs (CDs), enabling personalized treatment adapted to patient-specific pharmacokinetics with minimal invasiveness. This study aims to achieve simultaneous quantification of eight CDs in DBS, overcoming physicochemical challenges. A two-step protein precipitation method was used for simple and precise sample preparation. The drugs were analyzed using LC-MS/MS in ESI positive-ion mode, showing high sensitivity and linearity, with a correlation coefficient (r2) exceeding 0.999, after being separated on a reversed-phase chromatography by gradient elution of DW-acetonitrile containing 0.1 % formic acid + 2 mM ammonium formate. The validation results indicate good selectivity, with no observed matrix effect and carry-over. The intra- and inter-day accuracy and precision were within 6 % for most drugs, except for digoxin and deslanoside at low therapeutic levels where the variation was within 20 %. Stability tests confirmed suitable DBS handling and storage conditions, indicating drug stability for at least 30 days at room temperature. The analysis of whole spot has demonstrated remarkable precision and reliability in all target drugs. The analysis of 3 mm internal diameter discs, punched in and out of DBS, presumed to contain 3 µL of blood, showed acceptable accuracy for most drugs, with less polar drugs like digoxin and deslanoside showing lower accuracy, indicating a need for further correction due to non-uniform drug distribution. Consequently, the developed LC-MS/MS method enables the quantification of multiple CDs in a single DBS analysis, while suggesting the potential for accuracy-based analysis. [ABSTRACT FROM AUTHOR]

• The QuEChERS pretreatment method with LC-MS was used to monitor liver cancer drug concentrations in blood. • This simple, rapid, and efficient method detected 7 tyrosine kinase inhibitors in plasma within four minutes. • Most of the drugs selected in this study are second-line drugs. Tyrosine kinase inhibitors (TKIs) are commonly used to treat various cancers. Literature suggests that the blood concentration of TKIs strongly correlates with their efficacy and adverse effects. Therefore, establishing a Therapeutic Drug Monitoring (TDM) methodology for TKI drugs is crucial to improving their clinical efficacy and minimizing the treatment-related adverse effects. However, quantifying their concentrations in the plasma using existing methods to avoid potential toxicity is challenging. Herein, seven TKIs, namely sorafenib tosylate, axitinib, erlotinib, cediranib, brivanib, linifanib, and golvatinib, were successfully analyzed in human plasma by following a quick, easy, cheap, effective, rugged, and safe (QuEChERS) pretreatment method combined with ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Briefly, biological samples were extracted using 1 mL of methanol, followed by the sequential addition of 250 mg of anhydrous magnesium sulfate and 25 mg of N-propylethylenediamine (PSA) for salinization and purification by adsorption, respectively. In this study, dovitinib was used as the internal standard. The seven TKIs were detected by the gradient elution method for 4 min in the positive ion electrospray mode. The mobile phase comprised methanol (phase A) and 0.1 % aqueous formic acid solution (phase B) on the Agilent Zorbax RRHD Stablebond Aq, (2.1 × 50 mm; 1.8 μm). Brivanib, linifanib, axitinib, sorafenib tosylate, and golvatinib exhibited good linearity in the range of 5–500 ng/mL, and erlotinib and cediranib exhibited good linearity in the range of 10–1000 ng/mL, with linear correlation coefficients (R2) ≥ 0.99. The limits of detection and quantification were 0.60–0.18 ng/mL and 5–10 ng/mL, respectively. The intraday and interday accuracy values ranged from −6.12 % to 7.31 %, with a precision (RSD) of ≤ 10.57 %. The method was rapid, accurate, specific, simple, reproducible, and suitable for the quantitative determination of the seven TKIs in human plasma. [ABSTRACT FROM AUTHOR]

• Rapid and simultaneous determination of 6 volatile fatty acids (VFAs) via HPLC-MS/MS. • The derivatisation method solves the problems of fatty acids in the detection process. • High throughput analysis of samples can be determined high sensitivity and accuracy. • VFAs can be accurately quantified using only microsamples (faeces, serum and urine). A stable isotope dilution-liquid chromatography tandem mass spectrometry method based on a low-temperature derivatization strategy with 3-nitrophenylhydrazine (3-NPH) was developed for the determination of six volatile fatty acids (VFAs) in serum, urine, and feces. Ice acetonitrile was used to precipitate proteins and extract the target analytes. The extract was derivatized with 3-NPH methanol solution at 4 °C. BEH C8 (1.7 μm, 2.1 × 100 mm) column was used for chromatographic separation, and acetonitrile–water (both containing 0.01 % formic acid) were used as the mobile phase with a gradient elution of 10 min. Electrospray ionization source (ESI) in negative ion multiple reaction monitoring (MRM) mode were used for analyte detection. The regression coefficients R2 of the calibration curves for the six VFAs were in the range of 0.9963–0.9994, and the LOQs were in the range of 0.02–0.5 μg mL−1, with the recoveries in the range of 85.3–104.3 %, and the intra- and inter-day precision in the range of 1.8–9.1 %. The method is simple, accurate and reliable, and has been applied in the sensitive determination of VFAs in complex biological samples. [ABSTRACT FROM AUTHOR]

• Atenolol and metoprolol are the preferred drugs in the treatment of hypertension and heart failure. • Importance of assessing intestinal absorption and permeability of orally administered drugs by in-situ perfusion studies. • Use of HPLC as an analytical method in quantification and drug quality control. • Development of an HPLC method that simultaneously detects atenolol, metoprolol, and phenol red (zero permeability marker). Single-pass intestinal perfusion (SPIP) method is a widely used experimental model to determine the intestinal permeability of drugs. These studies are performed in the presence of a reference standard (metoprolol, MT) and a zero permeability marker (phenol red, PR). Therefore, it is important to develop a validated method for simultaneous determination of the investigated compound along with MT and PR. The aim of this study was to develop a reversed phase high-performance liquid chromatography (RP-HPLC) method with UV-detection for the simultaneous determination of atenolol (ATN), MT, and PR in the perfusion medium used in SPIP experiments. Separation of compounds were performed using an InertSustain C18 (250 × 4.6 mm, 5 µm) HPLC column at 35 °C. The mobile phase was a mixture of acetonitrile and phosphate buffer (pH 7.0, 12.5 mM) in gradient elution, and was delivered at a flow rate of 1 mL/min. The acetonitrile ratio of the mobile phase increased linearly from 10 to 35 % over 15 min. The injection volume was 20 µL, and ATN, MT and PR were detected at 224 nm. The retention times under optimum HPLC conditions were 5.028 min, 12.401 min, and 13.507 min for ATN, MT and PR, respectively. The developed RP-HPLC method was validated for selectivity, specificity, calibration curve and range, accuracy and precision, carry-over effect, stability, reinjection reproducibility, recovery and robustness. The method was linear for ATN (0.76–50 μg/mL), MT (1.14–50 μg/mL), and PR (0.47–20 μg/mL) with determination coefficients of 0.9999, 0.9994 and 0.9998, respectively. The results obtained for all validation parameters of the developed RP-HPLC method met the required limits of the ICH M10 Guideline. [ABSTRACT FROM AUTHOR]

• An LC-MS/MS method for simultaneous drug quantification was optimized. • The method covers 1st–3rd-generation antiepileptic drugs in a single analysis. • Cost-effective sample preparation by minimizing isotope-labeled internal standards. • The method was fully validated and stability was tested. • Reliability was confirmed in lipid-, glucose-, and protein-rich samples. This study aims to establish an LC-MS/MS method to simultaneously analyze 11 antiepileptic drugs with a particular focus on maintaining accuracy while reducing the number of isotope-labeled internal standards employed for cost-effectiveness. By applying a water/acetonitrile gradient elution containing 0.1 % formic acid and 2 mM ammonium formate as the mobile phase, optimal sensitivity for the target drugs could be obtained in positive ESI mode in LC-MS/MS. After optimizing various extraction techniques, extraction with 70 % acetonitrile was selected as it provided good recoveries (>93 %) for all targets without matrix effects. Accuracies within 3 % were achieved from the combination of six internal standards, while accuracies of 5 % and 10 % were obtained by reducing the number of internal standards to four and two, respectively, for more economical analysis. The accuracy of the established method was maintained in hyperglycemia, hyperlipidemia, and hyperalbuminemia sera, suggesting that it can be successfully applied to individual serum samples with various properties. [ABSTRACT FROM AUTHOR]

• A sensitive method was developed for letrozole and its carbinol metabolite in mouse plasma. • Limited sample volumes allow implementation in large-scale pharmacokinetic studies. • Letrozole levels in plasma are formulation- and strain-dependent. A liquid chromatography - electrospray ionization-mass spectrometry (LC-ESI-MS) method was developed for the quantification of letrozole, a third-generation aromatase inhibitor, and its main carbinol metabolite (CM) in support of murine pharmacokinetic studies. Using polarity switching, simultaneous ESI-MS measurement of letrozole and CM was achieved in positive and negative mode, respectively. The assay procedure involved a one-step protein precipitation and extraction of all analytes from mouse plasma requiring only 5 μL of sample. Separation was optimized on an Accucore aQ column with gradient elution at a flow rate of 0.4 mL/min in 5 min. Two calibration curves per day over four consecutive measurement days showed satisfactory linear responses (r 2 > 0.99) over concentration ranges of 5–1000 ng/mL and 20–2000 ng/mL for letrozole and CM, respectively. No matrix effect was found, and the mean extraction recoveries were 103–108 % for letrozole and 99.8–107 % for CM. Precision and accuracy within a single run and over four consecutive measurement days were verified to be within acceptable limits. Application of the developed method to preclinical pharmacokinetic studies in mice receiving oral letrozole at a dose 1 or 10 mg/kg revealed that the systemic exposure to letrozole was dose-, formulation-, and strain-dependent. These findings may inform the future design of preclinical studies aimed at refining the pharmacological profile of this clinically important drug. [ABSTRACT FROM AUTHOR]

• The simultaneous determination of 14 UV-filters in plasma were developed using LC-MS/MS. • Proteins and phospholipids in plasma are effectively removed. • The method can be applied to monitor human plasma exposure levels of UV-filters. • General population are exposure to UV-filters through multiple sources. Accurate monitoring of UV-filters exposure levels in human plasma is a challenge because of the significant differences in the physicochemical properties of UV-filters, as well as the matrix effect caused by abundant proteins and phospholipids in plasma. Therefore, an effective and rapid method for simultaneous determination of 14 UV-filters in human plasma using protein precipitation-solid phase extraction (SPE) coupled with liquid chromatography tandem mass spectrometry (LC-MS/MS) was developed. Acetonitrile with 0.1 % formic acid and 10 % isopropanol (v/v) were used as mobile phases. A gradient elution on an ACQUITY UPLC BEH-C18 column at 30 °C and 0.3 mL/min flow rate was applied for separation. The electrospray ionization positive or negative modes were selected to determine the corresponding analyte to increase selectivity and sensitivity. Results showed that acetonitrile-tetrahydrofuran (v/v, 8:2) as the extraction solvent can effectively precipitate protein in plasma and improve the solubility of UV-filters. The HybridSPE cartridge improved the removal efficiency of phospholipids, while 1 mL of methanol elution increased the extraction recoveries of targets. Fourteen UV-filters achieved good linearities, low detection limits (0.050 to 0.10 μg/L) and quantification limits (0.10 to 1.0 μg/L). Method accuracy and precision, extraction recoveries, and storage stabilities of all analytes met the criterion of 80–120 %. Moreover, this method was successfully applied for the determination of UV-filters in plasma randomly collected from adults. Nine of 14 UV-filters were determined and their concentrations were distributed widely, suggesting a big variation of individual UV-filters exposure. [ABSTRACT FROM AUTHOR]

• Antimicrobial pharmacokinetics is an emerging area of gut microbiome research. • Omadacyline is a tetracycline-class antibiotic with potent activity against Clostridioides difficile and a low propensity to cause C. difficile infection. • This study developed and validated a LC-MS/MS method to quantify omadacycline and its epimerization in stool. • A remarkable epimerization of omadacycline was observed in stool samples. This study developed and validated a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify omadacycline and its epimerization in stool to facilitate microbiome studies. Omadacycline was extracted in a methanol-water-ethylenediaminetetraacetic acid (ETDA) solvent containing deuterated omadacycline as internal standard, followed by dilution. In an optimal gradient elution mode, omadacycline and its C4 epimer were separated within 5 min on reversed-phase C18 column. The method showed a broad working range of 0.1–200 ng/ml with a limitation of detection (LOD) of 0.03 ng/ml, little fecal matrix effect, good intra-day and inter-day accuracy (90–101 %), precision (2–15 %), and recovery rate (99–105 %). The method was sufficiently sensitive to quantify omadacycline in human fecal samples (n = 82) collected during a 10-day therapy course and at follow-up (day 13 and day 30) that ranged from 1 to 4785 µg/g. Further analysis revealed that ∼9 % of omadacycline was epimerized in fecal matrix control while, on average, 37.4 % was epimerized in human fecal samples. This study developed and validated a novel, simple, sensitive, and accurate method utilizing LC-MS/MS to quantify omadacycline its epimerization in the human gut. This has important implications for future studies of omadacycline and other tetracycline-class antibiotics as part of gut microbiome studies. [ABSTRACT FROM AUTHOR]

• Develop and validate an LC-MS/MS method for the quantification of six small molecule Anti-SARS-CoV-2 drugs in plasma. • The devised methodology was effectively utilized in a pharmacokinetic study following the oral administration of six small molecule Anti-SARS-CoV-2 drugs in rats. • Presents a significant tool for preclinical and clinical investigations of six small molecule Anti-SARS-CoV-2 drugs, generating interest in further research on anti-Covid19 drugs. Antiviral treatment for COVID-19 is considered an effective tool in reducing the rate of severe cases and deaths. As of June 2023, a total of six small molecule antiviral drugs have been conditionally approved for marketing by the National Medical Products Administration (NMPA) within China. In this study, a method of HPLC-MS/MS was established and validated for the determination of six small molecule antiviral drugs in plasma using Lamivudine as an internal standard. The chromatographic separation was performed using gradient elution with an ACE 3 C 18 -PFP column (3.0 mm × 150 mm, 3 μm), and the mobile phase consisted of deionized water and acetonitrile/water (90:10, v/v), both with 10 mmol/L of ammonium acetate and 0.1 % ammonium hydroxide added. Quantitative analysis of the six small molecule drugs was carried out through selective reaction monitoring based on the positive ion spray ionization mode. The method exhibited excellent precision, accuracy, recovery, and linearity, and it was used to determine the pharmacokinetic characteristics in rats. Our work not only established a bioanalytical method for six small molecule antiviral drugs but also provided scientific references for clinical pharmacokinetic studies. [ABSTRACT FROM AUTHOR]

[Display omitted] • HF-LPME and nanoLC-HRMS provide high clean-up efficiency and detectability. • Lab-made columns minimize dead-volume and post-separation dilution. • The applicable concentration interval covers the therapeutic and toxic range. • This method can be applied for therapeutic drug monitoring and forensic purposes. Therapeutic drug monitoring (TDM) is a personalized care tool based on the determination of a target drug concentration in human serum. An antidepressant drug of interest for such investigations is fluoxetine (FXT), due to a severe impact of genetic polymorphisms on its metabolism. A bioanalytical method employed for TDM purposes must exhibit satisfactory selectivity and detectability, which becomes more difficult due to highly complex biological matrices. In this study, a highly selective bioanalytical method for the determination of FXT in human serum is proposed, which provides excellent clean-up efficiency based on a low cost hollow fiber liquid-phase microextraction (HF-LPME) sample preparation step and nano-liquid chromatography coupled to high-resolution mass spectrometry (nano-LC-HRMS). HF-LPME was performed using a two-phase "U" configuration, with 6 cm fiber, 20 µL of 1-octanol acting as supported liquid membrane, and ammonium hydroxide (pH 10) as the donor phase with NaCl (10 % m/v) and methanol (5 % v/v) as additives, requiring only 250 µL of the sample. The procedure was conducted for 30 min under a 750 rpm stirring rate. Gradient elution was carried out employing an acetonitrile–water as mobile phase, the composition of 30:70 to 100:00 (v/v) for 15 min, using formic acid 0.1 % (v/v) as an additive. MS1 was acquired in an Orbitrap mass analyzer, while MS2 was acquired in a linear trap quadrupole. Satisfactory linearity (Pearson's r = 0.99709) was obtained for a concentration range of 0.02 to 2.5 µg mL−1, which is compatible with the therapeutic and toxic range for FXT. The developed method presents adequate precision (1.61 to 7.45 %) and accuracy (95 to 114 %) and allows the dilution of high concentration samples in a 1:4 ratio (v/v), enabling its application for forensic serum samples. To our knowledge, this is the first study reporting a method based on HF-LPME and nano-LC-HRMS with any analytical purpose, especially with a TDM focus. [ABSTRACT FROM AUTHOR]

• A LC-MS/MS method for simultaneous quantification of insulin and liraglutide. • Method was developed and validated following FDA and EMA guidelines. • The method was applied to pharmacokinetics and biodistribution of drugs in rats. • Drugs were quantified in plasma, brain, lung, spleen, liver and kidneys. We present an ultra-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous detection of insulin degludec (I-Deg) and liraglutide (LIRA) in rat plasma and tissues, characterized by its sensitivity and selectivity. Chromatographic separation was achieved using an Acquity UPLC BEH C18 column, leveraging a mobile phase of acetonitrile and water (both with 0.1 % formic acid) under gradient elution over a run time of 7.5 min. The mass spectrometer operated in positive electrospray ionization multiple reaction monitoring (MRM) mode, tracking transitions of m / z 1221.6 → 641.6 for I-Deg, m / z 938.7 → 1064.1 for LIRA, and m / z 1184.7 → 454.4 for the internal standard. Validation ranged from 5 to 100 ng/mL, exhibiting robust linearity (r2 > 0.99) and limits of detection (LOD) of 1.63–2.02 ng/mL for I-Deg and 0.96–1.62 ng/mL for LIRA. Limits of quantification (LOQ) were 2.38–4.76 ng/mL for I-Deg and 3.22–4.40 ng/mL for LIRA. Notably, no significant matrix effects were detected. Stability was confirmed under various conditions, and precision metrics (intraday RSD 1.68–8.05 % for I-Deg and 1.11–7.69 % for LIRA; interday RSD 1.39–8.61 % for I-Deg and 1.06–8.83 % for LIRA) alongside accuracy (90.5–114.9 % for I-Deg and 92.7–113.7 % for LIRA) were within acceptable ranges. The method was successfully applied to pharmacokinetic and biodistribution studies following simultaneous subcutaneous administration of LIRA and I-Deg in rats. [ABSTRACT FROM AUTHOR]

• Evaporative light scattering detector was used to analyze low-calorie bulk sweeteners. • Six low-calorie bulk sweeteners were analyzed simultaneously for the first time and separated within 25 min. • Successful implementation of low-calorie bulk sweetener content/composition in food using HPLC-ELSD. A novel and sensitive method for the simultaneous analysis of six low-calorie bulk sweeteners (D-allulose, D-tagatose, D-mannitol, mycose, palatinose, and erythritol) without derivatisation was developed using high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD). Chromatographic separations were carried out on a Zorbax Original NH 2 (5 μm particle size, 250 mm×4.60 mm id, 70 Å) column with flow rate gradient elution with acetonitrile: water (80:20, v/v). Drift tube temperature was set at 50 ℃, the nebuliser carrier gas flow rate was 1.0 mL·min-1, and nitrogen pressure was regulated to 276 kPa with gain:3. The regression equation showed good linearity (R 2 = 0.9985-0.9998) for all six low-calorie bulk sweeteners in the tested range (0.060-0.60 mg·mL-1). The limits of detection (LOD) for the six low-calorie bulk sweeteners ranged from 0.02 to 0.06 mg·mL-1. The proposed HPLC-ELSD method was validated for the quantification of the low-calorie bulk sweeteners in 14 types of foods, and the results were satisfactory. In addition, the results showed that the number of sweeteners in each food product varied. The presence of multiple low-calorie bulk sweeteners in certain foods is interesting. This method is successful in monitoring low-calorie bulk sweeteners in food. [ABSTRACT FROM AUTHOR]

• Simultaneous determination method developed for six antiviral drugs based on LC-MS/MS. • Optimized and validated to apply in routine clinical and pharmaceutical testing. • Simple sample preparation and robust analysis for various applications. • Analyte stability assessed for temperature fluctuation in sample preparation/transportation. • Method is suitable for monitoring various antiviral drugs and others of similar character. We describe the simultaneous quantification of six antiviral drugs in serum based on high-performance liquid chromatography–tandem mass spectrometry (LC-MS/MS). The target drugs—hydroxychloroquine, chloroquine, favipiravir, umifenovir, ritonavir, and lopinavir—were extracted and purified from serum with 75 % v/v methanol as the precipitant reagent. The six analytes were clearly separated within 15 min using gradient elution and mixed-mode stationary phase. The measurement accuracy and precision were assured by adopting isotopes as internal standards. The optimized measurement procedure was strictly validated in linearity, sensitivity, accuracy, and precision. To confirm the robustness of the method in matrix, the method was additionally applied to various types of serum, namely hyperlipidemic and hyperglycemic serum. The method was then applied to assess the stability of the drugs in serum in order to set sample handling and storage guides for laboratory testing. Lastly, the method was implemented in different LC-MS systems to confirm its applicability across similar equipment commonly used in clinical testing laboratories. The overall results show that the optimized protocol is suitable for the accurate, simultaneous quantification of the six antiviral drugs in serum, and it is anticipated to satisfactorily serve as a reference protocol for the analysis of a wide range of other antiviral drugs for drug monitoring with various purposes. [ABSTRACT FROM AUTHOR]

• A quantification method for KRAS-inhibitors Adagrasib and Sotorasib was developed. • This method may support pharmacokinetic research for Adagrasib and Sotorasib. • Revealing overexposure may help reduce treatment toxicity in clinical practice. • The method was co-validated with brigatinib, lorlatinib, pralsetinib & selpercatinib. Recently, two small molecular inhibitors (SMIs) –adagrasib and sotorasib– have been introduced for targeting Kirsten rat sarcoma (KRAS) p.G12C mutations in patients with non-small cell lung cancer (NSCLC). In order to support pharmacokinetic research as well as clinical decision making, we developed and validated a simple and accurate liquid chromatography-tandem mass spectrometry method for the multiplexed quantification of adagrasib and sotorasib. This assay was co-validated with the quantification for brigatinib, lorlatinib, pralsetinib and selpercatinib. Methanol was used for single-step protein precipitation. Chromatographic separation was performed using an Acquity® HSS C18 UPLC column, with an elution gradient of ammonium formate 0.1 % v/ v in water and acetonitrile. In K2-EDTA plasma, adagrasib was found to be stable for at least seven days at room temperature and 4 °C, and at least 3 months at −80 °C. Sotorasib was found to be stable for at least three days at room temperature, seven days at 4 °C and at least 3 months at −80 °C. The method was validated over a linear range of 80–4000 ng/mL for adagrasib and 25–2500 ng/mL for sotorasib. The assay is therefore well-equipped for determining plasma concentrations in clinical practice. [ABSTRACT FROM AUTHOR]

A validated approach to determine various methionine cycle metabolites (S-adenosylmethionine, S-adenosylhomocysteine, and methylthioadenosine) in human blood plasma is offered. The approach is based on solid-phase extraction (with grafted phenylboronic acid) and derivatization with chloroacetaldehyde followed by ultra-performance liquid chromatography with fluorescence detection. We used a 100 × 2.1 mm × 1.8 μm C18 column for the selective separation of analytes. Chromatographic separation was achieved with gradient elution of acetonitrile (flow rate 0.2 mL/min) from 2 to 20%. The eluent was initially composed of 10 mM KH 2 PO 4 with 10 mM acetic acid and 25 μM heptafluorobutyric acid. The total analysis time was 11 min. Validation of the method included detection of the limit of detection (2 nM), limit of quantification (5 nM), accuracy (97.2–101%), and intra- and interday precision (2.2–9.0%). Analysis of plasma samples from healthy volunteers revealed that the average levels of S-adenosylmethionine, S-adenosylhomocysteine, and methylthioadenosine were 93.6, 20.9 and 14.8 nM, respectively. • Determination of S-adenosylmethionine and S-adenosylhomocysteine in plasma by UPLC-Fl. • Optimal balance of accuracy and performance were selected to optimize sample processing. • The method could be used for clinical researches. [ABSTRACT FROM AUTHOR]

A simple and fast LC-MS/MS method was developed and validated for simultaneous quantification of 20 proteinogenic l -amino acids (AAs) in a small volume (5 μL) of mouse plasma. Chromatographic separation was achieved on an Intrada Amino Acid column within 13 min via gradient elution with an aqueous solution containing 100 mM ammonium formate and an organic mobile phase containing acetonitrile, water and formic acid (v : v : v = 95:5:0.3), at the flow rate of 0.6 mL/min. Individual AAs and corresponding stable-isotope-labeled AAs internal standards were analyzed by multiple reaction monitoring (MRM) in positive ion mode under optimized conditions. Method validation consisted of linearity, sensitivity, accuracy and precision, recovery, matrix effect, and stability, and the results demonstrated this LC-MS/MS method as a specific, accurate, and reliable assay. This LC-MS/MS method was thus utilized to compare the dynamics of individual plasma AAs between healthy and orthotopic hepatocellular carcinoma (HCC) xenograft mice housed under identical conditions. Our results revealed that, 5 weeks after HCC tumor progression, plasma l -arginine concentrations were significantly decreased in HCC mice while l -alanine and l -threonine levels were sharply increased. These findings support the utilities of this LC-MS/MS method and the promise of specific AAs as possible biomarkers for HCC. • A simple and fast LC-MS/MS method was developed for simultaneous quantification of 20 natural amino acids in mouse blood. • Chromatographic separation was achieved on an Intrada Amino Acid column. • This LC-MS/MS method was validated as a specific, accurate, and reliable assay with minimal matrix effects. • This method revealed a lower arginine concentration and much higher alanine and threonine levels during HCC progression. [ABSTRACT FROM AUTHOR]

Talatisamine, as the efficacy ingredient of Aconitum , was known as a novel specific blocker for the delayed rectifier K+ channels in rat hippocampal neurons. In this study, a rapid, selective and reproducible UPLC-MS/MS separation method was established and fully validated for the quantitative determination of talatisamine levels in ICR (Institute of Cancer Research) mouse blood. A total of 24 healthy male ICR mice were divided into four groups that was administered talatisamine via intravenous at a dose of 1 mg/kg and oral administration of three doses (2, 4, 8 mg/kg). All blood samples were protein precipitate by using acetonitrile with an internal standard (IS) deltaline. The effective chromatographic separation was carried out through an UPLC BEH C18 analytical column (2.1 mm × 50 mm, 1.7 μm) with an initial mobile phase that consisted of acetonitrile and 10 mmol/L ammonium acetate aqueous solution (containing 0.1% formic acid) with a gradient elution pumped at a flow rate of 0.4 mL/min. Also, an electrospray ionization (ESI) was applied to quantify the talatisamine in the positive ions mode. The method validation demonstrated good linearity over the range of 1–1000 ng/mL (r2 ≥ 0.9993) for talatisamine in mouse blood with a lower limit of quantification (LLOQ) at 1 ng/mL. The accuracy values of the method were within 89.4% to 113.3%, and the matrix effects were between 103.2% and 106.3%. The mean extraction recoveries for talatisamine obtained from four concentrations of QC blood samples were exceeded 71.7%, and the relative standard deviation (RSD) both of intra- and inter-day precision values for replicate quality control samples did not exceed 15% respectively for all analytes during the assay validation. This method was successfully applied to the evaluation of the pharmacokinetic of talatisamine, regardless of intragastric or intravenous administration in mice. Based on the pharmacokinetics data, the bioavailability of talatisamine in mice was >65.0% after oral administration, exhibiting an excellent oral absorption. • UPLC-MS/MS method of talatisamine was developed for the first time in mouse blood. • The method was validated and stability studies were performed. • Lower limit of quantification of talatisamine for the UPLC-MS/MS method was 1 ng/mL. • Absolute bioavailability of talatisamine was 65.0% in mice. [ABSTRACT FROM AUTHOR]

A simple, accurate, and reliable liquid chromatography-mass spectrometry (LC-MS/MS) method was developed and validated for the determination of mangiferin in rat plasma and tissue homogenates using rutin as an internal standard (IS). Chromatographic separation was achieved on a Shiseido CAPCELL PAK C 18 column (150 × 2.0 mm, 5 μm) using a gradient elution of 1% acetic acid in water and methanol at a flow rate of 300 μL·min−1. Quantification was performed on an API 4000+ triple-quadrupole mass spectrometer equipped with a turbo electrospray ionization (ESI) source in positive ion multiple reaction monitoring (MRM) mode. Selected ion monitoring transitions of 423.1 → 273.1 and 611.4 → 303.3 were chosen to quantify mangiferin and IS. Biological samples were pretreated via protein precipitation with acetonitrile-acetic acid. The standard calibration curves were above the ranges of 2 to 500 ng·mL−1 and 5 to 2000 ng·mL−1 for tissues, and 1 to 600 ng·mL−1 for plasma. All calibration curves for tissue and plasma samples showed good linearity (r ≥ 0.9974) over the concentration ranges. Intra- and inter-day precisions were <14.0%, and accuracy ranged from 97.2% to 111.7%. The established method was successfully applied on mangiferin tissue distribution following the intragastric administration of mangiferin, Rhizoma Anemarrhenae (A. Rhizoma) decoction, or Rhizoma Anemarrhenae-Phellodendron (herb pair, HP) decoction under healthy or diabetic conditions. Mangiferin was detected from all the tested tissues (except for brain) after monomer administration, and the concentrations were lower compared with the decoction groups. Distributions in the HP group were lower than those in the A. Rhizoma group, but mangiferin content in pancreas was obviously higher than in other tissues and in the A. Rhizoma group. Compared with healthy rats, mangiferin distributions in pancreas and intestine were lower in diabetic rats administered with the same dose of the herb pair, but still higher than those in other tissues. In addition, distributions in liver, spleen, lung, kidney, stomach, and plasma were higher than those in the normal group. • A novel LC-MS/MS method was developed for determination of mangiferin in rat tissues. • Tissue distributions of mangiferin following single oral administration of mangiferin monomer, A. Rhizoma and HP were compared. • Tissue distributions of mangiferin following single oral administration of HP decoction in normal and diabetic rats were compared. • Mangiferin content was much higher after administrated with decoctions than monomer. • Diabetic pathology surroundings influenced mangiferin distribution. [ABSTRACT FROM AUTHOR]

Asthma and chronic obstructive pulmonary disease (COPD) are common respiratory disorders that have similar clinical presentation and misdiagnosis may lead to improper treatment. There is a need for a better, non-invasive test for the differentiation of asthma and COPD. In this study, we developed a new validated LC-MS/MS method for 17 urinary organic acids that could serve as potential biomarkers. Human urine samples were collected from adults with asthma or COPD. LC-MS/MS was performed using the differential isotope labeling approach. 4-(Dimethylamino) phenacyl bromide (DmPA) was used for derivatization using two different carbon isotopes, allowing for the formation of internal standard for each metabolite. Gradient elution was employed on a C 18 column while the LC-MS/MS operated in the multiple reaction monitoring mode (MRM). Regulatory guidelines were used for method validation. Partial Least Squares Discriminative Analysis (PLS-DA) was applied to the log-transformed values of metabolites in each group of asthma and COPD subjects. Full validation in targeted metabolomics is scarce with usually limited number of metabolites, unlike fit-for-purpose approach. Due to the endogenous nature of the metabolites, numerous challenges were encountered during method development and validation, such as the lactic acid interference from the surrounding environment. The required specificity, accuracy and precision was successfully achieved. The method was fully validated, ensuring robustness and reproducibility when analyzing patient samples. The method was applied to analyze human urine samples and PLS-DA analysis showed differentiation of asthma and COPD subjects (R2 0.89, Q2 0.68). As targeted metabolomics is expanding to the clinical sphere, more validated methods/strategies are needed. Our work will expand the current knowledge-base regarding targeted metabolomics. • A new validated LC-MS/MS method for the quantification of 17 urinary organic acids biomarkers was developed. • Target metabolites were derivatized with DmPA and gradient elution was employed on a C18 column. • Despite challenges, the LC-MS/MS method was fully validated as per regulatory guidelines. • The method was applied to analyze urine samples, showing differentiation of asthma and COPD subjects. [ABSTRACT FROM AUTHOR]

• A simple and rapid LC-MS/MS method was developed and validated for the quantification of tamsulosin in human plasma. • A smaller volume of plasma (100 μL) and a more economical and efficient protein precipitation method were used to achieve satisfactory sensitivity (0.05 ng/mL) and recovery. • The run time was shortened to 3.5 min, making it an attractive procedure for high-throughput bioanalysis of tamsulosin. • The bioequivalence study of a lower dosage (0.2 mg) tamsulosin hydrochloride sustained-release capsules in healthy Chinese male volunteers was first reported. Tamsulosin, the first highly selective α 1 -adrenoceptor antagonist, is widely used for urination disorders caused by benign prostatic hyperplasia (BPH). The pharmacokinetics and safety of 0.2 mg tamsulosin hydrochloride sustained-release capsules were evaluated in 60 healthy Chinese male subjects under fasting and fed conditions in this study. A simple, sensitive, and robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of tamsulosin in human plasma, which has been applied to pharmacokinetic study. The analyte and internal standard (tamsulosin-d5) were extracted by protein precipitation, and separated on a ZORBAX Eclipse XDB-C18 column (4.6 × 50 mm,1.8 µm; Agilent Tech) using a gradient elution with mobile phases methanol and 5 mM ammonium acetate. The linear range was 0.05-15.0 ng/mL. It showed good selectivity in normal, hyperlipidemic, and hemolyzed blank matrices. The CV (%) of intra-batch precision was <4.4% and the RE (%) of accuracy was in the range of −5.0%–6.7%; the CV (%) of inter-batch precision was <−5.8% and the RE (%) of accuracy was in the range of 1.2%-4.0%. The mean extraction recovery for the analyte was 102.1 ± 3.75% and for the IS was 102.2 ± 2.00%. Two formulations were considered bioequivalent under fasting and fed conditions, and the 90% confidence intervals for the geometric mean test/reference ratios were within the predetermined range of 80%-125%. A single oral dose of 0.2 mg tamsulosin hydrochloride sustained-release capsule was well-tolerated throughout the clinical trial, and no > Grade 1 adverse events (AEs) and serious AEs occurred during the trial. [ABSTRACT FROM AUTHOR]

• A simple LC-MS/MS method for the simultaneous quantification of sertraline and N-desmethylsertraline in human plasma is established. • The method requires a small plasma extraction volume of 200 μL. • The calibration ranges of the method covered the proposed therapeutic concentration ranges for the analytes. • The method was successfully applied to a clinical study to quantify analytes in maternal and umbilical cord plasma. A simple and sensitive LC-MS/MS method for the simultaneous quantification of the second-generation antidepressant sertraline, and its active metabolite, N-desmethylsertraline, in human plasma was developed and validated. The analytes were extracted from 200 µL human plasma using a simple protein precipitation method. A gradient elution mode of water and 0.1% formic acid and acetonitrile was used for chromatographic separation on a Poroshell EC-C18 column (3 × 100 mm, 2.7 µm) at a flow rate of 0.450 mL/min. MS/MS analysis was performed in positive ionization mode using the transitions of m / z 306.1 → 159.1, 309.1 → 275.2, 292.1 → 159.1, and 296.2 → 279.0 for sertraline, sertraline-d3, N-desmethylsertraline, and N-desmethylsertraline-d4, respectively. The calibration curves for sertraline and N-desmethylsertraline in human plasma ranged from 2.50–320 ng/mL and 10.0–1280 ng/mL, respectively, with correlation coefficients (r) of ≥ 0.9992. The intra- and inter-assay precisions for both analytes at four concentrations (LLOQ, QCL, QCM, and QCH) ranged between 2.2% and 12.2%, respectively, while their accuracies ranged between 92.0 and 111.7%, with the exception of LLOQ, which ranged between 84.3 and 106.0%. The mean percentage recovery and process efficiency at three concentrations (QCL, QCM, and QCH) was 94.2 and 87.9% for sertraline, and 95.7 and 95.2% for N-desmethylsertraline, respectively. Both analytes were stable in plasma at room temperature for 2 h, at −80 °C for 28 days and through three freeze/thaw cycles. This method was successfully applied to a clinical study investigating the use of sertraline during pregnancy. [ABSTRACT FROM AUTHOR]

Lupeol, a phytosterol and triterpene, possesses numerous medicinal properties against cancer, inflammation, arthritis, diabetes, heart diseases, etc. A novel, sensitive, specific and reproducible method for quantification of Lupeol in rat plasma using liquid chromatography combined with atmospheric pressure chemical ionization (APCI) tandem mass spectrometry (LC-MS/MS) was developed and validated as per regulatory guidelines. Sample preparation was simple and fast which consisted of one-step protein precipitation using acetonitrile. Testosterone was used as an internal standard. HyPurity Advance column was used to develop the chromatography method using 0.1% formic acid in water and acetonitrile as mobile phases by gradient elution. APCI positive ion mode was used for mass spectrometric detection. Multiple reaction monitoring (MRM) transitions of m/z 409.5 [M + H - H 2 O]+→137.3 for Lupeol and m/z 289.1 [M + H]+→97.1 for Testosterone were used for quantification. The method was validated over a linear concentration range of 5–5000 ng/mL with a correlation coefficient (r2) of ≥ 0.99. This method showed acceptable accuracy (89.52–97.10%), precision (%CV ≤ 10.75%) and recovery with a negligible matrix effect. Lupeol was found to be stable in the stock solution, autosampler condition and also in plasma for four freeze-thaw cycles, 6 h at ambient temperature and 30 days at −20°C. This method was successfully applied to measurement of Lupeol in plasma samples from pharmacokinetic study in rats and can be easily extended to human pharmacokinetic studies. • LC-MS/MS method was developed for measurement of Lupeol in plasma. • LC-MS/MS method was successfully validated in rat plasma as per guidelines. • A validated method was successfully applied to pharmacokinetic study in rats. • Pharmacokinetic parameters of Lupeol were reported using fully validated method. [ABSTRACT FROM AUTHOR]

An innovative fabric phase sorptive extraction-high performance liquid chromatography-diode array detection (FPSE-HPLC-DAD) method is developed and validated herein for the simultaneous extraction and analysis of five common antidepressants (venlafaxine, paroxetine, fluoxetine, amitriptyline, clomipramine) in human urine samples. Separation was obtained using a reverse phase column LiChroCART-LiChrospher®100 RP-8e (5 μm, 250 × 4 mm) with diode array detection monitoring at 235 nm. Gradient elution was applied at a flow rate of 1.2 mL/min with a mobile phase that consisted of acetonitrile and ammonium acetate (0.05 M). Fabric Phase Sorptive Extraction was evaluated for the first time with regards to the extraction of the examined antidepressants from human urine and proved to be a rapid, simple, environmentally friendly, efficient, and selective sample preparation approach. Sol-gel graphene sorbent, coated on cellulose FPSE media, were the most efficient among other with different polarities. The absolute recovery values were 25.5% for venlafaxine, 33.9% for paroxetine, 67.0% for fluoxetine, 43.0% for amitriptyline and 29.0% for clomipramine, while relative recoveries were higher than 90%. The developed method provides satisfactory limit of detection 0.15 ng/μL. • Innovative Fabric phase sorptive extraction is applied in biosamples. • Isolation of five antidepressants from urine is achieved. • Fast and simple sorptive extraction method is developed herein. • Green analytical principles are satisfied by proposed method. [ABSTRACT FROM AUTHOR]

Cyclopropylfentanyl has been encountered by law enforcement and public health officials beginning in mid-2017 and has been associated with numerous overdoses and fatalities. The detection and identification of cyclopropylfentanyl has become more challenging with the subsequent emergence of multiple structural isomeric fentanyl species, some of which have been detected in seized drug casework. These include crotonylfentanyl, methacrylfentanyl, para-methylacrylfentanyl, and ortho-methylacrylfentanyl; all of which have the same exact mass and similar fragmentation patterns. Since these compounds may be scheduled differently and pharmacologically act differently, it is important to be able to differentiate them analytically. Our method was developed and validated for the analysis and differentiation of cyclopropylfentanyl from its isomers by liquid chromatography tandem mass spectrometry (LC-MS/MS), in order to confirm the identify of cyclopropylfentanyl in biological specimens from death investigation casework; 36 postmortem blood samples were submitted for analysis. Cyclopropylfentanyl, crotonylfentanyl, methacrylfentanyl, and para-methylacrylfentanyl were successfully resolved using a 13-minute run time and a simple gradient elution. Ortho-methacrylfentanyl was resolved only in a 20-minute chromatographic run. The assay met validation performance characteristics, with an analytical range of 1–100 ng/mL and limits of detection of 0.1 ng/mL for all analytes. Analysis of commonly encountered substances showed no interferences. All samples previously reported positive for cyclopropylfentanyl were confirmed positive for cyclopropylfentanyl in the absence of its isomers. To our knowledge to date, no positive toxicological specimens have been encountered for any cyclopropylfentanyl isomers. • Cyclopropylfentanyl has several structural isomeric forms • Previous literature has failed to distinguish all isomers, providing doubt around confirmation of "cyclopropylfentanyl" • Chromatographic separation of five isomers was achieved during this study with analysis by LC-MS/MS • Cyclopropylfentanyl was confirmed in all blood samples (n =36); no other isomers were identified [ABSTRACT FROM AUTHOR]

Abstract BLU-554 is a potent, highly selective oral FGFR4 inhibitor. A bioanalytical assay for quantification of BLU-554 in mouse plasma and six tissue homogenates (brain, kidney, liver, lung, small intestine, and spleen) was developed and validated using liquid chromatography with tandem mass spectrometric detection and with erlotinib as internal standard. After protein precipitation with acetonitrile in a 96-well format and separation on an XBridge® Peptide BEH C18 column by gradient elution using 0.2% (v/v) ammonium hydroxide (in water) and methanol, analytes were ionized by positive electrospray and monitored in the selected reaction monitoring mode by triple quadrupole mass spectrometry. The assay was validated in a 1–1000 ng/ml concentration range using calibration in mouse plasma. Precisions (intra–day and inter–day) were in the range 2.8–10.1% and accuracies were in between 88.5 and 96.6% for all levels in all matrices. The assay was successfully applied for a pilot pharmacokinetic and tissue distribution study in wild-type mice. Highlights • First bioanalytical method developed for quantification of BLU-554 • New method validated in mouse plasma and six tissue homogenate • Simple sample preparation technique used in 96-well format • Assay applied for a pilot pharmacokinetic and tissue distribution study in mice [ABSTRACT FROM AUTHOR]

• A simple ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method in dynamic multiple reaction monitoring mode for simultaneous determination of as many as thirteen AAAs was developed for evaluating the distribution of toxicity phytochemicals in Aristolochiaceae plants. • More AAAs in Asarums were first demonstrated by UHPLC-MS/MS, and higher content of AA IVa, SHFA I, and Aristoxazole in the root than in the leaf of the collected batch verified Asarum heterotropoides. • Simple sample pretreatment step only by extracting Asarum and Aristolochia powder with methanol and wide lineal limit, high sensitivity, and precision of analytical method may be widely used in analyzing AAAs in complex systems. Asarum and Aristolochia are two large genera of Aristolochiaceae plants containing typical toxicant aristolochic acid analogs(AAAs), AAAs can be deemed as toxicity markers of Aristolochiaceae plants. Based on the least AAAs in dry roots and rhizomes of Asarum heterotropoides, Asarum sieboldii Miq and Asarum sieboldii var, all of which are enrolled in the Chinese pharmacopeia up to now. AAAs distribution in Aristolochiaceae plants, especially Asarum L. plants, is still obscure and controversial due to few AAAs measured, unverified species of Asarum, and complicated pretreatment in analytical samples making the results more challenging to reproduce. In the present study, a simple ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method in dynamic multiple reaction monitoring mode for simultaneous determination of thirteen AAAs was developed for evaluating the distribution of toxicity phytochemicals in Aristolochiaceae plants. The sample was prepared by extracting Asarum and Aristolochia powder with methanol, and the supernatant was analyzed using the Agilent 6410 system on an ACQUITY UPLC HSS PFP column with gradient elution of water and acetonitrile, containing 1% v/v formic acid (FA) each, at a flow rate of 0.3 mL/min. The chromatographic condition provided good peak shape and resolution. The method was linear over the specific ranges with the coefficient of determination (R 2) > 0.990. Satisfactory intra- and inter-day precisions were achieved with RSD less than 9.79%, and the average recovery factors obtained were in the range of 88.50%～105.49%%. The proposed method was successfully applied for simultaneous quantification of the 13 AAAs in 19 samples from 5 Aristolochiaceae species, especially three Asarum L. species enrolled in the Chinese Pharmacopoeia. Except Asarum heterotropoides, the results supported that the Chinese Pharmacopoeia (2020 Edition) adopting the root with rhizome as medicinal parts of Herba Asari instead of the whole herb for drug safety by providing scientific data. [ABSTRACT FROM AUTHOR]

• The first validated uHPLC-MS/MS method for the determination of metarrestin in human plasma. • Efficient one-step sample preparation. • Robust quantitation down to 1 ng/mL. • Successful bioanalysis of samples from metarrestin's first-in-human Phase IA/B trial demonstrates the assays clinical utility. Metarrestin is a first-in-class small molecule inhibitor targeting the perinucleolar compartment, a subnuclear body associated with metastatic capacity. Promising preclinical results led to the clinical translation of the compound into a first-in-human phase I trial (NCT04222413). To characterize metarrestin's pharmacokinetic profile in humans, a uHPLC-MS/MS assay was developed and validated to determine the disposition of the drug in human plasma. Efficient sample preparation was accomplished through one-step protein precipitation paired with elution through a phospholipid filtration plate. Chromatographic separation was achieved with gradient elution through an Acuity UPLC® BEH C18 column (50 × 2.1 mm, 1.7 µm). Tandem mass spectrometry facilitated the detection of metarrestin and tolbutamide, the internal standard. The effective calibration range spanned 1–5000 ng/mL and was both accurate (range −5.9 % to 4.9 % deviation) and precise (≤9.0 %CV). Metarrestin proved stable (≤4.9 % degradation) under various assay-imposed conditions. Matrix effects, extraction efficiency, and process efficiency were assessed. Further, the assay was successfully able to determine the disposition of orally administered metarrestin in patients from the lowest dose cohort (1 mg) for 48 h post-administration. Thus, the validated analytical method detailed in this work is simple, sensitive, and clinically applicable. [ABSTRACT FROM AUTHOR]

• ●The first methods for assay of EVT201 and its metabolites in human urine was set up. • ●The stability of metabolite M1 was investigated in kinds of matrices. • ●The methods utilized simple dilution, rapid analysis time and reproducible. • ●The methods have been validated following bioanalytical validation guidelines. • ●The methods were firstly applied to a mass balance study of EVT201 in human subjects. In this study, two simple and accurate LC–MS/MS methods were firstly developed and validated to quantify EVT201, a new partial GABA A receptor agonist used for the treatment of insomnia, and its metabolites comprising M1, M2, M3, M4 and M6 in human urine. The analytes in urine samples were determined after simple dilution, and ideal chromatographic separations were obtained on C18 columns using gradient elution. The assays were performed in MRM mode on AB QTRAP 5500 tandem mass spectrometry (ESI+). The concentration ranges (ng/mL) of analytes in human urine were as follows: EVT201, 1.00 to 36.0; M1, 1.40 to 308; M2, 2.00 to 72.0; M3, 5.00 to 1100; M4, 2.00 to 300; and M6, 2.80 to 420. The methods were fully validated including selectivity, carryover, matrix effect, recovery, linearity, accuracy, precision, dilution integrity and stability, and acceptable criteria were obtained. The methods were successfully applied to a mass balance study of EVT201. The results showed that the total cumulative urinary excretion rate of EVT201 and its five metabolites was 74.25 ± 6.50%, which suggested that EVT201 had high oral bioavailability, and urinary elimination was its major excretion pathway in human. [ABSTRACT FROM AUTHOR]

• An LC-MS/MS method was developed and validated for the quantitative determination of meta -iodobenzylguanidine (mIBG) concentrations in mouse plasma and tissues. • The developed method has a short run time and requires only micro-volumes of sample, allowing repeat blood sampling in the same animal. • The method was sufficiently sensitive and rapid to support large-scale pharmacokinetic studies in mice. • Comparative studies in wild-type mice and transporter-deficient mice revealed that the cardiac exposure to mIBG was dependent on functional expression of the organic cation transporter OCT3 and to a much lesser degree by OCT1, OCT2, and MATE1. A simple LC-MS/MS method for the quantitative determination of the norepinephrine analogue meta -iodobenzyl-guanidine (mIBG) was developed and validated for mouse plasma and tissues, including salivary gland and heart. The assay procedure involved a one-step solvent extraction of mIBG and the internal standard N-(4-fluorobenzyl)-guandine from plasma or tissue homogenates with acetonitrile. An Accucore aQ column was used to separate analytes using a gradient elution with a total run time of 3.5 min. Validation studies with quality control samples processed on consecutive days revealed values for intra-day and inter-day precision of < 11.3%, with values for accuracy ranging 96.8–111%. Linear responses were observed over the entire calibration curves range (up to 100 ng/mL), and the lower limit of quantification was 0.1 ng/mL, using sample volumes of 5 µL. The developed method was successfully applied to evaluate the plasma pharmacokinetics and tissue distribution of mIBG in wild-type mice and animals lacking the organic cation transporters OCT1, OCT2, OCT3, and/or MATE1 to further understand mechanisms contributing to drug distribution and elimination and causes of inter-individual pharmacokinetic variability. [ABSTRACT FROM AUTHOR]

• Ursolic acid sulfation metabolite was chemically synthesized and used as the authentic metabolite for ursolic acid. • An LC/MS method was developed and validated for UAS in human subcellular matrices. • The LC/MS assay can be applied for in vitro and in vivo drug metabolism and clinical disposition studies of ursolic acid. Ursolic acid (UA) is a naturally occurring pentacyclic triterpenoid that exhibits extensive pre-systemic metabolism from in vitro studies. However, there are no available authentic metabolite standards or validated analytical methods to quantitate UA metabolites. We have identified ursolic acid sulfate (UAS) as one of the major metabolites. We were able to identify and characterize its structure via comparison to the chemically synthesized UAS. A cyano (CN, 150 × 4.6 mm, 5 µm) column along with a gradient elution of acetonitrile and 0.08% (v/v) acetic acid, pH 3.0 were employed for chromatographic separation. Negative single ion recording mode (SIR) with electron-spray ionization (ESI) source at mass-to-charge ratios of 455.3 and 535.3 were monitored for UA and UAS, respectively. UAS linearity range was 0.010–2.500 µM. The absolute values of intra-day and inter-day precision (CV, %) and accuracy (DFN, %) were all below 15%. Thus, the analytical method has been validated in the human subcellular fractions to facilitate in vitro / in vivo DMPK and future clinical disposition studies on UA. [ABSTRACT FROM AUTHOR]

A high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method and an ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for determining eight coccidiostat (halofuginone, lasalocid, maduramicin, monensin, narasin, nigericin, robenidine and salinomycin) residues in beef were developed and compared. Samples were extracted with a mixture of acetic acid, acetonitrile and ethyl acetate and were then purified on a C 18 solid-phase extraction (SPE) column. The purified samples were analyzed by HPLC-MS/MS and UPLC-MS/MS, using 0.1% formic acid-water solution (A) and pure methanol (B) as the mobile phase. The samples were fractionated on a C 18 column using different gradient elution procedures, followed by qualitative analysis using a mass spectrometer operated in multiple reaction monitoring (MRM) mode with positive electrospray ionization; the external standard method was used for quantitation. At spiked levels that ranged from the limit of quantification (LOQ) to 100 μg/kg, the average recoveries were 71.96%–100.32% and 71.24%–89.24%, with relative standard deviations (RSDs) of 2.65%–12.38% and 2.98%–14.86% for UPLC-MS/MS and HPLC-MS/MS, respectively. The limits of detection (LODs) and LOQs of the eight coccidiostats were 0.14–0.32 μg/kg and 0.43–1.21 μg/kg, respectively, for UPLC-MS/MS analysis and 0.16–0.58 μg/kg and 0.53–1.92 μg/kg, respectively, for HPLC-MS/MS analysis. Both methods had good accuracy and precision, but UPLC-MS/MS had higher sensitivity than HPLC-MS/MS. [ABSTRACT FROM AUTHOR]

Chemical profiling of natural products by high performance liquid chromatography (HPLC) was critical for understanding of their clinical bioactivities, and sample pretreatment steps have been considered as a bottleneck for analysis. Currently, concerted efforts have been made to develop sample pretreatment methods with high efficiency, low solvent and time consumptions. Here, a simple and efficient online extraction (OLE) strategy coupled with HPLC–diode array detector–quadrupole time-of-flight tandem mass spectrometry (HPLC–DAD–QTOF-MS/MS) was developed for rapid chemical profiling. For OLE strategy, guard column inserted with ground sample (2 mg) instead of sample loop was connected with manual injection valve, in which components were directly extracted and transferred to HPLC–DAD–QTOF-MS/MS system only by mobile phase without any extra time, solvent, instrument and operation. By comparison with offline heat-reflux extraction for Fructus aurantii immaturus ( Zhishi ), OLE strategy presented higher extraction efficiency perhaps because of the high pressure and gradient elution mode. A total of eighteen flavonoids were detected according to their retention times, UV spectra, exact mass, and fragmentation ions in MS/MS spectra, and compound 9 , natsudaidain-3- O -glucoside, was discovered in Zhishi for the first time. It is concluded that the developed OLE–HPLC–DAD–QTOF-MS/MS system offers new perspectives for rapid chemical profiling of natural products. [ABSTRACT FROM AUTHOR]

Due to the difficulty and high cost for the preparation of triterpenes, one single standard for the simultaneous determination of multi-components (SSDMC) with high performance liquid chromatography (HPLC) is an advanced solution for multi-component analysis. Experiments were carried out to investigate the feasibility of SSDMC for the analysis of Ganoderma triterpenes, with external standard method (ESM) compared, and the samples of Ganoderma were classified by the content of Ganoderma triterpenes. The analysis was performed by using a Fortis Speed Core-C18 column (150 mm × 4.6 mm I.D., 2.6 μm) at gradient elution of 0.01% glacial acetic acid-water (V/V) and acetonitrile with diode array detection (252 nm), at a flow rate of 1 mL/min. The results showed that all calibration curves had good linearity (r 2 > 0.9999) within test ranges. The LOD and LOQ were lower than 2.52 ng and 6.43 ng, respectively. The RSD for intra-day and inter-day of the seventeen analytes were less than 3.12% at three levels, and the recoveries were 91.4–103.0%. The contents of other 16 triterpenes were determined with ganoderic acid A by SSDMC, which showed that there were few differences compared with the results obtained by ESM. Moreover, the classification of 25 different species and strains of Ganoderma by using the content of triterpenes intuitively reflected the distinction among Ganoderma . In summary, the developed method could be readily utilized as a method of quality evaluation for Ganoderma triterpenes. [ABSTRACT FROM AUTHOR]

As the stereoselective plasma protein binding is an important factor for the differences in pharmacokinetic or pharmacodynamic properties of chiral drugs, we elucidated the binding of ( R ) -/ ( S ) - HPABA to plasma protein. A UHPLC–MS/MS method was developed and validated to the quantitation and equilibrium dialysis method was applied to the separation of bound and unbound drugs. Protein binding reached equilibrium within 12 h of incubation at 37 °C. Liquid-liquid extraction with ethyl acetate was used for sample preparation. The separation was achieved through a Thermo Syncronis C 18 column (50 mm × 2.1 mm, 1.7 μm; Thermo, USA) using gradient elution at a flow rate of 0.4 ml/min. All of the recovery and matrix effect of the method met the requirements of analytical method validation. The plasma protein binding of ( R ) -/ ( S )-HPABA show significant species dependence in enantioselectivity. In addition, the results of molecular docking showed that the combining capacity of ( R ) - HPABA with HSA was higher than that of ( S ) - HPABA. [ABSTRACT FROM AUTHOR]

• Novel LC-MS/MS to determine semaglutide was developed for pharmacokinetic studies. • The method has superior sensitivity and fully validated in the plasma and brain. • Subcutaneous bioavailability of semaglutide was 76.65–82.85 % in rats. • Semaglutide is significantly distributed in hypothalamus among other brain regions. Semaglutide, one of the most potent glucagon-like peptide (GLP)-1 analogs, has widely been used to treat type II diabetes mellitus and obesity. Recent studies have shown that semaglutide also works on the brain, suggesting its potential utility for various diseases, including Parkinson's disease and Alzheimer's disease. This study aimed to develop a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of semaglutide in both plasma and brain to characterize the pharmacokinetics and brain distribution in rats. Semaglutide was extracted by simple protein precipitation with methanol from plasma and by solid phase extraction from brain tissue. Liraglutide was used as an internal standard. Gradient elution profiles with mobile phases comprising 0.1 % formic acid in water and acetonitrile were used for chromatographic separation. The lower limit of quantification (LLOQ) of the LC-MS/MS assay was 0.5 ng/mL for both rat plasma and brain. Intra- and inter-day accuracy ranged 89.20–109.50 % in the plasma and 92.00–105.00 % in the brain. Precision was within 8.92 % in the plasma and 7.94 % in the brain. Sprague-Dawley rats were given semaglutide by intravenous (IV, 0.02 mg/kg) and subcutaneous (SC, 0.1 and 0.2 mg/kg) injection. Plasma concentrations of semaglutide showed a multi-exponential decline with an average half-life of 7.22–9.26 hr in rats. The subcutaneous bioavailability of semaglutide was 76.65–82.85 %. The brain tissue to plasma partition coefficient (K p) value of semaglutide was estimated as <0.01. Among the different regions of the brain, semaglutide concentrations were significantly higher in the hypothalamus. The analytical method and pharmacokinetic information may be helpful toward a better understanding of the effect of semaglutide in the brain and further development of GLP-1 analogs for various brain diseases. [ABSTRACT FROM AUTHOR]

• A high-throughput method of amikacin in dried matrix spots by HPLC-MS/MS for therapeutic drug monitoring was established. • A stable isotope D5-amikacin internal standard was used instead of structure analogs to correct matrix effects in this method. • An excellent agreement of amikacin concentration in patients' samples was shown in DSS and serum methods. • DMS provides convenient choices for amikacin TDM. Individualized treatment of amikacin under the guidance of therapeutic drug monitoring (TDM) is important to reduce the occurrence of toxicity and improve clinical efficacy. In the present study, we developed and validated a simple and high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to determine the concentration of amikacin in dried matrix spots (DMS) which the matrix is serum. DMS samples were obtained by spotting volumetric blood onto Whatman 903® cards. Samples were punched into 3 mm diameter discs and extracted with 0.2 % formic acid in water. The HILIC column (2.1 mm × 100 mm, 3.0 µm) under gradient elution was applied, and the analysis time was 3 min per injection. The mass spectrometry transitions were m / z 586.3 → 163.0 for amikacin and m / z 591.4 → 163.1 for D5-amikacin. Full validation was conducted for DMS method, and the method was applied for the amikacin TDM and compared with serum method. The linearity was ranged from 0.5 to 100 mg/L. Both within-run and between-run accuracy and precision of DMS ranged from 91.8 % to 109.6 % and 3.6 % to 14.2 %, respectively. The matrix effect was 100.5 %–106.5 % of DMS method. Amikacin remained stable in DMS for at least 6 days at room temperature, 16 days at 4 °C, 86 days at −20 °C and −70 °C. A good agreement between the DMS method and serum method has been shown in Bland-Altman plots and Passing-Bablok regression. All of the results demonstrated that the DMS methods can be a favorable replacement for amikacin TDM. [ABSTRACT FROM AUTHOR]

• A sensitive APCI-LC-MS/MS method in dMRM analysis mode for simultaneous quantification of eleven nitrosamines in 17 min. • Dominantly DI water-based simple extraction within short time using a shaker at room temperature. • The validated method was applied to determine the concentrations of the nitrosamines in in active pharmaceutic ingredients (APIs) and final products (FPs) of valsartan and irbesartan. Nitrosamines (NAs) are potent genotoxic agents (GAs) in several animal species, and some are classified as probable or possible human carcinogens by the International Agency for Research on Cancer (IARC). In July 2018, angiotensin II receptor blockers (ARBs) which are used to treat high blood pressure have been recalled owing to contamination with NAs. In this study, a simple and sensitive method for the determination of eleven NAs in a single analysis was developed, using atmospheric pressure chemical ionisation source coupled liquid chromatography tandem mass spectrometer (LC-APCI-MS/MS). By performing the 17 min-run in dynamic multiple reaction monitoring (dMRM) mode, eleven NAs were separated on a Poroshell HPH C18 (4.6 × 150 mm, 2.7 µm) column with gradient elution implementing mobile phase A consisting of 0.2 % formic acid in water and mobile phase B consisting of methanol. The developed analytical method was successfully applied in both active pharmaceutical ingredients (APIs) and finished products (FPs) of valsartan and irbesartan with straightforward and effective extraction procedures. Good linearity with a correlation coefficient (R2) > 0.996 was achieved over the concentration in a range of 0.5–50 ng/mL. The limits of detection (LODs) ranged in 0.001–0.008 ppm and limits of quantitation (LOQs) ranged in 0.008–0.05 ppm of the method fulfilled thresholds of US Food and Drug Administration (US-FDA) and European Medicines Agency (EMA) for testing of GAs in valsartan and irbesartan. The accuracy of the proposed method ranged from 73.1 % to 115.2 % for APIs and the relative standard deviation (RSD %) was ≤11.3 while these validation parameters were in the range of 80.2–128.5 % and ≤ 10.6 for FPs, respectively. [ABSTRACT FROM AUTHOR]

• Volumetric Absorptive Microsampling (VAMS) is a biosampling technique with a small blood volume and minimum hematocrit effect. • Develop and validate an analytical method for quantifying favipiravir in VAMS using High Performance Liquid Chromatography – Photodiode Array with remdesivir as an internal standard. • Favipiravir is a prodrug of T-1105 made by modifying the pyrazine group as a COVID-19 therapy. Favipiravir is a prodrug of T-1105 made by modifying the pyrazine group as a COVID-19 therapy. During the pandemic, a safe and comfortable biosampling technique is needed for the subject or patient. Volumetric Absorptive Microsampling (VAMS) is a biosampling technique with a small blood volume and minimum hematocrit effect. The aims of this study were to develop and validate an analytical method for quantifying favipiravir extracted from VAMS using High Performance Liquid Chromatography – Photodiode Array with remdesivir as an internal standard. Analysis of favipiravir was performed using a C 18 column (Waters, Sunfire™ 5 µm; 250 × 4.6 mm), with injection volume of 50 µL, flow rate of 0.8 mL/min, column temperature 30 ℃, and wavelength 300 nm. The separation was conducted under gradient elution with mobile phase consists of acetonitrile-0.2 % formic acid-20 mM sodium dihydrogen phosphate pH 3.5 and run time 12 min. Sample preparation was carried out using a protein precipitation method with 500 µL of methanol as precipitating agent. Samples were mixed on vortex for 30 s, sonicated for 15 min, and centrifuged at 10,000 rpm for 10 min. Lower Limit of Quantification (LLOQ) obtained was 0.5 µg/mL and the calibration curve ranged from 0.5 to 160 µg/mL. Sensitivity, linearity, selectivity, carry-over, accuracy, precision, recovery, and stability were validated by the guideline from Food and Drug Administration 2018. The method developed has successfully met the full validation requirements by FDA 2018 with the LLOQ obtained was 0.5 µg /mL. [ABSTRACT FROM AUTHOR]

[Display omitted] • An LC-MS/MS method was developed for the simultaneous analysis of seven cardiovascular drugs in human serum. • A simple and economical organic extraction method was optimized for good extraction efficiency for all drugs. • The established method was fully validated for linearity, selectivity, accuracy, precision, carry-over, and matrix effects. • Stability was fully investigated to determine handling practices for the standard and serum samples. • The analytical method for cardiovascular drugs was successfully applied in various types of clinical samples. Therapeutic drug monitoring (TDM) of cardiovascular drugs is essential to improve treatment efficacy and minimize toxicity because of the usage of multiple drugs with a very limited therapeutic range and the high pharmacokinetic variation in patients. We developed and validated a reliable and economical liquid chromatography/tandem mass spectrometry (LC-MS/MS) method for the determination of seven cardiovascular drugs—procainamide, lidocaine, quinidine, deslanoside, digoxin, atorvastatin, and digitoxin—for clinical usage. Serum samples were prepared by simple protein precipitation with an organic solvent consisting of acetonitrile and methanol (2:1 v/v) and analyzed under optimized LC-MS/MS conditions. The chromatographic separations were accomplished within 15 min on a reversed-phase C18 column with a gradient elution of aqueous solvent and acetonitrile while maintaining 0.1 (v/v) % formic acid and 2 mM ammonium formate. The optimized MS/MS conditions in ESI-positive mode offered sufficient sensitivity for the seven cardiovascular drugs (LOQs between 0.5 and 1 ng/mL). This method was fully validated including linearity, selectivity, accuracy, precision, carry-over, and matrix effects. Additionally, stability under several conditions was tested to determine how to handle the standard solutions and serum samples. The seven cardiovascular drugs, simultaneously, were precisely and accurately analyzed in intra- and inter-day assays (RSD < 6 % and recovery between 96.3 and 102.8 %) using only two isotope-labeled internal standards (lidocaine-(diethyl-d10) and digoxin-21, 21, 22-d3). The presented method also showed good accuracy in analyzing the seven drugs in hyperlipidemia, hyperalbuminemia, and hyperglycemia serum, allowing it to be recommended as a common and routine analysis method for cardiovascular drugs in clinical practice. [ABSTRACT FROM AUTHOR]

• Automated online μSPE–LC–MS/MS developed and validated. • Developed method applied to detect cannabinoid metabolites in urine samples. • Isomeric separation achieved between Δ8-THCCOOH and Δ9-THCCOOH. • Metabolites detected in normalized concentrations in 54 urine samples. • Method useful to determine the legal boundaries for CBD, Δ8-THC, and Δ9-THC. In this study, an automated online micro-solid-phase extraction (μSPE)–liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the detection of metabolites of cannabidiol (CBD), Δ8-tetrahydrocannabinol (Δ8-THC), and Δ9-tetrahydrocannabinol (Δ9-THC), particularly 7-carboxy- cannabidiol (7-COOH-CBD), 11-nor-9-carboxy-Δ8-tetrahydrocannabinol (Δ8-THCCOOH), 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (Δ9-THCCOOH), and 11-nor-9-carboxy-Δ9- tetrahydrocannabinol-glucuronide (Δ9-THCCOOH-glu) in urine. An instrument top sample preparation (ITSP) cartridge was introduced to increase the sensitivity toward analytes and decrease the matrix effect of the urine. LC–MS/MS analysis was performed in the multiple-reaction monitoring mode, and the analytes were separated using an Acquity UPLC HSS T3 (2.1 × 100 mm, 1.8 µm) column and gradient elution with water containing 0.05 % acetic acid and methanol as the mobile phase. The calibration range was 0.5–200 ng/mL for all the analytes, with a correlation coefficient (r) of ≥0.996 and a weighting factor of 1/x2. The limits of detection for 7-COOH-CBD, Δ8-THCCOOH, Δ9-THCCOOH, and Δ9-THCCOOH-glu were 0.06, 0.02, 0.03, and 0.1 ng/mL, respectively. The intra- and inter-day accuracy ranged from −8.0 to 6.2 % and −7.3 to 7.8 % with a precision of ≤7.2 % and ≤6.2 %, respectively. The method was also validated for selectivity, recovery, matrix effect, stability, and dilution integrity. The developed method was successfully applied to the analysis of 78 urine samples, and 7-COOH-CBD, Δ8-THCCOOH, Δ9-THCCOOH, and Δ9-THCCOOH-glu were detected in 54 urine samples at normalized concentrations of 1.1, 0.6–939.1, 0.9–2595.0, and 1.3–527.6 ng/mg creatinine, respectively. [ABSTRACT FROM AUTHOR]

In this study, a simple and sensitive ultra performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS) method was developed and validated for quantitation of droxidopa in human plasma for the first time. A simple plasma protein precipitation method using methanol containing 3% formic acid was selected, and the separation was achieved by an Acquity UPLC™ BEH Amide column (2.1 mm × 50 mm, 1.7 μm) with a gradient elution using acetonitrile, ammonium formate buffer and formic acid as mobile phase. The detection of droxidopa and benserazide (internal standard, IS) was performed using positive-ion electrospray tandem mass spectrometry via multiple reaction monitoring (MRM). The precursor-to-product ion transitions m / z 214.2 → m / z 152.0 for droxidopa, and m / z 258.1 → m / z 139.1 for IS were used for quantification. A lower limit of quantification of 5.00 ng/mL was achieved and the linear curve range was 5.00–4000 ng/mL using a weighted (1/x 2 ) linear regression model. Intra-assay and inter-assay precision was less than 10.2%, and the accuracy ranged from 0.1% to 2.1%. Stability, recovery and matrix effects were within the acceptance criteria recommended by the regulatory bioanalytical guidelines. The method was successfully applied to a pharmacokinetic study of droxidopa in healthy Chinese volunteers. [ABSTRACT FROM AUTHOR]

• Basic hydrophilic polypeptides eluted with carbon dioxide mobile phases. • Best results obtained with methanesulfonic acid and water in methanol co-solvent. • Strong impact of the nature of counter-ion on chromatographic quality. • Strong impact of dilution solvent and injection volume on peak shapes. • Best results obtained with dilution solvent identical to co-solvent composition. The chromatographic analysis of long-chain hydrophilic therapeutic peptides, with molecular weight mostly in the 3500–4500 Da range (31–34 amino acids), is explored with pressurized CO 2 in the mobile phase. The optimal method was obtained on a Torus 2-PIC column, with a gradient elution of 50–90% co-solvent in CO 2 , which is relevant of enhanced-fluidity liquid chromatography (EFLC). Both UV (210 nm) and mass spectrometric detection modes were employed to assess the purity of the major peak and its resolution from impurities. Ten out of the eleven peptides in this set were basic, thus they were analyzed as acetate or trifluoroacetate salts. As significant peak distortion was observed in some cases, thorough examination of dilution solvent and injection volume was conducted to improve peak shape and resolution from impurities. Finally, the best injection volume was 1 µL, as any other volume (smaller or larger) yielded distorted peaks, and the best dilution solvent composition was the same as the mobile phase co-solvent (methanol comprising 5% water and 0.1 % methanesulfonic acid). However, not all peptide salts were fully soluble in this solvent so other alternatives (including more water in the dilution solvent), offering adequate dissolution but slightly inferior chromatographic performance should be chosen in such cases. [ABSTRACT FROM AUTHOR]

• Method for isolation of carpaine is proposed. • Presence of dihydroxy derivative of carpaine is reported for the first time. • Simple and fast HPLC method for quantification of carpaine is described. • Retention time of carpaine by given HPLC method is less than the reported method. • Proposed HPLC method can be easily scaled up for preparative isolation of carpaine. Carpaine, a major alkaloid of Carica papaya leaves, is widely studied for its anti-thrombocytopenic activity. The objective of present work was to isolate carpaine from dried leaves of Carica papaya. Isolation of carpaine was carried out by solvent extraction techniques followed by column chromatography by gradient elution using dichloromethane: methanol (80:20, v/v) as eluent. Dihydroxy derivative of carpaine was also identified for the first time from leaves of Carica papaya. Structures of carpaine and dihydroxy derivative of carpaine were confirmed by LC-MS/MS followed by IR studies. Fast HPLC method was developed using Sunniest C18 column (150 mm × 4.6 mm) as stationary phase and water (pH 8.5): acetonitrile (60: 40, v/v) as mobile phase at flow rate of 1.0 mL/min. Carpaine was eluted with mean retention time of 6.017 min. The developed HPLC method was used to estimate carpaine in extract and marketed formulations containing Carica papaya leaves. [ABSTRACT FROM AUTHOR]

• First reported of determination of Sinomenine derivative SWX in rats plasma by HPLC-MS/MS. • First reported of pharmacokinetic study of Sinomenine derivative SWX after intragastric and intravenous administration in rats. • The method was used to analyze samples from pharmacokinetics studies in Sprague–Dawley rats. The goal of this work was to develop a sensitive and accurate method based on high performance liquid chromatography with tandem mass spectrometry (LC-MS) detection for determining the concentration of the Sinomenine derivative SWX in plasma and tissue of rat. Chromatographic separation was achieved on an Agilent SB-C18 (2.1*50 mm, 2.7 µm) column. The mobile phase consisted of acetonitrile (solvent A) and 0.2 % formic acid in water (solvent B) with gradient elution as follows: 0–3 min, 50–90 % A, 3 min–3.01 min, 90–50 % A, 90 % A in 3.01 min–10 min. The flow rate was set to 0.3 mL/min. The column temperature was 40 °C, and the sample injection volume was 10 µL. The calibration curve had good linearity in the range of 10 ng/mL to 600 ng/mL. Biological samples were prepared by protein precipitation with acetonitrile. The area under the curve (AUC) and the peak drug concentration (C max) were linearly related to SWX dose. After oral administration of 25, 50 and 100 mg/kg SWX and intravenous administration of 0.5 mg/kg SWX, respectively, the absolute bioavailability of SWX was estimated as 12.4 %, 12.2 % and 10.6 %. SWX was widely distributed in heart, liver, spleen, lung, kidney, brain, breast, and fat, especially in lung, liver, and spleen. Distribution of SWX in brain suggested that it can pass the blood–brain barrier. The method established in this study has the advantages of high recovery and good reproducibility, and was suitable for the determination of the content of Sinomenine derivatives, providing a reliable scientific tool for carrying out pharmacokinetics research, providing a reliable scientific resource. [ABSTRACT FROM AUTHOR]

• First method for therapeutic drug monitoring in Paxlovid-treated COVID-19 patients. • Simultaneous quantification of nirmatrelvir and ritonavir (constituents of Paxlovid) • Sensitive and selective determination by LC-MS/MS. • Plasma sample preparation by simple protein precipitation. Nirmatrelvir is an antiviral agent active against SARS-CoV-2, the virus causing the pandemic disease COVID-19. It is administrated in combination with the protease inhibitor ritonavir, which acts in case of COVID-19 mainly as enzyme blocking agent preventing the premature metabolic elimination of nirmatrelvir. The combination of the two drugs in separate tablets is marketed under the brand name Paxlovid® and shows good effectivity in preventing the progression of COVID-19 to severe disease state. In this work, we described a LC-MS/MS method for the simultaneous quantification of nirmatrelvir and ritonavir in human plasma of patients treated for COVID-19 with Paxlovid®. After addition of D 6 -ritonavir as internal standard, plasma proteins were precipitated by the addition of methanol. The analytes were separated by gradient elution on a C18-column and were detected by tandem mass spectrometry. Calibration functions were linear in the ranges of 10 – 10000 ng/mL for nirmatrelvir and 2 – 2000 ng/mL for ritonavir. Inter-day and intra-day precision and accuracy was better than 15 % in the quality control samples and better than 20 % at the LLOQ. The method was successfully applied on samples of hospitalized patients treated for COVID-19 and proved to be capable in supporting therapeutic drug monitoring (TDM). [ABSTRACT FROM AUTHOR]

• The logic behind the implementation of shifting gradient programs is explained and exemplified. • The pros and cons compared to conventional approach with full gradients are discussed. • Main advantages include an increase in retention space coverage and peak capacity. • Major drawbacks include reduced peak heights and poor run-to-run repeatability. In on-line comprehensive two-dimensional liquid chromatography (LC × LC), the combination of similar chromatographic modes such as reversed-phase liquid chromatography × reversed-phase liquid chromatography (RPLC × RPLC) usually leads to the partial occupation of the available separation space. A possible solution to circumvent this issue may be to dynamically adjust the gradient elution in the second dimension (2D) throughout the LC × LC analysis. This allows the gradient elution to be tailored to the elution conditions of each fraction instead of using a conventional full gradient program in which the same gradient profile is repeated for each 2D-fraction. In this study, an online RPLC × RPLC method is optimized with shifting gradients in 2D. The logic behind implementing such programs in on-line LC × LC is explained. The optimized method consists of a combination of segmented and shifted gradient modes. It is shown that the retention space coverage can be increased by 50 % compared to a conventional full gradient program, leading to a significant increase in peak capacity (about 35 %). However, such an increase comes at the expense of larger peak widths in 2D and thus lower peak intensities. It is shown here that the use of shifting gradients raises another serious issue related to the repeatability of retention times between two successive 2D-separations. [ABSTRACT FROM AUTHOR]

• Multi-lab, multi-vendor validation of trace quantitation using sub-2 µm d p. • Characterize design differences between the major vendors in SFC. • All SFC's have excessive extra-column dispersion that is hard to fix. • Mass flow and composition vary between vendors with same set-points. • Critical need for dynamic compressibility compensation. Supercritical fluid chromatography has contributed significantly to chiral method development and semi-preparative purification in drug discovery, where sensitivity was not an issue. Now, analytical scale SFC's have been validated using a multi-vendor, inter-lab study for quantitation of achiral trace pharmaceutical impurities using sub-2 µm particles, with gradient elution, and UV detection. This should significantly increase the penetration of SFC into both chiral and achiral QA/QC applications. However, there is still work to be done. Extra-column dispersion is no better than previous generations, yet the technique is often superficially called "ultra high performance" SFC (UHPSFC), simply because sub-2 µm packings are used. Dispersion is far too high for use with sub-2 µm particles, requiring extensive hardware modification to use such particles with high efficiency. However, the most common means of reducing system dispersion in ultra high performance liquid chromatography (UHPLC) results in distortion of kinetic performance in SFC. Vendors need to specify or provide standardized plumbing schemes that allow the full use of sub-2 µm fully porous, and sub-3 µm superficially porous particles, with reduced plate height, h ≈ 2 when k' ≈ 2. There is no consensus on how to best perform dynamic compressibility compensation, since each vendor uses different CO 2 pump head temperatures, resulting in subtle differences in flow and composition between vendors. [ABSTRACT FROM AUTHOR]

• A novel LC-MS/MS method for the quantification of ceritinib, osimertinib, and crizotinib in human plasma. • A single-step protein precipitation was adopted in this method, which was free of matrix effect. • The method can be used for analyzing serum samples and is also a hemolysis and lipemia "insensitive" method. • The run time of 4.0 min was relatively shorter than previous methods. • The method is sensitive, time- and energy- efficient, providing an analytical tool for routine TDM. The objective of this study was to develop and validate a simple, rapid, and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of three tyrosine kinase inhibitors (ceritinib, osimertinib, and crizotinib) in human plasma using a single-step protein precipitation extraction. Chromatographic separation was achieved using a Waters X Bridge C18 (2.1 mm × 100 mm, 3.5 µm) and gradient elution with 0.2 % formic acid in water and acetonitrile. The total run time was 4.0 min, and the injection volume was 5 μL. The analytes were detected in the multiple reaction monitoring mode using electrospray ionization with positive ion mode. The m / z transitions of ceritinib, osimertinib, crizotinib and nilotinib were 558.0 → 433.2, 500.0 → 72.1, 450.0 → 259.3, and 530.0 → 289.1, respectively. The method was linear in the range of 2–500 ng/mL with lower limit of quantification of 2 ng/mL. Based on the guidelines on bioanalytical methods by the FDA, the validation studies demonstrated that the three analytes were both precise and accurate at four concentration levels, and the coefficient of variation was < 10.59 % and accuracy was > 88.26 %. We present a simple, rapid, and sensitive method for the simultaneous quantification of ceritinib, osimertinib, and crizotinib in human plasma by LC-MS/MS, which could be used in routine therapeutic drug monitoring. [ABSTRACT FROM AUTHOR]

• E7130 is a novel microtubule inhibitor and a promising ameliorator of the tumor microenvironment. • An analytical method was developed to quantify E7130 in mouse plasma. • A sensitive method was established at a LLOQ of 0.2 ng/mL for plasma microsamples (10 µL). • HRMS provided a high selectivity for quantification of E7130 in the biological matrix prepared via protein precipitation. E7130 is a novel microtubule inhibitor and a promising tumor microenvironment ameliorator. Since the amount of the administration in preclinical study is very small due to the high potency of E7130, this study aimed to establish a sensitive analytical method to measure E7130 concentration in mouse plasma samples obtained via microsampling. A sensitive and validated method was developed based on ultra-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC–HRMS). Chromatographic separation was achieved using a Waters ACQUITY UPLC BEH C18 1.7 µm (2.1 × 50 mm) column. Mobile phase A comprised 0.1% formic acid and 10 mM ammonium formate in water, and mobile phase B was methanol. A gradient elution was applied at a flow rate of 0.5 mL/min. The calibration curve drawn was linear in the 0.2–100 ng/mL E7130 concentration range for mouse plasma microsamples (10 µL). Analytical results demonstrated good precision (<6.7%) and accuracy (88.5%–100.0%) in E7130 quantitation, indicating that UHPLC–HRMS is a useful method for pharmacokinetic analysis and a valuable approach for the quantitation of hardly fragmented compounds. [ABSTRACT FROM AUTHOR]