Metabolites In Plasma Samples Research Articles

Human Mild Traumatic Brain Injury Decreases Circulating Branched-Chain Amino Acids and Their Metabolite Levels

The pathophysiology of traumatic brain injury (TBI) is complex and not well understood. Because pathophysiology has ramifications for injury progression and outcome, we sought to identify metabolic cascades that are altered after acute human mild and severe TBI. Because catabolism of branched-chain amino acids (BCAAs; i.e., valine, isoleucine, and leucine) leads to glucose and energy metabolism, and neurotransmitter synthesis and availability, we investigated BCAA metabolites in plasma samples collected within 24 h of injury from mild TBI (Glasgow Coma Scale [GCS] score >12), severe TBI (GCS ≤8), orthopedic injury, and healthy volunteers. We report decreased levels of all three BCAAs in patients with mild TBI relative to healthy volunteers, while these BCAAs levels in patients with severe TBI were further reduced compared with all groups. Orthopedic patients exhibited reductions in BCAA comparable to those in patients with mild TBI. The decrease in patients with mild and severe TBI persisted for derivatives of BCAA catabolic intermediates. Only plasma levels of methylglutarylcarnitine, a derivative of a leucine metabolite, were increased in patients with severe TBI compared with all other groups. Notably, logistic regression combination of three BCAA metabolites whose levels were changed by 24 h post-injury provided prognostic value (area under the curve=0.92) in identifying patients with severe TBI in whom elevated intracranial pressure (≥25 mm Hg) developed. These changes suggest alteration of BCAA metabolism after TBI may contribute to decreased energy production and neurotransmitter synthesis and may contribute to TBI pathophysiology. Supplementation of BCAAs and/or their metabolites may reduce TBI pathology and improve outcome.

Bio-generation of stable isotope labeled internal standards for absolute and relative quantitation of drug metabolites in plasma samples by LC–MS/MS

In order to achieve a better understanding of the toxicity of drug candidates, quantitative characterization of circulatory drug metabolites has been of increasing interest in current pharmaceutical research. Stable isotope labeled (STIL) internal standards (IS) are ideally used to simplify drug metabolite quantitation via liquid chromatography and tandem mass spectrometry (LC-MS/MS) analysis, primarily due to their capability to compensate matrix effects, thereby leading to faster method establishment by using generic assay conditions. However, chemical synthesis of STIL metabolites can often be resource intensive, requiring lengthy exploratory synthesis route development and/or extensive optimization to achieve the required stability for some metabolites. To overcome these challenges, we developed a general method that could generate STIL metabolites in a matter of hours from STIL parent drugs through the utilization of an appropriate in vitro metabolic incubation. This methodology can potentially save valuable synthesis resources, as well as provide timely availability of STIL IS. The following work demonstrates the proof-of-concept that multiple STIL metabolites can be generated simultaneously to provide satisfactory performance for both absolute quantitation of drug metabolites and for potential use in assessment of relative exposure coverage across species in safety tests of drug metabolites (MIST).

Development of a chiral micellar electrokinetic chromatography–tandem mass spectrometry assay for simultaneous analysis of warfarin and hydroxywarfarin metabolites: Application to the analysis of patients serum samples

The enantioseparation of warfarin (WAR) along with the five positional and optical isomers is challenging because of the difficulty to simultaneously separate and quantitate these chiral compounds. Currently, no effective chiral CE–MS methods exist for the simultaneous enantioseparation of WAR and all its hydroxylated metabolites in a single run. Polymeric surfactants (aka. molecular micelles) are particularly compatible with micellar electrokinetic chromatography–mass spectrometry (MEKC–MS) because they have a wider elution window for enantioseparation and do not interfere with the MS detection of chiral drugs. Using polysodium N-undecenoyl-l,l-leucylvalinate (poly-l,l-SULV) as a chiral pseudophase in MEKC–MS baseline separation of WAR, its five metabolites along with the internal standard was obtained in 45min. This is in comparison to 100min required for separation of the same mixture with packed column CEC–MS using a vancomycin chiral stationary phase. Serum samples were extracted with mixed-mode anion-exchange (MAX) cartridge with recoveries of greater than 85.2% for all WAR and hydroxywarfarin (OH-WAR) metabolites. Utilizing the tandem MS and multiple reaction monitoring mode, the MEKC–MS/MS method was used to simultaneously generate calibration curves over a concentration range from 2 to 5000ng/mL for R- and S-warfarin, 5 to 1000ng/mL for R- and S-6-, 7-, 8- and 10-OH-WAR and 10 to 1000ng/mL for R and S-4′-OH-WAR. For the first time, the limits of detection and quantitation for most WAR metabolites by MEKC–MS/MS were found to be at levels of 2 and 5ng/mL, respectively. The method was successfully applied for the first time to analyze WAR and its metabolites in plasma samples of 55 patients undergoing WAR therapy, demonstrating the potential of chiral MEKC–MS/MS method to accurately quantitate with high sensitivity.

Multicenter Study on the Clinical Effectiveness, Pharmacokinetics, and Pharmacogenetics of Mirtazapine in Depression

Pharmacogenetic tests and therapeutic drug monitoring may considerably improve the pharmacotherapy of depression. The aim of this study was to evaluate the relationship between the efficacy of mirtazapine (MIR) and the steady-state plasma concentrations of its enantiomers and metabolites in moderately to severely depressed patients, taking their pharmacogenetic status into account. Inpatients and outpatients (n = 45; mean age, 51 years; range, 19-79 years) with major depressive episode received MIR for 8 weeks (30 mg/d on days 1-14 and 30-45 mg/d on days 15-56). Mirtazapine treatment resulted in a significant improvement in mean Hamilton Depression Rating Scale total score at the end of the study (P < 0.0001). There was no evidence for a significant plasma concentration-clinical effectiveness relationship regarding any pharmacokinetic parameter. The enantiomers of MIR and its hydroxylated (OH-MIR) and demethylated (DMIR) metabolites in plasma samples on days 14 and 56 were influenced by sex and age. Nonsmokers (n = 28) had higher mean MIR plasma levels than smokers (n = 17): S(+)-enantiomer of MIR, 9.4 (SD, 3.9) versus 6.2 (SD, 5.5) ng/mL (P = 0.005); R(-)-enantiomer of MIR, 24.4 (SD, 6.5) versus 18.5 (SD, 4.1) ng/mL (P = 0.003). Only in nonsmokers, plasma levels of S(+)-enantiomer of MIR and metabolites depended on the CYP2D6 genotype. Therefore, high CYP1A2 activity seen in smokers seems to mask the influence of the CYP2D6 genotype. In patients presenting the CYP2B6 *6/*6 genotype (n = 8), S-OH-MIR concentrations were higher those in the other patients (n = 37). Although it is not known if S-OH-MIR is associated with the therapeutic effect of MIR, the reduction of the Hamilton scores was significantly (P = 0.016) more pronounced in the CYP2B6 *6/*6-genotyped patients at the end of the study. The role of CYP2B6 in the metabolism and effectiveness of MIR should be further investigated.

Automated method for determination of olive oil phenols and metabolites in human plasma and application in intervention studies

The interest for olive oil phenols (OOPs) is a growing trend thanks to their contribution to prevent or improve diseases associated to oxidative damage. OOPs ingested in the diet are found at low concentrations in blood either as free forms (e.g. hydroxytyrosol, tyrosol, vanillin, ferulic acid, coumaric acid) or conjugated as sulfate and glucuronide derivatives. Therefore, the identification/quantitation of OOPs in plasma to study their biological effects and elucidate their metabolism requires selective and sensitive methods. The present research describes the development, validation and application of an automated method based on on-line coupling of solid-phase extraction and liquid chromatography–tandem mass spectrometry (SPE–LC–MS/MS) for quantitation of conjugated and free OOPs in human plasma. This approach minimizes sample handling—thus reducing analyte losses and degradation by contact with the atmosphere—and increases analysis throughput, which is crucial in intervention studies dealing with cohorts formed by numerous individuals. The fundamental of the approach is the retention of OOPs and metabolites in an SPE anionic cartridge with subsequent on-line elution to an LC–MS/MS system. Quantitative analysis of OOPs (relative quantitation for conjugated OOPs) was carried out by selected reaction monitoring mode that reported relative limits of detection and quantitation between 0.02–0.28ng/mL (16.6–232pg on-column) and 0.05–0.83ng/mL (41.5–689pg on-column), respectively. The accuracy of the method, estimated as recovery factor, ranged from 84.2 to 99.4%, and precision, expressed as relative standard deviation, was below 3.8%. The resulting method has been applied to the determination of OOPs and metabolites in plasma samples from individuals who ingested a breakfast prepared with virgin olive oil. The proposed method has an excellent potential for high-throughput use in both clinical and research laboratories.

Schizophrenia shows a unique metabolomics signature in plasma.

Schizophrenia is a severe complex mental disorder affecting 0.5–1% of the world population. To date, diagnosis of the disease is mainly based on personal and thus subjective interviews. The underlying molecular mechanism of schizophrenia is poorly understood. Using targeted metabolomics we quantified and compared 103 metabolites in plasma samples from 216 healthy controls and 265 schizophrenic patients, including 52 cases that do not take antipsychotic medication. Compared with healthy controls, levels of five metabolites were found significantly altered in schizophrenic patients (P-values ranged from 2.9 × 10−8 to 2.5 × 10−4) and in neuroleptics-free probands (P-values ranging between 0.006 and 0.03), respectively. These metabolites include four amino acids (arginine, glutamine, histidine and ornithine) and one lipid (PC ae C38:6) and are suggested as candidate biomarkers for schizophrenia. To explore the genetic susceptibility on the associated metabolic pathways, we constructed a molecular network connecting these five aberrant metabolites with 13 schizophrenia risk genes. Our result implicated aberrations in biosynthetic pathways linked to glutamine and arginine metabolism and associated signaling pathways as genetic risk factors, which may contribute to patho-mechanisms and memory deficits associated with schizophrenia. This study illustrated that the metabolic deviations detected in plasma may serve as potential biomarkers to aid diagnosis of schizophrenia.

Innovative microwave-assisted oximation and silylation procedures for metabolomic analysis of plasma samples using gas chromatography–mass spectrometry

Analysis of plasma metabolomic samples by gas chromatography–mass spectrometry always requires comprehensive pretreatment including oximation and silylation. Although heating block (HB) is a commonly used method, it is time consuming. This study describes an extremely time-effective microwave-assisted (MA) oximation and silylation approach for metabolomic study of plasma samples. The Box–Behnken design was employed to optimize the MA conditions by means of oximation at 65W for 100s and then silylation through 180s incubation with 230W microwave irradiation. The results showed that microwave irradiation decreased the sample preparation time from approximately 180min to 5min without loss of information for the metabolites in plasma samples. Both the HB method and the developed MA method were applied in plasma metabolomic study of sulfur mustard intoxication. Partial least-squares discriminant analysis (PLS-DA) was used to globally understand the metabolic changes, and multi-criteria assessment was used to select the most significant and reliable variables as potential biomarkers. The data obtained by the MA method were in good correlation with the HB method. Compared with HB method, the newly developed MA oximation and silylation of plasma metabolome samples was more efficient and time-effective and may prove to be an attractive alternative for high-throughput sample preparation in plasma metabolomics.

Automated analysis of lidocaine and its metabolite in plasma by in‐tube solid‐phase microextraction coupled with LC‐UV for pharmacokinetic study

A sensitive, selective, and reproducible in-tube solid-phase microextraction and liquid chromatographic (in-tube SPME/LC-UV) method for determination of lidocaine and its metabolite monoethylglycinexylidide (MEGX) in human plasma has been developed, validated, and further applied to pharmacokinetic study in pregnant women with gestational diabetes mellitus (GDM) subjected to epidural anesthesia. Important factors in the optimization of in-tube SPME performance are discussed, including the draw/eject sample volume, draw/eject cycle number, draw/eject flow rate, sample pH, and influence of plasma proteins. The limits of quantification of the in-tube SPME/LC method were 50 ng/mL for both metabolite and lidocaine. The interday and intraday precision had coefficients of variation lower than 8%, and accuracy ranged from 95 to 117%. The response of the in-tube SPME/LC method for analytes was linear over a dynamic range from 50 to 5000 ng/mL, with correlation coefficients higher than 0.9976. The developed in-tube SPME/LC method was successfully used to analyze lidocaine and its metabolite in plasma samples from pregnant women with GDM subjected to epidural anesthesia for pharmacokinetic study.

Abstract B56: A phase 1, open-label, dose escalation, nonrandomized study to assess the maximum tolerated dose, dose limiting toxicity, and pharmacokinetics of OPB-31121 in subjects with advanced solid tumors.

Abstract Background: OPB-31121 is a novel compound exhibiting potent growth inhibition of cancer cell lines in vitro and xenografts in vivo. The exact mechanism of action of OPB-31121 has not been fully characterized, but studies indicate that a major effect is inhibition of STAT3 phosphorylation. STAT3 is frequently activated in a variety of solid and hematologic malignancies and may present an important target for antitumor therapy. Methods: Open-label, non-randomized, multi-center study in subjects with advanced solid tumors, using a 3+3 dose escalation design. OPB-31121 was administered orally for 21 days followed by 7 days rest per cycle (28-day cycle). The starting dose was 50 mg BID with escalations planned until the dose-limiting toxicity (DLT) was reached. The primary endpoint was determination of maximum tolerated dose (MTD). Additional endpoints included safety, pharmacokinetics, and anti-tumor effect of OPB-31121. Results: 30 subjects received treatment with OPB-31121. Most common tumor types were colorectal cancer (15), breast (3) and thyroid (2). Mean age was 55.9 (range 35–80) years and 17 of the patients were female. Most common adverse events (AEs) potentially attributed to treatment were gastrointestinal: nausea (80%), vomiting (73%), diarrhea (63%), anorexia (20%), and constipation (17%). Most AEs were CTCAE grades 1–2 and manageable with supportive treatment. Three DLTs were observed: one at 300 mg BID (grade 3 lactic acidosis), and two at 350 mg BID (a grade 3 diarrhea and a grade 3 vomiting); the MTD was 300 mg BID. All patients recovered from DLTs after discontinuing the drug. Six additional (9 total) subjects discontinued during the first cycle. Eight subjects completed only one cycle and 13 completed two cycles. No objective responses were observed. Disease progression was observed in all evaluable patients at first restaging. Pharmacokinetic measurements showed low and transient plasma levels of OPB-31121. Inter-patient variability was high. Exposure was low - area-under-the-curve (AUC) values were 2–3 orders of magnitude lower than those measured at active doses in mouse models. Analysis of metabolites in plasma samples indicates extensive CYP3A4 metabolism and suggests a large first-pass effect in humans, which had not been observed in rodents. Conclusions: OPB-31121 does not show potential as a therapeutic option for most malignancies. The extensive first-pass metabolism and dose-limiting gastrointestinal AEs limit the level of systemic exposure that can be achieved with this agent. Further studies may be warranted in cancers of organs where local concentrations of the compound may be higher (e.g., liver) and an exploratory study in hepatocellular carcinoma is ongoing in Asia. Because STAT3 remains an attractive antitumor target, chemically related compounds with similar pharmacologic activities are currently being evaluated preclinically. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B56.

Quantitative estimation of circulating metabolites without synthetic standards by ultra‐high‐performance liquid chromatography/high resolution accurate mass spectrometry in combination with UV correction

An early assessment of metabolite exposure in preclinical species can provide quantitative estimation on possible active or toxic metabolites. Frequently, synthetic metabolite standards are not available at the preclinical stage, precluding the quantitation of metabolites by means of calibration curves and quality control (QC) samples. We present here an approach to determine the extent of circulating metabolites using 'metabolite standards' generated by in vitro incubations in combination with the correction for mass spectrometry response based on UV response. The study was done by coupling ultra-high-performance liquid chromatography (UHPLC) to LTQ-Orbitrap high-resolution mass spectrometry, and the quantitation was based on full scan high-resolution accurate mass analysis in combination with retention time. First, we investigated the separation capacity of a 10.5 min UHPLC method and the quantitative capability of an LTQ-Orbitrap for full scan accurate mass quantitation by spiking chemical standards of buspirone and its six metabolites in blank plasma. Then we demonstrated the use of a UV correction approach to quantitatively estimate buspirone and its metabolites in plasma samples from a rat pharmacokinetics study. We compared the concentration versus time profiles of buspirone and its six metabolites in rat plasma samples obtained using three different approaches, including using UV correction, using individual standard curves for each metabolite prepared from the synthetic standard, and using a calibration curve of the parent compound buspirone. We demonstrated the estimated metabolite exposure of buspirone using this UV correction approach resulted in rank ordering of metabolite exposure within three-fold of the value obtained with metabolite standards, in contrast to eight-fold without UV correction. The approach presented in this paper provides a practical solution to an unmet bioanalytical need for quantitative information on metabolites without standards in preclinical in vivo studies.

Determination of ticagrelor and two metabolites in plasma samples by liquid chromatography and mass spectrometry

Rapid and sensitive analytical methods using liquid chromatography with tandem mass spectrometry (LC/MS/MS) were developed for the determination of ticagrelor, the first reversible oral platelet P2Y 12 receptor inhibitor, and its metabolites AR-C124910XX and AR-C133913XX in human plasma. Ticagrelor and its metabolites were extracted using protein precipitation with acetonitrile. Chromatographic separations were performed on reversed phase columns and detection using atmospheric pressure chemical ionization (APCI). Ticagrelor and AR-C124910XX were analyzed in the same assay, with the internal standard, d7-ZD6140, on a C18 column using negative ionization; AR-C133913XX analyzed separately on a phenyl column using positive ionization. Full validation of the methods was performed including selectivity, lower limit of quantification, accuracy, precision stability and incurred sample reproducibility and incurred sample stability. Total analytical run time was short (2 min). Calibration curves were established in the range 5–5000 ng/mL for ticagrelor, 2.5–2500 ng/mL for AR-C124910XX and 2–1000 ng/mL for AR-C133913XX. Lower limits of quantification for ticagrelor, AR-C124910XX and AR-C133913XX were determined to be 5, 2.5 and 2.0 ng/mL, respectively from 100 μL of human plasma. For ticagrelor, AR-C124910XX and AR-C133913XX, mean intra-batch accuracy was 91.9–109.0%, 86.8–109.2% and 100.5–112.0%, respectively; intra-batch precision was 4.0–8.4%, 5.2–16.9% and 3.9–12.3%, respectively. The methods were also applied to quantification of ticagrelor, AR-C124910XX and AR-C133913XX in rabbit, rat, mouse and marmoset, using 25 μL of animal plasma. A modified methodology was developed to quantify ticagrelor and AR-C124910XX in plasma from dog and cynomolgus monkey. Human incurred samples were found to generate consistent reproducibility and stability results. This method was successfully applied to determine plasma concentrations following administration of ticagrelor in human volunteers and patients, and animal safety evaluation studies. This validated methods has the advantages of being straightforward, robust and allows a fast throughput of samples.

Metabolites of puerarin identified by liquid chromatography tandem mass spectrometry: Similar metabolic profiles in liver and intestine of rats

Puerarin is a major active ingredient of Pueraria radix. Puerarin may exert its medicinal functions in part via its metabolites. In this study, we identified these metabolites to better understand and elucidate puerarin's metabolic pathway. Puerarin was intravenously administered to rats and then metabolites in plasma samples were identified by rapid resolution liquid chromatography electrospray ionization-collision induced dissociation tandem mass spectrometry (RRLC-ESI-CID-MS/MS). Chromatography was conducted on a Zorbax SB C18 column (2.1x100 mm, 1.8 microm) at 30 degrees C, with a gradient mobile phase consisting of 0.05% formic acid and acetonitrile, a flow rate of 0.2 mL min(-1), and a total run time of 14 min. MS/MS acquisition parameters were as follows: positive ionization mode, dry gas: nitrogen, 10 L min(-1), dry temperature: 350 degrees C, nebulizer: 40 psi, capillary: -3500 V, scan range: 250-800. The autoMS, manual, or multiple reaction monitoring mode was selected as required. Two glucuronidated metabolites of puerarin (M1 and M2) were detected. M1 and M2 are presumed to be puerarin-7-O-glucuronide and puerarin-4'-O-glucuronide, respectively, and M2 likely is suspected to be the major metabolite because it represented the predominate peak. Kinetic studies of metabolites demonstrated that M1 and M2 were detected in rat plasma at 5 min after intravenous administration of puerarin, the levels of M1 and M2 then reached their peaks at 10-15 and 15-30 min, respectively. The metabolic profiles were similar in rat liver and intestine investigated by in situ liver and intestine perfusion, indicating that no metabolic regioselectivity of puerarin occurs in the two organs.

Bioanalysis of pentoxifylline and related metabolites in plasma samples through LC‐MS/MS

Analytical aspects related to the assay of pentoxifylline (PTX), lisofylline (M1) and carboxypropyl dimethylxanthine (M5) metabolites are discussed through comparison of two alternative analytical methods based on liquid chromatography separation and atmospheric pressure electrospray ionization tandem mass spectrometry detection. One method is based on a 'pure' reversed-phase liquid chromatography mechanism, while the second one uses the additional polar interactions with embedded amide spacers linking octadecyl moieties to the silicagel surface (C-18 Aqua stationary phase). In both cases, elution is isocratic. Both methods are equally selective and allows separation of unknowns (four species associated to PTX, two species associated to M1) detected through specific mass transitions of the parent compounds and owning respective structural confirmation. Plasma concentration-time patterns of these compounds follow typical metabolic profiles. It has been advanced that in-vivo formation of conjugates of PTX and M1 is possible, such compounds being cleaved back to the parent ones within the ion source. The first method was associated with a sample preparation procedure based on plasma protein precipitation by strong organic acid addition. The second method used protein precipitation by addition of a water miscible organic solvent. Both analytical methods were fully validated and used to assess bioequivalence between a prolonged release generic formulation and the reference product, under multidose and single dose approaches.

Simultaneous PET Imaging of P-Glycoprotein Inhibition in Multiple Tissues in the Pregnant Nonhuman Primate

Studies in rodents indicate that the disruption of P-glycoprotein (P-gp) function increases drug distribution into the developing fetus and organs such as the brain. To simultaneously and serially evaluate the effect of P-gp activity and inhibition on the tissue distribution of drugs in a more representative animal model, we tested the feasibility of conducting whole-body PET of the pregnant nonhuman primate (Macaca nemestrina). We used (11)C-verapamil as the prototypic P-gp substrate and cyclosporine A (CsA) as the prototypic inhibitor. Four pregnant macaques (gestational age, 145-159 d; gestational term, 172 d) were imaged after the intravenous administration of (11)C-verapamil (30-72 MBq/kg) before and during intravenous infusion of CsA (12 or 24 mg/kg/h, n = 2 each). The content of verapamil and its metabolites in plasma samples was determined using a rapid solid-phase extraction method. The plasma and tissue time-radioactivity concentration curves of (11)C were integrated over 0-9 min after each verapamil injection. The tissue or arterial plasma area under the time-concentration curve (AUC(tissue)/AUC(plasma)) served as a measure of the tissue distribution of (11)C radioactivity. CsA effect on (11)C radioactivity distribution was interpreted as P-gp inhibition. The change in the fetal liver AUC ratio served as a reporter of placental P-gp inhibition. CsA effect on tissue distribution of (11)C radioactivity (AUC ratios) did not increase with the mean blood concentration of CsA, indicating a near-maximal P-gp inhibition. CsA increased maternal brain and fetal liver distribution of (11)C radioactivity by 276% +/- 88% (P < 0.05) and 122% +/- 75% (P < 0.05), respectively. Changes in other measured tissues were not statistically significant. These data demonstrate for the first time, to our knowledge, the feasibility of simultaneous, serial, noninvasive imaging of P-gp activity and inhibition in multiple maternal organs and the placenta in the nonhuman primate. Our findings, consistent with previous data in rodents, indicate that the activity of P-gp in the placenta and the blood-brain barrier is high and that the inhibition of P-gp facilitates drug distribution across these barriers.

Determination of procyanidins and their metabolites in plasma samples by improved liquid chromatography–tandem mass spectrometry

An off-line solid-phase extraction (SPE) and ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for determining procyanidins, catechin, epicatechin, dimer, and trimer in plasma samples. In the validation procedure of the analytical method, linearity, precision, accuracy, detection limits (LODs), quantification limits (LOQs), and the matrix effect were studied. Recoveries of the procyanidins were higher than 84%, except for the trimer, which was 65%. The LODs and LOQs were lower than 0.003 and 0.01 μM, respectively, for all the procyanidins studied, except for the trimers, which were 0.8 and 0.98 μM, respectively. This methodology was then applied for the analysis of rat plasma obtained 2 h after ingestion of grape seed phenolic extract. Monomers (catechin and epicatechin), dimer and trimer in their native form were detected and quantified in plasma samples, and their concentration ranged from 0.85 to 8.55 μM. Moreover, several metabolites, such as catechin and epicatechin glucuronide, catechin and epicatechin methyl glucuronide, and catechin and epicatechin methyl-sulphate were identified. These conjugated forms were quantified, in reference to the respective unconjugated form, showing concentrations between 0.06 and 23.90 μM.

Enantioselective determination of mexiletine and its metabolites p‐hydroxymexiletine and hydroxymethylmexiletine in rat plasma by normal‐phase liquid chromatography‐tandem mass spectrometry: Application to pharmacokinetics

Mexiletine (MEX), hydroxymethylmexiletine (HMM) and p-hydroxymexiletine (PHM) were analyzed in rat plasma by LC-MS/MS. The plasma samples were prepared by liquid-liquid extraction using methyl-tert-butyl ether as extracting solvent. MEX, HMM, and PHM enantiomers were resolved on a Chiralpak(R) AD column. Validation of the method showed a relative standard deviation (precision) and relative errors (accuracy) of less than 15% for all analytes studied. Quantification limits were 0.5 ng ml(-1) for the MEX and 0.2 ng ml(-1) for the HMM and PHM enantiomers. The validated method was successfully applied to quantify the enantiomers of MEX and its metabolites in plasma samples of rats (n = 6) treated with a single oral dose of racemic MEX.

Enantioselective determination of chloroquine and its n‐dealkylated metabolites in plasma using liquid‐phase microextraction and LC‐MS

A selective method using three-phase liquid-phase microextraction (LPME) in conjunction with LC-MS-MS was devised for the enantioselective determination of chloroquine and its n-dealkylated metabolites in plasma samples. After alkalinization of the samples, the analytes were extracted into n-octanol immobilized in the pores of a polypropylene hollow fiber membrane and back extracted into the acidic acceptor phase (0.1 M TFA) filled into the lumen of the hollow fiber. Following LPME, the analytes were resolved on a Chirobiotic V column using methanol/ACN/glacial acetic acid/diethylamine (90:10:0.5:0.5 by volume) as the mobile phase. The MS detection was carried out using multiple reaction monitoring with ESI in the positive ion mode. The optimized LPME method yielded extraction recoveries ranging from 28 to 66%. The method was linear over 5-500 ng/mL and precision (RSD) and accuracy (relative error) values were below 15% for all analytes. The developed method was applied to the determination of the analytes in rat plasma samples after oral administration of the racemic drug.

Validated method for the simultaneous determination of methadone and its main metabolites (EDDP and EMDP) in plasma of umbilical cord blood by gas chromatography–mass spectrometry

A sensitive and specific GC/MS method for the determination of methadone (MDN) and its two main metabolites, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and 2-ethyl-5-methyl-3,3-diphenylpyrroline (EMDP), in plasma samples obtained from venous and arterial umbilical cord blood and maternal blood has been developed, optimized and validated. Specimen preparation includes protein precipitation with acetonitrile and simultaneous solid-phase extraction of the three analytes. Methadone-d9 was used as internal standard for the determination of MDN and EMDP, while EDDP-d3 for EDDP. Limits of detection were 0.6 μg/L for MDN and 0.3 μg/L for EDDP and EMDP, while limits of quantification were 2.0 μg/L for MDN and 1.0 μg/L for EDDP and EMDP. The calibration curves were linear up to 2000 μg/L for MDN and up to 1000 μg/L for EDDP and EMDP. Absolute recovery ranged from 94.8 to 99.7% for all three analytes. Intra- and interday accuracy was less than 5.3 and 5.5%, respectively, while intra- and interday precision was less than 3.5 and 5.0%, correspondingly, for all analytes. The method proved suitable for the determination of MDN and its two main metabolites in plasma samples obtained from umbilical cord and maternal blood of a woman participating in a MDN maintenance program, during the prenatal and postpartum period.

Expansion of the Eicosanoid Profiling Methodology by the Addition of w‐3 Fatty Acid Metabolites

Eicosanoids, a large group of arachidonic acid (AA) metabolites, play an important role in several physiological processes such as inflammation or hypertension. They are produced via three different pathways by cytochrome P450, lipoxygenase and cyclooxygenase enzymes. It has been established that these enzymes metabolize not only AA but also other poly unsaturated fatty acids (PUFAs), i.e. eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA).Recent studies indicate that w‐3 fatty acids and their metabolites play an important protective role in multiple inflammatory conditions including IgAN. We are expanding our analytical method by adding separate platforms for lipid amides and leukotrienes but also by adding more targets to the oxylipin method. For example we incorporated metabolites of the three most important w‐3 fatty acids (EPA, DHA and a‐linolenic acid) in our existing eicosanoid profiling methodology. Thus, we developed a precise and sensitive method using LC‐MS/MS to identify and quantitate 88 oxylipin metabolites in plasma samples. This work was supported by NIEHS SBRP Grant P42 ES04699.

Chemosensitization of Cancer In vitro and In vivo by Nitric Oxide Signaling

Hypoxia contributes to drug resistance in solid cancers, and studies have revealed that low concentrations of nitric oxide (NO) mimetics attenuate hypoxia-induced drug resistance in tumor cells in vitro. Classic NO signaling involves activation of soluble guanylyl cyclase, generation of cyclic GMP (cGMP), and activation of cGMP-dependent protein kinase. Here, we determined whether chemosensitization by NO mimetics requires cGMP-dependent signaling and whether low concentrations of NO mimetics can chemosensitize tumors in vivo. Survival of human prostate and breast cancer cells was assessed by clonogenic assays following exposure to chemotherapeutic agents. The effect of NO mimetics on tumor chemosensitivity in vivo was determined using a mouse xenograft model of human prostate cancer. Drug efflux in vitro was assessed by measuring intracellular doxorubicin-associated fluorescence. Low concentrations of the NO mimetics glyceryl trinitrate (GTN) and isosorbide dinitrate attenuated hypoxia-induced resistance to doxorubicin and paclitaxel. Similar to hypoxia-induced drug resistance, inhibition of various components of the NO signaling pathway increased resistance to doxorubicin, whereas activation of the pathway with 8-bromo-cGMP attenuated hypoxia-induced resistance. Drug efflux was unaffected by hypoxia and inhibitors of drug efflux did not significantly attenuate hypoxia-induced chemoresistance. Compared with mice treated with doxorubicin alone, tumor growth was decreased in mice treated with doxorubicin and a transdermal GTN patch. The presence of GTN and GTN metabolites in plasma samples was confirmed by gas chromatography. Tumor hypoxia induces resistance to anticancer drugs by interfering with endogenous NO signaling and reactivation of NO signaling represents a novel approach to enhance chemotherapy.