Method development

A new Reverse Phase High Performance Liquid Chromatographic (RP-HPLC) method has been developed for estimation of Paracetamol and Alprazolam in bulk and tablet dosage forms using UV-detector. A RP Cell Pack C18 column (250 mm × 4.6 mm, 5 µ particle size) using acetonitrile and water (80:20 % V/V) as mobile phase by maintaining flow rate of 1 mL/min at 236 nm as detection wavelength. The peaks were eluted at 4.8 and 6.2 mins for Paracetamol and Alprazolam, respectively. The method was validated in accordance with ICH guidelines, the linearity curve for Paracetamol was obtained over the range of 50-175 µg/mL, and it was found to be linear with y = 1961x + 9226 (r2 = 0.999). The linearity curve for Alprazolam was obtained over the range of 0.25-1.5 µg/mL and was found to be linear with y =23328x + 939.3 (r2 = 0.998). The percentage recoveries were found to be 99-101% and 99-102%, respectively. The system suitability parameters such as number of theoretical plates and tailing factor were found to be 7242, 1.56 for PAR and 6755, 1.15 for ALP. Hence the developed RP-HPLC method was found to be simple, accurate, economical, rapid and can be applied for routine analysis of these drugs in their combined formulations.

Two novel methods having requisite precision, accuracy, specificity and robustness were developed and validated for quantitative determination of Apixaban in pharmaceutical dosage forms. The first method was based on isocratic reverse phase liquid chromatography using Sunfire C18, 150mm×4.6mm, 5µ and mobile phase consists of Buffer: acetonitrile (60:40) at a flow rate 1ml/min and detection was achieved photodiodide array detector set at 280 nm. The response was linear range of 5-50 µg/ml (R2 =0.9998). The second spectrophotometric method involves detection at 280 nm. The calibration curve range between 5-50 µg/ml (R2 =0.9999). Validation of method was carried out fulfilling ICH guidelines. Both the methods were applied without any interference from excipients, for determination of drug in coated tablets. It is suggested that the proposed HPLC and UV spectrophotometric methods could be used routine quality control and dosage form assay of Apixaban.

To develop a fast and sensitive  UV spectrophotometric method for the quantitative estimation of Formaldehyde in Rivastigmine tartrate drug substances and validate as per ICH guidelines. The method was based upon the observation, that a characteristic colourresults upon addition of  solution of Pentane-2,4-dione , also known as acetylacetone . In acetic acid and ammonium acetate buffer condition, acetylacetone and formaldehyde react to form dimethyl pyridine. Dimethyl pyridine is slightly yellow and its absorption maximum in aqueous solution is λ 420 nmin Rivastigmine tartrate drug substance.The developed method resulted in Formaldehyde exhibiting linearity in the range 0.975 to 234 µg/g. The Intraday and interday precision is exemplified by relative standard deviation of 0.562 % and 0.757%. Percentage Mean recovery was found to be in the range of 98‐101%, during accuracy studies. The limit of detection (LOD) and limit of quantitiation (LOQ) were found to be 1.3 µg/g and 3.9 µg/g respectively.The present work was aimed to develop a visible spectrophotometric method, which is simple, sensitive, accurate and cost effective to evaluate the quality of the bulk drugs.

A simple, rapid, precise LC-MS compatible method for the degradation impurities if any UHPLC method was developed for the determination of 1-deoxynojirimycin (DNJ) in Mulberry leaves. The DNJ in 200 mg dried leaves was extracted twice with 50 mL aqueous 0.05 mol/L HCl, then derivatized by 9 fluorenylmethyl chloroformate (9 FMOC-Cl) in Potassium Borate buffer, and analyzed using a high performance liquid chromatograph equipped with a fluorescence detector. Chromatographic separation of the same was performed by using a Shimpack XR ODS-III (150*3.0mm) 2.1µm as stationary phase with a mobile phase of 0.1% Acetic acid in water: Acetonitrile (60: 40, v/v) at the rate of 0.5 mL/min. Wavelengths used were 254 nm for excitation and 322 nm for emission, which were suitable for detecting the native fluorescence of all the pigments assayed. The derivatized DNJ was well dissolved from the hydrolysis products of 9 FMOC-Cl. The linearity ranged from 0.45 to 66 mg/L, and the detection limit was 0.2 mg/L (S/N = 3). The content of DNJ in Mulberry leaves was 0.09%, the recovery was 90.0%-110.0%.

The emerging new fixed dose combination of Glipizide (GLZ) as sustained release and Telmisartan (TEL) as immediate release were formulated as a bilayer matrix tablet using guggul. Guggul used as a binding agent with three different concentrations (70%, 80%, and 90%; F1-F6) was used for the preparation of first layer of tablet containing glipizide. The conventional tablet of telmisartan was prepared as a second layer. Prepared bilayer tablets were studied for the in vitro release study carried out at pH 1.2 for first two hours and then at pH 6.8 for 6 hours using USP dissolution apparatus II (Basket). All the evaluation tests were found to be within the acceptance limits specified in Indian Pharmacopeia. The assay and dissolution study of tablets were done by HPLC which was developed and validated according to the ICH Q2 (B) guidelines. The contents of assay of the tablets were found to be 90.92% - 103.84% for F2-F5. The release of glipizide from the tablet was extended up to 8 h. Tablet containing 2.5 mg of glipizide with 80% of guggul release of drug after 8 h was found to be 82.36% and tablet containing 5 mg of glipizide with 90% guggul release of glipizide after 8 h was found to be 74.67%. The release pattern of each formulation (F2-F5) was fitted into the dissolution kinetics models and all the formulations were best fitted in the Higuchi model kinetic. From the results, we have concluded that the guggul act as a binding agent as well as rate retarding agent.

The objective of the present research work is to develop a gradient, reversed-phase liquid chromatographic (RP-UPLC) method for the determination of Dutasteride in pharmaceutical bulk drugs for assay and its related impurities. The chromatographic separation was achieved on a Waters ACQUITY TM UPLC C8 Column (100mm×2.1mm, 1.7µm),. The isocratic LC method employs mixture of buffer and Acetonitrile in the ratio of (50:50 v/v) solutions as mobile phase. The buffer solution contains 1.0mM potassium di hydrogen orthophosphate pH adjusted to 5.0 with dil.Potassium hydroxide solution (Buffer) .The flow rate was 0.4 ml/min and the detection wavelength was 210 nm. In the developed UPLC method, the resolution between Dutasteride and its potential impurities, namely Imp-1, Imp-2 and Imp-3 was found to be greater than 4.0. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. Considerable degradation was found to occur in Acidic medium and mild degradation observed in base hydrolysis stress conditions. Degradation product formed during acidic hydrolysis was found to be Unknown impurity. The stress samples were assayed against a qualified reference standard and the mass balance was found close to 99.5%. The developed RP-UPLC method was validated with respect to linearity, accuracy, precision and robustness. The developed method was found to be linear in the range of 2.5-15µg/mL with correlation coefficient of 0.999 for assay procedures and found to be linear in the range of 0.05-3µg/mL with correlation coefficient of 0.999 for related impurities