Identification, isolation and characterization of dolutegravir forced degradation products and their cytotoxicity potential.

Dolutegravir was approved by USFDA, Canada and European regulatory authorities as antiretroviral medication. In this article, DLG forced degradation studies as per the International Council for Harmonization (ICH) prescribed stress conditions was conducted and the resulting degradants were fully characterized. DLG was stable in basic, thermal and photolytic stress conditions, whereas DLG was found to unstable in acidic and oxidative conditions. One degradant each from acid and peroxide treated solutions was resolved on LC-MS and labelled as DP-1 and DP-2 with RT 1.80 min and 1.41 min, respectively. DP-1 and DP-2 were isolated by preparative HPLC with C18 column using gradient elution method. Subsequently DP-1 and DP-2 peaks were subjected to HRMS for accurate mass. Molecular mass of DP-1 and DP-2 were m/z 420.1379 (positive mode) and m/z 214.0319 (negative mode), respectively. Further, DP-1 & DP-2 were subjected to NMR spectroscopic analysis (including 2D) for structural confirmation. DP-1 was identified as N-(2,4-difluorobenzyl)-9-hydroxy-2-(4-hydroxybutan-2-yl)-1,8-dioxo-2,8-dihydro-1H-pyrido[1,2-a]pyrazine-7-carboxamide and it is earlier reported by Gudisela et al. [19] as DLG process impurity. DP-2 was identified as 2-(2,4difluorobenzylamino)-2-oxoacetic acid which is novel DLG degradant and not reported earlier to the best of our knowledge. DLG along its forced degradation products were found to be non-cytotoxic in in vitro assay conditions using HepG2 cells.

Precolumn o-phthalaldehyde-N-acetyl-L-cysteine derivatization followed by RP-HPLC separation and fluorescence detection of sitagliptin enantiomers in rat plasma.

An indirect reversed-phase high-performance liquid chromatographic separation and fluorescence detection of sitagliptin enantiomers in rat plasma was developed and validated. Deproteinized rat plasma containing racemic sitagliptin was derivatized with o-phthalaldehyde and N-acetyl-L-cysteine under alkaline conditions, converted to diastereomers, and separated on a Lichrospher 100 RP-18e column using 20 mM phosphate buffer and methanol (45:55 v/v) as a mobile phase under isocratic mode of elution at a flow rate of 1.0 mL/min. Fluorescence detection was performed at 330 and 450 nm as excitation and emission wavelengths, respectively. The method was linear in the range of 50-5000 ng/ mL for both enantiomers. The intra- and interday accuracy and precision were within the predefined limits of ≤15% at all concentrations. The method was successfully applied to a pharmacokinetic study of sitagliptin after 5 mg/kg oral administration to Wistar rats. Robustness of the method was evaluated using design of experiments.

Development and validation of a stability indicating assay of doxofylline by RP-HPLC: ESI-MS/MS, ¹H and ¹³C NMR spectroscopic characterization of degradation products and process related impurities.

A validated stability indicating RP-HPLC assay of doxofylline was developed by separating its related substances and degradants on LichrocartC18 (250 mm × 4.6 mm; 5 µm) column using 10 mM ammonium acetate and acetonitrile as a mobile phase in a gradient mode of elution at a flow rate of 1.0 mL/min at 30 °C. The column effluents were monitored by a photo diode array detector set at 274 nm. The method was validated in terms of accuracy, precision and linearity as per ICH guidelines. The limits of quantification of doxofylline and impurities were obtained in the range of 0.19-0.36 µg/mL. Forced degradation of doxofylline was carried out under acidic, basic, thermal, photo, peroxide conditions and the degradation products were isolated and characterized by ESI-MS/MS, (1)H and (13)C spectroscopy. The method was successfully applied not only to quantify related substances and degradation products but also assay of doxofylline in bulk drugs. The recoveries of doxofylline and impurities were in the range of 99.00-100.05% and 97.83-99.86% respectively.

Development of a validated LC-MS/MS method for determination of doxofylline on rat dried blood spots and urine: application to pharmacokinetics.

A rapid and highly sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for determination of doxofylline on rat dried blood spots and urine was developed and validated. The chromatographic separation was achieved on a reverse phase C18 column (250 mm × 4.6 mm, 5 µm), using 20 mM ammonium acetate (pH adjusted to 3.5 with trifluoroacetic acid) and acetonitrile (75:25 v/v) as a mobile phase at 25 °C. LC-MS detection was performed with selective ion monitoring using target ions at m/z 267 and m/z 195 for doxofylline and caffeine used as internal standard respectively. The calibration curve showed a good linearity in the concentration range of 1-5000 ng/mL. The effect of hematocrit on extraction of doxofylline from DBS was evaluated. The mean recoveries of doxofylline from DBS and urine were 93.46 and 89.86% respectively. The intra and inter-day precisions were less than 4.28% in DBS as well as urine. The limit of detection and quantification were 0.24 and 0.84 ng/mL in DBS and 0.28 and 1.00 ng/mL in urine samples respectively. The method was validated as per ICH guidelines and successfully applied to a pharmacokinetic study of doxofylline in rats.