Major Metabolites In Human Plasma Research Articles

Development and validation of a sensitive UHPLC-MS/MS method for the simultaneous analysis of tramadol, dextromethorphan chlorpheniramine and their major metabolites in human plasma in forensic context: application to pharmacokinetics.

The prerequisites for forensic confirmatory analysis by LC/MS/MS with respect to European Union guidelines are chromatographic separation, a minimum number of two MS/MS transitions to obtain the required identification points and predefined thresholds for the variability of the relative intensities of the MS/MS transitions (MRM transitions) in samples and reference standards. In the present study, a fast, sensitive and robust method to quantify tramadol, chlorpheniramine, dextromethorphan and their major metabolites, O-desmethyltramadol, dsmethyl-chlorpheniramine and dextrophan, respectively, in human plasma using ibuprofen as internal standard (IS) is described. The analytes and the IS were extracted from plasma by a liquid-liquid extraction method using ethyl acetate-diethyl-ether (1:1). Extracted samples were analyzed by ultra-high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS). Chromatographic separation was performed by pumping the mobile phase containing acetonitrile, water and formic acid (89.2:11.7:0.1) for 2.0 min at a flow rate of 0.25 μL/min into a Hypersil-Gold C18 column, 20 × 2.0 mm (1.9 µm) from Thermoscientific, New York, USA. The calibration curve was linear for the six analytes. The intraday precision (RSD) and accuracy (RE) of the method were 3-9.8 and -1.7-4.5%, respectively. The analytical procedure herein described was used to assess the pharmacokinetics of the analytes in 24 healthy volunteers after a single oral dose containing 50 mg of tramadol hydrochloride, 3 mg chlorpheniramine maleate and 15 mg of dextromethorphan hydrobromide.

Determination of the rifamycin antibiotics rifabutin, rifampin, rifapentine and their major metabolites in human plasma via simultaneous extraction coupled with LC/MS/MS

A novel assay using high pressure liquid chromatography (HPLC) coupled to mass spectrometer (MS) detection was developed and validated for the rifamycin anti-tuberculosis antibiotics rifampicin (RIF), rifabutin (RBT), rifapentine (RPT) and their active desacetyl metabolites (dRIF, dRBT and dRPT, respectively) in human plasma. The assay uses 50μL of human plasma with a quick and simple protein-precipitation extraction to achieve a dynamic range of 75–30,000ng/mL for RIF, RBT and RPT and 37.5–15,000ng/mL for dRIF, dRBT and dRPT, respectively. The average %CV and %deviation were less than 20% at the lower limit of quantitation and less than 15% over the range of the curve. The method was fully validated according to FDA criteria for bioanalytical assays and has successfully been used to support three large international tuberculosis trials.

Simultaneous determination of flurbiprofen and its hydroxy metabolite in human plasma by liquid chromatography-tandem mass spectrometry for clinical application.

Flurbiprofen (FLB) is one of the phenylalkanoic acid derivatives of non-steroidal anti-inflammatory drugs used for the management of pain and inflammation in patients with arthritis. We developed and validated a rapid and sensitive high-performance liquid chromatography analytical method utilizing tandem mass spectrometry (HPLC-MS/MS) for the simultaneous determination of FLB and its major metabolite, 4'-hydroxyflurbiprofen (4'-OH-FLB), in human plasma. Probenecid was used as an internal standard (IS). After liquid-liquid extraction with methyl t-butyl ether, chromatographic separation of the two analytes was achieved using a reversed-phase Luna C18 column (2.0mm×50mm, 5μm particles) with a mobile phase of 10mM ammonium formate buffer (pH 3.5)-methanol (15:85, v/v) and quantified by MS/MS detection in ESI negative ion mode. The flow rate of the mobile phase was 250μl/min and the retention times of FLB, 4'-OH-FLB, and IS were 1.1, 0.8, and 0.9min, respectively. The calibration curves were linear over a range of 0.01-10μg/ml for FLB and 0.01-1μg/ml for 4'-OH-FLB. The lower limit of quantifications using 100μl of human plasma was 0.01μg/ml for both analytes. The mean accuracy and precision for intra- and inter-run validation of FLB and 4'-OH-FLB were all within acceptable limits. The present HPLC-MS/MS method showed improved sensitivity for quantification of the FLB and its major metabolite in human plasma compared with previously described analytical methods. The validated method was successfully applied to a pharmacokinetic study in humans.

LC-MS/MS determination of apixaban (BMS-562247) and its major metabolite in human plasma: an application of polarity switching and monolithic HPLC column.

apixaban (BMS-562247) (Eliquis(®)) is a novel, orally active, selective, direct, reversible inhibitor of the coagulation factor Xa (FXa). A sensitive and reliable method was developed and validated for the measurement of apixaban (BMS-562247) and its major circulating metabolite (BMS-730823) in human citrated plasma for use in clinical testing. A 0.100 ml portion of citrated plasma sample was extracted and analyzed by LC-MS/MS. Run times were approximately 3 min. The lower limit of quantification (LLOQ) was 1.00 ng/ml for BMS-562247 and 5.00 ng/ml for BMS-730823. Intra- and inter-assay precision values for replicate QC control samples were within ≤5.36% for both analytes (≤7.52% at the LLOQ). The accuracy for both analytes was within ±9.00%. The method was demonstrated to be sensitive, selective and robust, and was successfully used to support clinical studies.

Simultaneous determination of bambuterol and its two major metabolites in human plasma by hydrophilic interaction ultra-performance liquid chromatography–tandem mass spectrometry

In this study, a rapid and sensitive hydrophilic interaction ultra-performance liquid chromatography-tandem mass spectrometry (HILIC-UPLC-MS/MS) method was developed for simultaneous determination of bambuterol and its two major metabolites monocarbamate bambuterol and terbutaline in human plasma. All samples were simply precipitated using acetonitrile and separated on a UPLC-HILIC column under gradient elution with a mobile phase consisting of acetonitrile and water with the addition of 10mm ammonium acetate and 0.1% formic acid at 0.4 mL/min. The analytes were detected by a Xevo TQ-S tandem mass spectrometer with positive electrospray ionization in multiple reaction monitoring mode. The established method was highly sensitive with the lower limit of quantification (LLOQ) of 10.00 pg/mL for each analyte, and the intra- and inter-day precisions were <12.8%. The analytical runtime within 4.0 min per sample made this method suitable for high throughput determination. The validated method was successfully applied to a clinical pharmacokinetic study of bambuterol in eight healthy volunteers. Furthermore, the effects of the chromatographic conditions on the retention of the analytes on HILIC were investigated, and the benefits of HILIC were evaluated by comparing with a C18 column. The results indicated that liquid-liquid partition and the electrostatic interactions played an important role in the retention of the analytes on HILIC in this study. And HILIC offered particular advantages over RPLC approach in the aspects of the peak symmetry, the column efficiency, and the column pressure.

Pharmacokinetics, Metabolism, and Excretion of [14C]Axitinib, a Vascular Endothelial Growth Factor Receptor Tyrosine Kinase Inhibitor, in Humans

The disposition of a single oral dose of 5 mg (100 μCi) of [(14)C]axitinib was investigated in fasted healthy human subjects (N = 8). Axitinib was rapidly absorbed, with a median plasma Tmax of 2.2 hours and a geometric mean Cmax and half-life of 29.2 ng/ml and 10.6 hours, respectively. The plasma total radioactivity-time profile was similar to that of axitinib but the AUC was greater, suggesting the presence of metabolites. The major metabolites in human plasma (0-12 hours), identified as axitinib N-glucuronide (M7) and axitinib sulfoxide (M12), were pharmacologically inactive, and with axitinib comprised 50.4%, 16.2%, and 22.5% of the radioactivity, respectively. In excreta, the majority of radioactivity was recovered in most subjects by 48 hours postdose. The median radioactivity excreted in urine, feces, and total recovery was 22.7%, 37.0%, and 59.7%, respectively. The recovery from feces was variable across subjects (range, 2.5%-60.2%). The metabolites identified in urine were M5 (carboxylic acid), M12 (sulfoxide), M7 (N-glucuronide), M9 (sulfoxide/N-oxide), and M8a (methylhydroxy glucuronide), accounting for 5.7%, 3.5%, 2.6%, 1.7%, and 1.3% of the dose, respectively. The drug-related products identified in feces were unchanged axitinib, M14/15 (mono-oxidation/sulfone), M12a (epoxide), and an unidentified metabolite, comprising 12%, 5.7%, 5.1%, and 5.0% of the dose, respectively. The proposed mechanism to form M5 involved a carbon-carbon bond cleavage via M12a, followed by rearrangement to a ketone intermediate and subsequent Baeyer-Villiger rearrangement, possibly through a peroxide intermediate. In summary, the study characterized axitinib metabolites in circulation and primary elimination pathways of the drug, which were mainly oxidative in nature.

Validation of A Method for Quantifying Enzalutamide and its Major Metabolites in Human Plasma by LC–MS/MS

Enzalutamide is an androgen receptor inhibitor that targets multiple steps in the androgen receptor signaling pathway. Oral enzalutamide was recently approved by the US FDA and health authorities in other regions for the treatment of patients with metastatic castration-resistant prostate cancer who previously received docetaxel. The objective of this study was to validate a method for quantification of enzalutamide and its two major metabolites in human plasma. The analytes were extracted from plasma by an LLE procedure, separated by reversed phase HPLC and detected by MS/MS in positive mode ESI. The quantitation range was 0.0200-50.0 µg/ml. The method proved to be rapid and simple, and met FDA validation criteria.

A Rapid and Sensitive HPLC-Fluorescence Method for Determination of Mirtazapine and Its two Major Metabolites in Human Plasma.

A rapid and sensitive HPLC method has been developed for the quantification of mirtazapine (MRZ), a noradrenergic and specific serotonergic inhibitor antidepressant (NaSSA) and its two major metabolites N-desmethyl mirtazapine (NDM) and 8-hydroxymirtazapine (8-OHM) in human plasma. The separation was achieved using Chromolith C18 column and a mobile phase of acetonitrile: phosphate buffer (pH = 3, 20:80, v/v) in isocratic mode at a flow rate of 2 mL/min. A fluorescence detector was set at 290 and 350 nm for excitation and emission, respectively. Zolpidem was used as the internal standard. Liquid-liquid extraction was applied for sample clean up. All analytes were eluted in less than 5 minutes with LOQ of 1 ng/mL for MRZ and 2 ng/mL for both NDM and 8-OHM. The developed method was successfully applied to quantify MRZ and its metabolites in plasma of a healthy volunteer.

Identification of Drug Metabolites in Human Plasma or Serum Integrating Metabolite Prediction, LC–HRMS and Untargeted Data Processing

Comprehensive identification of human drug metabolites in first-in-man studies is crucial to avoid delays in later stages of drug development. We developed an efficient workflow for systematic identification of human metabolites in plasma or serum that combines metabolite prediction, high-resolution accurate mass LC-MS and MS vendor independent data processing. Retrospective evaluation of predictions for 14 (14)C-ADME studies published in the period 2007-January 2012 indicates that on average 90% of the major metabolites in human plasma can be identified by searching for accurate masses of predicted metabolites. Furthermore, the workflow can identify unexpected metabolites in the same processing run, by differential analysis of samples of drug-dosed subjects and (placebo-dosed, pre-dose or otherwise blank) control samples. To demonstrate the utility of the workflow we applied it to identify tamoxifen metabolites in serum of a breast cancer patient treated with tamoxifen. Previously published metabolites were confirmed in this study and additional metabolites were identified, two of which are discussed to illustrate the advantages of the workflow.

LC–MS/MS assay for the quantitation of the HDAC inhibitor belinostat and five major metabolites in human plasma

The histone deacetylase inhibitor belinostat is being evaluated clinically as a single agent in the treatment of peripheral T-cell lymphomas and in combination with other anticancer agents to treat a wide range of human cancers including acute leukemias and solid tumors. To determine the pharmacokinetics of belinostat in the NCI ODWG liver dysfunction study, we developed and validated an LC–MS/MS assay for the quantitation of belinostat and five major metabolites in 0.05mL human plasma. After protein precipitation, chromatographic separation was achieved with a Waters Acquity BEH C18 column and a linear gradient of 0.1% formic acid in acetonitrile and water. Detection with an ABI 4000Q mass spectrometer utilized both electrospray positive and negative mode ionization. The assay was linear from 30 to 5000ng/mL for all six analytes and proved to be accurate (92.0–104.4%) and precise (CV <13.7%), and fulfilled FDA criteria for bioanalytical method validation. We demonstrated the suitability of this assay for measuring parent drug and five major metabolites in plasma from a patient who was administered belinostat IV at a dose of 400mg/m2. The LC–MS/MS assay that has been developed will be an essential tool to further define the metabolism and pharmacology of belinostat in the ongoing liver organ dysfunction as well as other studies that investigate belinostat with other anticancer agents.

Stable-Isotope Dilution LC–MS/MS Assay for Determination of Iloperidone and its Two Major Metabolites, P 88 and P 95, in Human Plasma: Application to A Bioequivalence Study

Iloperidone is a novel antipsychotic drug with high affinity for serotonin and D(2) dopamine receptors. A sensitive and rapid isotope dilution LC-MS/MS method has been developed for the simultaneous determination of iloperidone and its two major metabolites in human plasma. The analytes and their deuterated analogs as internal standards were quantitatively extracted from 100-µl human plasma by SPE. The method was validated over the concentration range of 0.01-6 ng/ml for all the analytes. Baseline separation of analytes was possible within 3 min on ACE 5 C8 column. The accuracy and precision (%CV) of the method varied from 96.2 to 105%, and 1.17 to 4.75%, respectively. The extraction recovery was >84%, while the internal standard-normalized matrix factors ranged from 0.97-1.03 for all three analytes. The developed method was successfully applied to support a bioequivalence study in healthy volunteers.

Simultaneous quantitation of 3-n-butylphthalide (NBP) and its four major metabolites in human plasma by LC–MS/MS using deuterated internal standards

3-n-Butylphthalide (NBP) is a cardiovascular drug widely used in China for the treatment of cerebral ischemic stroke. Our previous study showed that NBP underwent extensive metabolism in humans and that 10-keto-NBP (M2), 3-hydroxy-NBP (M3-1), 10-hydroxy-NBP (M3-2) and NBP-11-oic acid (M5-2) were the major circulating metabolites. A better understanding of the plasma exposures of NBP and its four major metabolites is crucial to supporting the safety evaluation, good clinic practice and discovery of new antistroke drugs. Herein, a liquid chromatography–tandem mass spectrometry method was developed and validated for the simultaneous determination of NBP, M2, M3-1, M3-2, and M5-2 in human plasma. To improve assay sensitivity and achieve simultaneous analysis, M3-1 and M5-2 were monitored in (−)ESI (electrospray) mode within the first 3.5min and NBP, M2, and M3-2 thereafter in (+)ESI mode. Deuterated internal standards for all analytes were synthesized to compensate for the impact of matrix effects. Based on the vast differences in physicochemical properties among the five analytes and the necessary baseline separation of two isomers (i.e., M3-1 and M3-2), gradient elution comprising a mobile phase of methanol–acetonitrile–5mM ammonium acetate was used after methanol-induced protein precipitation of plasma samples. The developed method was linear in the concentration range of 3.00–800ng/ml for NBP and M2, and 3.00–2400ng/ml for M3-1, M3-2, and M5-2. The lower limit of quantitation was 3.00ng/ml for each analyte. The intra- and inter-day accuracy and precision were within acceptable limits (±15%) at all concentrations. The method was successfully applied to characterize the pharmacokinetic profiles of NBP and its major metabolites following a single oral administration of 200mg NBP to healthy volunteers.

Study on paclitaxel and its metabolites in human plasma by electrospray ionization tandem mass spectrometry

Objective: To investigate the ionization effect,structural fragmentation pathways of paclitaxel and its major metabolites in human plasma by electrospray ionization tandem mass spectrometry(ESI-MSn).Methods:The reference solution of paclitaxel was infused into mass spectrometer for studying its ionization and fragmentation in MS and MS2 mode.Paclitaxel metabolites in human body were separated and identified by LC-MS/MS method.Results: The ionization efficiency of paclitaxel was greatly enhanced by the additives in the solution.The protonated ion(~) at m/z 854 was the most abundant under positive ESI condition.Dehydration and ester bond cleavage were the main fragmentation procedures of paclitaxel.Various fragment ions were formed,and the fragment ion at m/z 286 was the most prominent and stable.Three metabolites of paclitaxel were found in human plasma and 6α-hydroxy paclitaxel was the main metabolite.Conclusion: The study on mass spectrometric behavior of paclitaxel and its primary metabolites in human plasma provided an important evidence for qualitative and quantitative analysis of paclitaxel by mass spectrometry and research on its metabolism.

High-sensitivity liquid chromatography–tandem mass spectrometry method for the simultaneous determination of sodium picosulfate and its three major metabolites in human plasma

Sodium picosulfate (PICO-Na) is a member of the polyphenolic group of stimulant laxatives. Its major metabolites in humans are its active aglycone BHPM (bis-(p-hydroxyphenyl)-pyridyl-2-methane), the monoglucuronide (M1) and the monosulfate (M2) of BHPM. A sensitive, specific and rapid liquid chromatography–tandem mass spectrometry method was established and validated for the simultaneous determination of picosulfate (PICO) and its three major metabolites in human plasma to investigate the pharmacokinetics of PICO and its major metabolites. Following protein precipitation with acetonitrile, chromatographic separation was achieved on a Luna 5u C18(2) column using gradient elution starting with 10% of 10mM ammonium acetate followed by increasing percentages of acetonitrile to eliminate interferences due to in-source conversion of the conjugated metabolites. Detection was performed on a tandem mass spectrometer equipped with an electrospray ionization source operated in the positive mode, using the transitions of m/z 438.1→m/z 278.1 for PICO, m/z 278.1→m/z 184.2 for BHPM, m/z 454.1→m/z 184.2 for M1, and m/z 358.1→m/z 184.2 summed with m/z 358.1→m/z 278.1 for M2. Deuterium labeled compounds of the analytes were used as the internal standard, two of which, M1-d12 and M2-d12, were synthesized in-house. The method was validated in concentration ranges of 0.150–40.0ng/mL for PICO and M2, 0.600–160ng/mL for BHPM, and 0.045–12.0ng/mL for M1 with acceptable accuracy and precision. The method was successfully applied to characterize the pharmacokinetic profiles of PICO and its metabolites in healthy volunteers after a single oral administration of 5mg PICO-Na.

Development and Validation of a Sensitive Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Assay for the Simultaneous Quantification of Midostaurin (PKC412) and Its Two Major Metabolites: A Suitable Tool for the Monitoring of the Drug in Patients Suffering From Advanced Systemic Mastocytosis

Abstract 4913 Background:Among tyrosine kinase inhibitors, midostaurin (PKC412) takes place in the treatment of patients presenting with advanced systemic mastocytosis and, specifically aggressive systemic mastocytosis (ASM) or mast cell leukemia (MCL) with or without associated clonal hematological non-mast cell lineage disease (AHNMD). Our knowledge of the pharmacokinetic (PK) behavior of this major drug remains unfortunately sporadic. We developed a powerful liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay, which is able to sensitively detect PKC412 as its two major metabolites CGP52421 and CGP62221 in human plasma. The method was validated in clinical situation and, especially in the aim to monitor plasma concentrations of PKC412 in 11 patients, already enrolled in a Phase II clinical trial (Novartis Lab.). In addition, we also determined the PK profile of PKC412 at steady-state for one specific patient. Methods:A sensitive LC-MS/MS assay was developed for the simultaneous quantification of the product PKC412 and its two major metabolites in human plasma. Midostaurin-D5 was used as internal standard. The method was then validated in accordance with international guidelines and within the bioanalytical ±15% acceptance limits.In any case, PKC412 regimen was 100 mg bid. A total of 61 blood samples were collected from 11 patients and, between 15 min to 12 hrs following oral administration. Sampling took place over a period of 1 to 924 days following the beginning of treatment. PKC412 residual concentration at steady-state, range and mean concentration and, intra- and inter-individual variability coefficients were also calculated. On the other hand, a model independent method was used to determine the PK profile of PKC412 for a 12th patient (using 22 samples collected within 24 hrs): maximal concentration post-dosing (Cmax – ng/mL), time to Cmax (Tmax - hrs) were experimental values; terminal half-life (t1/2lz - hrs) and AUC0–12 were calculated during each 12 hrs interval post-dosing. Data are expressed as means ± SD. Results:This LC-MS/MS method is satisfactory for the key validation criteria i. e. trueness, bias, precision (repeatability and intermediate precision), accuracy and, linearity, over a range of concentrations of 75 to 2500 ng/mL for both PKC412, CGP52421 and, CGP62221. Patients monitored (8/11 males) were 57±14 yrs-old. All except one were treated for ASM±AHNMD (the last one had MCL), 10/11 patients carrying the ckit D816V mutation. The main characteristics at inclusion were as followed: body weight = 65±11 kg, BMI = 22±3 kg/m2, albumin = 31±6 g/L, GFR = 64±12 mL/min, bilirubin = 14±6 mg/L, GGT = 103±78 UI/L, ALP = 264±158 UI/L, tryptase = 412±411 μg/L. At the last follow-up visit i. e. between 54 to 924 day after initiation therapy, 8/11 patients were either in major or partial response with the following clinical picture: body weight 74±16 kg, BMI 24±4 kg/m2, albumin 40±2 g/L, DFG 59±11 mL/min, bilirubin 12±8 mg/L, GGT 56±57 UI/L, ALP 128±59 UI/L, tryptase 256±264 μg/L. It is important to point out that the tolerability profile was very good. The PKC412 residual concentrations value determined at steady-state were between 487 to 3254 ng/mL (1333±867 ng/mL). The intra- and inter-individual coefficients of variations were 18 and 65% respectively. About our 12th subject, major PK parameters were: Cmax = 3692±447 ng/mL, Tmax = 2, 5±2 hrs, t1/2lz = 95±88 hrs, AUC0–12= 34326±2197 ng/mL. hrs. Conclusions:The LC-MS/MS method is efficient and enables a sensitive and discriminative analysis of PKC412 and its major metabolites in human plasma. These first results highlight the high intra- and inter-individual variability of the PK behavior of the drug and, suggest the interest of its routine therapeutic monitoring vs. both adherence to the treatment and, its tolerability profile. Further, it has been shown that the 2 major metabolites of PK412 are active; under these conditions, we are working to the simultaneous therapeutic drug monitoring of the 3 compounds. Disclosures:No relevant conflicts of interest to declare.

Simultaneous determination of apatinib and its four major metabolites in human plasma using liquid chromatography–tandem mass spectrometry and its application to a pharmacokinetic study

Apatinib, also known as YN968D1, is a novel antiangiogenic agent that selectively inhibits vascular endothelial growth factor receptor-2. Currently, apatinib is undergoing phase II/III clinical trials in China for the treatment of solid tumors. Apatinib is extensively metabolized in humans, and its major metabolites in circulation include cis-3-hydroxy-apatinib (M1-1), trans-3-hydroxy-apatinib (M1-2), apatinib-25-N-oxide (M1-6), and cis-3-hydroxy-apatinib-O-glucuronide (M9-2). To investigate the pharmacokinetics of apatinib and its four major metabolites in patients with advanced colorectal cancer, a sensitive and selective liquid chromatography–tandem mass spectrometry method was developed and validated for the simultaneous determination of apatinib, M1-1, M1-2, M1-6, and M9-2 in human plasma. After a simple protein precipitation using acetonitrile as the precipitation solvent, all the analytes and the internal standard vatalanib were separated on a Zorbax Eclipse XDB C18 column (50mm×4.6mm, 1.8μm, Agilent) using acetonitrile: 5mmol/L ammonium acetate with 0.1% formic acid as the mobile phase with gradient elution. A chromatographic total run time of 9min was achieved. Mass spectrometry detection was conducted through electrospray ionization in positive ion multiple reaction monitoring modes. The method was linear over the concentration range of 3.00–2000ng/mL for each analyte. The lower limit of quantification for each analyte was 3.00ng/mL. The intra-assay precision for all the analytes was less than 11.3%, the inter-assay precision was less than 13.8%, and the accuracy was between −5.8% and 3.3%. The validated method was successfully applied to a clinical pharmacokinetic study following oral administration of 500mg apatinib mesylate in patients with advanced colorectal cancer.

Quantitation of paclitaxel and its two major metabolites using a liquid chromatography–electrospray ionization tandem mass spectrometry

A sensitive and selective liquid chromatographic–tandem mass spectrometric (LC–MS/MS) method for the determination of paclitaxel (Taxol) and its two major metabolites in human plasma has been developed. Samples were prepared after liquid–liquid extraction and analyzed on a C 18 column interfaced with a Q-Trap tandem mass spectrometer. Positive electrospray ionization was employed as the ionization source. The mobile phase consisted of acetonitrile–water (0.05% formic acid) (65:35) at the flow rate of 0.25 mL/min. The analytes and internal standard docetaxel were both detected by use of multiple reaction monitoring mode. The method was linear in the concentration range of 0.5–500.0 ng/mL for paclitaxel, 6α-hydroxypaclitaxel and p-3′-hydroxypaclitaxel, respectively. The lower limit of quantification (LLOQ) was 0.5 ng/mL for paclitaxel, 6α-hydroxypaclitaxel and p-3′-hydroxypaclitaxel, respectively. The intra- and inter-day relative standard deviation across three validation runs over the entire concentration range was less than 8.18%. The accuracy determined at three concentrations was within ±10.8% in terms of relative error. The total run time was 7.0 min. This assay offers advantages in terms of expediency, and suitability for the analysis of paclitaxel and its metabolites in various biological fluids.

LC–MS/MS method for simultaneous determination of valproic acid and major metabolites in human plasma

A rapid and sensitive method using liquid chromatography-tandem mass spectroscopy (LC–MS/MS) was developed and validated for simultaneous quantitative determination of valproic acid and three major metabolites (3-OH-valproic acid, 4-ene-valproic acid and 5-OH-valproic acid) in human plasma. The analytes and internal standard were isolated from 200 μL samples by solid phase extraction using a ZORBAX SB-C 8 column (3.5 μm, 2.1 × 100 mm) with an isocratic mobile phase consisting of methanol-10 mM ammonium acetate (80:20, v/v) containing 0.1% formic acid at a flow rate of 0.3 mL/min. The method had a chromatographic total run time of 2.0 min. The lower limit of quantification of valproic acid, 3-OH-valproic acid, 4-ene-valproic acid and 5-OH-valproic acid of the method was 2030, 51.5, 50.15 and 51.25 ng/mL, respectively. The method was linear for valproic acid and the three metabolites with correlation coefficients >0.995 for all analytes. The intra-day and inter-day accuracy and precision of the assay were less than 15.0%. This analytical method was successfully used to assay plasma concentrations of valproic acid and the three metabolites in human plasma from epileptic patients.

Simple and sensitive HPLC method for the fluorometric determination of methotrexate and its major metabolites in human plasma by post‐column photochemical reaction

A simple and sensitive HPLC method has been developed for the determination of methotrexate (MTX) and its major metabolites, 7-hydroxymethotrexate (7-OH-MTX) and 2,4-diamino-N(10-) methylpteroic acid (DAMPA), in human plasma. After deproteinization of the plasma with 5% aqueous acetonitrile solution containing 5% trichloroacetic acid, MTX, 7-OH-MTX, DAMPA and 2,4-diaminopteroic acid (DAPA) as an internal standard were separated on a reversed-phase column, and the eluent was subsequently irradiated with UV light (245 nm), producing fluorescent photolytic degradation products. The analytes were then detected spectrofluorometrically at 452 nm with excitation at 368 nm. The extraction efficiencies of MTX, 7-OH-MTX and DAMPA from plasma at 100 pmol/mL were 81.5±5.4, 82.5±5.3 and 56.2±7.0%, respectively. The limits of quantification for MTX, 7-OH-MTX and DAMPA in plasma were 5 pmol (2.3 ng), 0.8 pmol (0.38 ng) and 10 pmol (3.4 ng)/mL, respectively. The within- and between-day variations for MTX, 7-OH-MTX and DAMPA were reliable (each was lower than 6.3%). This method was also used to monitor the concentrations of MTX and its metabolites in a patient on MTX therapy.

Abstract P5-11-04: Phenotype-Genotype Correlations in Breast Cancer Patients Treated with Tamoxifen

Abstract Background: Personalized medicine strategies are especially relevant for drugs which show a high inter-individual variability regarding their pharmacokinetic and -dynamic properties. Considerable inter-patient variations have been described regarding the plasma concentrations of tamoxifen and its metabolites. Due to the observation that tamoxifen metabolites can show a high pharmacological activity, tamoxifen metabolism has received considerable attention. The metabolism of tamoxifen is complex resulting in a huge number of different products. Important metabolic transformation reactions include demethylation catalyzed by Cytochrome P450 3A4 and hydroxylation mainly catalyzed by Cytochrome P450 2D6 (CYP2D6). The impact of the CYP2D6 activity, which can be predicted from genetic information, on plasma levels and the related pharmacological effects has controversially been discussed. To get a better understanding of the inter-individual differences of the plasma levels of tamoxifen and some of its metabolites we have used liquid chromatography-tandem mass spectrometry (LC/MS/MS) to screen the plasma samples of 106 breast cancer patients. This method enabled the quantification of tamoxifen as well as a relative quantification of demethyltamoxifen and hydroxylated tamoxifen metabolites. To study the impact of enzyme activity on plasma levels, CYP2D6 genotypes were determined as well. Material and methods: For metabolic profiling 1 ml plasma was treated with a solid phase extraction procedure. Chromatographic separation was accomplished on a reversed-phase column. Analytes were detected by mass spectrometry. For genotyping, the CYP2D6 gene was amplified by a long range PCR followed by a multiplexed PCR to generate specific short amplicons. The amplicons were directly analysed by LC/MS. The information necessary to determine the allelic state were obtained from the measured molecular masses. Results: We determined the CYP2D6 genotype of 106 breast cancer patients treated with tamoxifen. We found 19% “Poor Metabolizers", 51% “Intermediate Metabolizers", 29% “Extensive Metabolizers” and 7% “Ultrarapid Metabolizers”. The observed plasma concentrations of tamoxifen and its metabolites showed a high inter-patient variability For instance, plasma concentrations between 26-307 ng/ml (mean 125 ng/ml) were observed for tamoxifen. We did not find a significant impact of the CYP2D6 status on tamoxifen plasma levels. However, the demethyltamoxifen/tamoxifen as well as the hydroxytamoxifen/tamoxifen ratios appear to be influenced by the CYP2D6 status: the higher the metabolic activity, the higher the hydroxytamoxifen content and the lower the demethyltamoxifen content. Conclusions: We have developed analytical tools, which allow the determination of the content of tamoxifen and its major metabolites in human plasma and the genotyping of the CYP2D6 gene. Phenotypic and genotypic data of 106 patients were acquired. Correlation of the data revealed that the genotype had no significant impact on tamoxifen plasma levels. Nevertheless, patients with a predicted higher CYP2D6 activity showed lower demethyltamoxifen/tamoxifen ratios as well as higher hydroxytamoxifen/tamoxifen ratios. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-11-04.