A NOVEL VALIDATED HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC STABILITY-INDICATING ASSAY METHOD IN REVERSE PHASE FOR ESTIMATION OF HYDROXYPROGESTERONE CAPROATE IN AN INJECTABLE FORMULATION.

A high-performance liquid chromatographic stability-indicating assay method in reverse-phase has been developed and validated for the quantitative measurement of Hydroxyprogesterone corporate in injectable formulation prepared in castor oil base containing excipient benzyl benzoate as preservative. The reverse-phase method was developed by using Sunfire C18 column having dimensions (150 x 4.6 mm x 5μm) with a mobile phase[A] comprised of water, acetonitrile, and Methanol mixed in 40:30:30 v/v ratio and mobile phase [B] comprised of water, acetonitrile, and Methanol mixed in 20:40:40 v/v ratio pumped gradient program at a flow rate of 1.0 ml/min where the thermostat of the column oven controlled at 30°C and sample compartment at 5°C. The injection volume is set at 20μL. Hydroxyprogesterone corporate has a retention time of 10 minutes with UV detection at 240 nm. The detector was showing linear response in a range of 0.25 μg/ml to 150 μg/ml for Hydroxyprogesterone corporate. The guidelines of the International Conference on Harmonization on guidance for industry (2005) validation of analytical procedures, text, and methodology Q2(R1) were followed for the validation of this novel method. For quantification of Hydroxyprogesterone corporate the developed method was found to be genuinely exact precise, accurate, linear, robust, rugged, stable. and cost-effective.

An analytical “quality by design” approach in RP-HPLC method development and validation for reliable and rapid estimation of irinotecan in an injectable formulation

The objective of the present study was to develop a robust, simple, economical and sensitive HPLC-UV method using the “quality-by-design” approach for the estimation of irinotecan (IRI) in marketed formulations. RP-HPLC method was developed by applying Box-Behnken design with Hyper-Clone (Phenomenex®) C18 column (250 × 4.6 mm id, particle size 5 µm, ODS 130 Å) as a stationary phase. Acetonitrile and 20 mmol L−1 potassium phosphate buffer (pH 2.5) containing 0.1 % triethylamine in a ratio of 45:55 % (V/V) was used as a mobile phase. The sample was injected in a volume of 20 µL into the HPLC system. UV detector at 254 nm was used to estimate and quantify IRI. Isocratic elution was opted while the flow rate was maintained at 0.75 mL min−1. The retention time of IRI was found to be 4.09 min. The responses were found to be linear for concentration range of 0.5 to 18.0 µg mL−1 and the coefficient of determination value was found to be 0.9993. Percent relative standard deviation for intra- and inter-day precisions was found in the range of 0.1 to 0.4 %. LOD and LOQ values were found to be 4.87 and 14.75 ng mL−1, resp. Robustness studies confirmed that the developed method is robust with RSD of a maximum 0.1 %. The method is simple, precise, sensitive, robust and economical making it applicable to the estimation of IRI in an injectable formulation.

IMPURITY PROFILING IMPURITY PROFILING OF THIAMINE HYDROCHLORIDE INJECTION BY RP-HPLC AND CHARACTERIZATION OF DEGRADATION PRODUCT BY LC-MS/MS/QTOF

Objective: To propose a comprehensive, simple, and affordable RP-HPLC method for impurity profiling and characterization of unknown degradation products of thiamine hydrochloride injectable formulation. Methods: The chromatographic separation employs gradient mode using the octadecyl silane column using a mobile phase consisting of phosphate buffer with ion pair reagent, acetonitrile, and methanol delivered flow rate at 1.2 ml/min. The detection was carried out at 248 nm using empower software. LC-MS/MS/QTOF hyphenated technique was used for isolation and characterization of unknown degradation impurity. The performance of the method was systematically validated as per ICH Q2 (R1) guidelines. Results: Degradation product observed in accelerated stability was characterized by LC-MS/MS/QTOF hyphenated technique and found m/z value 351.1604 and postulated as an oxidative degradation product of thiamine due to excipient interaction. The validated method was sensitive, selective, and specific data proves the method is precise and accurate from LOQ to 150% level and results are within 95-108% and less than 4.5% RSD. The developed method is linear from 0.03-58.83 µg/ml with a correlation coefficient of more than 0.990 and LOD and LOQ value ranged from 0.03 to1.51 μg/ml. Conclusion: An efficient RP-HPLC method for impurity profiling of thiamine injectable formulation was successfully developed and unknown degradation product observed instability condition samples characterized by LC-MS/MS/QTOF technique. The validated method can be successfully employed for the impurity profiling of thiamine injectable in the quality control department.