Rajendra Pratap Singh

A sensitive and selective liquid chromatographic tandem mass spectrometric assay for simultaneous quantification of novel trioxane antimalarials in different biomatrices using sample-pooling approach for high throughput pharmacokinetic studies

In the present studies, to give momentum to traditionally low throughput pharmacokinetic screening, a bioanalytical method based on the concept of sample pooling for simultaneous bioanalysis of multiple compounds is discussed. A sensitive, selective, specific and rapid HPLC/ESI-MS/MS assay method was developed and validated for the simultaneous quantitation of three novel trioxane antimalarials (99-357, 99-408 and 99-411) in rat plasma using trioxane analogue as internal standard. The suitably validated bioanalytical method was then further extrapolated to rabbit and monkey plasma by performing partial validation. Extraction from the plasma involves a simple two-step liquid-liquid extraction with n-hexane. The analytes were chromatographed on a cyano column by isocratic elution with acetonitrile:ammonium acetate buffer (pH 6) (85:15, v/v) and analyzed by mass spectrometry in multiple reaction-monitoring (MRM) mode. The chromatographic run time was 5.5 min and the weighted (1/x(2)) calibration curves were linear over a range of 1.56-200 ng/ml. The limit of detection (LOD) and lower limit of quantification (LLOQ) in rat plasma, rabbit plasma and monkey plasma were 0.78 and 1.56 ng/ml, respectively, for all three analytes. The intra- and inter-batch accuracy and precision in terms of % bias and % relative standard deviation were found to be well within the acceptable limits (< 15%). The average absolute recoveries of 99-357, 99-408 and 99-411 from spiked plasma samples were > 90%, > 70% and > 60%, respectively. The assay method described here could be applied to study the pharmacokinetics of 99-357, 99-408 and 99-411 using sample-pooling technique.

Liquid chromatographic tandem mass spectrometric assay for quantification of 97/78 and its metabolite 97/63: a promising trioxane antimalarial in monkey plasma.

The present manuscript describes development and validation of LC-MS/MS assay for the simultaneous quantitation of 97/78 and its active in-vivo metabolite 97/63 in monkey plasma using alpha-arteether as internal standard (IS). The method involves a single step protein precipitation using acetonitrile as extraction method. The analytes were separated on a Columbus C(18) (50 mm x 2 mm i.d., 5 microm particle size) column by isocratic elution with acetonitrile:ammonium acetate buffer (pH 4, 10 mM) (80:20 v/v) at a flow rate of 0.45 mL/min, and analyzed by mass spectrometry in multiple reaction-monitoring (MRM) positive ion mode. The chromatographic run time was 4.0 min and the weighted (1/x(2)) calibration curves were linear over a range of 1.56-200 ng/mL. The method was linear for both the analytes with correlation coefficients >0.995. The intra-day and inter-day accuracy (% bias) and precisions (% RSD) of the assay were less than 6.27%. Both analytes were stable after three freeze-thaw cycles (% deviation <8.2) and also for 30 days in plasma (% deviation <6.7). The absolute recoveries of 97/78, 97/63 and internal standard (IS), from spiked plasma samples were >90%. The validated assay method, described here, was successfully applied to the pharmacokinetic study of 97/78 and its active in-vivo metabolite 97/63 in Rhesus monkeys.

Pharmacokinetic study of the novel, synthetic trioxane antimalarial compound 97-78 in rats using an LC-MS/MS method for quantification

The present study has been designed to investigate the pharmacokinetic parameters of the novel trioxane antimalarial 97-78 (US Patent 6316493 B1, 2001) in male and female rats after single oral and intravenous administration. The pharmacokinetic profile of 97-78 was investigated in the form of its completely converted metabolite 97-63 after dose administration. Quantification of metabolite 97-63 in rat plasma was achieved using a simple and rapid LC-MS/MS method. The LC-MS/MS method has been validated in terms of accuracy, precision, sensitivity and recovery for metabolite 97-63 in rat plasma. The intra- and interday accuracy (% bias) and precision (% RSD) values of the assay were less than 10% for metabolite 97-63. The chromatographic run time was 4.0 min and the weighted (1/x2) calibration curves were linear over the range 1.56-200 ng/ml. This method was successfully applied for analysis of pharmacokinetic study samples. Maximum plasma concentrations of 97-63 at 47 mg/kg oral administration in male and female rats were 1986.6 ng/ml and 4086.7 ng/ml at time (Tmax) 0.92 h and 0.58 h, respectively. The area under the curve (AUC(0-infinity)), elimination half-life (t(1/2) beta) and mean residence time (MRT) were 4669.98 ng x h/ml, 2.8 h and 4.2 h in male and 11786.0 ng x h/ml, 4.52 h and 4.32 h in female rats respectively. After single oral and intravenous administration of 97-78 to male and female rats significant differences were observed in pharmacokinetic parameters (AUC and t (1/2) beta) for metabolite 97-63.

Liquid chromatographic-tandem mass spectrometry assay for quantitation of a novel antidiabetic S002-853 in rat plasma and its application to pharmacokinetic study.

A sensitive and selective LC-MS/MS method has been developed and validated for the estimation of novel antidiabetic synthetic flavonoid S002-853 in rat plasma using centchroman as an internal standard. The method involves a simple two-step liquid-liquid extraction with diethyl ether. The analyte was chromatographed on a Pierce Spheri-5, guard cyano column (30 x 4.6 mm i.d., 5 microm) with isocratic mobile phase consisting of methanol-ammonium acetate buffer (pH 4.6, 10 mm; 90 : 10, v/v) at a flow rate of 0.75 mL/min. The API 4000 triple-quadrupole LC-MS/MS system was operated under multiple reaction-monitoring mode. The ionization was performed by electrospray ionization technique in positive ion mode. The chromatographic run time was 6 min and the weighted (1/x(2)) calibration curves were linear over the range 0.78-400 ng/mL. The limit of detection and lower limit of quantification were 0.195 and 0.78 ng/mL, respectively. The intra- and inter-batch accuracy (%bias) and precision (%RSD) were found to be less than 8.47 and 11.6% respectively. The average absolute recoveries of S002-853 and internal standard from spiked plasma samples were >90%. S002-853 was stable for 8 h at ambient temperature, 4 weeks at -60 degrees C and after three freeze-thaw cycles. The assay was successfully applied to determine the pharmacokinetic parameters in male Sprague-Dawley rats after an oral dose administration at 25 mg/kg.

Association of interleukin-1beta C + 3953T gene polymorphism with human male infertility

Cytokines are involved in the regulation of spermatogenesis likely mediating the crosstalk among Sertoli and germ cells to facilitate germ cell movement across the seminiferous epithelium during cellular events such as germ cell differentiation. Members of the Interleukin-1 (IL-1) family are pleiotropic cytokines that are involved in inflammation, immunoregulation, and other homeostatic functions. Interleukin-1 alpha (IL-1α), IL-1β, and the IL-1 antagonistic molecule (IL-1 Ra) are present in the testis under normal homeostasis and they further increase upon infection/inflammation. In the present study we have examined the association of Cu2009+u20093953T polymorphism of the human IL-1B gene with human male infertility. The case control study comprised of two groups: 222 infertile patients and 230 fertile healthy control men. Genotyping for SNP Cu2009+u20093953T IL-1B was carried out by polymerase chain reaction followed by analysis with specific endonucleases (PCR-RFLP). DNA sequencing was used to validate the PCR-RFLP results. The genotype frequencies of the IL-1B Taq C/T polymorphism were compared between infertile men and controls. The frequency was significantly higher in asthenozoospermic patients compared to fertile control men (odds ratiou2009=u200910.4, CI: 2.50- 43.96, pu2009=u20090.001). The Cu2009+u20093953T of the IL-1B gene is associated with male infertility risk in the asthenozoospermic patients from an Indian population.

Liquid chromatography tandem mass spectrometry method for determination of antidiabetic chalcones derivative S001-469 in rat plasma, urine and feces: application to pharmacokinetic study.

A sensitive and selective liquid chromatography tandem mass spectrometry assay was developed for quantitation of a novel antidiabetic chalcones derivative S001-469 in rat matrices. Plasma and urine samples were prepared by double liquid-liquid extraction with diethyl ether and feces by protein precipitation using acetonitrile. Chromatographic elution was carried on cyano guard column (30 mm × 4.6 mm i.d., 5 µm) in isocratic mode at a flow rate of 0.75 mL/min using mobile phase comprising of methanol: ammonium acetate buffer (pH 4.6, 10 mM) (90:10, v/v). Run time was 6 min. Detection was achieved by employing positive ionization mode on a triple-quadrupole LC-MS/MS system with an electrospray ionization (ESI) source. The calibration curves were linear over the range of 0.78-400 ng/mL for all 3 matrices. The method was validated and proved reliable through high and consistent intra- and inter- day accuracy and precision (<15%) values. Recoveries was >85% from spiked plasma, urine and feces samples. S001-469 was stable in plasma at room temperature till 8 h and at -60 °C for 30 d and 3 freeze-thaw cycles.

Interspecies comparison of the pharmacokinetics and oral bioavailability of 99-357, a potent synthetic trioxane antimalarial compound

The pharmacokinetic data obtained in lower animals is of considerable importance in drug discovery and development. The objective of the present study was to generate in vitro and in vivo preclinical pharmacokinetic data of 99-357, a synthetic trioxane antimalarial, in rats and rabbits and to scale-up the data in order to apply for further studies. The pharmacokinetic profile of 99-357 was investigated after both intravenous and oral dose in rats and rabbits. Oral studies were carried out at three dose levels 6, 12 and 24mg/kg in rats while in rabbit only one dose level was selected. Both compartmental and non-compartmental approaches were used to calculate the pharmacokinetic parameters following intravenous and oral doses in both the species. The clearance in rat and rabbit was 45-57% and 60-67% respectively of hepatic blood flow. The plasma protein binding in rats was approximately 75%. In vitro studies showed high RBC partitioning and low to moderate hepatic clearance. Linearity was observed in terms of dose and AUCs suggesting linear pharmacokinetics at the dose levels studied in rats. The oral bioavailability of compound 99-357 in rat and rabbit at 12mg/kg dose level was comparable and 39% and 41% respectively.