Active Metabolite In Human Plasma Research Articles

Simultaneous quantification of baloxavir marboxil and its active metabolite in human plasma using UHPLC-MS/MS: Application to a human pharmacokinetic study with different anticoagulants

Baloxavir marboxil (BXM) is a cap-dependent nucleic acid endonuclease inhibitor, which exerts its antiviral effects after being metabolized to its active form baloxavir acid (BXA). Ethylenediamine tetra-acetic acid (EDTA) and heparin are the two most used anticoagulants in clinical blood sample collection to estimate drug levels in plasma. However, compared to heparin plasma, there is a lack of clinical pharmacokinetic data of BXA using EDTA anticoagulant tubes for blood collection. In the present study, an efficient, rapid, and sensitive ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for simultaneous quantification of BXM and its active metabolite BXA in human plasma with its isotopic baloxavir-d5 (BXA-d5) as internal standard (IS). Plasma samples (50 μL) were undergone using acetonitrile containing 0.1 % formic acid a precipitant. Chromatographic separation was achieved by a Waters XBridge®C8 (2.1 mm × 50 mm, 2.5 µm) column. The gradient mobile phase was 0.1 % formic acid in water (A, pH 2.8) and 0.1 % formic acid in acetonitrile (B) and delivered at a flow rate of 0.6 mL/min for 4.5 min. BXM and BXA were monitored using a positive electrospray triple quadrupole mass spectrometer (TRIPLE QUAD™ 6500+) via multiple reaction monitoring mode. The mass-to-charge ratios (m/z) were 572.2→247.0, 484.2→247.0 and 489.2→252.0 for BXM, BXA, and BXA-d5 (IS). Calibration curves exhibited excellent linearity in the range of 0.1–10 ng/mL for BXM (r2 > 0.996), and 0.3–300 ng/mL for BXA (r2 > 0.998). Within-run and between-run precisions in coefficients of variations were less than 11.62 % for BXM and less than 7.47 % for BXA, and accuracies in relative error were determined to be within −7.78 % to 5.70 % for BXM and −6.67 % to 8.56 % for BXA. Extraction recovery efficiency was 92.76 % for BXM, 95.32 % for BXA, and 99.26 % for BXA-d5, respectively. The matrix effect of BXM and BXA was in line with the requirements, where the relative deviation of the accuracy was less than 6.67 % and the precision was less than 6.69 %. The validated efficient and simple UHPLC-MS/MS method was successfully used in the pharmacokinetic study of BXM and BXA in healthy human volunteers with K2EDTA and heparin tubes for blood collection. EDTA might compete with BXA for chelating metal ions and thereby decrease the plasma ratio in whole blood, leading to approximately 50 % lower measurement of pharmacokinetic parameters as compared with those obtained from heparin plasma anticoagulant tubes.

Simultaneous determination of 24 antiepileptic drugs and their active metabolites in human plasma by UHPLC-MS/MS

Antiepileptic drugs (AEDs) have narrow therapeutic ranges with large individual variability. Routine therapeutic drug monitoring of AEDs was useful for dose optimization, but the common immunoassays could not meet the detection requirements of AEDs, especially for new generation AEDs. The aim of this study was to validate an ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for simultaneously quantification of 24 AEDs and their active metabolites in human plasma and comparison with a chemiluminescent immunoassay (Simens ADVIA Centaur). The method validation was performed according to FDA and EMEA guidelines. A one-step protein precipitation by acetonitrile followed a five-fold dilution was performed for sample pretreatment. A 5.2 min gradient separation by methanol and 10 mM ammonium acetate was used for separation at 0.6 mL/min under 45 °C. Both positive and negative electrospray ionization were used. Isotopic internal standard was used for all analytes. The inter-day (36 days) accuracy and precision of quality control samples were − 1.07–13.69% and < 6.70% for all analytes. The stability was acceptable for all analytes under routine storing conditions. A total of 436 valproic acid, 118 carbamazepine, and 65 phenobarbital samples were determined twice by each of the UHPLC-MS/MS and immunoassay. Evaluated by Bland-Altman plot, the mean overestimation of the immunoassay compared to UHPLC-MS/MS was 16.5% for valproic acid, 5.6% for carbamazepine, and 40.3% for phenobarbital.

Simultaneous Determination of Rifamycin Antibiotics and Their Active Metabolites in Human Plasma Using UHPLC-MS/MS to Evaluate Their Impact on Target Peak Concentrations: A Short Communication.

Standard and proper antituberculosis (anti-TB) treatment is essential for patients with TB, and rifamycin antibiotics are key components of anti-TB therapy. Therapeutic drug monitoring (TDM) of rifamycin antibiotics can shorten the time to response and complete treatment of TB. Notably, antimicrobial activities of the major active metabolites of rifamycin are similar to those of their parent compounds. Thus, a rapid and simple assay was developed for simultaneous determination of rifamycin antibiotics and their major active metabolites in plasma to evaluate their impact on target peak concentrations. Here, the authors have developed and validated a method for simultaneous determination of rifamycin antibiotics and their active metabolites in human plasma using ultrahigh-performance liquid chromatography tandem mass spectrometry. Analytical validation of the assay was performed in accordance with the bioanalytical method validation guidance for industry described by the US Food and Drug Administration and the guidelines for bioanalytical method validation described by the European Medicines Agency. The drug concentration quantification method for rifamycin antibiotics, including rifampicin, rifabutin, and rifapentine, and their major active metabolites was validated. Significant differences in the proportions of active metabolites in rifamycin antibiotics may affect the redefinition of their effective concentration ranges in the plasma. The method developed herein is expected to redefine the ranges of "true" effective concentrations of rifamycin antibiotics (including parent compounds and their active metabolites). The validated method can be successfully applied for high-throughput analysis of rifamycin antibiotics and their active metabolites for TDM in patients receiving anti-TB treatment regimens containing these antibiotics. Proportions of active metabolites in rifamycin antibiotics markedly varied among individuals. Depending on the clinical indications of patients, the therapeutic ranges for rifamycin antibiotics may be redefined.

Quantitative determination of the content of venlafaxine and its active metabolite in human plasma using liquid chromatography-mass spectrometry

A validated method for the quantitative determination of venlafaxine, O-desmethylvenlafaxine in human plasma by tandem liquid chromatographymass spectrometry (HPLC/MS/MS) has been proposed. Sample preparation was carried out by liquid-phase extraction with support (SLE, from «supported liquid extraction») using HyperSEP cartridges and washing with 3-methylbutyl ether. The measure of the target analytes was carried on a triple quadrupole mass spectrometer under multiple-reaction monitoring (+MRM) mode with the electrospray ionization. The calibration curves were linear with regression coefficient r2 &gt; 0.999. The method validation was showed high sensitivity, specificity, accuracy and reproducibility, as well as stability of analytes after freeze/thaw cycles and storage at –20 °C. The method is developed for using in therapeutic drug monitoring.

Development of a Sensitive and High-Throughput Assay for Simultaneous Quantification of 5 Tyrosine Kinase Inhibitors and 2 Active Metabolites in Human Plasma Using Ultra-high Performance Liquid Chromatography Coupled to Tandem Mass Spectrometry.

Breakpoint cluster region-Abelson (BCR-ABL) tyrosine kinase inhibitors (TKIs) demonstrate improved therapeutic efficacy in chronic myeloid leukemia (CML). However, drug-drug interactions, nonadherence, and host-related factors may influence plasma concentrations. Therefore, therapeutic drug monitoring may be necessary for patients presenting inadequate treatment responses or adverse events. Herein, the authors aimed to develop a more sensitive and high-throughput method than those previously reported to simultaneously quantify 5 TKIs (imatinib, nilotinib, dasatinib, bosutinib, and ponatinib) and 2 active metabolites (N-desmethyl imatinib and N-desmethyl ponatinib) using ultra-performance liquid chromatography coupled with tandem mass spectrometry. Plasma samples were prepared according to a solid-phase extraction protocol using an Oasis MCX µElution plate. The assay fulfilled the requirements of the US Food and Drug Administration for assay validation, with a lower limit of quantification of 0.2 ng/mL for dasatinib, 0.3 ng/mL for N-desmethyl ponatinib, 0.5 ng/mL for N-desmethyl imatinib, bosutinib, and ponatinib, and 2.5 ng/mL for imatinib and nilotinib. Within-batch and batch-to-batch precision at the lower limit of quantification and quality control levels were within 14.3% and 10.9%, respectively. Within-batch and batch-to-batch accuracies ranged from 15.5% to 13.0% and 5.70% to 7.03%, respectively. A positive electrospray ionization mode was used with a run time of 6.0 minutes. The assay applicability was verified by the successful measurement of 78 clinical samples from patients undergoing CML therapy. The method allows assessment of trough concentrations of TKIs and active metabolites in patients with CML, and hence can be used to assess blood samples in routine clinical settings.

A three-step stacking capillary electrophoresis of field-amplified sample injection, sweeping, and micellar collapse for determination of dabigatran and its active metabolite in human plasma.

A three-step stacking capillary electrophoresis (CE) composed of field-amplified sample injection, sweeping, and analyte focusing by micellar collapse (FASI-sweeping-AFMC) was developed to determine dabigatran (D) and its major active metabolite, dabigatran acyl-beta-d-glucuronide (DAG), in human plasma. After optimization and validation, this novel approach was further applied to monitor 5 real samples, and the 25.2-186.8 ng mL-1 D could be observed among those. Based on these results, the novel CE stacking strategy was successfully applied for the analysis of D and DAG in human plasma and could be served as a tool for clinical assays.

A novel LC-MS/MS method for simultaneous estimation of acalabrutinib and its active metabolite acalabrutinib M27 in human plasma and application to a human pharmacokinetic study.

A simple, specific, selective and accurate bioanalytical method was developed and validated for simultaneous estimation of acalabrutinib and its active metabolite in human plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Deuterated analogs of both the analytes were used as internal standards. The extraction of analytes and internal standards were evaluated from the human plasma by liquid–liquid extraction technique using methyl tertiary butyl ether (TBME). The separation of the analytes was carried out on Zorbax Eclipse XDB-C18 (150 × 4.6 mm, 5 μm) column with a mixture of acetonitrile and 10 mM ammonium formate in 0.1% formic acid buffer (65 : 35, v/v) as mobile phase at a flow rate of 1 mL min−1. The method linearity was determined in the widen concentration range from 5.000 ng mL−1 to 1600 ng mL−1 with r2 > 0.99. The entire method validation was carried out as per the USFDA guidelines on bioanalytical method validation and all validation experiment results were found within acceptable limits. Clinical pharmacokinetic study of both the parent drug and its active metabolite was successfully performed on six healthy volunteers under fasting conditions by applying the present method.

Shifting the linear range of the calibration curves for the quantification of oral molecular-targeted anticancer drugs and the active metabolite in human plasma using LC-ESI-MS/MS

Shifting the linear range of the calibration curves for the quantification of oral molecular-targeted anticancer drugs and the active metabolite in human plasma using LC-ESI-MS/MS

DDRE-08. A BIOANALYTICAL LC-MS/MS ASSAY TO QUANTIFY TOTAL AND UNBOUND LY3214996, ABEMACICLIB AND ITS ACTIVE METABOLITES IN HUMAN PLASMA, CEREBROSPINAL FLUID AND HUMAN GLIOBLASTOMA

Abstract BACKGROUND Here, we report on our development and validation of a sensitive and rapid LC-MS/MS method for the determination of total and unbound concentrations of ERK inhibitor LY3214996, CDK4/6 inhibitor abemaciclib and its M2 and M20 active metabolites in human plasma, cerebrospinal fluid and human glioblastoma tissue. METHODS Analytes were extracted from patient plasma, CSF and glioblastoma homogenate samples by protein precipitation with methanol. Four levels of quality controls were used during validation. The method was validated according to FDA guidelines and CAP/CLIA regulations. Equilibrium dialysis was performed to determine unbound fraction of four analytes in plasma and brain tissue. The detection was performed on Sciex QTRAP 6500+ mass spectrometer in positive electrospray ionization mode. RESULTS The method was validated over a concentration range from 0.2-500 nmol/L for all four analytes. The analytical separation was optimized on Phenomenex Kinetex™ F5 column with total run time of 3.8 min using gradient elution. For all the analytes the maximum coefficient of variation for intra- and inter-day precision was 12.0% and the accuracy was within 90.2-119.7% in all matrixes. The analytes are stable in plasma and brain homogenate for at least 19 hours and 6 hours at room temperature (RT), respectively. Stability of stock and working solutions was demonstrated for at least 15 hours (RT) and 28 days (2-8°C). The unbound fraction of the analytes in pooled human plasma and brain were determined to be 0.371 and 0.065 for LY3214996, 0.026 and 0.013 for abemaciclib, 0.052 and 0.008 for M2, 0.024 and 0.021 for M20, respectively. CONCLUSIONS A bioanalytical method to quantify LY3214996, abemaciclib and its M2 and M20 metabolites is successfully developed and validated. The method is currently applied to evaluate plasma pharmacokinetics and CNS penetration of LY3214996 and abemaciclib in recurrent glioblastoma patients in an ongoing Phase 0 clinical trial (NCT04391595).

High-throughput simultaneous quantification of five azole anti-fungal agents and one active metabolite in human plasma using ultra-high-performance liquid chromatography coupled to tandem mass spectrometry

ObjectivesFor patients with hematological malignancy, triazole antifungal agents such as fluconazole (FLCZ), itraconazole (ITCZ), voriconazole (VRCZ), posaconazole (PSCZ) and isavuconazole (ISCZ) are often used for prophylaxis of deep mycosis. Since these azoles exhibit large pharmacokinetic variability, dose adjustment by therapeutic drug monitoring is recommended for some azoles. This study aimed to develop and validate a novel method for simultaneous determination of plasma concentrations of FLCZ, ITCZ, VRCZ, PSCZ, ISCZ and ITCZ-OH, an active metabolite of ITCZ, using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). Design & methodsA high-throughput solid-phase extraction method using 96-well MCX µElution Plate was selected as the pretreatment procedure. ResultsThe calibration curves for FLCZ, ITCZ, ITCZ-OH, VRCZ, PSCZ and ISCZ showed good linearity (back-calculation of calibrators: relative error ≤ 15% [LLOQ: ≤ 20%]) over wide ranges of 100–100000, 20–20000, 40–40000, 20–20000, 5–5000 and 50–50000 ng/mL, respectively. The validation results of all six drugs fulfilled the criteria of the guidance for bioanalytical method validation of the US Food and Drug Administration for within-batch and batch-to-batch precision and accuracy. The extraction recovery rates were good at ≥ 74.9%, and almost no matrix effects were found for all the drugs. The trough (10 h post-dose in 1 patient on PSCZ) drug concentrations in patients with hematologic malignancy who received oral FLCZ, ITCZ, VRCZ or PSCZ were quantified using the method developed. The measurements for all samples were within the ranges of the calibration curves, demonstrating the feasibility of clinical application of the novel method. ConclusionsWe have succeeded in developing a novel high-throughput method using UHPLC-MS/MS for simultaneous quantification of plasma concentrations of FLCZ, ITCZ, ITCZ-OH, VRCZ, PSCZ and ISCZ.

Itraconazole and rifampicin, as CYP3A modulators but not P-gp modulators, affect the pharmacokinetics of almonertinib and active metabolite HAS-719 in healthy volunteers.

Almonertinib is a novel third-generation EGFR tyrosine kinase inhibitor. It is mainly metabolized by CYP3A in vitro, and N-desmethylated almonertinib (HAS-719) is the major active metabolite in human plasma. In this study, we investigated the effects of CYP3A inhibitor itraconazole and CYP3A inducer rifampicin on the pharmacokinetics of almonertinib and HAS-719 in 64 healthy volunteers. We found that when co-administered with itraconazole, the maximal plasma concentration (Cmax) and the plasma exposure (AUC0-t) of almonertinib were increased by 56.3% and 2.38-fold, respectively, whereas the Cmax and AUC0-t of HAS-719 were reduced by 86.8% and 71.8%, respectively. Co-administration with rifampicin reduced the Cmax and AUC0-t of almonertinib by 79.3% and 92.6%, but the AUC0-t of HAS-719 was unexpectedly decreased by 72.5%. In vitro assays showed that both almonertinib and HAS-719 were substrates of CYP3A and P-gp. Co-administration of rifampicin in Beagle dogs reduced the fecal recovery of almonertinib and HAS-719, and markedly increased the levels of metabolites derived from further metabolism of HAS-719, which was consistent with human plasma data, suggesting that although rifampicin was also a potent inducer of P-gp, the pharmacokinetic alternation of HAS-719 was mainly due to its further metabolism but not excretion changes. Moreover, we revealed that almonertinib was a moderately sensitive substrate of CYP3A in vivo. Special attention should be paid to the interaction between almonertinib and drugs or food affecting CYP3A activity in the clinical application of almonertinib.

Development and validation of LC–MS/MS method for simultaneous determination of dabigatran etexilate and its active metabolites in human plasma, and its application in a pharmacokinetic study

Dabigatran is a direct thrombin inhibitor widely used for preventing various thrombotic events. Although there are several established LC–MS/MS based methods for quantification of plasma dabigatran etexilate and its active metabolites (dabigatran and dabigatran acylglucuronide), so far, there are no studies for simultaneous quantification of dabigatran etexilate, dabigatran, and dabigatran acylglucuronide in human plasma samples. In the present study, a novel and sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed and validated for assessment of dabigatran pharmacokinetics in human plasma samples according to FDA guidelines. We used the new method to simultaneously quantify dabigatran etexilate, dabigatran, and dabigatran acylglucuronide in human plasma samples. After deproteinization using acetonitrile, the supernatants were evaporated, dissolved in a mobile phase, and finally injected onto an HPLC system with a silica-based C18 reverse-phase column. Mass spectrometer system was operated in multiple reaction monitoring mode (MRM) (dabigatran etexilate: m/z 629.464→290.100; dabigatran: m/z 472.300→289.100, and dabigatran acylglucuronide: m/z 648.382→289.100) and all the components were confirmed using positive electrospray ionization (ESI). Correlation coefficients > 0.999 were achieved for all the calibration curves with linear regression. The intra-day and inter-day accuracies of dabigatran etexilate, dabigatran, and dabigatran acylglucuronide were 95.84–109.44 %, 99.4–103.42 %, and 98.37–104.42 %, respectively, while their corresponding precisions were 3.84–9.79, 1.07–8.76 %, and 2.56–4.51 %, respectively. We successfully applied the new method to determine the pharmacokinetic profiles of dabigatran etexilate, dabigatran, and dabigatran acylglucuronide in humans. Our findings demonstrated the method as robust, reliable, and sensitive.

A new simple method for quantification of cilostazol and its active metabolite in human plasma by LC-MS/MS: Application to pharmacokinetics of cilostazol associated with CYP genotypes in healthy Chinese population.

A simple, sensitive, and fully automated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous quantification of cilostazol (CIL) and its active metabolite, 3,4-dehydro cilostazol (CIL-M), in human plasma. Plasma samples were processed by protein precipitation in 2 mL 96-deep-well plates, and all liquid transfer steps were performed through robotic liquid handling workstation, enabling the whole procedure fast, compared to the reported methods. Separation of analytes was successfully achieved on a UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm) with mobile phase A (5 mM ammonium formate containing 0.1% formic acid) and mobile phase B (methanol) at a flow rate of 0.30 mL min-1 . The total run time was 3.5 min per sample. Mass spectrometric detection was conducted by electrospray ion source in positive ion multiple reaction monitoring mode. Calibration curves were linear over the concentration range of 1.0-800 ng·mL-1 for CIL and 0.05-400 ng·mL-1 for CIL-M. The coefficient of variation for the assay's precision was 12.3%, and the accuracy was 88.8-99.8%. It was fully validated and successfully applied to assess the influence of CYP genotypes on the pharmacokinetics of CIL after oral administration of 50 mg tablet formulations of CIL to healthy Chinese volunteers. The results suggest that, in Chinese population, the genotype of CYP3A5 affects the plasma exposure of CIL.

Validation of an HPLC-MS/MS Method for the Determination of Plasma Ticagrelor and Its Active Metabolite Useful for Research and Clinical Practice.

Ticagrelor is an antiplatelet agent which is extensively metabolized in an active metabolite: AR-C124910XX. Ticagrelor antagonizes P2Y12 receptors, but recently, this effect on the central nervous system has been linked to the development of dyspnea. Ticagrelor-related dyspnea has been linked to persistently high plasma concentrations of ticagrelor. Therefore, there is a need to develop a simple, rapid, and sensitive method for simultaneous determination of ticagrelor and its active metabolite in human plasma to further investigate the link between concentrations of ticagrelor, its active metabolite, and side effects in routine practice. We present here a new method of quantifying both molecules, suitable for routine practice, validated according to the latest Food and Drug Administration (FDA) guidelines, with a good accuracy and precision (<15% respectively), except for the lower limit of quantification (<20%). We further describe its successful application to plasma samples for a population pharmacokinetics study. The simplicity and rapidity, the wide range of the calibration curve (2–5000 µg/L for ticagrelor and its metabolite), and high throughput make a broad spectrum of applications possible for our method, which can easily be implemented for research, or in daily routine practice such as therapeutic drug monitoring to prevent overdosage and occurrence of adverse events in patients.

Method Development and Validation for Simultaneous Determination of Six Tyrosine Kinase Inhibitors and Two Active Metabolites in Human Plasma Using UPLC–MS/MS for Therapeutic Drug Monitoring

Over the past decades, therapeutic drug monitoring (TDM) of tyrosine kinase inhibitors (TKIs) and their main active metabolites has shown benefits in improving treatment efficacy and safety. Therefore, a sensitive, simple and economical ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the determination of six oral tyrosine kinase inhibitors (TKIs) and two active metabolites, including imatinib (IMA), N-desmethyl imatinib (NDIMA), sunitinib (SUNI), N-desethyl sunitinib (NDSUNI), regorafenib (REGO), nilotinib (NILO), dasatinib (DASA) and osimertinib (OSI) in human plasma for therapeutic drug monitoring. The plasma samples were deproteinated with acetonitrile after spiking with two deuterated internal standards (ISs, [ 2 H 8 ]-imatinib and [ 2 H 10 ]-sunitinib) and separated on a 40 °C ACQUITY UPLC® BEH C 18 column (1.7 μm, 2.1 mm×50 mm). The mobile phase was composed of acetonitrile (solution A) and water-formic acid-ammonium acetate (1M)(994:1:5, v/v/v, solution B). Gradient elution was applied at a flow rate of 0.4 mL/min. Detection was carried out using a Triple Quad 5500 tandem mass spectrometer coupled with an electrospray ionization (ESI) source in positive mode. The method was validated over the calibration curve (CV) range of 2-400 ng/mL for NDSUNI and DASA, 2.5-500 ng/mL for SUNI, 10-2000 ng/mL NDIMA and OSI, 20-4000 ng/mL for NILO, 30-6000 ng/mL for REGO and 50-10000 ng/mL for IMA using linear regression and 1/x 2 weighting. No difference was observed in the matrix effect (ME) among blank human plasma, hemolytic plasma and lipemic plasma samples in general. And the intra- and inter-batch precision and accuracy of all eight components were acceptable. To provide a clinical reference for the operation, the stability of the whole process from sample collection to drug detection was verified. SUNI and NDSUNI showed obvious photoisomerization under light exposure. Therefore, strict light protection was applied for all sample collection and handling steps of SUNI and NDSUNI. Compared with heparin anticoagulant tubes, the stability of the eight compounds in both whole blood and plasma was better in K3-EDTA and sodium citrate anticoagulant tubes. Given that all the analytes were stable in plasma at 4 °C for 48 h and in whole blood at room temperature for 48 h but OSI and REGO were unstable in whole blood and plasma at room temperature, samples should be centrifuged as soon as possible to be preserved as plasma at 4 °C when OSI or REGO is detected. In conclusion, this validated method can provide support for clinical practice, such as therapeutic drug monitoring (TDM) and pharmacokinetic studies of these six TKIs and two active metabolites.

HPLC-MS/MS Method for Quantitation of Sildenafil and Its Active Metabolite in Human Plasma

A high demand for sildenafil-based drugs puts a premium on the development of methods for quantitation of sildenafil in bio-substrates to facilitate pharmacokinetic analysis in bioequivalence studies. High performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) was proposed for the method development due to its high sensitivity, selectivity, reproducibility, and performance. The aim of the study was to develop and validate an HPLC-MS/MS method for quantitative determination of sildenafil and its active metabolite N-desmethyl sildenafil in human plasma. Approbation of the developed technique in the study of the pharmacokinetic profiles of sildenafil and N-desmethyl sildenafil in healthy volunteers in the study of the bioequivalence of the drug sildenafil in the form of a spray. Materials and methods : the method was implemented using the Agilent 1200 high-performance liquid chromatography system with the Agilent 6440 triple quadrupole system, and Poroshell 120 EC-C18 chromatographic column, 4.6 m × 150 mm × 2.7 μm. Calibration samples were prepared using sildenafil citrate and N-desmethyl sildenafil reference standards with 99.5 and 98.5% purity, respectively. Vardenafil was used as an internal standard. Pharmacokinetic profiles of sildenafil and N-desmethyl sildenafil were studied in 44 healthy male volunteers as part of a bioequivalence study approved by the Ministry of Health of the Russian Federation. The primary data processing was performed using Mass Hunter software version B 06.00, and statistical processing was performed using Microsoft Office Excel 2010 and Statistica 6.1. Results : the authors developed and validated a method for quantitation of sildenafil and its active metabolite N-desmethyl sildenafil in human plasma. The developed method was used successfully to study pharmacokinetic profiles of the discussed compounds in healthy volunteers. Conclusions : the developed method of quantitative determination of sildenafil and its active metabolite N-desmethyl sildenafil in human plasma is simple, reproducible, fast, and robust. The results of the pharmacokinetic studies using the developed method demonstrated bioequivalence of the test product and the reference product.

Simultaneous Determination of Amlodipine, Losartan, and the Losartan Carboxylic Acid Active Metabolite in Human Plasma by LC-MS/MS

Simultaneous Determination of Amlodipine, Losartan, and the Losartan Carboxylic Acid Active Metabolite in Human Plasma by LC-MS/MS

LC-MS/MS Method for the Quantitative Determination of Tanespimycin and its Active Metabolite in Human Plasma: Method Validation and Overcoming an Insidious APCI Source Phenomenon

LC-MS/MS Method for the Quantitative Determination of Tanespimycin and its Active Metabolite in Human Plasma: Method Validation and Overcoming an Insidious APCI Source Phenomenon

Development and validation of a liquid chromatography coupled to tandem mass spectrometry method for the simultaneous quantification of five analgesics and sedatives, and six of their active metabolites in human plasma: Application to a clinical study on the determination of neurological death in the intensive care unit

A sensitive and selective high-performance liquid chromatographic method coupled to tandem mass spectrometry was developed and validated for the quantification of morphine, hydromorphone, fentanyl, midazolam and propofol and their metabolites morphine-3-β-d-glucuronide, morphine-6-β-d-glucuronide, hydromorphone-3-β-d-glucuronide, 1′-hydroxymidazolam-β-d-glucuronide, α-hydroxymidazolam and 4-hydroxymidazolam in human plasma using potassium oxalate/sodium fluoride mixture as anticoagulant. Human plasma samples (0.4 mL) to which were added a mixture of eleven deuterated internal standards were subjected to solid phase extraction using a mixed-mode polymeric Oasis PRiME MCX in 96-well format. Propofol was selectively eluted and further derivatized using 2-Fluoro-1-methylpyridinium p-toluenesulfonate, whereas the remaining 10 analytes were eluted separately and further concentrated. The derivatized propofol was analyzed separately in a second injection. The analytes were chromatographically separated on a Kinetex phenyl-hexyl analytical column in gradient elution mode, using a mobile phase consisting of aqueous ammonium formate/formic acid buffer and methanol. The overall run time was 8 min. Detection was performed using an AB/SCIEX 4000 QTRAP instrument with positive electrospray ionization employing scheduled multiple reaction monitoring mode. The lower limits of quantification ranged from 0.02 to 5 ng/mL depending on the analyte. Calibration curves covered a concentration range of 1000× in all cases but 1′-hydroxymidazolam-β-d-glucuronide where it covered a range of 500 × . The validated method was accurate and precise, the intra-day accuracy and precision of quality control samples (4 concentration levels, n = 6 each) being within 91.5–112 % and 1.3–13.2 % (coefficient of variation), respectively, and inter-day (n = 24; 4 days) accuracy and precision of quality control samples (3 concentration levels) being within 94.8–103.5 % and 3.2–11.2 % (coefficient of variation). Mean absolute extraction recoveries were above 60 % for all compounds, except for hydromorphone-3-β-d-glucuronide (44 %) and for 1′-hydroxymidazolam-β-d-glucuronide (33 %). Internal standard corrected matrix effect ranged from −4.8 to 3.8 % in normal plasma and in plasma containing 1 % hemolyzed blood. Analytes were stable (above 90 %) in plasma and blood for 19 h at 22 °C, in blood for 90 h at 5 °C, in plasma for 60 days at −20 °C, for 4 months at −70 °C and after three freeze-thaw cycles, and in the injection solvent for at least 3 days in the autosampler. The present method is successfully being applied in a multicenter clinical study for the analysis of plasma samples from patients in intensive care units from a number of Canadian hospitals.

Simultaneous determination and determination of sildenafil and its active metabolite in human plasma using LC-MS/MS method.

A sensitive and selective high-performance liquid chromatography-tandam mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous quantification of sildenafil and its metabolite N-desmethyl sildenafil in human plasma. Sildenafil-d8 was used as an internal standard. The analytes were extracted by precipitation extraction and chromatographed on a C18 column using mobile phase A of water (containing 0.1% formic acid) and mobile phase B of acetonitrile (containing 0.1% formic acid) with gradient elution. Quantification was done using multiple reaction monitoring mode to monitor the precursor-to-product ion transitions of m/z 475.4 → m/z 283.3 for sildenafil, m/z 461.4 → m/z 283.2 for N-desmethyl sildenafil and m/z 483.3 → m/z 108.1 for IS in positive ionization mode. The calibration curve was established over the range of 2.00-1,000 ng/ml and the correlation coefficient was >0.99. The intra-day and inter-day relative standard deviations were <6.5% for sildenafil and 6.3% for N-desmethyl sildenafil respectively. Accuracy determinaed at four concentrations was 86.50-105.67% for sildenafil and 96.83-114.40% for N-desmethyl sildenafil. This method was successfully applied to a pharmacokinetic description of sildenafil and the effect of food intake on the pharmacokinetics of sildenafil was also demonstrated in healthy Chinese volunteers.