Ultrasound-assisted dispersive liquid-liquid microextraction for determination of three gliflozins in human plasma by HPLC/DAD.

A novel, highly sensitive ultrasound-assisted dispersive liquid-liquid microextraction (UA-DLLME) high performance liquid chromatography with diode-array detection (HPLC/DAD) method was developed for the determination of empagliflozin, dapagliflozin and canagliflozin in human plasma using methanol as protein precipitating agent/disperser and 1-dodecanol as extracting solvent. The analytes were eluted with an isocratic mobile phase consisting of acetonitrile:aqueous 0.1% trifluoroacetic acid pH 2.5, (40:60, v/v), at a flow rate of 1 mL/min and UV detection at 210 nm. The microextraction conditions were optimized regarding type and volume of extractant, type of disperser, sample pH, extraction time and centrifugation time. Under the optimal conditions, the enrichment factors were 19 for empagliflozin, 27 for dapagliflozin and 50 for canagliflozin. Linearity ranges were 2-2500 ng/mL, 3.5-2500 ng/mL and 1.1-2500 ng/mL for empagliflozin, dapagliflozin and canagliflozin, respectively. The developed method employs very small volumes of organic solvents in sample extraction and allows determination of small concetrations of gliflozins in human plasma.

HPTLC Method for Determination of R, R-Glycopyrronium Bromide and its Related Impurities.

An innovative high-performance thin layer chromatographic (HPTLC) method was designed, optimized and validated for the quantification of R, R-glycopyrronium bromide (GLY) and its related impurities in drug substance and drug product. Separation was performed on HPTLC plates pre-coated with silica gel 60 F254 by dichloromethane:methanol:formic acid (10:0.5:0.5, v/v/v) as a developing system. GLY and its related impurities namely, glycopyrronium impurity G and glycopyrronium impurity J, were separated giving compact well-resolved spots with significant retardation factor (Rf) values of 0.17 ± 0.02, 0.34 ± 0.02 and 0.69 ± 0.02, respectively. Quantification was done at 220 nm in the ranges of 0.3-10 and 0.2-4.0 µg/spot with limits of detection and quantification of 0.1, 0.3 and 0.05, 0.2 µg/spot for GLY and its related impurities, respectively. Good accuracy was obtained with mean percentage recovery of 99.48 ± 1.36, 100.04 ± 1.32 and 99.61 ± 0.80 and R2 ≥ 0.9968 for GLY and its impurities, consecutively. Validation parameters were presented according to the International Conference on Harmonization. The method was used to investigate impurity profile of GLY in drug substance and drug product and could be applied in routine analysis of the drug. Comparison between the developed method and the reported method revealed no statistical difference.

A UPLC/DAD method for simultaneous determination of empagliflozin and three related substances in spiked human plasma.

A simple, rapid and sensitive ultrahigh performance liquid chromatographic method was developed for the determination of the anti-diabetic drug: empagliflozin (EMPA) and three related substances in spiked human plasma, using dapagliflozin (DAPA) as an internal standard and tetrahydrofuran as a plasma protein precipitating agent. The chromatographic separation was achieved on an Acquity "UPLC® BEH" C18 column (50 mm × 2.1 mm i.d, 1.7 µm particle size), and a mobile phase consisting of aqueous trifluoroacetic acid (0.1%, pH 2.5): acetonitrile (60:40, v/v) at a flow rate of 0.5 mL/min. Upon using the UPLC system, the run time could be reduced to less than 1.2 min, and the solvents consumption decreased to 0.36 mL of acetonitrile per run. The response was linear over a concentration range of 50-700 ng/mL and 40-200 ng/mL (r2 = 0.9994-0.9999) with lower limits of detection and quantification (LOD/LOQ) of 15/50, 11.5/40, 12/40 and 12.5/40 ng/mL for EMPA and the three related substances, respectively. Good accuracy was obtained with mean percentage recoveries ≥ 96.97% for the studied compounds. The method was validated according to the ICH guidelines and was found suitable for routine analysis of EMPA and its related substances in human plasma.

Spectrophotometric and spectrodensitometric determination of paracetamol and drotaverine HCl in combination.

Thin-layer chromatography, first derivative, ratio spectra derivative spectrophotometry and Vierordt's method have been developed for the simultaneous determination of paracetamol and drotaverine HCl. TLC densitometric method depends on the difference in Rf values using ethyl acetate:methanol:ammonia (100:1:5 v/v/v) as a mobile phase. The spots of the two drugs were scanned at 249 and 308 nm over concentration ranges of 60-1200 microg/ml and 20-400 microg/ml with mean percentage recovery 100.11%+/-1.91 and 100.15%+/-1.87, respectively. The first derivative spectrophotometric method deals with the measurements at zero-crossing points 259 and 325 nm with mean percentage recovery 99.25%+/-1.08 and 99.45%+/-1.14, respectively. The ratio spectra first derivative technique was used at 246 and 305 nm with mean percentage recovery 99.75%+/-1.93 and 99.08%+/-1.22, respectively. Beer's law for first derivative and ratio spectra derivative methods was obeyed in the concentration range 0.8-12.8 and 0.4-6.4 microg/ml of paracetamol and drotaverine HCl, respectively. Vierordt's method was applied to over come the overlapping of paracetamol and drotaverine HCl in zero-order spectra in concentration range 2-26 and 2-40 microg/ml respectively. The suggested methods were successfully applied for the analysis of the two drugs in laboratory prepared mixtures and their pharmaceutical formulation. The validity of the methods was assessed by applying the standard addition technique. The obtained results were statistically agreed with those obtained by the reported method.