Search

• Developed & validated LC-MS/MS to detect T-CPT-11, NE-CPT-11, and SN-38 in plasma. • Reproducible SPE-HLB protocol robustly separated NE-CPT-11 from plasma. • Extensive studies performed to ensure NE-CPT-11 stability in plasma during storage. • Developed methods applied to clinical pharmacokinetic study of Onivyde® in a pediatric patient. • First report of NE-CPT-11 concentrations in a pediatric patient with recurrent solid malignancy. Preclinical studies have demonstrated that liposomal irinotecan (CPT-11), a topoisomerase I inhibitor, has broad activity against adult cancers, including pancreatic, gastric, colon, lung, glioma, ovarian, and breast cancer. Encapsulation of irinotecan into liposomes can modify its pharmacokinetic properties dramatically. Also, the pharmacokinetic profiles of liposomal drug formulations are not fully understood; thus, bioanalytical methods are needed to separate and quantify nonencapsulated vs. encapsulated concentrations. In this study, two robust, specific, and sensitive LC-MS/MS methods were developed and validated to separate and quantify the nonencapsulated CPT-11 (NE-CPT-11) from the sum-total CPT-11 (T-CPT-11) and its major metabolite, SN-38, in human plasma after intravenous administration of liposomal irinotecan. NE-CPT-11 and SN-38 were separated from plasma samples by using solid-phase extraction, and T-CPT-11 was measured by protein precipitation. The liposomal CPT-11 formulation was unstable during sample storage and handling, resulting in elevated NE-CPT-11 concentration. To improve the stability of liposomal CPT-11, a cryoprotectant solution was added to human plasma samples prior to storage and processing. CPT-11, SN-38, and their respective internal standards, CPT-11-d10 and SN-38-d3, were chromatographically separated on a reversed-phase C 18 analytical column. The drugs were detected on a triple quadrupole mass spectrometer in the positive MRM ion mode by monitoring the transitions 587.3 > 124.1 (CPT-11) and 393.0 > 349.1 (SN-38). The calibration curves demonstrated a good fit across the concentration ranges of 10–5000 ng/mL for T-CPT-11, 2.5–250 ng/mL for NE-CPT-11, and 1–500 ng/mL for SN-38. The accuracy and precision were within the acceptable limits, matrix effects were nonsignificant, recoveries were consistent and reproducible, and the analytes were stable under all tested storage conditions. Finally, the LC-MS/MS methods were successfully applied in a phase I clinical pharmacokinetic study of nanoliposomal irinotecan (Onivyde®) in pediatric patients with recurrent solid malignancies or Ewing sarcoma. [ABSTRACT FROM AUTHOR]

• First reported determination of eupafolin by UPLC-MS/MS in rat plasma. • First reported pharmacokinetics of eupafolin in rats. • The bioavailability of eupafolin was relatively low (8.3%). In this experiment, a rapid and highly sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) technology was established and validated for the quantitation and pharmacokinetic analysis of eupafolin in rat plasma, utilizing licochalcone B as internal standard (IS). After liquid–liquid extraction of the analyte samples by ethyl acetate, chromatographic separation was achieved using a UPLC HSS T3 column under gradient elution conditions, with the mobile phase consisting of acetonitrile and water (with 0.1 % formic acid). Eupafolin was quantified by multiple reaction monitoring (MRM) in electrospray positive-ion mode (ESI+), employing the mass transition m / z 315.2 → 300.3 for eupafolin and m / z 285.4 → 270.3 for IS. Eupafolin demonstrated excellent linear relationship (r > 0.99) over the concentration range of 1.25–1250 ng/mL, with the lower limit of quantification (LLOQ) of the UPLC-MS/MS assay determined as 1.25 ng/mL. Method validation followed the bioanalytical method validation criteria outlined by the FDA. The accuracy of eupafolin ranged from 86.7 % to 111.2 %, and the precision was less than 12 %. The matrix effect was observed at 92.8 %-98.6 %, while the recoveries exceeded 83.2 %. The established UPLC-MS/MS assay was successfully employed for the pharmacokinetic evaluation of eupafolin in rats. The half-lives (t 1/2z) were determined to be 1.4 ± 0.4 h and 2.5 ± 1.4 h for intravenous and oral administration, respectively. Notably, the bioavailability of eupafolin was relatively low (8.3 %). The optimized UPLC-MS/MS technology showed highly sensitive, selective, and effective, rendering it suitable for the pharmacokinetics of eupafolin in preclinical practice. [ABSTRACT FROM AUTHOR]

• A high efficacy and reproducible LC-MS/MS analytical method for rapid quantification of FP-025 (Aderamastat) in human plasma have been developed and validated. This LC-MS/MS bioanalytical method has been demonstrated and discussed for the first time. • Quantification method was successfully validated with respect to linearity, sensitivity, accuracy, precision, dilution, selectivity, hemolyzed plasma, lipemic plasma, batch size, recovery, matrix effect, stability and carry-over. Aderamastat (FP-025) is a small molecule, selective matrix metalloproteinase (MMP)-12 inhibitor, under development for respiratory conditions which may include chronic inflammatory airway diseases and pulmonary fibrosis. To support evaluation of the pharmacokinetic parameters of Aderamastat in humans, we developed and validated a high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) analytical method for the quantification of Aderamastat in human plasma. This assay was validated in compliance with the Food and Drug Administration (FDA) Good Laboratory Practice Regulations (GLP) and European Medicines Agency (EMA) guidelines. K 2 EDTA human plasma samples were spiked with internal standard, processed by liquid–liquid extraction, and analyzed using reversed-phase HPLC with Turbo Ion Spray® MS/MS detection. Separation was done using a chromatographic gradient on 5 µm C6-Phenyl 110 Å, 50*2 mm analytical column at a temperature of 35 °C. The LC-MS/MS bioanalytical method, developed by QPS Taiwan to determine the concentration of Aderamastat in K 2 EDTA human plasma, was successfully validated with respect to linearity, sensitivity, accuracy, precision, dilution, selectivity, hemolyzed plasma, lipemic plasma, batch size, recovery, matrix effect, and carry-over. These data indicate that the method for determination of Aderamastat concentrations in human K 2 EDTA plasma can be used in pharmacokinetics studies and subsequent clinical trials with Aderamastat. Authors declare that, this novel data is not published and not under consideration for publication by another journal than this journal. All data will be made available on request. [ABSTRACT FROM AUTHOR]

• An LC-MS/MS method for VAMS was developed for quantifying Lurasidone using a liquid–liquid extraction technique. • The method was validated over a concentration range of 5.0 to 1200.0 ng/mL and achieved acceptable precision and accuracy. • The recovery for analyte from VAMS was approximately 40% at four different concentrations. • A comparative study between both conventional (plasma) and microsampling (DBS and VAMS) techniques. • The study supports the advantages of microsampling while maintaining scientific judgement. The ecological impact of biological, chemical, and analytical research practices, including toxic reagents and biohazardous waste, has led to the development of alternative sampling and extraction techniques for bioanalysis. Microsampling (sample volume < 50 µL) aligns with the 3Rs principle, allowing multiple sampling points from the same animal at different time points and improving animal welfare. A bioanalytical method was developed to investigate factors related to bioanalytical challenges and the implementation of microsampling techniques. An LC-MS/MS method for Volumetric Absorptive Microsampling (VAMS), 20 µL, was developed for quantifying Lurasidone using a liquid–liquid extraction technique. The method uses a C18, Phenomenex column for chromatographic separation and a mobile phase composition of Methanol, Acetonitrile, and Water with 0.1 % HFBA. The method was validated over a concentration range of 5.0 to 1200.0 ng/mL and achieved acceptable precision and accuracy. The recovery for analyte from VAMS was approximately 40% at four different concentrations and is consistent (%CV < 15), with no significant differences among HCT levels. The matrix factor ranged between 85.00 and 115.00 %, showing no substantial issues with reduced or enhanced signal. The stability data showed no significant degradation of LUR in VAMS samples when stored at room temperature for 15 days. The newly established method for Lurasidone confirmed the use of VAMS sampling method and its analysis on LC-MS/MS. Further, the data obtained from microsampling techniques was compared with conventional (plasma) technique, as proof-of-concept, and it confirms the agreement between the two methods. The study supports the advantages of microsampling in protecting the environment and animals while maintaining scientific judgement. [ABSTRACT FROM AUTHOR]

• Four analytes are detected simultaneously in the DBSs by UPLC-MS/MS. • Only only 12 μL of whole blood are used for sample preparation. • The analysis intervals are only three minutes. Homocysteine, methionine, methylmalonic acid and 2-methylcitric acid are clinically relevant markers in the methionine, propionate, and cobalamin metabolism. This study aimed to develop and validate an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for simultaneously determining total homocysteine, methionine, methylmalonic acid and 2-methylcitric acid in dried blood spots. Three 3.2 mm discs were punched from each calibrator, quality control, and sample dried blood spot into a 96-well U-plate. Each sample was spiked with internal standards and extracted. Then the supernatant was transferred to another 96-well U-plate. After nitrogen drying, the dried residues were reconstituted, centrifuged, and the resulting supernatant was transferred to another 96-well plate for analysis. The method was performed using UPLC-MS/MS within 3 min, validated according to guidance documents, and applied to 72 samples from confirmed patients with methionine, propionate, and cobalamin metabolism disorders. The UPLC-MS/MS method provided satisfactory separation of the four analytes. The R2 values were ≥ 0.9937 for all analytes. The recoveries ranged from 94.17 to 114.29 %, and the coefficients of variation for intraday and interday precision were 0.19 % to 5.23 % and 1.02 % to 6.89 %, respectively. No significant carry-over was detected for the four analytes, and most of confirmed samples exhibited biomarker patterns characteristic of the relevant disorders. A simple and fast UPLC-MS/MS method was successfully developed, validated, and applied to clinical samples for the simultaneous determination of total homocysteine, methionine, methylmalonic acid, and 2-methylcitric acid in dried blood spots. [ABSTRACT FROM AUTHOR]

• MrgX2-HALO-tag/CMC-HPLC-MS system was used to screen MrgX2-targeted ligands from A. capillaris extract. • Interactions between ligands and MrgX2 were studied by CMC model and molecular docking. • Seven chlorogenic acid analogs in A. capillaris extract were screened out as potential pseudo‑allergic compounds. Artemisia capillaris Thunb. (A. capillaris) is a well-known traditional Chinese herbal medicine with a wide range of pharmacological effects, such as soothing the liver and gallbladder, heat clearance, and detoxifying. Hence, its extract is commonly added to various traditional Chinese medicine formulas. Traditional Chinese medicine injection (TCMI) is a mature pharmaceutical dosage form developed using TCM theory combined with modern science and technology. Notably, allergic reactions, especially pseudo‑allergic reactions (PARs), greatly limited the use of these injections. Therefore, screening pseudo‑allergic components in A. capillaris extract is clinically significant. In the present study, we proposed a two-dimensional screening and identification system based on mas-related G protein-coupled receptor X2-HALO-tag/cell membrane chromatography (MrgX2-HALO-tag/CMC) high performance liquid chromatography mass spectrometry (HPLC-MS); seven potential active components were screened from 75 % ethanol extract of A. capillaris : NCA, CA, CCA, 1,3-diCQA, ICA-B, ICA-A, and ICA-C. The receptor-ligand interactions between these seven compounds and MrgX2 protein were analyzed using frontal analysis and molecular docking technology. Furthermore, a mast cell degranulation-related assay was used to assess the pseudo‑allergic activity of these compounds. The screened compounds can serve as ligands of MrgX2, and this study provides a research basis for pseudo‑allergic reactions caused by TCMIs containing A. capillaris. [ABSTRACT FROM AUTHOR]

• A sensitive and accurate method was developed to measure tetrodotoxin in human biological sample. • Sample cleanup using online MCX SPE was of good purification and less labour-intensive. • The matrix effects in plasma and urine matrices were significantly improved with on-line cleanup. • The method had been successfully applied to the detection of actual poisoning samples. An efficient technique for quantitative analysis of tetrodotoxin (TTX) in human plasma and urine has been developed, which combines liquid chromatography-tandem mass spectrometry (LC-MS/MS) with online MCX solid phase extraction (SPE) cleanup. Sample preparation, including extraction with acetonitrile containing 0.5 % acetate acid, centrifugation, and filtration, was followed by online SPE cleanup. The whole run-time was less than 15 min, including online cleanup, chromatographic separation, and re-equilibration of the online SPE − LC-MS/MS system. The parameters of sample extraction, purification, separation, and detection were optimized. The matrix-matched internal standard calibration standard curves with linear regression coefficients larger than 0.9990 were established for quantification. The LOD and LOQ for this approach were determined to be 0.1 ng/mL and 0.3 ng/mL, respectively. The recoveries for varied concentrations of TTX in human plasma and urine were 84.9–104.2 % and 89.2–109.6 %, respectively. The matrix effects of TTX in human plasma and urine matrices were 85.5 % and 74.3 %, respectively, and both the inter- and intra-day precision values were less than 9.5 %. This analytical method was successfully employed for detecting TTX in biological samples from a poisoned patient who accidentally ingested the nassarius glans. [ABSTRACT FROM AUTHOR]

• A nano liquid chromatography-orbitrap fusion mass spectrometer for peptides was firstly established. • The highly specific and thermal sensitive peptides in milk heating process was screened and identified. • This study provides new insights for distinguishing overheated pasteurized milk. Milk is one of the most widely consumed foods globally. To protect consumer interests, it is essential to establish an analytical method to detect the degree of heating in milk. A novel approach using nano liquid chromatography-orbitrap fusion mass spectrometer was developed for screening and identifing thermally sensitive peptides markers in the milk heating process (below 100 °C). This method integrates untargeted proteomics and chemometric tools to analyze protein quantitation data from differently heat-treated milk. Thirteen potential markers were screened out and identified, and further confirmed using by standard substances. Then, the accurate concentrations of 13 potential markers determined by isotope-dilution ultra-performance liquid chromatography-tandem triple quadrupole mass spectrometry were further mining the highly specific and thermally sensitive peptides markers. And Four peptides—INLFDTPLETQYVR, FELLGCELNGCTEPLGLK, QFQFIQVAGR, and GEADALNLDGGYIYTAGK—were selected as marker peptides to differentiate normal pasteurized milk from overheated pasteurized milk. The concentrations of INLFDTPLETQYVR ranges from 150 ± 11 µg/L to 350 ± 23 µg/L, while the concentrations of FELLGCELNGCTEPLGLK ranges from 40 ± 5 µg/L to 92 ± 3 µg/L, can distinguish normal pasteurized milk from overheated pasteurized milk. QFQFIQVAGR indicates overheated pasteurized milk at 230 ± 21 µg/L, and GEADALNLDGGYIYTAGK signifies 750 ± 43 µg/L. This study provides new insights for distinguishing overheated pasteurized milk. [ABSTRACT FROM AUTHOR]

• Quantification of amiodarone, desethylamiodarone and mexiletine in one single run. • Efficient and robust LC-MS/MS method for guiding anti-arrhythmic therapy in ventricular arrhythmia. • The validated method was applied and evaluated in clinical practice for therapeutic drug monitoring. Amiodarone and mexiletine are used for ventricular arrhythmias, for which a combination therapy of both anti-arrhythmic drugs (AADs) is not uncommon. Therapeutic drug monitoring (TDM) can be beneficial for clinical guidance of therapy, especially to correctly identify adverse events. Desethylamiodarone, the active metabolite of amiodarone, accumulates over time and is associated with serious adverse events. Therefore, simultaneous TDM for amiodarone, desethylamiodarone and mexiletine is advantageous in clinical practice. The presented LC-MS/MS method was validated for selectivity, matrix effect, linearity, accuracy, precision, carry-over and stability. The method was continuously evaluated during eight months of clinical use. The method was shown to be linear within the measured range of 0.1 to 10 mg/L for each component. The matrix effect was considered negligible. No interfering responses were found for amiodarone, desethylamiodarone and the isotopic-labeled internal standards. A constant and reproducible within-run contribution of 45.3 %, originating from the system, was identified for mexiletine. The systemic contribution to the peak area of the lowest quantifiable concentration of mexiletine affected the selectivity and carry-over effect measurements. Multiple measurements showed that regression adjusted concentrations were accurate and reproducible, indicating calibration correction was applicable. Sample stability was found to be within limits for all storage conditions and freeze–thaw cycles. Furthermore, long-term method evaluation with external controls resulted in stable measurements with a percentage coefficient of variance between 1.3 % and 6.3 %. The presented practical and reliable method is applicable for clinical TDM and will allow clinical practitioners to guide drug therapy of amiodarone and mexiletine. [ABSTRACT FROM AUTHOR]

• A UPLC-ICPMS method for quantification of intact oxaliplatin was developed. • The UPLC runtime was 2.5 min and injection volume was 1 µL. • ICPMS at m / z 195 detected intact oxaliplatin and other platinum species. • The method was validated for quantification of intact oxaliplatin in human plasma. • This efficient, specific and reliable method will inform cancer chemotherapy. Interest is increasing in the use of different liquid chromatography techniques coupled online to mass spectrometry for the quantification of platinum anticancer drugs in human plasma to inform cancer chemotherapy. We developed, validated and studied the application of a method for quantification of intact oxaliplatin in human plasma using ultra high performance liquid chromatography hyphenated to inductively coupled plasma mass spectrometry (UHPLC-ICP-MS). Plasma samples were processed instantly after collection from patients to preserve oxaliplatin speciation by methanol-deproteinization, and storage of diluted supernatants (plasma:methanol 1:2 v/v) at −80 °C. UHPLC separation of intact oxaliplatin and internal standard (carboplatin) was achieved using a C18 column and linear gradient mobile phase (Mobile phase A: water-methanol (97:3 v/v), 0.075 mM sodium dodecyl sulfate, 9.79 nM thallium adjusted to pH 2.5 with trifluoromethanesulfonic acid; Mobile phase B: 100 % methanol (v/v)) with ICP-MS detection to monitor platinum and thallium at m / z 195 and 205, respectively. The limit of quantification was 50 nM in methanol-deproteinized diluted plasma (1:2 v/v). Linearity was established for calibration standards ranging from 50 to 500 nM made in methanol-deproteinized diluted plasma (1:2 v/v), and for dilution of higher concentration samples in blank matrix containing internal standard (final dilution 1:29 v/v). Intra-day and inter-day accuracy ranged from 96.8 to 103 % of nominal concentration and precision from 0.62 to 2.49 % coefficient of variation. Recovery was complete and a matrix effect confirmed the requirement for matrix-matched standards. Intact oxaliplatin was stable during storage for at least 473 days, and during analysis, in methanol-deproteinized diluted plasma (1:2 v/v). The method was applied to determining the plasma concentrations of intact oxaliplatin in patients undergoing cancer chemotherapy, and studies of oxaliplatin degradation in vitro. This improved method based on UHPLC-ICP-MS will allow more specific, efficient and reliable quantification of intact oxaliplatin in human plasma. [ABSTRACT FROM AUTHOR]

• We have developed a rapid and stable method for the simultaneous quantification of choline, L-carnitine, betaine, trimethylamine, trimethylamine oxide and creatinine in plasma, liver and feces of biological samples. • This method was applied to quantify choline, L-carnitine, betaine, trimethylamine, trimethylamine oxide and creatinine levels in plasma, liver and feces of hyperlipidemic rats. • The effects of Rosa Roxburghii Tratt juice on choline, L-carnitine, betaine, trimethylamine, trimethylamine oxide and creatinine in plasma, liver and feces of hyperlipidemic rats were investigated. Due to the close correlation between choline, L-carnitine, betaine and their intestinal microbial metabolites, including trimethylamine (TMA) and trimethylamine N-oxide (TMAO), and creatinine, there has been an increasing interest in the study of these compounds in vivo. In this study, a rapid stable isotope dilution (SID)-UHPLC-MS/MS method was developed for the simultaneous determination of choline, L-carnitine, betaine, TMA, TMAO and creatinine in plasma, liver and feces of rats. The method was validated using quality control (QC) samples spiked at low, medium and high levels. Second, we applied the method to quantify the effects of Rosa Roxburghii Tratt juice (RRTJ) on plasma, liver, and fecal levels of choline, L-carnitine, betaine, TMA, TMAO, and creatinine in high-fat diet-induced hyperlipidemic rats, demonstrating the utility of the method. The limits of detection (LOD) were 0.04–0.027 µM and the limits of quantification (LOQ) were 0.009–0.094 µM. The linear ranges for each metabolite in plasma were choline1.50–96 µM; L-carnitine: 2–128 µM; betaine: 3–192 µM; TMA: 0.01–40.96 µM; TMAO: 0.06–61.44 µM and creatinine: 1–64 µM (R2 ≥ 0.9954). The linear ranges for each metabolite in liver were Choline: 12–768 µM; L-carnitine: 1.5–96 µM; betaine: 10–640 µM; TMA: 0.5–32 µM; TMAO: 0.02–81.92 µM and creatinine: 0.2–204.8 µM (R2 ≥ 0.9938). The linear ranges for each metabolite in feces were choline: 1.5–96 µM; L-carnitine: 0.01–40.96 µM; Betaine: 1.5–96 µM; TMA: 1–64 µM; TMAO: 0.02–81.92 µM and Creatinine: 0.02–81.92 µM (R2 ≥ 0.998). The intra-day and inter-day coefficients of variation were < 8 % for all analytes. The samples were stabilized after multiple freeze–thaw cycles (3 freeze–thaw cycles), 24 h at room temperature, 24 h at 4 °C and 20 days at −80 °C. The samples were stable. The average recovery was 89 %-99 %. This method was used to quantify TMAO and its related metabolites and creatinine levels in hyperlipidemic rats. The results showed that high-fat diet led to the disorder of TMAO and its related metabolites and creatinine in rats, which was effectively improved after the intervention of Rosa Roxburghii Tratt juice（RRTJ）. A method for the determination of choline, L-carnitine, betaine, TMA, TMAO and creatinine in plasma, liver and feces samples was established, which is simple, time-saving, high precision, accuracy and recovery. [ABSTRACT FROM AUTHOR]

• This is a sensitive method (1 ng/ml). • This method generates the shortest run time <1 min (rapid analysis). • This is the only published method that utilized Thiorphan_D7 as an internal standard. • There is an important application to prove the reliability of the new method, which is bioequivalence study in Jordanian subjects. Racecadotril, an anti-secretory medication, has been used as an adjuvant in an oral rehydration therapy for children experiencing severe diarrhea. Racecadotril is quickly converted to thiorphan, an active metabolite, after oral treatment, which mediates all subsequent activities. An efficient and rapid liquid chromatography-tandem mass spectrometry method was developed and fully validated to measure thiorphan in human plasma, using thiorphan-d7 as an internal standard. The extraction method used was protein precipitation while chromatographic separation was achieved using InertSil CN-3 (50 × 2.1 mm, 5 µm column). The assay was linear over the concentration range of 1–200 ng/ml with correlation coefficients of ≥0.9991. The intra- and inter-day precisions were less than 10.0 % for all concentrations investigated. 0.02 % aqueous formic acid and methanol (30:70 v: v) were used as mobile phases, with an analysis time of less than 1 min. This method proved stable under several conditions. The developed method worked well in a three-period pharmacokinetic bioequivalence study after a single oral administration of 100 mg racecadotril to 15 healthy Jordanian volunteers under fasting conditions. [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]

• 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]

• A UPLC-MS/MS method for simultaneous quantification of OPC-61815 and its three metabolites was developed for the first time. • The method was proved to be efficient and robust with high sensitivity and selectivity. • The pharmacokinetics of OPC-61815 in a phase I trial was studied using this method. OPC-61815 is an intravenous formulation vasopressin antagonist designed to treat heart failure patients, especially who have difficulty in oral intake. Tolvaptan together with DM-4103 and DM-4107 are considered as the major metabolites of OPC-61815 biotransformed in the liver via cytochrome P450 (CYP) 3A. An efficient and robust ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for quantification of OPC-61815 and its three metabolites in human plasma was developed and fully validated. To our best knowledge, it was the first published method that simultaneously quantified all of these four analytes in only one run. Simple and rapid sample preparation procedure and very short UPLC-MS/MS run time (3.5 min) offered OPC-61815 and its metabolites relatively high throughput detection, which was greatly beneficial to further clinical bio-sample analysis. The method showed good linearity and sufficient sensitivity in the range of 2.00–1000 ng/mL with a low limit of quantitation (2.00 ng/mL) for each analyte. For samples with concentrations above 1000 ng/mL, 100-fold dilution with blank plasma before sample preparation was accepted. High precision and accuracy, high selectivity and satisfactory recovery of this method were demonstrated. For all of the four analytes, no significant matrix effect or carry-over was observed. The stability of analytes and internal standards under different conditions were evaluated to ensure they were stable during the whole period of storage, preparation and detection. Also, re-injection reproducibility was investigated. In addition, the conversion test showed that almost no OPC-61815 converted into DM-4103 and DM-4107 during sample processing, while attention should be paid to the concentration difference between OPC-61815 and tolvaptan in bioanalysis. The developed UPLC-MS/MS method was successfully applied to an open, single and multiple dose administration phase I trial for monitoring the pharmacokinetics of OPC-61815. This work provided a promising way for further pharmacokinetic study of OPC-61815. [ABSTRACT FROM AUTHOR]

• Accurate quantitation of reactive aldehydes in brain. • 3-NPH improve sensitivity for detecting aldehydes. • Sample pretreatment and derivatization were optimized and analysis was achieved in a 7 min LC-MS/MS method. • The accumulation of HHE, HNE and acrolein was observed at 28 days post TBI. Reactive aldehydes are a class of electrophilic low molecular weight compounds that play an essential role in physiological function and lipid peroxidation. These molecules are implicated in many diseases, especially cardiovascular and neurodegenerative diseases, and are potential endogenous markers of lipid peroxidation. However, there are limited options to accurately quantify multiple reactive aldehydes in brain tissue. This study developed and validated a 3-nitrophenylhydrazine derivatization-based LC-MS/MS method to quantify four reactive aldehydes: malondialdehyde, acrolein, 4-hydroxy-2-hexenal and 4-hydroxy-2-nonenal. Method development involved comparing the sensitivity of detection between widely used derivatization reagents: 2,4-dinitrophenylhydrazine and 3-nitrophenylhydrazine. Our data showed that 3-nitrophenylhydrazine resulted in greater sensitivity. Additional method development included evaluation of hydrolysis sample pretreatment, selection of protein precipitation reagent, and optimization of derivatization conditions. The optimized conditions included no hydrolysis and use of 20 % trichloroacetic acid as the protein precipitation reagent. The optimized derivatization condition was 25 mM 3-nitrophenylhydrazine reacted at 20 °C for 30 min. The chromatographic conditions were optimized to reduce matrix effects, ion suppression, and efficient analysis time (<7-minute analytical run). The four aldehyde species were accurately quantified in brain tissue using stable-labeled internal standards. Application of this assay to a traumatic brain injury mouse model revealed significant accumulation of acrolein, 4-hydroxy-2-hexenal, and 4-hydroxy-2-nonenal at 28 days post injury. Overall, a validated method was developed to rapidly quantify the most prominent reactive aldehydes associated with lipid peroxidation during injury progression following acute brain trauma. [ABSTRACT FROM AUTHOR]

• A sensitive method for quantifying decitabine in mouse plasma was developed and validated. • Robust, accurate, and precise results were demonstrated, with a lower limit of quantitation of 0.4 ng/mL. • Decitabine was determined in microvolumes of mouse plasma. • Increased oral bioavailability of decitabine was observed when given with cedazuridine. • The mouse model was found useful for evaluating pharmacokinetic drug-drug interactions with decitabine. Decitabine is a DNA methyltransferase inhibitor used in the treatment of acute myeloid leukemia and myelodysplastic syndrome. The notion that ongoing trials are presently exploring the combined use of decitabine, with or without the cytidine deaminase inhibitor cedazuridine, and other antileukemic drugs necessitates a comprehensive understanding of pharmacokinetic properties and an evaluation of drug-drug interaction liabilities. We report here the development and validation of a sensitive UHPLC-MS/MS method for quantifying decitabine in mouse plasma, which should be useful for such studies. The method involved a one-step protein precipitation extraction, and chromatographic separation on an XBridge HILIC column using gradient elution. The method was found to be robust, accurate, precise, and sufficiently sensitive (lower limit of quantitation, 0.4 ng/mL) to determine decitabine concentrations in microvolumes of plasma from mice receiving the agent orally or intravenously in the presence or absence of cedazuridine. [ABSTRACT FROM AUTHOR]

• An analytical method for β-agonist residues in six matrices was developed. • The method was fully validated according to CIR EU 2021/808. • LOD and LOQ were in the range of 0.004–0.048 μg/kg and 0.010 to 0.075 μg/kg, respectively. • Out of the 180 samples collected from Egyptian markets, 21.11 % had β-agonist residues. • 90 % of liver samples (n = 30) from Egyptian markets had terbutaline residues. A reliable liquid chromatography coupled to quadrupole-Orbitrap high-resolution mass spectrometry (LC-Q-Orbitrap HRMS) method was developed for the simultaneous identification and quantification of 13 β-agonist residues in bovine liver, meat, milk, kidney, poultry, and egg. Dispersive-solid phase extraction (d-SPE) using acetonitrile (ACN) was used to prepare the samples. The analyte in the extracts was separated on a reversed-phase Accucore aQ (50 mm × 2.1 mm, 2.6 μm) using a mobile phase of an aqueous solution containing 2 mM ammonium acetate and acetonitrile (ACN) 0.1 % formic acid. The method was validated in accordance with Commission Implementing Regulation (CIR) EU 2021/808 at six different concentrations ranging from 0.1 to 5 μg/kg. The mean recoveries ranged from 65 to 94 %, while repeatability and reproducibility values were all below 13 %. The linearity, as correlation coefficients (R2) ranged from 0.9955 to 0.9999. The decision limit (CCα) and detection capability (CCβ) ranges were 0.11–0.13 µg/kg and 0.12–0.15 µg/kg, respectively. The limits of detection (LOD) and limits of quantification (LOQ) were in the range of 0.004–0.048 μg/kg and 0.010–0.075 μg/kg, respectively. Of the 180 samples that were collected from local markets in Egypt, 21.11 % had β-agonist residues. The mean concentration (µg/kg) and detection frequency (%) of the most frequently found β-agonist in the samples were as follows: terbutaline (2.63 µg/kg and 90 %), ractopamine (5.14 µg/kg and 23.3 %). The method's applicability was verified by successfully completing two rounds of proficiency testing (PT). [ABSTRACT FROM AUTHOR]

[Display omitted] • A fast and sensitive method was established for determination of 12 analytes. • The pharmacokinetics of Zingiberis Rhizoma − Jujubae Fructus herb pair were profiled. • The absorption of the analytes was decreased in the stomach cold syndrome rats. • The compatibility of the herb pair promoted the absorption of analytes in model rats. The Zingiberis Rhizoma − Jujubae Fructus herb pair (ZJHP) is a classic herb pair in traditional Chinese medicine. The herb pair shows the effect of dispelling cold, harmonizing the middle and improving gastrointestinal function, and is widely used for patients with stomach cold syndrome (SCS), stomachache and anemofrigid cold. The gingerols, shogaols, flavonoids and triterpenic acids are the important bioactive ingredients of ZJHP. However, few pharmacokinetic studies have been investigated in vivo for the above compounds. To comprehend the kinetics of active components and promote their curative application, a fast and sensitive ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC-MS/MS) method was established for simultaneous determination of 12 analytes in normal and SCS rats in this study. The results showed that the pharmacokinetic parameters (C max , T max , t 1/2z , MRT 0-t , AUC 0-t and AUC 0-∞) in SCS model were significantly different from those in normal rats. In addition, the pharmacokinetics of rats given ZJHP were also varied from single herb oral administration, especially in model condition. These results indicated that the in vivo processes of the above analytes changed under pathological conditions and the compatibility of the herb pair could significantly influence the absorption of active components, which might provide an insight and further supports for the clinical application of ZJHP. [ABSTRACT FROM AUTHOR]

• A novel LC-MS/MS method was developed for the quantification of 35 drugs in blood. • Analysis targets toxicologically relevant antihypertensive and antiarrhythmic drugs. • Special emphasis was placed on the analysis of post mortem samples. • Validation demonstrated satisfactory results for all analytes. Antiarrhythmic and antihypertensive drugs are frequently encountered in post mortem analysis, and the question may arise as to whether they were administered in therapeutic doses, and if they were taken in accidental, intentional, or suicidal overdose scenarios. Therefore, a novel analytical method was developed and validated for the quantification of 35 drugs with toxicological relevance, including antihypertensive and antiarrhythmic drugs (ajmaline, amlodipine, amiodarone, atenolol, bisoprolol, carvedilol, clonidine, desethylamiodarone, diltiazem, donepezil, doxazosin, dronedarone, esmolol, flecainide, lercanidipine, lidocaine, metoprolol, nebivolol, nimodipine, pindolol, prajmaline, propafenone, propranolol, sotalol, urapidil, and verapamil), as well as other medications commonly found in combination (sildenafil, tadalafil, atorvastatin, clopidogrel, dapoxetine, memantine, pentoxifylline, rivastigmine, and ivabradine). The method enables simultaneous identification and quantification in blood samples using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Validation exhibited excellent linearity across the concentration range for all analytes. Precision and accuracy were within acceptable limits, with bias and relative standard deviation (RSD) values consistently below 9 % and 10 %, respectively. Selectivity and specificity assessments confirmed the absence of any interference from contaminants or co-extracted drugs. The method demonstrated very high sensitivity, with limits of detection (LOD) as low as 0.01 ng/ml and limits of quantification (LOQ) as low as 0.04 ng/ml. Extraction recovery exceeded 57.5 % for all analytes except atenolol, and matrix effects were <17 % for all analytes except pindolol. Processed sample stability evaluations revealed consistent results with acceptable deviations for all analytes. In addition, the method was specifically tested for the use in post mortem analysis. The applicability of our method was demonstrated by the analysis of two authentic human autopsy blood samples. [ABSTRACT FROM AUTHOR]

[Display omitted] • Quantification of SCFAs in preadolescent children plasma was performed. • Sex, age, and body weight-specific changes in plasma SCFA were revealed. • Acetic acid was found to be the most abundant SCFA in children's plasma. • Increased propanoic acid was observed in the plasma of the overweight group. • No sex-specific differences were observed in plasma SCFA concentrations. The purpose of this study is to explore the plasma short-chain fatty acid (SCFA) concentrations in 9–12-year-old Japanese children collected in the Hokkaido study, focusing on how factors such as age, sex, and body mass index (BMI) correlate with these levels. The Hokkaido Study on Children's Health is an ongoing longitudinal study since 2002, encompassing 20,926 pregnant women in Hokkaido Prefecture, Japan, between 2003 and 2012. We contacted 1881 children aged 9–12 born between April 2006 and January 2010, and 342 non-fasting plasma samples (boys = 181, girls = 161) were obtained from this cohort, alongside assessments of their height and weight. Plasma SCFA concentrations were determined using N,N -dimethylethylenediamine derivatization method coupled with liquid chromatography-mass spectrometry. Ethyl acetate was used to extract SCFAs from plasma, and the recovery ranged from 83 % to 108 %. Our findings indicate that acetic acid had the highest concentration across all age groups and sexes. The concentrations of butyric acid, valeric acid, and hexanoic acid increased with age, peaking in 12-year-old children. Conversely, the level of 4-hydroxy valeric acid showed a decreasing trend with increasing age groups. This study also explored the correlation between BMI and SCFA concentrations, comparatively higher level of propionic acid was observed in the overweight group. The results obtained in this study enhance our understanding of the role of SCFAs in the growth and development of children and provide a foundation for future nutritional intervention and health promotion strategies. [ABSTRACT FROM AUTHOR]

• The chromatographic parameters were optimized using a Box-Behnken experimental design. • The optimized HPLC–UV method was applied to simultaneously quantify co-delivery of RHT and AS loaded niosomes. • HPLC–UV method was validated according to the ICH guidelines. Rivastigmine hydrogen tartrate (RHT), a reversible cholinesterase inhibitor, is considered as the first-line therapy for mild to moderate Alzheimer's disease. Asiaticoside (AS), a pentacyclic triterpenoid saponin, is well known as cognitive enhancer due to its antioxidant effect. Based on the hypothesis of their synergistic therapeutic potential, RHT and AS were co-encapsulated in niosomal formulation. A simple, precise, and accurate high-performance liquid chromatography method was developed for simultaneous quantitative analysis. The chromatographic parameters were optimized by Box-Behnken experimental design. The separation was performed on a reversed-phase Phenomenex C18 (150 mm × 4.6 mm, 5 μm) column at 30 °C under the UV detection of 210 nm. The optimized mobile phase consisted of a mixture of 20 mM potassium dihydrogen phosphate buffer (pH 2.6) and acetonitrile (72:28 % v/v) under the isocratic mode at the flow rate of 0.9 mL/min. The developed method was fully validated under the ICH guidelines and could be successfully applied for simultaneous quantitative analysis of RHT and AS in niosomal formulation. [ABSTRACT FROM AUTHOR]

• Developed an LC-MS/MS method for the simultaneous determination of new FDA approved anticancer drugs in rat plasma. • The developed method was validated as per USFDA guidelines including stability parameters. • The developed LC-MS/MS method was efficiently employed in an in vivo pharmacokinetic study of anticancer drugs in rat plasma. • The developed LC-MS/MS can act as the therapeutic drug monitoring in combined therapy of selected drugs in non-small cell lung cancer. Non-small cell lung cancer (NSCLC) is a significant subtype of lung cancer, and poses a dangerous global threat. One of the current approaches of NSCLC treatment is a combination therapy of adagrasib and pembrolizumab. Accurate monitoring of these drug concentrations in biological fluids is critical for treatment efficacy. Since no method was reported for simultaneous estimation of these drugs, this study focuses on the development of a validated LC-MS/MS bioanalytical method for simultaneous quantification of Adagrasib and Pembrolizumab in rat plasma. The analytes were extracted from the biological matrix through liquid–liquid extraction techniques using acetonitrile as extraction solvent. The analytes were separated on a Waters X-bridge phenyl C 18 column, with a mixture of acetonitrile: 0.1 % TFA in water (50: 50 v/v) as mobile phase at an isocratic flow rate of 1.0 mL/min with a runtime of about 5 min. Adagrasib (m / z 605.12 → 201.62), Pembrolizumab (m / z 146.32 → 85.15), and Sotorasib (m / z 561.59 → 218.92) were determined by recording the mass spectra through multiple reaction monitoring in positive mode. The method was validated according to USFDA guidelines. The results demonstrate satisfactory linearity with an r2 value of 0.9998 in the ranges of 40–800 and 10–200 ng/mL, accuracy with mean percentage recovery of 95.22–98.59 % and 96.98–98.57 %, precision indicated with %RSD ranged between 0.39–1.91 % and 0.85–9.03 % for Adagrasib and Pembrolizumab respectively, and other key parameters. The developed method can determine the pharmacokinetic parameters to indicate the efficacy and safety of the drugs, and also can quantify selected drugs simultaneously in biological samples. [ABSTRACT FROM AUTHOR]

• In this work, we established and assessed a streamlined nSEC-MS workflow with a benchtop LC–MS platform, to quantitatively monitor DAR and DLD of different chemotype and drug load level cysteine-linked ADCs. • To deploy this workflow in QC environment, complete method validation was conducted in three independent laboratories, adhering to the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) Q2(R1) guidelines. • The results met the predefined analytical target profile (ATP) and performance criteria, encompassing specificity/selectivity, accuracy, precision, linearity, range, quantification/detection limit, and robustness. • The method validation design offers a reference for other nSEC-MS methods that are potentially used to determine the DAR and DLD on cysteine-linker ADCs. • To the best of our knowledge, this study is the first reported systematic validation of the nSEC-MS method for detecting DAR and DLD. The results indicated that the co-validated nSEC-MS workflow is suitable for DAR and DLD routine analysis in ADC quality control, release, and stability testing. The development and optimization of Antibody-Drug Conjugates (ADCs) hinge on enhanced analytical and bioanalytical characterization, particularly in assessing critical quality attributes (CQAs). The ADC's potency is largely determined by the average number of drugs attached to the monoclonal antibody (mAb), known as the drug-to-antibody ratio (DAR). Furthermore, the drug load distribution (DLD) influences the therapeutic window of the ADC, defining the range of dosages effective in treating diseases without causing toxic effects. Among CQAs, DAR and DLD are vital; their control is essential for ensuring manufacturing consistency and product quality. Typically, hydrophobic interaction chromatography (HIC) or reversed-phase liquid chromatography (RPLC) with UV detector have been used to quantitate DAR and DLD in quality control (QC) environment. Recently, Native size-exclusion chromatography-mass spectrometry (nSEC-MS) proves the potential as a platformable quantitative method for characterizing DAR and DLD across various cysteine-linked ADCs in research or early preclinical development. In this work, we established and assessed a streamlined nSEC-MS workflow with a benchtop LC–MS platform, to quantitatively monitor DAR and DLD of different chemotype and drug load level cysteine-linked ADCs. Moreover, to deploy this workflow in QC environment, complete method validation was conducted in three independent laboratories, adhering to the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) Q2(R1) guidelines. The results met the predefined analytical target profile (ATP) and performance criteria, encompassing specificity/selectivity, accuracy, precision, linearity, range, quantification/detection limit, and robustness. Finally, the method validation design offers a reference for other nSEC-MS methods that are potentially used to determine the DAR and DLD on cysteine-linker ADCs. To the best of our knowledge, this study is the first reported systematic validation of the nSEC-MS method for detecting DAR and DLD. The results indicated that the co-validated nSEC-MS workflow is suitable for DAR and DLD routine analysis in ADC quality control, release, and stability testing. [ABSTRACT FROM AUTHOR]

• This is the first described quantification method for the simultaneous determination of cefuroxime and flucloxacillin in synovial tissue and bone using UPC2-MS/MS. • Two of the commonly administered antimicrobial agents during the one-stage or two-stage arthroplasty exchange are researched in this analysis. • A fast and simple sample preparation is applied. • The method has already successfully been applied in a clinical study, to relate target-site concentrations to serum concentrations, which ultimately can lead to more precision dosing. After a revision surgery, approximately 1–2 % of patients will develop a periprosthetic joint infection (PJI). During the revision surgery, the infected prosthesis is removed, a debridement is performed and a new or temporary spacer is placed. Additionally, patients are treated with antibiotics during and after the surgery. Adequate exposure of the administered antibiotic to the pathogen is of crucial importance during the treatment of any infection. Inadequately low concentrations are associated with an increase in antibiotic resistance, antibiotic related side effects, treatment failures and prolonged infections. While high concentrations may lead to serious adverse events and potential lasting damage. Despite the importance of optimal dosing, there is a lack of knowledge with respect to the correlation between the plasma concentrations and target site concentrations of the antibiotics. Two of the commonly administered antimicrobial agents during the arthroplasty exchange are cefuroxime and flucloxacillin. Therefore, an accurate, specific, and sensitive quantification method is required in order to assess pharmacokinetics of cefuroxime and flucloxacillin in synovial tissue and bone. The aim of this study is to develop and validate a quantification method for the measurement of cefuroxime and flucloxacillin in human synovial tissue and bone using the UPC2-MS/MS conform Food and Drug Administration guidelines. The method was found linear for both compounds in both matrices (r 2 > 0.990) from 1 µg/g to 20 µg/g, except for cefuroxime in bone, which was validated from 1 µg/g to 15 µg/g. We developed and validated a quantification method for cefuroxime and flucloxacillin in synovial tissue and bone using a simple sample preparation and a short analysis run time of 5.0 min, which has been already successfully applied in a clinical study. To our knowledge, no methods have been described earlier for the simultaneous quantification of cefuroxime and flucloxacillin in synovial tissue and bone. [ABSTRACT FROM AUTHOR]

• HILIC-MS/MS method was developed to detect 18 amino acids in pregnant woman plasma. • Surrogate matrix was used to plot quantitative curves, and validated in human plasma. • Ala, Lys, Trp, Glu and Phe levels in subjects with sleep disorders had obvious changes. • The method supports to study plasma amino acid levels at different pregnancy stages. Many pregnant women experience sleep disorders, and amino acid levels could play a crucial role in affecting maternal sleep. To explore this potential relationship, an accurate and effective UHPLC-MS/MS method has been developed to monitor 18 amino acids in the plasma samples of pregnant women. This method aims to assess how plasma amino acid levels might be linked to sleep disorders during pregnancy. Plasma samples were precipitated with acetonitrile containing 0.2% formic acid. We used 5% seralbumin as the surrogate matrix to establish quantitative curves for amino acid determination in human plasma. The method was validated in both the surrogate matrix and human plasma. The optimized UHPLC-MS/MS method was validated, showing that that the analytes had comparable recovery and negligible matrix effects in both 5% seralbumin and human plasma. The linearity, lower limit of quantification, precision, accuracy, and stability all met the acceptance criteria. The validated method was successfully applied to determination of the plasma levels of 18 amino acids in pregnant women with or without sleep disorders, indicating that alanine, lysine, tryptophan, glutamic acid, and phenylalanine levels had significant changes which may be related to sleep disorders during early pregnancy. An accurate, reliable, and efficient UHPLC-MS/MS method was successfully developed and support to find the specific amino acids as potential biomarkers for sleep disorders in pregnant women. [ABSTRACT FROM AUTHOR]

• Despite the success of antiretroviral therapy, HIV persists in tissues. • Adequate antiretroviral concentrations in tissues are necessary to suppress virus. • Quantifying concentrations in tissues ensures adequate antiretroviral exposure. • A UHPLC-MS/MS method was validated to quantify a panel of five antiretrovirals in human tissues. Although antiretroviral therapy (ART) is highly effective for the treatment of HIV-1 infection to suppress virus in the blood, HIV persists in tissues. HIV persistence in the tissues is due to numerous factors, and one of those factors are antiretroviral (ARV) concentrations. ARV concentrations in tissues must be adequate to suppress HIV at the sites of action. While therapeutic drug monitoring in the plasma is well-known, drug monitoring in the tissues provides local assessments of adequate ARV exposure to prevent localized HIV resistance formation. Towards these efforts, we validated an ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS/MS) method in human tissues (cervical, rectal, and vaginal tissues) for the simultaneous quantification of five ARVs: bictegravir, cabotegravir, dolutegravir, doravirine, and raltegravir. For this assay, protein precipitation with acetonitrile with stable, isotopically-labeled internal standards followed by supernatant pre-concentration was performed. Analyte separation was accomplished using a multistep UPLC gradient mixture of 0.1 % formic acid in water (A) and acetonitrile (B) with a Waters Cortecs T3 (2.1x100 mm) column. The assay was extensively validated as per the United States Food and Drug Administration Bioanalytical Method Validation Guidance over a clinically observed range (0.05–50 ng/mL) with superb linearity (R2 > 0.99 across all ARVs). The assay run time was 8.5 min. This analytical method achieves appropriate performance of trueness (85.5–107.4 %), repeatability, and precision (CV < 15 %). Our method will be employed for the therapeutic monitoring of guideline-recommended ARVs in human tissues for monitoring therapeutic efficacy in HIV treatment and prevention research efforts. [ABSTRACT FROM AUTHOR]

• The method successfully separated Obeticholic acid from its stereo isomeric metabolites. • The method successfully separated the interferors with close retention time in blank plasma from the target analytes. • The amount of plasma required for detection was reduced. • The sensitivity was higher and the quantitation limit was lower. • The method has been verified by a complete methodology and applied in portraying the pharmacokinetics of Obeticholic acid and its two active metabolites in healthy subjects. Obeticholic acid (OCA), a semisynthetic bile acid derivative, was approved for its therapeutic use in primary biliary cirrhosis. OCA has a enterohepatic circulation and host-gut microbiota metabolic interaction, which produce various metabolites. Such metabolites, especially structural isomers of OCA, together with the need to achieve idea lower limit of quantitation (LLOQ) with minimum matrix interference, bring about significant difficulties to the bioanalysis of OCA. Herein, by applying a combination of solid-phase extraction (SPE) and ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), we introduced an approach for the bioanalysis of OCA along with its two major metabolites—glyco-OCA (GOA) and tauro-OCA (TOA) in human plasma, the full validation results of which showed excellent performance. The quantitative range is 0.2506 ∼ 100.2 ng/mL for OCA, 0.2500 ∼ 100.0 ng/mL for GOA, as well as 0.1250 ∼ 50.00 ng/mL for TOA, respectively. This method was successfully applied to the pharmacokinetic studies in healthy subjects following administration of OCA tablets. [ABSTRACT FROM AUTHOR]

• Florfenicol metabolites synthesized with standard lab techniques. • High linearity (>0.99) and ±10 % accuracy confirmed in fish analysis. • Consistent detection of florfenicol metabolites in treated fish fillets. • Developed synthesis and SPE-UHPLC-MS/MS methods aids fish drug metabolism studies. This study concerns the synthesis of the florfenicol (FF) metabolites florfenicol amine (FFA), florfenicol alcohol (FFOH), and monochloroflorfenicol (FFCl), for their subsequent use as reference standards in On-line solid-phase extraction-ultra high-performance liquid chromatography-tandem mass spectrometry (SPE-UHPLC-MS/MS) analysis. The metabolites were characterized using 1H and 13C NMR, as well as HRMS, and their purities were confirmed by quantitative NMR to ensure analytical reliability. Validation of the developed analytical method showed that it presented acceptable performance, with linearity >0.99 for all the target analytes, accuracies within ±10 % of nominal concentrations, and intra- and inter-day precisions within 15 %. Application of this method to fillets from fish that had been treated with florfenicol (dose of 10 mg/kg bw daily) demonstrated its effectiveness in consistently detecting FF and its metabolites throughout the treatment. The results emphasized the utility of the method for enhancing pharmacokinetic and residue depletion research. The ability to precisely monitor the drug and its metabolites in treated fish provides important insights into florfenicol metabolism, laying the groundwork for further comprehensive profiling studies of metabolites in fish tissue. [ABSTRACT FROM AUTHOR]

• A novel LC-MS/MS method can simultaneously test multiple TKIs and triazoles. • A simpler and more time-saving method was established in this study. • Therapeutic drug monitoring was performed in CML patients co-treated with TKIs and triazoles. • Drug-drug interactions were found between TKIs and triazoles. Tyrosine kinase inhibitors (TKIs) and triazole antifungals are the first-line drugs for treating chronic myeloid leukemia (CML) and fungal infections, respectively, but both suffer from large exposure differences and narrow therapeutic windows. Moreover, these two types of drugs are commonly used together in CML patients with fungal infections. Multiple studies and guidelines have suggested the importance of therapeutic drug monitoring (TDM) of TKIs and triazoles. Currently, methods for the simultaneous determination of both types of drugs are limited. We developed a simple, rapid, and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous quantification of three commonly used TKIs (imatinib, dasatinib, and nilotinib) and three commonly used triazoles (voriconazole, itraconazole, and posaconazole) in human plasma. The analytes were eluted on a Welch XB-C 18 analytical column (50 × 2.1 mm, 5 µm) at 0.7 mL/min, using a gradient elution of 10 mM ammonium formate (A) and methanol–acetonitrile-isopropanol (80:10:10, v / v / v) containing 0.2 % formic acid (B) with a total analysis time of 3.5 min. The calibration curves were linear over the range from 20 to 4000 ng/mL for imatinib and nilotinib, from 2 to 400 ng/mL for dasatinib, and from 50 to 10,000 ng/mL for voriconazole, itraconazole, and posaconazole. Selectivity, accuracy, precision, recovery, matrix effect, and stability all met the validation requirements. The method was successfully used for TDM in CML patients who co-treated with both TKIs and triazoles. Drug-drug interaction analysis between TKIs and triazoles showed that a significant positive correlation was observed between imatinib and voriconazole, as well as dasatinib and voriconazole. Therefore, this method can be well applied in clinical TDM for patients receiving TKIs, triazoles, or both simultaneously. [ABSTRACT FROM AUTHOR]

• The CF-UF coupled with HPLC-MS/MS method was developed and validated for unbound lenvatinib analysis. • Significant factors affecting CF-UF process was optimized. • Significant differences existed in the unbound fraction between individuals. • Please contact me if any possible requestion. In clinical practice, the determination of unbound drug concentration is very important for dose adjustment and toxicity prediction because only the unbound fraction can achieve a pharmacological effect. A fast, sensitive and accurate analytical method of centrifugal ultrafiltration coupled with high performance liquid chromatography-tandem mass spectrometry method was developed and applied to allow the quantification of unbound lenvatinib concentration. The application of linear regression analysis was used to examine the effects of centrifugal force, centrifugal time, and protein content on ultrafiltrate volume (V u). The results indicated that the centrifugal force and centrifugal time have an influence on V u that is significantly positive (P < 0.05). This developed method with good linearity (r 2 = 0.9996), good accuracy (bias % ≤ 2.24 %), good precision (CV % ≤ 7.10 %), and good recovery (95.46 %−106.46 %) was suitable for routine clinical practice and studies. Particularly, the ultrafiltration membrane had no non-specific binding to lenvatinib. The unbound fractions can be separated in just 15 min. This method was applied to quantify clinical samples and to determine the plasma protein binding and unbound fraction of lenvatinib. This study provides a more effective and promising method for determination of unbound lenvatinib. It could be beneficial to measure the unbound concentration of lenvatinib in personalized medicine and therapeutic drug monitoring in routine clinical practice. [ABSTRACT FROM AUTHOR]

• Evaluation of an alternative specimen for drugs of abuse testing. • New validated automated multipanel LC-MS/MS method. • Successful clinical application comparing exhaled breath with urine. Aerosol microparticles in exhaled breath carry non-volatile compounds from the deeper parts of the lung. When captured and analyzed, these aerosol microparticles constitute a non-invasive and readily available specimen for drugs of abuse testing. The present study aimed to evaluate a simple breath collection device in a clinical setting. The device divides a breath sample into three parallel "collectors" that can be individually analyzed. Urine was used as the reference specimen, and parallel specimens were collected from 99 patients undergoing methadone maintenance treatment. Methadone was used as the primary validation parameter. A sensitive multi-analyte method using tandem liquid chromatography – mass spectrometry was developed and validated as part of the project. The method was successfully validated for 36 analytes with a limit of detection of 1 pg/collector for most compounds. Based on the validation results tetrahydrocannabinol THC), cannabidiol (CBD), and lysergic acid diethylamide (LSD) are suitable for qualitative analysis, but all other analytes can be quantitively assessed by the method. Methadone was positive in urine in 97 cases and detected in exhaled breath in 98 cases. Median methadone concentration was 64 pg/collector. The methadone metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) was detected in 90 % of the cases but below 10 pg/collector in most. Amphetamine was also present in the urine in 17 cases and in exhaled breath in 16 cases. Several other substances were detected in the exhaled breath and urine samples, but at a lower frequency. This study concluded that the device provides a specimen from exhaled breath, that is useful for drugs of abuse testing. The results show that high analytical sensitivity is needed to achieve good detectability and detection time after intake. [ABSTRACT FROM AUTHOR]

• Successful development and validation the method to monitor the whole blood concentration of cyclosporine A in pediatric patients with minimum time and small-volume plasma consumption. • The analysis method minimized medical costs by optimizing extraction processing and allowed the analysis with a simple one-step protein precipitation method. • 657 cases of CsA concentrations were determined from 197 pediatric patients by a routine EMIT assay and by a validated in-house LC-MS/MS method. • The correlation between the EMIT and LC-MS/MS in different ranges was analyzed, including < 40 ng/mL, > 500 ng/mL, excluding < 40 ng/mL and > 500 ng/mL, 40–––200 ng/mL, and 200–––500 ng/mL, respectively. • This is the first study reporting the comparison of the EMIT and LC-MS/MS for the measurement of whole blood CsA concentration in children. Cyclosporine A (CsA) is a widely used immunosuppressive drug with a narrow therapeutic index and large individual differences. Its therapeutic and toxic effects are closely related to blood drug concentrations, requiring routine therapeutic drug monitoring (TDM). The current main methods for TDM of CsA are enzyme multiplied immunoassay technique (EMIT) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). However, few study on the method comparison of the EMIT and LC-MS/MS for the measurement of whole blood CsA concentration in children has been reported. In this study, we developed a simple and sensitive LC-MS/MS assay for the determination of CsA, and 657 cases of CsA concentrations were determined from 197 pediatric patients by a routine EMIT assay and by the validated in-house LC-MS/MS method on the same batch of samples, aimed to address the aforementioned concern. Consistency between the two assays was evaluated using linear regression and Bland-Altman analysis. The linear range of LC-MS/MS was 0.500–2000 ng/mL and that of the EMIT was 40–500 ng/mL, respectively. Overall, the correlation between the two methods was significant (r-value ranging from 0.8842 to 0.9441). Unsatisfactory consistency was observed in the concentrations < 40 ng/mL (r = 0.7325) and 200–500 ng/mL (r = 0.6851). Bland-Altman plot showed a mean bias of −18.0 % (±1.96 SD, −73.8 to 37.8 %) between EMIT and LC-MS/MS. For Passing-Bablok regression between EMIT and LC-MS/MS did not differ significantly (p > 0.05). In conclusion, the two methods were closely correlated, but the CsA concentration by LC-MS/MS assay was slightly higher than that by EMIT method. Switching from the EMIT assay to the LC-MS/MS method was acceptable, and the LC-MS/MS method will receive broader application in clinical settings due to its better analytical capabilities, but the results need to be further verified in different laboratories. [ABSTRACT FROM AUTHOR]

[Display omitted] • Identification and absolute quantification of naturally derived prostate-specific antigen (PSA). • First absolute quantification of PSA that uses amino acid hydrolysis coupled with isotope-dilution mass spectrometry (IDMS), and enzymatic hydrolysis-IDMS simultaneously. • Support the development of high-order reference materials of PSA to ensure the accuracy, comparability, and traceability of testing in medical laboratories. Prostate-specific antigen (PSA) is a diagnostic marker for prostate cancer; however, because it is a macromolecular glycoprotein with complex and diverse isoforms, it is difficult to standardize clinical PSA detection results. To overcome this limitation, herein, naturally extracted PSA was characterized as free PSA (fPSA), and the PSA solution was successfully quantified by amino acid analysis coupled with isotope-dilution mass spectrometry (AAA-IDMS) and enzymatic hydrolysis-IDMS; the results could be traced to the International System of Units (SI) through absolutely quantified amino acids and peptides. After protein hydrolysis or digestion condition optimization, amino acids and signature peptides were detected by liquid chromatography-mass spectrometry with the multiple reaction monitoring mode. The mass concentrations of PSA obtained through AAA-IDMS and enzymatic hydrolysis-IDMS were (75.3 ± 1.5) µg/g (k = 2) and (74.7 ± 1.7) µg/g (k = 2), respectively. The PSA weighted average mass concentration was (75.0 ± 1.6) µg/g (k = 2). The consistency assessment between the two methods was successfully validated, ensuring absolute quantitative accuracy. This study lays the foundation for the development of high-order reference materials for the clinical detection of PSA, which can improve the accuracy, reliability, and consistency of clinical PSA test results. [ABSTRACT FROM AUTHOR]

• A dilute and shoot method was proposed for determination of 24-hour urinary cortisol by LC-MS/MS. • The total run time was 3 min for fast analysis. • The preservatives for urine collection were studied to ensure good clinical practice. • The reference interval and diagnostic performance were investigated for clinical testing. 24-hour urinary free cortisol (UFC) is considered as the first-line test for screening and diagnosis of Cushing's syndrome. Although 24-hour UFC assay has been extensively studied by liquid chromatography-tandem mass spectrometry (LC-MS/MS), an accurate assay coupled with a reliable sample preparation procedure and a method-specific reference interval would be very important for reasonable diagnosis. In this study, a simple dilute and shoot method has been proposed for UFC determination by LC-MS/MS. Namely, 50 µL of urine sample was mixed with 200 µL of a 50 % methanol/water solution containing the internal standard cortisol-13C3. The mixture was centrifuged and the supernatant was used for direct analysis by LC-MS/MS. This method was validated with wide linear range from 0.625 to 500 ng/ml with coefficients of variation (CVs) ≤ 3.64 %, excellent precision (intra-day CVs ≤ 5.70 % and inter-day CVs ≤ 5.33 %) and good recovery in the range of 93.3–109 %. The preservatives were further evaluated for urine storage. It was recommended that no preservatives could be used in collection of 24-hour urine for good detecting peaks. The investigation of reference interval and diagnostic performance finally confirmed the potential usage of this LC-MS/MS assay in routing clinical testing. [ABSTRACT FROM AUTHOR]

• New magnetic sorbent based on MIL-101Cr (NH 2) MOF and NiFe 2 O 4 was prepared for the first time. • Dispersive solid phase extraction method was done for extraction of sunitinib from biological samples. • The sample preparation was effectively performed prior to HPLC-MS/MS analysis. • High efficiency, broad linear range, and sensitive determinations were the advantages of the method. Herein, a simple, sensitive, and reliable dispersive solid phase extraction was reported for the efficient extraction of sunitinib from biological samples. To facilitate the extraction of the desired analyte from urine and plasma samples, magnetic MIL-101Cr (NH 2) @SiO 2 @ NiFe 2 O 4 was synthesized by a hydrothermal method and applied as an effective sorbent during the extraction process. After adsorption of the drug using 10 mg of MIL-101Cr (NH 2) @ SiO 2 @ NiFe 2 O 4 nanoparticles through vortexing (1 min), the sorbent was separated from the sample solution using a magnet. To eluate the drug, the sorbent containing the sunitinib was contacted with 100 µL dimethylformamide. The eluent was analyzed by high performance liquid chromatography-tandem mass spectrometry. Reasonable validation data consisting of low limits of detection (0.14, 0.35, and 0.70 ng mL−1 in deionized water, plasma, and urine) and quantification (0.48, 1.2, and 2.4 ng mL−1 in deionized water, plasma, and urine, respectively), a wide linear range of the calibration curve (0.48–200, 1.2–200, and 2.4–100 ng mL−1 in deionized water, plasma, and urine, respectively) good extraction recovery (76 %), and low relative standard deviations for inter- and intra-day precisions (6.9 %) were obtained by the method. Eventually, the proposed procedure was effectively implemented on both plasma and urine samples, yielding successful outcomes. [ABSTRACT FROM AUTHOR]

• Simultaneous quantification and confirmation of oxycodone/metabolites in equine urine. • The method is validated for quantification analysis. • The method is verified for equine doping control analysis. • Oxycodone metabolites have longer detection periods compared to oxycodone. • Oxymorphone is the primary metabolite of oxycodone in equine urine. Oxycodone, an opioid commonly used to treat pain in humans, has the potential to be abused in racehorses to enhance their performance. To understand the pharmacokinetics of oxycodone and its metabolites in horses, as well as to detect the illegal use of oxycodone in racehorses, a method for quantification and confirmation of oxycodone and its metabolites is needed. In this study, we developed and validated an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method that can simultaneously quantify and confirm oxycodone and eight metabolites in equine urine. Samples were subjected to enzymatic hydrolysis and then liquid–liquid extraction using ethyl acetate. The analyte separation was achieved on a Hypersil Gold C18 sub-2 µm column and analytes were detected on a triple quadrupole mass spectrometer. The limit of detection (LOD) and lower limit of quantification (LLOQ) were 25–50 pg/mL and 100 pg/mL, respectively. Excellent linearity of the calibration curves was observed over a range of 100–10000 pg/mL for all nine analytes. Retention time, signal-to-noise ratio, and product ion ratios were utilized as confirmation criteria, with the limits of confirmation (LOC) ranging from 100 to 250 pg/mL. The data from a pilot pharmacokinetic (PK) study suggested that oxycodone metabolites have longer detection periods in equine urine compared to oxycodone itself; thus, the detection of metabolites in equine urine extends the ability to detect oxycodone exposure in racehorses. [ABSTRACT FROM AUTHOR]

• First validated LC-MS/MS method for quantification of polyphenols in commercial fish feed ingredients. • Evaluation of the effect of the storage time, temperature, vial color and concentration on the stability of polyphenols by means of a factorial design. • Comprehensive study of the matrix effects to guide the selection of an appropriate calibration method. • Evaluation of the method on real commercial fish feed ingredient samples. The perception of polyphenols as a safe, healthy, and sustainable solution for replacing synthetic antioxidants has been an important factor for their rapid growing in the global food market. Therefore, it is essential to use reliable methods for their quantification in commercial products intended for animal or human consumption. The purpose of this study is to evaluate the performance of some solvents used for the extraction of selected polyphenols, explore their stability under different experimental conditions, and validate a liquid chromatography tandem mass-spectrometry method for their quantification in commercial fish feed ingredients by using the standard addition method. The regression models for gallic acid, hydroxytyrosol, catechin, oleuropein, carnosol and carnosic acid were linear in the range 0–30 μg/mL, limit of detection and quantification around 0.03 and 0.1 μg/mL, respectively, and accuracy within ± 15 % of the nominal concentrations. The method was successfully applied to the determination of specific polyphenols in commercial fish feed ingredients supplemented with polyphenols from olive and rosemary extracts. [ABSTRACT FROM AUTHOR]

• An easy-to-use LC–MS method to quantify racemic MDMA and MDA was developed. • An enantioselective LC–MS method for MDMA and MDA was developed using a chiral column. • Both bioanalytical methods allow sensitive and rapid sample measurement. • Chiral pharmacokinetics were assessed in subjects dosed with MDMA or its enantiomer. 3,4-Methylenedioxymethamphetamine (MDMA) is an entactogen with therapeutic potential. The two enantiomers of MDMA differ regarding their pharmacokinetics and pharmacodynamics but the chiral pharmacology of MDMA needs further study in clinical trials. Here, an achiral and an enantioselective high performance liquid chromatography–tandem mass spectrometry method for the quantification of MDMA and its psychoactive phase I metabolite 3,4-methylenedioxyamphetamine (MDA) in human plasma were developed and validated. The analytes were detected by positive electrospray ionization followed by multiple reaction monitoring. The calibration range was 0.5–500 ng/mL for the achiral analysis of both analytes, 0.5–1,000 ng/mL for chiral MDMA analysis, and 1–1,000 ng/mL for chiral MDA analysis. Accuracy, precision, selectivity, and sensitivity of both bioanalytical methods were in accordance with regulatory guidelines. Furthermore, accuracy and precision of the enantioselective method were maintained when racemic calibrations were used to measure quality control samples containing only one of the enantiomers. Likewise, enantiomeric calibrations could be used to reliably quantify enantiomers in racemic samples. The achiral and enantioselective methods were employed to assess pharmacokinetic parameters in clinical study participants treated with racemic MDMA or one of its enantiomers. The pharmacokinetic parameters assessed with both bioanalytical methods were comparable. In conclusion, the enantioselective method is useful for the simultaneous quantification of both enantiomers in subjects treated with racemic MDMA. However, as MDMA and MDA do not undergo chiral inversion, enantioselective separation is not necessary in subjects treated with only one of the enantiomers. [ABSTRACT FROM AUTHOR]

• Simultaneous detection of youkenafil and M1 in different human matrixes by LC-MS/MS. • First report of concentrations of youkenafil and M1 in seminal plasma. • Youkenafil has no significant acute effect on semen quality. • No dose-proportional relationship for both youkenafil and M1 within 100–400 mg. Youkenafil is a novel Phosphodiesterase type 5 inhibitor used for treating erectile dysfunction. N-desethyl compound of youkenafil (M1) is its main active metabolite. In this study, two methods were developed and validated for the simultaneous determination of youkenafil and M1 by HPLC-MS/MS in human matrices including seminal plasma and plasma, in which the multiple reaction monitoring and electrospray ionization in positive mode were adopted, and the deuterated youkenafil (youkenafil-d5) was selected as the internal standard. The collected semen sample was kept at room temperature for approximately 30 min until fully liquefied. The volume of the liquefied semen was measured and then divided into two parts. One part was centrifuged to obtain the seminal plasma for the content detection of youkenafil and M1, while the other part was used for routine semen analysis. The chromatographic separation was accomplished with the column of Poroshell 120 EC-C18 (5 × 2.1 mm, 2.7 μm, Agilent). Protein precipitation with methanol was used for the pretreatment of seminal plasma and plasma. The intra-run and inter-run precisions were less than 6.4 % (relative standard deviation) and accuracies were all within −4.7 %–6.8 % (relative error) in both matrices. All other validated bioanalytical parameters were within the acceptance criteria set by the FDA. The methods were successfully applied to different clinical studies of youkenafil. In the clinical study of the acute effect of youkenafil on semen quality in healthy males, the content of youkenafil in seminal plasma was extremely low. Concentrations of youkenafil and M1 in seminal plasma were lower than those in plasma, at 20.7 % and 4.49 % of the plasma concentration, respectively. There was no significant acute effect of youkenafil on semen quality. In the pharmacokinetic study of youkenafil after single dose-escalation administration, the exposure to youkenafil and M1 was non-linear with the dose in the range of 100–400 mg. [ABSTRACT FROM AUTHOR]

• FDA-validated LC-MS/MS method for the bioanalysis of diamorphine and metabolites. • A non-laborious method facilitating rapid measurement of large sample quantities. • The method requires only small volumes of plasma (50 μL) for analysis. • Applicable for pharmacokinetic, drug-drug interaction, forensic, and metabolic studies. • Pharmacokinetic analysis of clinical study samples from an opioid-dependent patient. Diamorphine, commonly known as heroin, is a semi-synthetic opioid analgesic. In the context of heroin-assisted treatment for opioid-dependent patients, diamorphine is mostly administered intravenously. However, recent attention has shifted towards intranasal administration as a better-tolerated alternative to the intravenous route. Here, we developed and validated a rapid bioanalytical method for the simultaneous quantification of diamorphine and its major metabolites 6-monoacetylmorphine, morphine, morphine-3-glucuronide, and morphine-6-glucuronide in human plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS). A straightforward protein precipitation extraction step was used for sample preparation. Chromatographic analyte separation was achieved using a Kinetex EVO C 18 analytical column and a mobile phase gradient comprising an aqueous solution of ammonium hydrogen carbonate and methanol supplied with formic acid. Employing positive electrospray ionization and scheduled multiple reaction monitoring, we established a quantification range of 1–1,000 ng/mL for all analytes. Our validation results demonstrate a mean intra-assay accuracy of 91–106% and an intra-assay precision (CV) between 2 and 9% for all analytes and over three validation runs. The method exhibits a high extraction recovery (> 87%) and a negligible matrix effect (99–125%). Furthermore, no interferences with endogenous plasma compounds were detected. Lastly, we applied the method to assess the plasma concentrations of an opioid-dependent patient after the intranasal administration of diamorphine in a clinical study. In summary, we have successfully developed a rapid, highly reliable, and straightforward bioanalytical method for quantifying diamorphine and its metabolites in low amounts of clinical plasma samples. [ABSTRACT FROM AUTHOR]

• The serum levels of vitamins were determined by High Performance Liquid Chromatography (HPLC). • 16S rRNA gene sequencing and Ultra Performance Liquid Chromatography-tandem Mass Spectrometry (UPLC-MS/MS) targeted metabolomics were used for microbiome and metabolome analysis. • 25 (OH) VD3 and (VD2 + VD3), vitamins A and B9 are significantly lower in the training cohort of septic patients and have potential predictive value for the prognosis of septic patients. • The intestinal flora and flora metabolites of septic patients differed from those of healthy adults and were strongly associated with altered serum vitamin levels. Vitamins are essential micronutrients that play key roles in many biological pathways associated with sepsis. The gut microbiome plays a pivotal role in the progression of sepsis and may contribute to the onset of multi-organ dysfunction syndrome (MODS). The aim of this study was to investigate the changes in serum vitamins, and their correlation with intestinal flora and metabolomic profiles in patients with sepsis. The serum levels of vitamins were determined by Ultra Performance Liquid Chromatography (UPLC). 16S rRNA gene sequencing and Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) targeted metabolomics were used for microbiome and metabolome analysis. In the training cohort: After univariate, multivariate (OPLS-DA) and Spearman analyses, it was concluded that vitamin levels of 25 (OH) VD3 and (VD2 + VD3), as well as vitamins A and B9, differed significantly among healthy controls (HC), non-septic critical patients (NS), and sepsis patients (SS) (P < 0.05). The validation cohort confirmed the differential vitamin findings from the training cohort. Moreover, analyses of gut flora and metabolites in septic patients and healthy individuals revealed differential flora, metabolites, and metabolic pathways that were linked to alterations in serum vitamin levels. We found for the first time that vitamin B9 was negatively correlated with g_Sellimonas. Sepsis patients exhibited significantly lower levels of 25 (OH) VD3 and (VD2 + VD3), vitamins A and B9, which hold potential as predictive markers for sepsis prognosis. The changes in these vitamins may be associated with inflammatory factors, oxidative stress, and changes in gut flora. [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]

• Vitamin D 2 , D 3 , and 13 metabolites were separated using a 10 min UHPSFC method. • 1,25-(OH) 2 -D 3 , 1,24-(OH) 2 -D 3, and 24,25-(OH) 2 -D 3 were separated on baseline in UHPSFC method. • APPI and APCI techniques were compared in UHPSFC-MS/MS with regard to sensitivity. • Vitamin D esters were separated in UHPLC for the first time. • UHPLC using 1-AA instead of C18 was examined for the first time. • UHPSFC and UHPLC methods were verified and compared. Fat-soluble vitamin D is an essential bioactive compound important for human health. Insufficient vitamin D levels can result not only in bone disease but also in other disorders, such as cancer, metabolic disorders, and diseases related to poor immune function. The current methods commonly used for vitamin D analysis are often applied to determine the levels of the most abundant metabolite in plasma, i.e., 25-OH-D 2 /D 3. These methods do not consider the presence of other hydroxylated and esterified metabolites, including isomers and epimers, which are typically found in low concentrations. In this study, we developed a fast and selective ultra-high performance supercritical fluid chromatography (UHPSFC) method using a 150 mm long 1-amino anthracene (1-AA) column and a mobile phase consisting of carbon dioxide and methanol/isopropanol (1/1, v/v) mixed with 8 % water. After thorough optimization of column temperature and back pressure, the separation of four vitamin D 3 esters, vitamin D 3 and D 2 , and eight mono- and di-hydroxylated metabolites, including three groups of isomers, was achieved in 10 min. Two ion sources, atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization optimized within this study, were compared in tandem mass spectrometry (MS/MS) detection. No significant sensitivity differences were observed. Subsequently, the same 1-AA column chemistry was examined in ultra-high performance liquid chromatography (UHPLC) as the stationary phase that could hypothetically bring different selectivity in the separation of vitamin D and its metabolites. However, this hypothesis was rejected, and C 18 was used as a stationary phase in the final optimized UHPLC-MS/MS method. Despite detailed optimization, the final 15 min UHPLC method was not able to separate di-hydroxylated isomers of vitamin D 3 , while it enabled better resolution of esterified forms compared to UHPSFC. Optimized methods provided similar repeatability of retention times and peak areas, with RSD < 2 % and 10 %, respectively. The lowest limits of quantification were in the range of 1.2 – 4.9 ng/mL for UHPSFC-APCI-MS/MS, while for UHPLC-APCI-MS/MS, they were typically in the range of 2.6 – 9.6 ng/mL. Based on the obtained results, the UHPSFC-APCI-MS/MS method was the most promising approach for fast, selective, and sensitive analysis that could be applied in the analysis of biological samples with emphasis on the separation of both hydroxylated and esterified metabolites, including isomeric forms. [ABSTRACT FROM AUTHOR]

• 4-ANPP, acetyl fentanyl, fentanyl, p- fluorofentanyl, norfentanyl, and xylazine quantitated. • O - and m -fluorofentanyl qualitatively identified. • Analytes extracted from postmortem blood using solid-phase extraction. • Analytes separated and detected by liquid chromatography tandem mass spectrometry. • Results provide valuable information to the forensic toxicology community. The purpose of this study was to develop and validate a method to quantitate the veterinary sedative xylazine as well as 4-anilino-N-phenethylpiperidine (4-ANPP), acetyl fentanyl, fentanyl, norfentanyl, and p- fluorofentanyl in blood utilizing liquid chromatography tandem mass spectrometry. This method also qualitatively monitors for the presence of o -fluorofentanyl and m -fluorofentanyl isomers. UCT Clean Screen® DAU extraction columns were utilized to isolate the analytes in postmortem blood samples. The extracts were eluted, evaporated, reconstituted, and then analyzed using a Waters Acquity™ UPLC coupled a triple quadrupole mass spectrometer. The lower limit of quantitation was determined to be 0.1 ng/mL for all analytes, except for xylazine (0.2 ng/mL). The upper limit of quantitation for all analytes was 100 ng/mL. No interferences from matrix, internal standard, or common drug analytes were observed. Bias (-13.1–4.6 %) and precision (-13.14–10.3 %) fell within the acceptable ± 20 % criteria range. Dilution integrity at x2, x10, and x100 was evaluated and all results were within ± 20 % of the target concentration. Processed extract stability was evaluated after 72 h and all results were within ± 20 % of the analyte initial concentration. Matrix effects were the most prominent with xylazine, but deemed acceptable as the deuterated internal standard also observed comparable enhancement. Analysis of 89 postmortem blood samples by this method resulted in positive results for fentanyl (0.27–66 ng/mL, n = 82), xylazine (0.24–958 ng/mL, n = 21), 4-ANPP (0.10–38 ng/mL, n = 72), acetyl fentanyl (0.18–1.5 ng/mL, n = 3), p- fluorofentanyl (0.11–33 ng/mL, n = 30), and norfentanyl (0.10–98 ng/mL, n = 73). [ABSTRACT FROM AUTHOR]

• Saliva model consisting of nine components. • LC-MS/MS-based method for determination of leachables from biomaterials. • Method validation showed that the model is a suitable surrogate for native saliva. This study aimed to prove the validity of a mixture of chemicals, including salts, small organic molecules, mucin, and α-amylase, as saliva surrogate ("artificial saliva") for assessing leakage of methacrylate monomers and other constituents from dental materials. To achieve this, we developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of 2-hydroxyethyl methacrylate (HEMA), triethylene glycol dimethacrylate (TEGDMA), diurethane dimethacrylate (UDMA), bisphenol A glycerolate dimethacrylate (BisGMA), diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide (TPO), bisphenol A (BPA), and five homologues of ethoxylated bisphenol A dimethacrylate (BisEMA EO2–6) in unstimulated and artificial saliva, and compared their concentrations in the two saliva media following either spiking with a mixture of the compounds or incubation of test specimens of printed biomaterials. Test specimens were immersed in unstimulated/artificial saliva, incubated at 37 °C for 24 h, and saliva aliquots were extracted with methanol and subsequently analyzed by LC-MS/MS. The method was validated with regard to matrix effects, linearity, selectivity, lower limits of quantification (LLOQ), precision, bias and combined measurement uncertainty (u′). The performance characteristics of the method were comparable for unstimulated and artificial saliva samples. The combined u' for individual chemicals at a concentration of 10 × LLOQ were within the range of 5.3–14 % for unstimulated saliva and 6.9–16 % for artificial saliva, except for the BisEMA homologues. Combined u' for the latter were 27–74 % in unstimulated saliva, and 27–79 % in artificial saliva. There was no detectable release of BPA from the test specimens, and the TPO concentrations were mainly below the LLOQ. TEGDMA and UDMA were detected in the highest quantities, and at comparable concentrations in the unstimulated and artificial saliva. For all BisEMA homologues, the release was higher in unstimulated saliva than in artificial saliva. The study showed that the artificial saliva model can be a suitable replacement for native saliva, but might underestimate leakage of more lipophilic methacrylates. [ABSTRACT FROM AUTHOR]

• Japanese Azadinium poporum produces AZA2 as dominant toxin and several AZAs. • Twelve new AZAs were discovered from Japanese Azadinium poporum. • New AZAs detected as bi-charged ions were discovered. • AZAs detected as bi-charged ions were sulfated AZAs and some had hexoses. Lipophilic marine biotoxin azaspiracids (AZAs) are produced by dinoflagellates Azadinium and Amphidoma. Recently, several strains of Azadinium poporum were isolated from Japanese coastal waters, and detailed toxin profiles of two strains (mdd421 and HM536) among them were clarified by several detection techniques on liquid chromatography-tandem mass spectrometry (LC-MS/MS) and liquid chromatography-quadrupole time of flight mass spectrometry (LC-QTOFMS). In our present study, AZA analogues in seven strains of A. poporum from Japanese coastal waters (including two previously reported strains) were determined by these detection techniques. The dominant AZA in the seven strains was AZA2 accompanied by small amounts of several known AZAs and twelve new AZA analogues. Eight of the twelve new AZA analogues discovered in our present study were detected as bi-charged ions on the positive mode LC/MS/MS. This is the first report describing AZA analogues detected as bi-charged ions with hexose and sulfate groups in their structures. [ABSTRACT FROM AUTHOR]

• Cytotoxic anticancer drugs are increasingly recognized to be valid candidates for therapeutic drug monitoring (TDM). • Numerous LC-MS/MS methods have been developed to that end for the quantification in patients' plasma samples of single or combination of chemotherapeutic drugs. • However, there is a need for a more systematic use of stable isotopically labelled internal standards of cytotoxic drugs to circumvent patients' biological matrices variability, that would increase the reliability of cytotoxic drugs quantification. • LC-MS/MS assays can accommodate multiplexed analyses of cytotoxic drugs with optimal selectivity and specificity as well as short analytical times. Yet, there is still several organisational and clinical constraints to address before TDM of cytotoxic drugs could be more largely adopted. In the era of precision medicine, there is increasing evidence that conventional cytotoxic agents may be suitable candidates for therapeutic drug monitoring (TDM)- guided drug dosage adjustments and patient's tailored personalization of non-selective chemotherapies. To that end, many liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) assays have been developed for the quantification of conventional cytotoxic anticancer chemotherapies, that have been comprehensively and critically reviewed. The use of stable isotopically labelled internal standards (IS) of cytotoxic drugs was strikingly uncommon, accounting for only 48 % of the methods found, although their use could possible to suitably circumvent patients' samples matrix effects variability. Furthermore, this approach would increase the reliability of cytotoxic drug quantification in highly multi-mediated cancer patients with complex fluctuating pathophysiological and clinical conditions. LC-MS/MS assays can accommodate multiplexed analyses of cytotoxic drugs with optimal selectivity and specificity as well as short analytical times and, when using stable-isotopically labelled IS for quantification, provide concentrations measurements with a high degree of certainty. However, there are still organisational, pharmacological, and medical constraints to tackle before TDM of cytotoxic drugs can be more largely adopted in the clinics for contributing to our ever-lasting quest to improve cancer treatment outcomes. [ABSTRACT FROM AUTHOR]