Simultaneous quantification of empagliflozin, linagliptin and metformin hydrochloride in bulk and synthetic mixture by RP–LC method

Background Extensive literature review revealed that no RP–LC method has been developed for simultaneous estimation of EMPA, LINA and MET in combined dosage form. This is a newer combination approved by USFDA on 4th June 2019 and it is launch in the United State Market on 27th January 2020. Result A simple, sensitive, specific, precise and accurate reverse phase—high performance liquid chromatography (RP- HPLC) method has been developed for simultaneous estimation of Empagliflozin, Linagliptin and Metformin HCl in bulk and synthetic mixture. Phenomenex C18 column (250 mm × 4.6 mm, 5 µm) was used as stationary phase for chromatographic separation through isocratic elution using Acetonitrile: Methanol: Water in a ratio (27: 20: 53, v/v/v) pH 4 adjusted with 1% Ortho-phosphoric acid as mobile phase at flow rate 1 ml/min. PDA detector was used for simultaneous analysis of all three drugs at common wavelength 223 nm and the each injection volume was 20 µl. The linearity range for Empagliflozin, Linagliptin and Metformin HCl was found to be 0.5–5 µg/ml, 0.25–2.5 µg/ml, and 50–500 µg/ml, respectively. The retention time for Empagliflozin, Linagliptin and Metformin HCl was found to be 14.5 min, 3.4 min and 2.01 min, respectively. The percentage (%) recovery was found to be 99.98–100.81% for Empagliflozin, 99.33–100.57% for Linagliptin and 100.65–101.35% for Metformin HCl respectively. Conclusion As per the international Conference on Harmonisation (ICH) Q2 (R1) guideline, proposed RP–LC method validation has been carried out. The proposed RP–LC method was repeatable and selective as per statistical analysis and it can be use for simultaneous estimation of Empagliflozin, Linagliptin and Metformin HCl in bulk and synthetic mixture. The proposed method might be applied for simultaneous estimation of all three drugs in pharmaceutical formulation.

Development and validation of UV-Spectrophotometric and RP-HPLC method for simultaneous estimation of Metformin and Doxycycline in bulk and synthetic mixture

A simple, rapid, sensitive, accurate and precise UV spectrophotometric and isocratic RP-HPLC method have been developed for simultaneous estimation of Metformin and Doxycycline in bulk and synthetic mixture. Spectrophotometric estimation was done by two methods. First method was Q-absorbance ratio method, where two wavelengths 236 nm (λmax of Metformin) and 248 nm (Iso-absorptive point) were used. The second method was first derivative method. In this method the zero-crossing point of Metformin was selected at 282 nm and for Doxycycline, it was 232 nm. The solvent used was methanol in both the above UV-spectrophotometric methods. Metformin and Doxycycline showed good linearity in the series of 1-9 µg/ml and 2-20 µg/ml respectively by both the two methods with an excellent correlation coefficient (r2≥0.998). In RP-HPLC method, the chromatographic separation was achieved on Luna Phenomenex C18 (150 mmХ6 mm, 5 µm) analytical column. A mixture of Acetonitrile: Phosphate buffer (50 mM): Triethylamine (TEA): Tetrahydrofuran (THF) (30:66:2:2) pH adjusted to 2.1 with orthophosphoric acid was used as the mobile phase, at a flow rate of 1 ml/min and at detector wavelength 248 nm. The retention time of Metformin and Doxycycline was found to be 3.561±0.0017 and 5.574±0.0131 minutes respectively. A linear response was observed over the concentration range 4-64 µg/ml of Metformin and 5-80 µg/ml of Doxycycline. All the three methods were validated in accordance to ICH guidelines for linearity, accuracy, precision, LOD and LOQ. The proposed methods were effectively utilized for the concurrent estimation of Metformin and Doxycycline in synthetic mixture. Keywords: Metformin, Doxycycline, Q-absorbance ratio method, First derivative method, RP-HPLC

Development and Validation of Novel RP-HPLC Method for the Simultaneous Determination of Remogliflozin and Vildagliptin in Bulk and in synthetic Mixture

Vildagliptin which is DPP-4 inhibitor and Remogliflozin which is SGLT2 inhibitor in single dose regimen lower blood glucose by separate, complementary mechanisms. Both are glucose dependent, accounting for the low risk of hypoglycaemia during treatment. There is no risk factors associated with this combination and moreover it is single dose regimen. The aim of the present study was to develop and validate a simple, rapid and reproducible gradient high performance reverse phase liquid chromatography method for the estimation of Remogliflozin and Vildagliptin in bulk drug sample and in synthetic mixture using Xterra® Waters C18 column (150 mm×4.6 mm, 5 µm) at 25°C with UV detection at 210 nm and for this gradient mode was used. The compounds were eluted gradiently at a flow rate of 1.0ml/min. The average retention times for Remogliflozin and Vildagliptin were 4.881 and 6.334 min, respectively. The calibration curves were linear (r2 =0.988) over the concentration range 10-200 µg/ml for Remogliflozin and 10-200 µg/ml for Vildagliptin. No spectral or chromatographic interferences from formulation excipients were found and hence it was successfully applied for the determination of Remogliflozin and Vildagliptin in bulk and in synthetic mixture. The accuracy of the proposed method was determined by recovery studies and found to be 98-101%. The proposed method was validated and results conformed to ICH parameters.

COMPOUNDS FROM PEPPER FLOWERS AND FRUITS AS POTENTIAL ATTRACTANTS FOR THE CAPTURE OF PEPPER WEEVIL

Two field experiments were conducted to evaluate synthetic attractants derived from pepper flowers, flower buds and fruits, alone or in combination with the aggregation pheromone. The evaluation was carried out with the release and recapture of Anthonomus eugenii adults at different distances from the four cardinal points in separate trials. The volatility of the synthetic mixture and aggregation pheromone was determined by gas chromatographic analysis of the volatiles captured by dynamic headspace. The traps with synthetic mixture and essential oil captured insects at 10 m, while the aggregation pheromone trapped up to 60 m. The combination of synthetic mixture or essential oil with the aggregation pheromone did not increase the number of recaptures compared to the single pheromone. The synthetic mixture together with geranic acid recaptured adults up to 15 m, although they were not significantly different from the control. The exclusion of geranic acid from the aggregation pheromone significantly reduced the number of recaptured insects (P<0.05), while geranic acid alone failed to capture weevils. The results could be improved by increasing the concentrations of the compounds or by adding other compounds released during the reproductive stages of pepper. These results could guide future efforts for the development of tools based on synthetic plant volatiles for the monitoring of this pest.

Development and Validation of a New RP-HPLC Analytical Method for the Simultaneous Determination of Luliconazole and Clobetasol Propionate in Synthetic Mixture

Aim: To develop new selective and sensitive reverse-phase high-performance liquid chromatography (RP-HPLC) approach for the quantification of antifungal drug Luliconazole integrate with corticosteroid drug Clobetasol Propionate in a synthetic mixture. Methods: The method was validated to achieve International Conference Harmonization (ICH) requirements. Chromatographic separation was carried out by isocratic technique on a reversed-phase Inertsil C18 column (5 µm, 250mm x 4.6mm i.d with the mixture of Acetonitrile: Water pH adjusted with H3PO4 (60: 40) and UV detection at 264 nm. The compounds were eluted at a flow rate of 1.0 mL/min with an injection volume of 20μL. Results: The calibration curves were linear (r2 > 0.999) over the concentration range 10-200 μg/mLfor Luliconazole and 5-100 μg/mL for Clobetasol Propionate. The average retention times for Luliconazole and Clobetasol Propionate were 3.16 and 6.94 min, respectively. The % RSD for the proposed method was found to be less than %2. The % recovery was found to between 99.22-99.48% for the developed method. Conclusion: The developed method is simple, rapid, precise, and accurate and hence was successfully applied for the determination of Luliconazole and Clobetasol Propionate in a synthetic mixture.