A Newly Developed HPLC-UV/Vis Method Using Chemical Derivatization with 2-Naphthalenethiol for Quantitation of Sulforaphane in Rat Plasma.

Sulforaphane (SFN), a naturally occurring isothiocyanate, has received significant attention because of its ability to modulate multiple biological functions, including anti-carcinogenic properties. However, currently available analytical methods based on high-performance liquid chromatography (HPLC)-UV/Vis for the quantification of SFN have a number of limitations, e.g., low UV absorbance, sensitivity, or accuracy, due to the lack of a chromophore for spectrometric detection. Therefore, we here employed the analytical derivatization procedure using 2-naphthalenethiol (2-NT) to improve the detectability of SFN, followed by HPLC separation and quantification with UV/Vis detection. The optimal derivatization conditions were carried out with 0.3 M of 2-NT in acetonitrile with phosphate buffer (pH 7.4) by incubation at 37 °C for 60 min. Separation was performed in reverse phase mode using a Kinetex C18 column (150 mm × 4.6 mm, 5 µm) at a flow rate of 1 mL/min, with 0.1% formic acid as a mobile phase A, and acetonitrile/0.1% formic acid solution as a mobile phase B with a gradient elution, with a detection wavelength of 234 nm. The method was validated over a linear range of 10-2000 ng/mL with a correlation of determination (R2) > 0.999 using weighted linear regression analysis. The intra- and inter-assay accuracy (% of nominal value) and precision (% of relative standard deviation) were within ±10 and <15%, respectively. Moreover, the specificity, recovery, matrix effect, process efficiency, and short-term and long-term stabilities of this method were within acceptable limits. Finally, we applied this method for studying in vivo pharmacokinetics (PK) following oral administration of SFN at doses of 10 or 20 mg/kg. The Cmax (µg/mL), Tmax (hour), and AUC0-12h (µg·h/mL) of each oral dose were 0.92, 1.99, and 4.88 and 1.67, 1.00, and 9.85, respectively. Overall, the proposed analytical method proved to be reliable and applicable for quantification of SFN in biological samples.

Development and validation of a high throughput bioanalytical UPLC-MS/MS method for simultaneous determination of tamoxifen and sulphoraphane in rat plasma: Application to an oral pharmacokinetic study.

Tamoxifen (TAM) is the choice of a drug approved by the Food and Drug Administration (FDA) for the treatment of estrogen-positive receptor (ER+) breast cancer. Sulphoraphane (SFN), a natural plant antioxidant compound, also acts on estrogen-positive breast cancer receptor. Thus, a combination of TAM with SFN is preferred as it helps to minimize the drug-related toxicity and increases the therapeutic efficacy by providing synergistic anticancer effects of both drugs. In the present study, a new simple, sensitive, precise, and selective UPLC-MS/MS method was developed for the simultaneous quantification of tamoxifen and sulphoraphane using propranolol as an internal standard (IS) in rat plasma. Chromatographic separation was achieved on reverse phase Acquity UPLC BEH C18 column (50 mm × 2.1 mm, i.d., 1.7 µm) with an isocratic mobile phase composed of solvent A (0.1% formic acid in acetonitrile) and B (0.1% formic acid in water) (80:20, v/v) at a flow-rate of 0.4 mL/min. The detection and quantification of analytes was performed on Waters ZsprayTM Xevo TQD using selected-ion monitoring operated under a positive electrospray ionization mode. The transitions were m/z = 372.0 [M+H]+ → 71.92 for tamoxifen, m/z = 177.9 [M+H]+ → 113.9 for sulphoraphane and m/z = 260.3 [M+H]+ → 116.1 for propranolol. The method was linear over the concentration range of 8-500 ng/mL (r2 = 0.9996) for tamoxifen, 30-2000 ng/mL (r2 = 0.9998) for sulphoraphane with insignificant matrix effect and high extraction recovery on spiked quality control (QC) samples. The intra- and inter-batch precisions and accuracy were within the acceptable limits, and both the analytes were found to be stable throughout the short term, long term and freeze thaw stability studies. The validated method was successfully applied for the simultaneous estimation of TAM and SFN in an oral pharmacokinetic study in female Wistar rats. This developed UPLC-MS/MS method could be a valuable tool for future pharmacokinetic interaction, therapeutic drug monitoring and pharmacokinetic characterization of novel formulations.

Development of a liquid chromatography/tandem-mass spectrometry assay for the simultaneous determination of teneligliptin and its active metabolite teneligliptin sulfoxide in human plasma.

Teneligliptin is a recently developed dipeptidyl peptidase-4 (DPP-4) inhibitor for the treatment of type 2 diabetes mellitus. To study simultaneous pharmacokinetics of teneligliptin and its major active metabolite, teneligliptin sulfoxide in human plasma, we developed and validated a LC-MS/MS method. The analytes were detected in the positive mode using multiple reaction monitoring (teneligliptin: m/z 427.2→243.1; teneligliptin-d8 : m/z 435.2→251.3; teneligliptin sulfoxide: m/z 443.2→68.2). The method demonstrated accuracy, precision, and linearity over the concentration range of 5 to 1000 ng/mL for teneligliptin and 2.5 to 500 ng/mL for teneligliptin sulfoxide. The developed method is the first fully validated method capable of simultaneous determination of teneligliptin and its active metabolite, teneligliptin sulfoxide in plasma. The suitability of the method was successfully demonstrated in terms of quantification of teneligliptin and teneligliptin sulfoxide pharmacokinetics in plasma samples collected from healthy volunteers. The measurement of plasma metabolite/parent ratio of teneligliptin was feasible by this method.

Evaluation of redox profiles in exogenous subclinical hyperthyroidism at two different levels of TSH suppression.

OBJECTIVE: Evaluate the relationship between exogenous subclinical hyperthyroidism and oxidative stress through the analysis of the redox profile of patients with subclinical hyperthyroidism exogenous (SCH) grade I (TSH = 0.1 to 0.4 IU/mL) and grade II (TSH < 0.1 IU/mL). SUBJECTS AND METHODS: We analyzed 46 patients with SCH due to the use of TSH suppressive therapy with LT4 after total thyroidectomy along with 6 control euthyroid individuals (3M and 3W). Patients were divided into two groups, G1 with TSH ≥ 0.1-0.4 IU/mL (n = 25; and 7M 14W) and G2 with TSH < 0.1 IU/mL (n = 25; and 4M 21W). Venous blood samples were collected to measure the levels of markers for oxidative damage (TBARS, FOX and protein carbonylation), muscle and liver damage (CK, AST, ALT, GGT) and antioxidants (GSH, GSSG and catalase). RESULTS: Individuals in G2 showed a GSH/GSSG ratio ~ 30% greater than those in G1 (p = 0.004) and a catalase activity that was 4 times higher (p = 0.005). For lipid peroxidation, the levels measured in G2 were higher than both control and G1 (p = 0.05). No differences were observed for both protein carbonyl markers. G1 and G2 presented with greater indications of cell injury markers than the control group. CONCLUSION: TSH suppression therapy with LT4 that results in subclinical hyperthyroidism can cause a redox imbalance. The greater antioxidant capacity observed in the more suppressed group was not sufficient to avoid lipid peroxidation and cellular damage.

Evaluation of redox profiles in exogenous subclinical hyperthyroidism at two different levels of TSH suppression

ABSTRACT Objective: Evaluate the relationship between exogenous subclinical hyperthyroidism and oxidative stress through the analysis of the redox profile of patients with subclinical hyperthyroidism exogenous (SCH) grade I (TSH = 0.1 to 0.4 IU/mL) and grade II (TSH < 0.1 IU/mL). Subjects and methods: We analyzed 46 patients with SCH due to the use of TSH suppressive therapy with LT4 after total thyroidectomy along with 6 control euthyroid individuals (3M and 3W). Patients were divided into two groups, G1 with TSH ≥ 0.1-0.4 IU/mL (n = 25; and 7M 14W) and G2 with TSH < 0.1 IU/mL (n = 25; and 4M 21W). Venous blood samples were collected to measure the levels of markers for oxidative damage (TBARS, FOX and protein carbonylation), muscle and liver damage (CK, AST, ALT, GGT) and antioxidants (GSH, GSSG and catalase). Results: Individuals in G2 showed a GSH/GSSG ratio ~ 30% greater than those in G1 (p = 0.004) and a catalase activity that was 4 times higher (p = 0.005). For lipid peroxidation, the levels measured in G2 were higher than both control and G1 (p = 0.05). No differences were observed for both protein carbonyl markers. G1 and G2 presented with greater indications of cell injury markers than the control group. Conclusion: TSH suppression therapy with LT4 that results in subclinical hyperthyroidism can cause a redox imbalance. The greater antioxidant capacity observed in the more suppressed group was not sufficient to avoid lipid peroxidation and cellular damage.

Investigating Sulfoxide-to-Sulfone Conversion as a Prodrug Strategy for a Phosphatidylinositol 4-Kinase Inhibitor in a Humanized Mouse Model of Malaria.

The in vivo antimalarial efficacies of two phosphatidylinositol 4-kinase (PI4K) inhibitors, a 3,5-diaryl-2-aminopyrazine sulfoxide and its corresponding sulfone metabolite, were evaluated in the NOD-scid IL2Rγnull (NSG) murine malaria disease model of Plasmodium falciparum infection. We hypothesized that the sulfoxide would serve as a more soluble prodrug for the sulfone, which would lead to improved drug exposure with oral dosing. Both compounds had similar efficacy (90% effective dose [ED90], 0.1 mg kg-1 of body weight) across a quadruple-dose regimen. Pharmacokinetic profiling revealed rapid sulfoxide clearance via conversion to sulfone, with sulfone identified as the major active metabolite. When the sulfoxide was dosed, the exposure of the sulfone achieved was as much as 2.9-fold higher than when the sulfone was directly dosed, thereby demonstrating that the sulfoxide served as an effective prodrug for the treatment of malaria.

Pharmacokinetics and Pharmacodynamics of Azeloprazole Sodium, a Novel Proton Pump Inhibitor, in Healthy Japanese Volunteers.

The pharmacokinetics (PK) and pharmacodynamics (PD) of proton pump inhibitors differ among cytochrome P450 (CYP) 2C19 genotypes. Therefore, we developed azeloprazole sodium (Z-215), a novel proton pump inhibitor, whose metabolism is not affected by CYP2C19 activity in vitro. However, the PK and PD of azeloprazole sodium have not been evaluated in Japanese subjects. We conducted an open-label, crossover study in healthy Japanese male volunteers to evaluate the plasma concentration and intragastric pH with respect to CYP2C19 genotype after repeated administration of 10, 20, and 40 mg azeloprazole sodium and 10 and 20 mg rabeprazole sodium (rabeprazole). The plasma concentration profile of azeloprazole sodium was similar among genotypes, whereas that of rabeprazole differed. The 24-hour intragastric pH ≥ 4 holding time ratio (pH ≥ 4 HTR) of azeloprazole sodium was similar among genotypes. The pH ≥ 4 HTR was 52.5%-60.3%, 55.1%-65.8%, and 69.4%-77.1% after administration of 10, 20, and 40 mg azeloprazole sodium, respectively, and 59.2%-72.3% and 64.4%-91.2% after administration of 10 and 20 mg rabeprazole, respectively, on the fifth day of dosing. The maximum plasma concentration (Cmax ), area under the plasma concentration-time curve (AUC), and pH ≥ 4 HTR of azeloprazole sodium were proportional to dose. The Cmax , AUC, and pH ≥ 4 HTR on day 5 were slightly higher following administration of 20 mg azeloprazole sodium before comparison with after a meal. No serious adverse events were observed. These results suggest that azeloprazole sodium is useful for treating gastroesophageal reflux disease in all CYP2C19 genotypes.

Range of therapeutic prothipendyl and prothipendyl sulfoxide concentrations in clinical blood samples.

Due to a lack of reference blood concentrations in the literature, the forensic evaluation of prothipendyl findings in blood samples is difficult. Interpretations with regard to the assessment of blood concentrations as well as an estimation of the ingested prothipendyl amounts were often vague. To describe a concentration range in clinical samples, prothipendyl and prothipendyl sulfoxide concentrations were determined in serum samples of 50 psychiatric patients receiving 40 mg, 80 mg, or 160 mg doses of prothipendyl. The analyses of prothipendyl and prothipendyl sulfoxide were carried out using validated methods of high performance liquid chromatography coupled to triple quadrupole mass spectrometry (LC-QQQ-MS), respectively. 40 mg doses caused average prothipendyl serum concentrations of 18.0 ng/mL (1 hour after intake) and 7.9 ng/mL (10.5 hours after intake), while 80 mg doses caused averages of 42.6 ng/mL and 15.2 ng/mL at the mentioned times of sampling. Irrespective of the given dose, prothipendyl concentrations below 30 ng/mL were observed in 80% of the patient samples taken 1 hour after ingestion as well as in 90% of the samples collected 10.5 hours after administration. Serum concentrations of the Phase I metabolite prothipendyl sulfoxide averaged 4.3 ng/mL (1 hour after intake) and 3.6 ng/mL (10.5 hours after intake). Possible drug-drug interactions regarding absorption and metabolism of prothipendyl are discussed. Results of the herein presented study are useful for the interpretation of analytical prothipendyl findings in forensic toxicology. The utility of the described concentration range is demonstrated by discussing two death cases involving prothipendyl findings.

LC-MS/MS assay for the simultaneous quantitation of the ATM inhibitor AZ31 and the ATR inhibitor AZD6738 in mouse plasma.

The ATM kinase inhibitor AZ31 and ATR kinase inhibitor AZD6738 are in various phases of preclinical and clinical evaluation for their ability to potentiate chemoradiation. To support the preclinical evaluation of their pharmacokinetics, we developed and validated an LC-MS/MS assay for the simultaneous quantification of AZ31 and AZD6738 in mouse plasma. A "dilute and shoot" method was used to precipitate proteins from a sample volume of 50µL. Chromatographic separation was achieved using a Phenomenex Polar-RP column and a gradient mobile phase consisting of methanol-water with 0.1% formic acid. Detection was accomplished using a Waters Quattro Micro mass spectrometer in positive ionization mode. The assay utilizing 50µL sample was linear from 10 to 5000ng/mL and determined to be both accurate (-8.2 to 8.6%) and precise (<5.4% CV) and achieved the criteria for U.S. FDA guidance for bioanalytical method validation. Quantification was achieved in mouse tissue homogenate using a separate 200µL sample preparation. This LC-MS/MS assay will be essential for determining the tissue distribution and pharmacokinetics in future mouse studies.

A sensitive LC-MS/MS method for analysis of pericyazine in presence of 7-hydroxypericyazine and pericyazine sulphoxide in human plasma and its application to a comparative bioequivalence study in Chinese healthy volunteers.

A robust and highly sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the determination of pericyazine in human plasma. The plasma sample was alkalized with sodium hydroxide solution and handled by liquid-liquid extraction with ethyl acetate after adding perphenazine as an internal standard (IS). The analytes were separated on an Ultimate™ AQ-C18 analytical column at 40°C, with a gradient elution consisting of A (aqueous phase: 5mM ammonium acetate buffer solution containing 0.1% formic acid) and B (organic phase: acetonitrile) at a flow rate of 0.350mL/min. The detection was conducted on an API 4000 tandem mass spectrometer coupled with electrospray ionization (ESI) source in positive ion mode. The multiple reaction monitoring (MRM) transitions, m/z 366.5>142.4 for pericyazine, m/z 382.5>142.4 for its 7-hydroxy and sulphoxide metabolites and m/z 404.3>171.3 for IS were chosen to achieve high selectivity in the simultaneous analyses. The method exhibited great improvement in sensitivity (LLOQ of 0.021ng/mL) and good linearity over the concentration range of 0.021-9.90ng/mL. The intra- and inter-day precision, accuracy, and stability results were within the acceptable limits and no matrix effect was observed. This method was successfully applied in a bioequivalence study to evaluate the pharmacokinetics in 20 healthy male Chinese volunteers. Additional exploratory analyses of 7-hydroxy and sulphoxide metabolites of pericyazine in the same samples suggest that the unchanged drug is predominant in the plasma and suitable for the bioequivalence comparison after a single oral administration of 10mg pericyazine.

Simultaneous determination of sulfur mustard and related oxidation products by isotope-dilution LC-MS/MS method coupled with a chemical conversion.

Sulfur mustard (SM) is a highly reactive alkylating vesicant with high toxicity and complicated metabolism, the in vivo profile of its oxidation metabolism is not still fully known and urgently needs to be clarified well. In this work, an isotope-dilution high performance liquid chromatography-tandem mass spectrometric method coupled with chemical conversion was developed for the simultaneous quantification of SM and its oxidation products, i.e., mustard sulfoxide (SMO) and mustard sulfone (SMO2). The accurate measurement of SM and its oxidation products with high reaction activity was achived via the method of chemical conversion of 2-(3,5-bis(mercaptomethyl)phenoxy) acetic acid into stable derivative products. Method validation was performed in whole blood matrix, the linear range of the method was between 0.2 and 1000µg/L with correlation coefficients (r(2))>0.99, and the lower limits of quantification for SM, SMO and SMO2 were 1, 1, 0.2µg/L, respectively. The validated method was successfully applied to a toxicokinetics research of SM and its oxidation products after SM dermal exposed rats in a single dose. All three target analytes were found in whole blood samples from poisoned rats, and significant time-dependent responses were also observed. Among them, SMO2 with relatively high toxicity was identified and quantified in vivo for the first time, while SMO was the major product in whole blood and some of them continued to be oxidized to SMO2in vivo. These results give a direct experimental evidence to support that a large amount of SM is converted into the corresponding SMO and SMO2, and these oxidation products might cause potential combined toxic effects.

Conformational Adaption May Explain the Slow Dissociation Kinetics of Roniciclib (BAY 1000394), a Type I CDK Inhibitor with Kinetic Selectivity for CDK2 and CDK9.

Roniciclib (BAY 1000394) is a type I pan-CDK (cyclin-dependent kinase) inhibitor which has revealed potent efficacy in xenograft cancer models. Here, we show that roniciclib displays prolonged residence times on CDK2 and CDK9, whereas residence times on other CDKs are transient, thus giving rise to a kinetic selectivity of roniciclib. Surprisingly, variation of the substituent at the 5-position of the pyrimidine scaffold results in changes of up to 3 orders of magnitude of the drug-target residence time. CDK2 X-ray cocrystal structures have revealed a DFG-loop adaption for the 5-(trifluoromethyl) substituent, while for hydrogen and bromo substituents the DFG loop remains in its characteristic type I inhibitor position. In tumor cells, the prolonged residence times of roniciclib on CDK2 and CDK9 are reflected in a sustained inhibitory effect on retinoblastoma protein (RB) phosphorylation, indicating that the target residence time on CDK2 may contribute to sustained target engagement and antitumor efficacy.

Effects of triclabendazole on secretion of danofloxacin and moxidectin into the milk of sheep: role of triclabendazole metabolites as inhibitors of the ruminant ABCG2 transporter.

ATP-binding cassette transporter G2/breast cancer resistance protein (ABCG2/BCRP) mediates drug-drug interactions that affect the secretion of drugs into milk. The aims of this study were: (1) to determine whether the major plasma metabolites of the flukicide triclabendazole (TCBZ), triclabendazole sulfoxide (TCBZSO) and triclabendazole sulfone (TCBZSO2), inhibit ovine and bovine ABCG2 and its Y581S variant in vitro, and (2) to examine whether coadministration of TCBZ with the ABCG2 substrates danofloxacin (a fluoroquinolone) and moxidectin (a milbemycin) affects the secretion of these drugs into the milk of sheep. TCBZSO and TCBZSO2 inhibited ruminant ABCG2 in vitro by reversing the reduced mitoxantrone accumulation and reducing basal to apical transport of nitrofurantoin in cells transduced with bovine variants (S581 and Y581) and the ovine variant of ABCG2. Coadministration of TCBZ with moxidectin or danofloxacin to sheep resulted in significantly reduced levels of moxidectin, but not danofloxacin, in the milk of TCBZ-treated sheep compared to sheep administered moxidectin or danofloxacin alone. The milk area under concentration time curve (AUC 0-48 h) was 2.99±1.41 µg h/mL in the group treated with TCBZ and moxidectin, and 7.75±3.58 µg h/mL in the group treated with moxidectin alone. The AUC (0-48 h) milk/plasma ratio was 37% lower in the group treated with TCBZ and moxidectin (7.34±1.51) than in the group treated with moxidectin alone (11.68±3.61). TCBZ metabolites appear to inhibit ruminant ABCG2 and affect the secretion of ABCG2 substrates into milk of sheep.

Determination of imidol hydrochloride in human plasma and urine by high-performance liquid chromatography-tandem mass spectrometry and its application to clinical pharmacokinetic study.

Imidol hydrochloride is a novel drug for the treatment of hepatitis B virus infection. A simple, special and sensitive solid-phase extraction liquid chromatography­tandem mass spectrometry method for determination of imidol in human plasma and urine was developed for the first time and applied to a pharmacokinetic study. The chromatographic separation was achieved on a C18 column (50 x 2.1 mm, 3.5 µm) using gradient elution with acetonitrile and water both containing 0.1% acetic acid at a flow rate of 0.25 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring mode via a positive eletrospray ionization source. The mass transition pairs of m/z 517.8 --> 325 and m/z 298 --> 174 were used to detect imidol and the (-)-clausenamide (internal standard), respectively. The retention times of imidol and (-)-clausenamide were 2.5 and 2.7 min, respectively. Linearity, accuracy, precision, recovery, matrix effect, dilution test and stability were evaluated during method validation over the range of 0.2-500 ng/mL in human plasma and 0.5-500 ng/mL in urine. The method was successfully applied to a clinical pharmacokinetic study of imidol in healthy volunteers following oral administration.

Pharmacokinetics of toltrazuril and its metabolites, toltrazuril sulfoxide and toltrazuril sulfone, after a single oral administration to pigs.

Toltrazuril (TZR) is a triazine-based antiprotozoal agent. Following a single oral administration of TZR at 10 and 20 mg/kg to male pigs, the mean TZR concentration in plasma peaked at 4.24 and 8.18 microg/ml at 15.0 and 12.0 hr post-dose, respectively. TZR absorbed was rapidly converted to the short-lived intermediary metabolite toltrazuril sulfoxide (TZR-SO), and then metabolized to the reactive toltrazuril sulfone (TZR-SO2). TZR-SO2 was actually more slowly eliminated, with average half-lives of 231 and 245 hr, compared with TZR (48.7 and 68.9 hr) or TZR-SO (51.9 and 53.2 hr) in the 10 and 20 mg/kg groups, respectively. This study demonstrates that TZR metabolizes to TZR-SO2 having a long-terminal half-life, enabling the persistent clinical efficacy in the treatment of I. suis infection. In contrast, special consideration should be given to the residual of TZR-SO2.

Unchanged triclabendazole kinetics after co-administration with ivermectin and methimazole: failure of its therapeutic activity against triclabendazole-resistant liver flukes.

BACKGROUND: The reduced drug accumulation based on enhanced drug efflux and metabolic capacity, identified in triclabendazole (TCBZ)-resistant Fasciola hepatica may contribute to the development of resistance to TCBZ. The aim of this work was to evaluate the pharmacokinetics and clinical efficacy of TCBZ administered alone or co-administered with ivermectin (IVM, efflux modulator) and methimazole (MTZ, metabolic inhibitor) in TCBZ-resistant F. hepatica-parasitized sheep. Sheep infected with TCBZ-resistant F. hepatica (Sligo isolate) were divided into three groups (n = 4): untreated control, TCBZ-treated (i.r. at 10 mg/kg) and TCBZ+IVM+MTZ treated sheep (10 i.r., 0.2 s.c. and 1.5 i.m. mg/kg, respectively). Plasma samples were collected and analysed by HPLC. In the clinical efficacy study, the animals were sacrificed at 15 days post-treatment to evaluate the comparative efficacy against TCBZ-resistant F. hepatica. RESULTS: The presence of IVM and MTZ did not affect the plasma disposition kinetics of TCBZ metabolites after the i.r. administration of TCBZ. The AUC value of TCBZ.SO obtained after TCBZ administration (653.9 +/- 140.6 microgxh/ml) was similar to that obtained after TCBZ co-administered with IVM and MTZ (650.7 +/- 122.8 microgxh/ml). Efficacy values of 56 and 38% were observed for TCBZ alone and for the combined treatment, respectively. No statistical differences (P > 0.05) were observed in fluke counts between treated groups and untreated control, which confirm the resistant status of the Sligo isolate. CONCLUSIONS: The presence of IVM and MTZ did not affect the disposition kinetics of TCBZ and its metabolites. Thus, the combined drug treatment did not reverse the poor efficacy of TCBZ against TCBZ-resistant F. hepatica.

Development and validation of an LC-MS/MS method for the determination of quetiapine and four related metabolites in human plasma.

Pharmacokinetic measurement of the psychotropic compound quetiapine and four related metabolites in human plasma was conducted using a sensitive and specific liquid-chromatography tandem mass spectrometry (LC-MS/MS) assay that has been developed and validated for this purpose. The assay employs a single liquid-liquid extraction of quetiapine and its N-desalkyl (norquetiapine, M211,803, M1), 7-hydroxy (M214,227, M2), 7-hydroxy N-desalkyl (M236,303, M3), and sulfoxide (M213,841, M4) metabolites from human plasma, and utilizes dual-column separation, using Luna C(18) columns (50mmx2.0mm, 5microm) and positive ionization tandem MS detection in the multiple reaction monitoring (MRM) mode of the analytes and their respective stable labeled internal standards. The method provides a linear response from a quantitation range of <0.70ng/ml to at least 500ng/ml for each analyte using 40microl of plasma. The applicable range was extended by dilution up to 100-fold with blank matrix. The accuracy and precision for quetiapine were less than 6.0% and 6.4% for quetiapine, respectively. The accuracy (and precision) was less than 9.4% (5.9%) for norquetiapine; 6.4% (6.2%) for M2; and 10.0% (6.4%) for M3; and 8.6% (9.5%) for M4. This methodology enabled the determination of the pharmacokinetics of quetiapine and its metabolites in human plasma, and an example of its application is presented.

An improved LC-MS/MS method for quantitative determination of ilaprazole and its metabolites in human plasma and its application to a pharmacokinetic study.

AIM: To improve and validate an analytical method based on liquid chromatography and electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) for the quantitative measurement of ilaprazole and its two metablites in human plasma. METHODS: Separation of analytes and the internal standard (IS), omeprazole, was performed on a Thermo HyPURITY C18 column (150x2.1 mm, 5 microm) with a mobile phase consisting of 10 mmol/L ammonium formate water-acetonitrile solution (50:50, v/v) at a flow rate of 0.25 mL/min. The API4000 triple quadruple mass spectrometer was operated in multiple reactions monitoring mode via positive electrospray ionization interface using the transition m/z 367.2 --> m/z184.0 for ilaprazole, m/z 383.3 --> m/z 184.1 for ilaprazole sulfone, m/z 351.2 --> m/z 168.1 for ilaprazole thiol ether and m/z 346.2 --> m/z 198.0 for omeprazole. RESULTS: The method was linear over the concentration range of 0.23-2400.00 ng/mL for ilaprazole, 0.05-105.00 ng/mL for ilaprazole thiol ether and 0.06-45.00 ng/mL for ilaprazole sulfone. The intra- and inter-day precisions were all less than 15% in terms of relative standard deviation (RSD), and the accuracy was within 15% in terms of relative error (RE) for ilaprazole, ilaprazole sulfone and ilaprazole thiol ether. The lower limit of quantification (LLOQ) was identifiable and reproducible at 0.23, 0.05 and 0.06 ng/mL with acceptable precision and accuracy for ilaprazole, ilaprazole sulfone and ilaprazole thiol ether, respectively. CONCLUSION: The validated method offered sensitivity and a wide linear concentration range. This method was successfully applied for the evaluation of the pharmacokinetics of ilaprazole and its two metabolites after single oral doses of 5 mg ilaprazole to 12 healthy Chinese volunteers.

Development of PBPK model of molinate and molinate sulfoxide in rats and humans.

Molinate has been widely used as a pre-emergent herbicide in the rice fields of California's Central Valley. In rat studies, the metabolite molinate sulfoxide is suspected of causing testicular toxicity after exposure to molinate. The sulfoxide is generated in the liver and can circulate in the blood, eventually reaching the testis. Man qualitatively produces the same molinate metabolites as the rat. To extrapolate the reproductive risk to man, the present study outlines the development of a preliminary PBPK (physiologically-based pharmacokinetic) model, validation in the rat and extrapolation to man. The preliminary seven-compartment PBPK model for molinate was constructed for the adult, male Sprague-Dawley rat that employed both flow-limited (blood, kidney, liver, rapid-perfused tissues and slowly perfused tissues) and diffusion-limited (fat) rate equations. The systemic circulation connects the various compartments. The simulations predict the molinate blood concentrations of the rat blood and testes compartment favorably with the profiles obtained from 10 and 100mg/kg po or 1.5 and 15mg/kg iv doses. Human physiological parameters were substituted into the oral dosed model and the simulations closely predicted the molinate blood concentration obtained from 5.06mg oral dose. A sensitivity analysis determined for an oral dose that peak blood molinate concentrations were most responsive to the blood flows to kidney and fat compartments while testicular molinate sulfoxide concentrations depended on molinate sulfoxide partition coefficients for the testes compartment and the K(m) for glutathione conjugation of molinate sulfoxide in the liver compartment.

Pharmacokinetics of the new proton pump inhibitor ilaprazole in Chinese healthy subjects in relation to CYP3A5 and CYP2C19 genotypes.

BACKGROUND: PPIs are widely used in peptic diseases, and this paper is to investigate the kinetic characteristics of a new PPI ilaprazole in Chinese healthy subjects and the association with CYP3A5 and CYP2C19 polymorphisms. METHODS: 21 subjects were selected and treated with 10mg ilaprazole according to their CYP3A5*3 genotypes (including 7 of CYP3A5*1/*1, 7 of *1/*3, and 7 of *3/*3). The plasma concentrations of ilaprazole and its metabolites were monitored by LC-MS/MS method. RESULTS: The C(max), AUC((0-6)), AUC((0-48)) and AUC((0-infinity)) of ilaprazole were all significantly different across the 3 CYP3A5 genotypes (including 4 of CYP3A5*1/*1, 4 of *1/*3, 3 of *3/*3; P<0.05) in CYP2C19 wild-type subjects (CYP2C19 wt/wts), similar variety of C(max) and AUC((0-6)) among CYP3A5 genotypes (including 3 of CYP3A5*1/*1, 3 of *1/*3, 4 of *3/*3; P<0.05) were also observed in CYP2C19 heterozygous subjects (CYP2C19 wt/mts). The sulfoxidation metabolic index (measure of collective CYP3A activity) indicates that the CYP3A5*1/*1, (high-expressers), *1/*3, (low-expressers), and *3/*3 (no-expressers) groups have medium, lowest and highest activities on ilaprazole metabolism, inconsistent with genotype-based CYP3A5 enzymatic activity. Further analysis showed no correlation between ilaprazole metabolism and CYP2C19 genotypes, evidenced by that the AUC((0-infinity)) of ilaprazole from either CYP3A5*1/*1 or CYP3A5*1/*3 groups was much higher in CYP2C19 wt/wts (n=4) than that in CYP2C19 wt/mts (n=3) (P<0.001), but the C(max) and AUC((0-6)) of ilaprazole from CYP3A5*3/*3 groups, were significantly lower in CYP2C19 wt/wts (n=3) compared to CYP2C19 wt/mts (n=4) (P<0.01). CONCLUSIONS: There was no demonstrated relationship between ilaprazole metabolism and CYP3A5 polymorphisms.