ABSTRACT
Darunavir, an anti-HIV drug was subjected to forced degradation under acid, base, thermal and neutral hydrolysis, oxidation and photolysis as prescribed by ICH guidelines. Four major degradation products were formed under acid and base hydrolysis, while stable under neutral and thermal hydrolysis, oxidative and photolysis. The drug and its degradation products were separated on Hiber, LiChrospher® 60, RP-select B, C8 column (250mm×4.6mm i.d., 5µm) using 10mM ammonium acetate: acetonitrile (52:48, v/v) as mobile phase in an isocratic elution mode by LC. The degradation products were characterized by LC-MS/MS and fragmentation pathways were proposed. The proposed structures of degradation products were confirmed by HRMS and the LC method was validated with respect to specificity, linearity, accuracy, recovery, LOD and LOQ.
Subject(s)
Anti-HIV Agents/chemistry , Sulfonamides/chemistry , Biological Assay/methods , Chromatography, Liquid/methods , Darunavir , Drug Stability , Hydrogen-Ion Concentration , Hydrolysis , Oxidation-Reduction , Photolysis , Tandem Mass Spectrometry/methodsABSTRACT
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.
Subject(s)
Acetylcysteine/chemistry , Fluorescent Dyes/chemistry , Pyrazines/blood , Triazoles/blood , o-Phthalaldehyde/chemistry , Animals , Chromatography, High Pressure Liquid , Drug Stability , Limit of Detection , Molecular Structure , Pyrazines/chemistry , Rats , Sitagliptin Phosphate , Spectrometry, Fluorescence , Time Factors , Triazoles/chemistryABSTRACT
A simple and selective liquid chromatography-tandem mass spectrometric method for determination of pramipexole on rat dried blood spots was developed and validated. Chromatographic separation was achieved on a Synergy polar-RP column using 10mM ammonium acetate and methanol (50:50, v/v) as mobile phase in an isocratic mode of elution at a flow rate of 1.0mL/min. LC-MS was performed in a positive ion electro spray ionization mode and the MS/MS ion transitions 212.10â153.03 for PRX and 198.10â153.03 for internal standard (2-amino-4,5,6,7-tetrahydro-6-ethyl-amino-benzthiazole) were monitored. The developed method exhibited a linear dynamic range over 100-5000pg/mL for PRX on dried blood spots. The overall extraction recovery of PRX from DBS was 96.7%. The intra- and inter-day accuracy and precision were within the pre-defined limits of ≤15% at all concentrations. Influence of hematocrit and spot volume on dried blood spot was also evaluated and found to be well within the acceptable limits. The method was successfully applied to pharmacokinetic studies of PRX in rats.
