Main Metabolites In Plasma Research Articles

Pharmacokinetic study of a new 4,5-dihydroisoxazole-5-carboxamide derivative in rats

Introduction. 3-(2-butyl-5-chloro-1H-imidazole-4-yl)-N-[4-methoxy-3-(trifluoromethyl)phenyl]-4,5-dihydro-1,2-oxazole-5-carboxamide (R004) is a prospective new molecule for the treatment of rheumatoid arthritis. This compound is at the stage of preclinical research, during which its pharmacokinetics must be studied along with efficacy and safety. In this case, it is necessary to establish the main pharmacokinetic parameters of the drug and its metabolites in blood plasma after a single administration.Aim. Evaluation of linearity of pharmacokinetics and relative bioavailability of 3-(2-butyl-5-chloro-1H-imidazole-4-yl)-N-[4-methoxy-3-(trifluoromethyl)phenyl]-4,5-dihydro-1,2-oxazole-5-carboxamide and its main metabolites in plasma after a single administration of the substance to rats.Materials and methods. Substance R004 was administered to rats once orally at dosages of 10 mg/kg, 20 mg/kg and 40 mg/kg and intraperitoneally at a dosage of 10 mg/kg. The study was carried out using 24 Wistar rats: 6 rats were used for each of the dosage. Blood samples were collected before administration and 30 min, 1 h, 1 h, 30 min, 2 h, 3 h, 4 h, 6 h, 8 h, 10 h, 12 h, 24 h after administration of the drug. The obtained plasma was stabilized with a 250 mM ammonium acetate solution to prevent hydrolysis of R004. Plasma samples were prepared by means proteins precipitation by acetonitrile solution of internal standards. Measurement of the concentration of R004 and its metabolites 3-(2-butyl-5-chloro-1H-imidazole-4-yl)-4,5-dihydro-1,2-oxazole-5-carboxylic acid (M1) and 4-methoxy-3-(trifluoromethyl)aniline (M2) was performed using HPLC-MS/MS. A non-compartment approach was applied for evaluation pharmacokinetic parameters.Results and discussion. The developed bioanalytical method has been fully validated in accordance with the requirements of the EAEU guideline for conducting bioequivalence study and ICH M10 guideline. The analytical range of determination in plasma of R004 was 2–2000 ng/ml, M1 and M2 – 1–1000 ng/ml. The dependence of the parameters Cmax and AUC0–t of the studied compound and its metabolites on the administered dose was linear. Thus, the value of the correlation coefficient of Cmax R004 was 0.9995, and value of the correlation coefficient of AUC0–t was 0.9991. The presence of enterohepatic recirculation R004 has been determined. The relative bioavailability of R004 was 21.26%.Conclusion. The developed method was successfully applied for the pharmacokinetic research. R004 and its metabolites have linear pharmacokinetics in case of oral administration.

A systematic UHPLC-Q-TOF-MS/MS-based strategy for analysis of chemical constituents and related in vivo metabolites of Buyang Huanwu decoction

Ethnopharmacological relevanceBuyang Huanwu Decoction (BYHWD), a traditional Chinese medicine, is one of the classic prescriptions for the treatment of ischemic stroke in clinical practice. It has the effects of tonifying qi, activating blood circulation, and promoting meridian circulation. However, its chemical analysis has not been clarified, which greatly hinders its further clinical application. Therefore, it is necessary to clarify the chemical constituents and metabolites profile of BYHWD in vivo. Aim of the studyCharacterizing the chemical basis of BYHWD in vitro, and combing studies of related metabolism in vivo to screen out the potential active components of BYHWD with pharmacological effects in vivo. Materials and methodsTwelve male rats weighed 200 ± 20 each were selected for the experiments. According to the fragmentation of different structural types of components and diagnostic ions, UHPLC-Q-TOF-MS/MS was used to classify and clarify the unknown components of BYHWD and identify the material basis of BYHWD in vitro. Then, rat plasma, tissues, feces, and urine were collected for analysis. Based on the similarity of MS responses (accurate molecular weight and secondary fragmentation) and chromatographic behavior (retention time), the in vivo prototype and metabolites were analyzed. Through the phase I and phase II metabolism law, a metabolite library was established to analyze the prototype-matched metabolites. ResultsA total of 121 in vitro compounds and 55 in vivo prototypes of BYHWD were identified, corresponding to 123 matched prototypes. It was mainly composed of flavonoids, triterpene saponins, nucleosides and lactones both in vitro and in vivo. Quercetin, ligustilide, astragaloside IV, calycosin, paeoniflorin and ferulic acid were the main prototypes and metabolites in plasma and urine. ConclusionQuercetin, ligustilide, astragaloside IV, calycosin, paeoniflorin and ferulic acid were the main active ingredients of BYHWD.

LC-MS-MS quantification of Δ8-THC, Δ9-THC, THCV isomers and their main metabolites in human plasma.

In recent years, potential therapeutic applications of several different cannabinoids, such as Δ9-tetrahydrocannabinol (Δ9-THC), its isomer Δ8-THC and Δ9-tetrahydrocannabivarin (Δ9-THCV), have been investigated. Nevertheless, to establish dose-effect relationship and to gain knowledge of their pharmacokinetics and metabolism, sensitive and specific analytical assays are needed to measure these compounds in patients. For this reason, we developed and validated an online extraction high-performance liquid/liquid chromatography-tandem mass spectrometry (LC/LC-MS-MS) method for the simultaneous quantification of 13 cannabinoids and metabolites including the Δ8 and Δ9 isomers of THC, THCV and those of their major metabolites in human plasma. Plasma was fortified with cannabinoids at varying concentrations within the working range of the respective compound and 200 µL was extracted using a simple one-step protein precipitation procedure. The extracts were analyzed using online trapping LC/LC-atmospheric pressure chemical ionization-MS-MS running in the positive multiple reaction monitoring mode. The lower limit of quantification ranged from 0.5 to 2.5 ng/mL, and the upper limit of quantification was 400 ng/mL for all analytes. Inter-day analytical accuracy and imprecision ranged from 82.9% to 109% and 4.3% to 20.3% (coefficient of variance), respectively. Of 534 plasma samples following controlled oral administration of Δ8-THCV, 236 were positive for Δ8-THCV (median; interquartile ranges: 3.5 ng/mL; 1.8-11.9 ng/mL), 383 for the major metabolite (-)-11-nor-9-carboxy-Δ8-tetrahydrocannabivarin (Δ8-THCV-COOH) (95.4 ng/mL; 20.7-328 ng/mL), 260 for (-)-11-nor-9-carboxy-Δ9-tetrahydrocannabivarin (Δ9-THCV-COOH) (5.8 ng/mL; 2.5-16.1 ng/mL), 157 for (-)-11-hydroxy-Δ8-tetrahydrocannabivarin (11-OH-Δ8-THCV) (1.7 ng/mL; 1.0-3.7 ng/mL), 49 for Δ8-THC-COOH (1.7 ng/mL; 1.4-2.3 ng/mL) and 42 for Δ9-THCV (1.3 ng/mL; 0.8-1.6 ng/mL). We developed and validated the first LC/LC-MS-MS assay for the specific quantification of Δ8-THC, Δ9-THC and THCV isomers and their respective metabolites in human plasma. Δ8-THCV-COOH, 11-hydroxy-Δ8-THCV and Δ9-THCV-COOH were the major Δ8-THCV metabolites in human plasma after oral administration of 98.6% pure Δ8-THCV.

Exploring the effect of Wuzhi capsule on the pharmacokinetics of regorafenib and its main metabolites in rat plasma using liquid chromatography-tandem mass spectrometry.

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

Digestive interaction between dietary nitrite and dairy products generates novel nitrated linolenic acid products

Nitrated fatty acids are important anti-inflammatory and protective lipids formed in the gastric compartment, with conjugated linoleic acid (rumenic acid, RA, 9Z,11E-18:2) being the primary substrate for lipid nitration. The recently reported identification of nitrated rumelenic acid (NO2-RLA) in human urine has led to hypothesize that rumelenic acid (RLA, 9Z,11E,15Z-18:3) from dairy fat is responsible for the formation of NO2-RLA. To evaluate the source and mechanism of NO2-RLA formation, 15N labeled standards of NO2-RLA were synthesized and characterized. Afterward, milk fat with different RA and RLA levels was administered to mice in the presence of nitrite, and the appearance of nitrated fatty acids in plasma and urine followed. We confirmed the formation of NO2-RLA and defined the main metabolites in plasma, urine, and tissues. In conclusion, RLA obtained from dairy products is the main substrate for forming this novel electrophilic lipid reported to be present in human urine.

LC-MS/MS assisted biomonitoring of ropivacaine and 3-OH-ropivacaine after plane block anesthesia for cardiac device implantation.

Introduction: Ropivacaine is a popular local anesthetic used for regional anesthesia or for pain management. Although designed as an enantiomerically pure drug, an aspect that reduces the adverse effects, its toxicological effects are still a risk. As such, biomonitoring to assure appropriate dosage and bioavailability are essential to avoid complications during or post-surgery. Methods: The study focused on developing a sensitive, selective, and accurate liquid chromatography-mass spectrometry (LCMS/MS) method which facilitates the biomonitoring of ropivacaine and its main metabolite in plasma after regional anesthesia using ropivacaine. Results and Discussion: The method was validated with regards to all relevant parameters, such as sensitivity, selectivity, accuracy, precision, and the effect of sample matrix. The method was successfully used in a pilot study, which included one patient undergoing plane block anesthesia for cardiac device implantation. The results showed the method is appropriate for its intended purpose and could even be used in other, similar applications.

Human blood plasma biomarkers of diet and weight loss among centrally obese subjects in a New Nordic Diet intervention.

The New Nordic Diet (NND) has been shown to promote weight loss and lower blood pressure amongst obese people. This study investigates blood plasma metabolite and lipoprotein biomarkers differentiating subjects who followed Average Danish Diet (ADD) or NND. The study also evaluates how the individual response to the diet is reflected in the metabolic differences between NND subjects who lost or maintained their pre-intervention weight. Centrally obese Danes (BMI >25) followed NND (90 subjects) or ADD (56 subjects) for 6 months. Fasting blood plasma samples, collected at three time-points during the intervention, were screened for metabolites and lipoproteins (LPs) using proton nuclear magnetic resonance spectroscopy. In total, 154 metabolites and 65 lipoproteins were analysed. The NND showed a relatively small but significant effect on the plasma metabolome and lipoprotein profiles, with explained variations ranging from 0.6% for lipoproteins to 4.8% for metabolites. A total of 38 metabolites and 11 lipoproteins were found to be affected by the NND. The primary biomarkers differentiating the two diets were found to be HDL-1 cholesterol, apolipoprotein A1, phospholipids, and ketone bodies (3-hydroxybutyric acid, acetone, and acetoacetic acid). The increased levels of ketone bodies detected in the NND group inversely associated with the decrease in diastolic blood pressure of the NND subjects. The study also showed that body weight loss among the NND subjects was weakly associated with plasma levels of citrate. The main plasma metabolites associated with NND were acetate, methanol and 3-hydroxybutyrate. The metabolic changes associated with the NND-driven weight loss are mostly pronounced in energy and lipid metabolism.

Monitoring of Levosimendan Administration in Patients with Pulmonary Hypertension Undergoing Cardiac Surgery and Effect of Two Different Dosing Schemes on Hemodynamic and Echocardiographic Parameters.

The perioperative management of patients with pulmonary hypertension (PH) undergoing cardiac surgery represents one of the most challenging clinical scenarios. This fact mainly depends on the relationship existing between PH and right ventricular failure (RVF). Levosimendan (LS) is an inodilator that might be an effective agent in the treatment of PH and RVF. The aim of this study was to examine the impact of the duration of cardiopulmonary bypass (CPB) on the therapeutic drug monitoring of LS and to evaluate the effect of preemptive administration of LS on perioperative hemodynamic and echocardiographic parameters in cardiac surgical patients with preexisting PH. In this study, LS was administered in adult patients undergoing cardiac surgery before CPB in order to prevent exacerbation of preexisting PH and subsequent right ventricular dysfunction. Thirty cardiac surgical patients with preoperatively confirmed PH were randomized to receive either 6 μg/kg or 12 μg/kg of LS after the induction of anesthesia. The plasma concentration of LS was measured after CPB. In this study, a low sample volume was used combined with a simple sample preparation protocol. The plasma sample was extracted by protein precipitation and evaporated; then, the analyte was reconstituted and detected using specific and sensitive bioanalytical liquid chromatography with mass spectrometry (LC-MS/MS) methodology. The clinical, hemodynamic, and echocardiographic parameters were registered and evaluated before and after the administration of the drug. A fast bioanalytical LC-MS/MS methodology (a run time of 5.5 min) was developed for the simultaneous determination of LS and OR-1896, its main metabolite in human plasma. The LC-MS/MS method was linear over a range of 0.1-50 ng/mL for LS and 1-50 ng/mL for its metabolite OR-1896. Measured plasma concentrations of LS were inversely related to the duration of CPB. LS administration before CPB during cardiac surgery was effective in reducing pulmonary artery pressure and improving hemodynamic parameters after CPB, with a more pronounced and durable effect of the drug at the dose of 12 μg/kg. Additionally, administration of LS at a dose of 12 μg/kg in cardiac surgical patients with PH before CPB improved right ventricular function. LS administration decreases pulmonary artery pressure and may improve right ventricular function in patients with PH undergoing cardiac surgery.

Development and validation of a bioanalytical method for the quantification of CHF6550 and its metabolite (CHF6671) in rat plasma and lung homogenate using LC–MS/MS

A selective and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for accurate determination of CHF6550 and its main metabolite in rat plasma and lung homogenate samples. All biological samples were prepared by simple protein precipitation method using deuterated internal standards. The analytes were separated on a HSS T3 analytical column with 3.2 min run time at flow rate of 0.5 mL/min. The detection was performed on a triple-quadrupole tandem mass spectrometer equipped with positive-ion electrospray ionization by selected-reaction monitoring of the transitions at m/z 735.3 → 98.0 for CHF6550 and m/z 638.3 → 319.2 and 638.3 → 376.2 for CHF6671. The calibration curves for plasma samples were linear between 50 and 50000 pg/mL for both analytes. The calibration curves for lung homogenate samples were linear within 0.1–100 ng/mL for CHF6550 and 0.3–300 ng/mL for CHF6671. The method was successfully applied to a 4-week toxicity study.

Biotransformation of the Novel Myeloperoxidase Inhibitor AZD4831 in Preclinical Species and Humans.

We report herein an in-depth analysis of the metabolism of the novel myeloperoxidase inhibitor AZD4831 ((R)-1-(2-(1-aminoethyl)-4-chlorobenzyl)-2-thioxo-2,3-dihydro-1H-pyrrolo[3,2-d]pyrimidin-4(5H)-one) in animals and human. Quantitative and qualitative metabolite profiling were performed on samples collected from mass balance studies in rats and humans. Exposure of circulating human metabolites with comparable levels in animal species used in safety assessment were also included. Structural characterization of 20 metabolites was performed by liquid chromatography high-resolution mass spectrometry, and quantification was performed by either 14C analysis using solid phase scintillation counting or accelerator mass spectrometry and, where available, authentication with synthesized metabolite standards. A complete mass balance study in rats is presented, while data from dogs and human are limited to metabolite profiling and characterization. The metabolism of AZD4831 is mainly comprised of reactions at the primary amine nitrogen and the thiourea sulfur, resulting in several conjugated metabolites with or without desulfurization. A carbamoyl glucuronide metabolite of AZD4831 (M7) was the most abundant plasma metabolite in both human healthy volunteers and heart failure patients after single and repeated dose administration of AZD4831, accounting for 75%-80% of the total drug-related exposure. Exposures to M7 and other human circulating metabolites were covered in rats and/or dogs, the two models most frequently used in the toxicology studies, and were also highly abundant in the mouse, the second model other than rat used in carcinogenicity studies. The carbamoyl glucuronide M7 was the main metabolite in rat bile, while a desulfurized and cyclized metabolite (M5) was abundant in rat plasma and excreta. SIGNIFICANCE STATEMENT: The biotransformation of AZD4831, a novel myeloperoxidase inhibitor inhibiting xanthine derivative bearing thiourea and primary aliphatic amine functions, is described. Twenty characterized metabolites demonstrate the involvement of carbamoylation with glucuronidation, desulfurization, and cyclization as main biotransformation reactions. The carbamoyl glucuronide was the main metabolite in human plasma, likely governed by a significant species difference in plasma protein binding for this metabolite, but this and other human plasma metabolites were covered in animals used in the toxicity studies.

Simultaneous determination of dacomitinib and its major metabolite, O-desmethyl dacomitinib in human plasma by LC-MS/MS and its application to clinical testing in patients with non-small cell lung cancer

Dacomitinib, an irreversible pan-ErbB tyrosine kinase inhibitor targeting the human epidermal growth factor receptor, is used for the treatment of metastatic non-small cell lung cancer. To facilitate the investigations on its metabolism and other relevant studies, based on high-performance liquid chromatography-tandem mass spectrometry (HPLC–MS/MS), a rapid and sensitive bioanalytical technique was established and fully validated for simultaneous quantification of dacomitinib and its main metabolite in human plasma. The plasma samples were treated with acetonitrile containing 0.1% formic acid and the liquid supernatant was collected, dried and dissolved in methanol-water-formic acid (200:800:1, v/v) before injection. The chromatographic separation was performed on an ACE Excel C18 column (2.1 mm × 50.0 mm, i.d., 5 μm) by gradient elution with a mixture of buffer (5 mM ammonium acetate in 0.1% formic acid) and acetonitrile, serving as the mobile phase, with an overall run time of 4 min. Dacomitinib, O-desmethyl dacomitinib and IS were subsequently detected on an AB QTRAP 5500 mass spectrometer in positive ion and multiple reaction monitoring modes at the precursor-to-product transitions of m/z 470.4 → 385.0, m/z 456.0 → 370.9 and m/z 480.2 → 385.1, respectively. The accuracy and precision of determinations were guaranteed within the concentration ranges of 0.25–100 ng/mL for dacomitinib and 0.20–80 ng/mL for O-desmethyl dacomitinib. The intra- and inter-assay accuracy ranged from 92.00% to 104.50% and the intra- and inter-assay precision was less than 8.20% for each analyte. The method was validated and the relevant parameters, including selectivity, interference among analytes and internal standard, carry-over effect, dilution integrity, extraction recovery, matrix effect, and stability, all satisfied the requirements formulated by the US Food and Drug Administration and the European Medicines Agency. The clinical applicability of the fully-validated method was evaluated in medicated samples from patients on dacomitinib.

Abstract 1369: Predicting clinical pharmacokinetics and toxicity of current and emerging oncology therapeutics by normothermic perfusion of isolated human-sized organs

Abstract Novel cancer therapeutics have less than a 12% probability of translating from bench to bedside. Unwarranted toxicity and inadequate therapeutic delivery due to uptake by clearance organs, not predicted by current preclinical methods, have contributed towards this high rate of attrition. In the present work, we propose normothermic machine perfusion of human or human-sized organs as a more predictive, closer-to-human model to investigate drug pharmacokinetics and toxicity. Over the past decade, developments in the field of organ preservation for transplantation have enabled prolonged (>12 hours) normothermic machine perfusion (NMP) of isolated porcine or human organs ex vivo, maintaining quasi-physiological haemodynamic, synthetic and metabolic function using a packed red cell perfusate with physiological oxygenation and nutrient levels at normal body temperature. This preserves physiological processes such as metabolism and drug elimination, and enables easy access to tissue, blood and excreted biological fluids, with NMP livers producing bile and NMP kidneys producing urine. We hypothesise that this will provide a physiologically relevant platform to investigate drug pharmacokinetics and toxicity. We selected a widely used small-molecule chemotherapeutic (Irinotecan hydrochloride 2mg/ml, Medac, UK) which has seen decades of clinical use and benefits from extensive clinical pharmacokinetic data. The small-molecule drug was infused into isolated porcine and human livers and kidneys, with quantification of concentration time profiles of the prodrug and its main metabolites in plasma, bile and urine over 16-24 hours of NMP. In addition to irinotecan (CPT11), three of its metabolites (APC, SN38G, SN38) were successfully detected and quantified, demonstrating peak plasma concentrations (Cmax ~ 10,000 ng/mL, 1000 ng/mL, 100 ng/g, 30 ng/g), plasma decay rates and percentages of injected dose in bile (%ID ~20%, 8%, 25%, 1%) and urine (%ID ~20%, 0.06%, 0.5%,0.1%) that are comparable to clinical data. Drug-tissue toxicity could also be adequately replicated in the NMP model. In conclusion, we have demonstrated that human-sized isolated, normothermically perfused livers and kidneys accurately represent the clinically observed pharmacokinetic and toxicity profiles of an established small-molecule therapeutic. Further model validation is ongoing for biologics and other nanomedicines which are susceptible to clearance by the mononuclear phagocytic system or are hepato- or nephrotoxic. If this proves successful, normothermic machine perfusion of isolated porcine and human organs could greatly aid the early screening of candidate therapeutics and significantly enhance the pace and success rate with which they are translated into patients. Citation Format: Tamsyn Clark, Luca Bau, Fungai Dengu, Daniel Voyce, Robert Carlisle, Peter Friend, Constantin Coussios. Predicting clinical pharmacokinetics and toxicity of current and emerging oncology therapeutics by normothermic perfusion of isolated human-sized organs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1369.

Identification of Robust Metabotypes Associated With Increased Cardiometabolic Disease Risk- an Approach for Improved Prevention Through Precision Nutrition

Abstract Objectives We hypothesize that individuals can be grouped into robust metabolic phenotypes (metabotypes) based on biochemical, anthropometric, gut microbial and metabolomics data and that such metabotypes will reflect differences in cardiometabolic disease risk and can act as targets for tailored nutritional prevention. We furthermore hypothesize that diet-gut microbiota interactions will be major determinants of the metabotypes. Methods Metabotyping is currently performed based on baseline data from 628 Danish adults from a validation sub-study of the Danish Diet Cancer and Health-Next Generation cohort. Participants were followed for one year, also providing data at 6 and 12 months. Dietary data was obtained by food frequency questionnaire and repeated 24h recalls and data on physical activity, smoking, sociodemographic factors, disease prevalence and use of medication was collected by questionnaires. Untargeted metabolomics and gut microbiota are currently determined. Metabotypes will be identified using clustering algorithms, variable optimization and data integration of the plasma metabolome, the 16S rRNA microbiota and 14 biochemical and anthropometric variables. Differences in habitual diet and physical activity across metabotypes will be determined as well as main metabotype determinants and potential plasma metabolite biomarkers. We will also assess the reproducibility of the metabotypes and biomarkers over time. Results Two clusters of individuals were identified by using the currently available clinical and anthropometric data. One of the clusters presented with mean values consistent with overweight, hypertension and dyslipidemia. Next, we will integrate the plasma metabolomics and gut microbial data into the analysis to determine the metabotypes. Conclusions We have identified one higher risk group and one lower risk group for cardiometabolic disease and will identify and characterize metabotypes based on more extensive data. Future research will assess whether individuals belonging to different metabotypes respond differently to dietary interventions. Funding Sources Formas, Sweden.

LC-MS/MS method for determination of seneciphylline and its metabolite, seneciphylline N-oxide in rat plasma, and its application to a rat pharmacokinetic study.

A rapid and sensitive ultra-performance liquid chromatography-tandem electrospray ionization mass spectrometry (UPLC-ESI/MS) method was established and validated for simultaneous determination of seneciphylline and its main metabolite in rat plasma. The plasma sample was prepared by simple methanol-mediated precipitation. Chromatographic separation was achieved within 3min by gradient elution using acetonitrile and water containing 0.1% formic acid as mobile phase on a Waters ACQUITY BEH C18 column (100 × 2.1mm, i.d. 1.7 μm). Quantitation was conducted in a positive multiple reaction monitoring mode. The linearity of the method was over the range of 1-1,000 ng/mL, with the lower limit of quantification of 1ng/mL. The intra- and inter-day precision and accuracy, extraction recovery, and matrix effect of analytes were within the acceptable limit. The analytes were stable during the process of sample collection, preparation, and analysis. The validated method was further applied to a pharmacokinetic study of seneciphylline in rats after oral and intravenous administration. The results revealed that seneciphylline was quickly absorbed into plasma (Tmax , 0.23-0.32 h) and reached the maximum concentration of 0.82-1.75 μg/mL after oral administration. Both seneciphylline and seneciphylline N-oxide were eliminated from plasma quickly. The low system exposure (oral bioavailability, 5.43-10.31%) was related to the extensive metabolism in the liver and microflora.

Development of an LC–MS/MS method for quantifying ASK120067, a novel mutant-selective inhibitor of the epidermal growth factor receptor (EGFR) as well as its main metabolite in human plasma and its application in a pharmacokinetic study

ASK120067, an oral irreversible tyrosine kinase inhibitor (TKI) targeting the epidermal growth factor receptor (EGFR), is formulated for the management of patients with non-small cell lung cancer (NSCLC) who harbor T790M resistant and EGFR active mutations. Two rapid and high-throughput methods based on liquid chromatography-tandem mass spectrometry to detect ASK120067 and its primary metabolite CCB4580030 in human plasma were developed and applied in the clinical trials. A protein precipitation method using acetonitrile coupled with a gradient elution separation in a BEH C18 column (1.7 µm, 2.1 × 50 mm) was used to process plasma and separation analytes. The chromatographic separation was performed on the mobile phase of 5 mM ammonium acetate in water with 0.1% formic acid (A) and acetonitrile (B), and the flow rate was 0.4 mL/min. The multiple reaction monitoring (MRM) mode was selected to monitor the precursor-to-product ion transitions of m/z 546.2 → m/z 431.2 for ASK120067 and m/z 532.1 → m/z 420.2 for CCB4580030 at the positive ionization mode. The precision and accuracy of the two methods for ASK1200067 and CCB4580030 were within acceptable range for the linear range in 5.00–5000 ng/mL and 0.500–500 ng/mL, respectively. Further stabilities for the two analytes and internal standard were also investigated covered the entire experimental process beginning from harvesting whole blood to plasma extraction and analysis. ASK120067 was then administered without issue onto a dose-escalation, the first-in-human Phase I clinical trial in Chinese NSCLC patients to determine the pharmacokinetics of oral ASK120067 administration.

Development and validation of GC–MS method for determination of methcathinone and its main metabolite in mice plasma and brain tissue after SPE: Pharmacokinetic and distribution study

Only focusing on the plasma levels is inadequate for the full consideration of the physiological disposition of illicit drugs in vivo. Therefore, we conducted the inclusive study on the in vivo dynamic process of intraperitoneal administration of methcathinone (MET), a well-known member of the synthetic cathinone derivatives, which is structurally similar to amphetamine analogs. This study described a validated, selective and sensitive GC–MS method for the simultaneous quantification of MET and its main metabolite, ephedrine (EPD), in the plasma and brain tissue of mice, after solid phase extraction (SPE). Ephedrine-d3 was used as an internal standard (IS). The developed method was validated following US-FDA guidelines within a concentration range of 5–1000 ng/mL for both drugs (r2 > 0.998) in the mice plasma and brain. The recoveries of MET and EPD from the mice plasma and brain ranged from 108.5 to 112.1%. The intra- and inter-day RSDs were ≤ 11.0 %. The proposed method was applied, for the first time, to investigate the pharmacokinetic (PK) and distribution study of MET and EPD following intraperitoneal administration of MET (1.4 mg/kg) to Swiss albino mice. The results exhibited that the Cmax and Tmax of MET in mice plasma was 517.1 ng/mL and 15 min as compared to 3.6 ng/mL and 2 h of EPD. Moreover, MET rapidly passed the blood brain barrier with Cmax of 1444.5 ng/mL achieved at 15 min, whereas, EPD monitored Cmax of 43.6 ng/mL at 4 h in mice brain. The highest concentration of MET in the mice brain followed by plasma was reported, with a necessity to perform more detailed clinical investigations.

Absorption, metabolism, and excretion of orange juice (poly)phenols in humans: The effect of a controlled alcoholic fermentation

The consumption of orange juice provides high concentrations of health-promoting bioactive compounds, the amount of which may increase upon alcoholic fermentation. Although fermentation may offer new prospects for the industry of orange-related products, there is a lack of studies reporting the influence of controlled alcoholic fermentation on the bioavailability of orange juice (poly)phenols in humans. The aim of this study was to evaluate the absorption profile, pharmacokinetic parameters, and urinary excretion of orange juice (poly)phenols in nine volunteers after acute administration of an orange juice and a beverage prepared after controlled alcoholic fermentation of the juice. Plasma and urine samples were analysed through a UHPLC-ESI-MS/MS targeted approach. A total of 24 (poly)phenol metabolites including both flavanone and phenolic acid derivatives were quantified, most of them being recorded only in urine. Phase II conjugates of hesperetin and naringenin were the main metabolites in plasma, while phenolic acids, in particular hydroxybenzoic acids, were the main compounds in urine. (Poly)phenols in both beverages were highly bioavailable (between 46 and 59%) and a notable inter-individual variability was seen. Significant treatment × time interactions were recorded for the sum of flavanones and phenolic acids in plasma, the (poly)phenols in the fermented juice being absorbed faster than after orange juice intake. Nevertheless, despite the food matrix having an impact on the absorption profile of orange juice (poly)phenols, this did not influence the pharmacokinetic parameters and urinary excretion of the (poly)phenol metabolites.

A simple and fast LC-MS/MS method for the routine measurement of cabozantinib, olaparib, palbociclib, pazopanib, sorafenib, sunitinib and its main active metabolite in human plasma

Targeted therapies such as cabozantinib (CABO), pazopanib (PAZO), sorafenib (SORA), sunitinib (SUNI) and its main active metabolite N-desethyl-sunitinib (DST-SUNI), olaparib (OLA) and palbociclib (PALBO) display large pharmacokinetics variability impacting their responses in terms of efficacy or toxicity. For the monitoring of these drugs, an analytical method allowing to routinely measure their concentrations in human plasma is needed. Such a method has been developed and validated and is presented here.The chromatographic separation is achieved on a Zorbax Bonus-RP analytical column using an isocratic elution of 92% V/V of acetonitrile and 8% of water in 0.1% formic acid at a flow rate of 500 µl/min for 0.5 min and then 300 µl/min for 2 min. After a liquid-liquid extraction of plasma samples, a step of filtration is performed. This method was validated based on the EMA and French committee of accreditation guidelines.The analysis time is 2.5 min per run, and all analytes eluted within 0.53–1.61 min. The standard curves are linear over the range from 1 to 380 ng/ml for SUNI; from 4.3 to 450 ng/ml for DST-SUNI; from 6 to 1000 ng/ml for PALBO; from 75 to 5000 ng/ml for CABO, from 0.17 to 20 µg/ml for OLA; from 0.35 to 40 µg/ml for SORA and from 1.7 to 200 µg/ml for PAZO. The method also showed satisfactory results in terms precision (below 9.5% for within-run and below 13% for between-run) and accuracy (below 13.5% for within-run and below 14% for between-run). After sampling, all the compounds are stable in whole blood at ambient temperature at least for 6 h and plasma are stable for 48 h at ambient temperature or 4 °C.The method presented here allows to measure the concentrations of 7 targeted therapies in a routine setting. We moreover present here a method that is, to our knowledge, one of the first detailed method aimed at the measurement of palbociclib in human plasma in a routine setting, together with data useful for the management of samples in routine hospital practice.

Simultaneous determination of the pharmacokinetics of A-type EGCG and ECG dimers in mice plasma and its metabolites by UPLC-QTOF-MS

A-type epigallocatechin-3-gallate (EGCG) and epicatechin-3-O-gallate (ECG) dimers have multiply biological activities. In this study, the pharmacokinetics of them were investigated in mice after a single dose intravenous administration, and the metabolites in mice plasma and urine were investigated by ultra-performance liquid chromatography-Quadrupole-time of flight mass spectrometer (UPLC-QTOF-MS). Our results showed that the half-life (t1/2) of A-type EGCG and ECG dimers were 116.37 min and 33.04 min, respectively, and the maximal concentration in plasma was 32.81 μg/mL and 55.59 μg/mL, respectively. It was found that two dimers were firstly experienced by quinone methide (QM) fission to form the EGCG and ECG analogue, and the phase II metabolites were generated subsequently. The main metabolites in plasma and urine were glucuronidation and sulphation derivatives. In addition, small molecule weight of phenolic acids were detected in urine.

Development of a thin layer chromatography method for plasma correction of [18F]fluorocholine metabolites in positron emission tomography quantification studies in humans

IntroductionAfter its intravenous injection, [18F]fluorocholine is oxidized by choline-oxidase into its main plasma metabolite, [18F]fluorobetaine. If PET kinetic modeling quantification of [18F]fluorocholine uptake is intended, the plasma input time-activity-curve of the parent tracer must be obtained, i.e., the fraction of the total plasma radioactivity corresponding to the nonmetabolized [18F]fluorocholine at each time has to be known. Hence our aim was to develop an easy-routine Thin-Layer-Chromatography (TLC) method to separate and quantify the relative fractions of [18F]fluorocholine and [18F]fluorobetaine as a function of time during PET imaging in humans. MethodsFirst, we tested several combinations of solvents systems and layers to select the one showing the best resolution on non-radioactive standards. Thereafter, [18F]fluorobetaine was obtained through chemical oxidation of an [18F]fluorocholine sample at diferent incubation times and we applied the selected TLC-system to aliquots of this oxidation solution, both in a saline and in human deproteinized plasma matrices. The plates were detected by a radio-TLC-scanner. This TLC-system was finally applied to arterial plasma samples from 9 patients with high-grade-glioma undergoing brain PET imaging and a parent fraction curve was obtained in each of them. ResultsA TLC-system based on Silica-Gel-60//MeOH-NH3 was selected from the choline/betaine non-radioactive standards assay. Radiochromatograms of [18F]fluorocholine oxidation solution yielded two separated and well-defined peaks, Rf = 0,03 ([18F]fluorocholine) and Rf = 0.78 (18F]fluorobetaine) consistent with those observed on non-radioactive standards. During the oxidation, the [18F]fluorocholine radioactivity peak decreased progressively at several incubation times, while the other peak ([18F]fluorobetaine) increased accordingly. The mean values of the parent fraction of [18F]fluorocholine of the 9 patients studied (mean+/-SD) were 94% ± 6%, 58% ± 15%, 43% ± 10%, 39% ± 6% and 37% ± 6% at 2.8 min, 5.8 min, 8.8 min, 11.7 min and 14.7 min post-injection, respectively. ConclusionsWe have developed a TLC-system, easy to perform in a standard radiopharmacy unit, that enables the metabolite correction of arterial input function of [18F]fluorocholine in patients undergoing PET oncologic quantitative imaging.