Measurement Uncertainty and Validation for Quantitation of Salbutamol in Human Urine by Gas Chromatography-Tandem Mass Spectrometry.

A simple, rapid, sensitive and specific gas chromatography-tandem mass spectrometry (GC-MS/MS) method was developed for quantitation of salbutamol in human urine using salbutamol-d3 as the internal standard. The processing of urines samples includes deconjugation with enzymatic hydrolysis, solid phase extraction procedure utilizing XAD2 column and liquid-liquid extraction accompanied by the derivatization by means of MSTFA/IODO-TMS/DTE mixture. The GC column was a HP Ultra-1 (17 m × 0.22 mm × 0.11 µm) used to separate the peak of interest. The data for GC-MS/MS were acquired and processed utilizing GC Labs Solution and Insight GCMS Software. The detection of spectra was performed on TQ 8050. This method included a chromatographic run of 13.67 min and the linearity was found over the concentration range of 250-2000 ng/mL with a regression coefficient (r2) of 0.99. The coefficient of variation for intra and interday assay precision was between 1.85 and 2.85% and the accuracy was between 95.50 and 107.04% for low quality control (QC), medium QC and high QC. The recovery was adequate to reliable detect the analyte at or below the level recommended by the World Anti-Doping Agency i.e., threshold 1000 ng/mL. The limit of detection and limit of quantification were found to be 10 and 100 ng/mL, respectively. The expanded measurement uncertainty (Uexp%) was found to be 8.28%.

Process development for the total synthesis of the novel drug metabolite Carboxy toremifene as a standard reference material along with characterization and purity assessment for the Antidoping quality Control Purposes.

Tamoxifen and toremifene are two selective estrogen receptor modulators (SERMs) commonly used to treat breast cancer in women. Toremifene is well-known as a triphenylethylene derivative. Carboxy toremifene is a common metabolite of toremifene and tamoxifen. Since 2005, the World Anti-Doping Agency (WADA) has banned the SERMs category during in and out of competition. These substances are in the S4 category in the WADA prohibited list as "agents with anti-oestrogenic activity." However, there is no commercially accessible carboxy toremifene reference material in the market. This research highlights the novel synthetic procedure, the development of a carboxy toremifene HPLC method, and validation, along with detailed characterization using advanced analytical techniques using 1 H NMR, HRMS, FT-IR-ATR and UV-visible spectroscopy. RP-HPLC-DAD method was developed and validated to assess the purity of carboxy toremifene. Developed reference material has shown 100% purity. Therefore, we recommend that this synthesized carboxy toremifene may be used as reference material to strengthen the WADA-accredited lab to maintain a clean sports mission during sports competitions.

Design and synthesis of new of 3-(benzo[d]isoxazol-3-yl)-1-substituted pyrrolidine-2, 5-dione derivatives as anticonvulsants.

A series of 3-(benzo[d]isoxazol-3-yl)-N-substituted pyrrolidine-2, 5-dione (7a-7d, 8a-8d, 9a-9c) have been prepared and evaluated for their anticonvulsant activities. Preliminary anticonvulsant activity was performed using maximal electroshock (MES) and subcutaneous pentylenetetrazole (scPTZ) tests after intraperitoneal (ip) injection into mice, which are the most widely employed models for early identification of anticonvulsant candidate. The acute neurological toxicity (NT) was determined applying rotorod test. The quantitative evaluation after oral administration in rats showed that the most active was 3-(benzo[d]isoxazol-3-yl)-1-(4-fluorophenyl) pyrrolidine-2, 5-dione (8a) with ED50 values of 14.90 mg/kg. Similarly the most potent in scPTZ was 3-(benzo[d]isoxazol-3-yl)-1-cyclohexylpyrrolidine-2, 5-dione (7d) with ED50 values of 42.30 mg/kg. These molecules were more potent and less neurotoxic than phenytoin and ethosuximide which were used as reference antiepileptic drugs.

Pharmacokinetic interaction between febuxostat and morin in rats.

BACKGROUND: Due to wide consumption of flavonoids in the dietary supplement, and an imperative role of CYPs and P-glycoprotein inhibition in drug disposition. So there is increasing scientific interest in drug-flavonoid interactions. OBJECTIVE: The present study aims to investigate the effect of morin, a flavonoid, on the pharmacokinetics of febuxostat in rats. METHODS: A simple ultra-performance liquid chromatography method has been developed for the calculation of febuxostat in 100 µl rat plasma using febuxostat D7 as an internal standard (IS). The assay procedure involved a single-step, liquid-liquid extraction of febuxostat and IS from plasma with methanol. Pharmacokinetic parameters of febuxostat were determined in rats after an oral administration of febuxostat (5 mg/kg) to rats in the control, coadministered and pretreated groups of morin (10 mg/kg). RESULTS: Compared to the control rats given febuxostat alone, the Cmax and AUC of febuxostat increased by 18 - 20 and 47 - 50%, respectively, in rats pretreated with morin. The plasma half-life (t1/2) of the pretreated group is increased by 2.5-fold compared with the control group. Consequently, relative bioavailability values of febuxostat in the rats pretreated with morin were significantly higher (p < 0.05) than those from the control and coadministered groups. Increased bioavailability indicates that the presence of morin could be effective in inhibiting CYP1A1, CYP1A2 and CYP3A4-mediated metabolism and/or effective in inhibiting P-glycoprotein-mediated cellular efflux of febuxostat. CONCLUSION: The presence of morin significantly enhanced the oral exposure of febuxostat, suggesting that concurrent use of morin or morin-containing dietary supplements with febuxostat should be verified to avoid drug-flavonoid interactions.

Chromatographic separation of piracetam and its metabolite in a mixture of microsomal preparations, followed by an MS/MS analysis.

A rapid bioanalytical method was evaluated for the simultaneous determination of piracetam and its metabolite (M1) in human microsomal preparations by fast ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS). In addition, a validated method of M1 in rat plasma was developed and successfully applied on pharmacokinetic studies. The present study was carried out to determine the metabolic pathways of piracetam for phase I metabolism and used cytochrome P450 isoforms responsible for the piracetam metabolism in human liver microsomes (HLMs). While additional potential metabolites of piracetam were suggested by computer-modeling. The resulting 2-(2-oxopyrrolidin-1-yl) acetic acid was the sole metabolite detected after the microsomal treatment. The amide hydrolysis mainly underwent to form a metabolite i.e., 2-(2-oxopyrrolidin-1-yl) acetic acid (M1).

Synthesis, characterization of (Z)-N-(1-(2-(2-amino-3-(dimethylamino) methyl)phenyl)-5-phenyl-1,3,4, oxadiazol-3(2H)-yl)ethanone analogs as potent antimicrobial and hydrogen peroxide scavenging agents.

A series of (Z)-N-(1-(2-(2-amino-3-((dimethylamino) methyl) phenyl)-5-phenyl-1,3,4,oxadiazol-3(2H)- yl)ethanone derivatives was prepared and studied for its antimicrobial and antioxidant activities. Among the synthesized derivatives, compounds (7c) (Z)-N-(1-(2-(2-amino-3-((dimethylamino)methyl)phenyl)-5-phenyl-1,3,4-oxadiazol-3(2H)- yl)ethylidene)-4-chloroaniline, (7g) (Z)-N-(1-(2-(2-amino-3-((dimethylamino)methyl)phenyl)-5-phenyl-1,3,4-oxadiazol- 3(2H)-yl)ethylidene)-4-nitroaniline and (7i) (Z)-N-(1-(2-(2-amino-3-((dimethylamino)methyl)phenyl)-5-phenyl-1,3,4- oxadiazol-3(2H)-yl)ethylidene)-4-methoxyaniline were found to be the most effective antimicrobial compounds. While the compounds 7c and 7g were the most potent antioxidant compounds with significant hydrogen peroxide scavenging activity.

Effect of morin on pharmacokinetics of piracetam in rats, in vitro enzyme kinetics and metabolic stability assay using rapid UPLC method.

The aim of this study was to investigate the effect of Morin on the pharmacokinetics of Piracetam in rats, in vitro enzyme kinetics and metabolic stability (high throughput) studies using human liver microsomes in UPLC. For pharmacokinetics studies, male Wistar rats were pretreated with Morin (10 mg/kg) for one week and on the last day, a single dose of Piracetam (50 mg/kg) was given orally. In another group, both Morin and Piracetam were co-administered to evaluate the acute effect of Morin on Piracetam. The control group received oral distilled water for one week and administered with Piracetam on the last day. As Morin is an inhibitor of P- Glycoprotein (P-gp) and CYP 3A, it was anticipated to improve the bioavailability of Piracetam. Amazingly, relative to control, the areas under the concentration time curve and peak plasma concentration of Piracetam were 1.50- and 1.45-fold, respectively, greater in the Morin-pretreated group. However, co-administration of Morin had no significant effect on these parameters. Apart from the aforementioned merits, the results of this study are further confirmed by clinical trials; Piracetam dosages should be adjusted to avoid potential drug interaction when Piracetam is used clinically in combination with Morin and Morin-containing dietary supplements. The in vitro enzyme kinetics were performed to determined km, Vmax & CLins . The in vitro metabolic stability executed for the estimation of metabolic rate constant and half-life of Piracetam. These studies also extrapolate to in vivo intrinsic hepatic clearance (Clint, in vivo ) from in vitro intrinsic hepatic clearance (CLint, in vitro ).