International Conference On Harmonization Guidelines Research Articles

LC and LC-MS/MS Studies for the Separation and Identification of Impurities and Degradation Products of Betaxolol.

Betaxolol (Bx) is a selective β1 receptor blocker used in the treatment of hypertension and glaucoma. The aim of the present work was to demonstrate an approach involving use of liquid chromatography (LC) and liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS) for the simultaneous separation, identification and characterization of impurities and of degradation products of betaxolol without their isolation from the reaction mixtures. At optimum condition, and according to ICH guidelines, the limit of detection (LOD) and limit of quantification (LOQ) for Bx are found to be 5.46 and 16.54μgmL-1, respectively. However, the LOD and LOQ for the major degradation product P6 were 2.15 and 6.53μgmL-1. Betaxolol was subjected to hydrolytic (acidic and basic) and oxidative, stress conditions according to International Conference on Harmonization (ICH) guideline Q1A (R2), and as results, the drug was found to be labile in acidic, basic and oxidative stress conditions. Based on LC-ESI/MS analysis, the found results revealed that Bx decomposes in acidic, basic and oxidizing environments. All degradation products were identified with the help of their fragmentation pattern and the masses obtained upon the MS analysis.

Validated chromatographic methods for determination of teriflunomide and investigation of its intrinsic stability

Two rapid, precise, and sensitive stability-indicating high performance chromatographic methods for the measurement of Teriflunomide in its degradation products’ existence were developed. These were RP-HPLC and HPTLC using UV detector. HPLC separation was accomplished utilizing Thermo BDS hypercil C18 column (250 × 4.6 mm, 5 μm) and acetonitrile: 0.03 M potassium dihydrogen phosphate: triethylamine (50:50:0.1%, by volume) as mobile phase at flow rate of 1mL/min. The separated peaks were detected at 250.0 nm. The densitometric approach was conducted utilizing HPTLC 60 F254 silica gel plates, and a developing system of benzene: ethanol: acetic acid (7.5:1:0.25, by volume) and detection was done at 250.0 nm. The developed approaches were evaluated regarding the International Conference on Harmonization (ICH) instructions. The calibration curves of both techniques were constructed with linearity ranges of (5-100) µg/mL and (2–10) µg /band, for HPLC and densitometric determination, consecutively. Teriflunomide was exposed to base and acid hydrolysis, oxidation using H2O2 and finally, thermal degradation as stated in ICH guidelines. The degradation product structures’ elucidation was achieved through LC-MS.

Development And Validation Of Rp-Hplc Method For The Estimation Of Tenofovir And Emtricitabine In Bulk And Pharmaceutical Dosage Form.

A novel, simple, efficient, rapid, and precise reverse-phase high-performance liquid chromatography (RP-HPLC) method was developed for the estimation of Tenofovir and Emtricitabine in the bulk and pharmaceutical dosage form. The currently developed method was subsequently validated according to ICH guidelines in terms of linearity, accuracy, precision, the limit of detection, the limit of quantification, robustness, etc. Methods: The separation of the selected drugs was optimized after several trials including change of mobile phase and its composition, stationary phase, flow rate, column temperature, etc. The separation was performed by using an Inertsil ODS C18 column (250 mm x 4.6 mm, 5μ) and UV absorption was measured at 231 nm. Methanol: Acetonitrile: Water was selected as the mobile phase in the ratio of 50:20:30 (V/V/V) at a flow rate of 1 mL/min. As per International Conference on Harmonization (ICH) Q2 R1 guidelines, several validation parameters were evaluated which include specificity, linearity, precision, accuracy, the limit of detection (LOD), and the limit of quantitation (LOQ). The acceptable degree of linearity range was found to be 40-100 µg/mL. The standard solution exhibited retention times of 3.06 minutes and 5.07 minutes for Tenofovir and Emtricitabine respectively. The LOD and LOQ obtained were 0.05µg/ml and 0.02µg/mL, 15µg/mL, and 0.08µg/mL for Tenofovir and Emtricitabine respectively. The percent recovery was found to be 98 to 102%. Hence, the proposed method is simple, selective, and specifically meets the requirements of ICH guidelines for the validation of the analytical method.

Stress degradation study of Etoricoxib, isolation, and characterization of major degradation impurity by preparative high‐performance liquid chromatography, liquid chromatography‐mass spectrometry, and nuclear magnetic resonance: Validation of ultra‐performance liquid chromatography method

AbstractEtoricoxib (ETO) is a selective cyclooxygenase‐2 inhibitor, used as a nonsteroidal anti‐inflammatory drug. In this study, a detailed investigation was conducted on the degradation behavior of ETO under acidic, basic, neutral, photolytic, oxidative, and thermal degradation conditions as per the International Conference on Harmonization (ICH) guideline Q1A (R2). A gradient “ultra‐performance liquid chromatography (UPLC)” method was developed for separating the degradation products formed under various degradation conditions along with process‐related impurities. Acquity BEH phenyl column was used for the separation of analytes and 0.1% formic acid in water and 0.05% formic acid in acetonitrile:methanol (8:2 %v/v) were used as mobile phase‐A and mobile phase‐B. A major degradant namely, N‐oxide impurity was observed in oxidative degradation along with two minor degradants where N‐oxide was confirmed based on LC‐mass spectrometry (LC‐MS) studies. Further, the major degradant was enriched and isolated using preparative HPLC and the conformation of the N‐oxide impurity was established using nuclear magnetic resonance techniques including two‐dimensional studies and high‐resolution MS. In addition, the method for related substances by UPLC has been validated by considering a major degradant, namely N‐oxide along with process‐related impurities as per the ICH guidelines validation parameters.

Simultaneous UV Spectrophotometric Method for Estimation of Berberine and Withanolide in Polyherbal Immunity Booster Dosage Form

The majority of commercial herbal preparations used to improve immunity lack standardization with regard to the active ingredients marker molecules. The objective of present study is the simultaneous estimation of berberine and withanolide in herbal immunity booster tablet using UV spectrophotometric method. The method was validated according to ICH guidelines. The results indicated that berberine and withanolide have maximal absorption wavelength i.e. λmax at 348 nm and 231 nm, respectively. Withanolide and berberine were found to behave linearly over the concentration range of 5–25μg/mL with R2 value of 0.9998 and 0.9999, respectively. Withanolide and berberine have shown % relative standard deviation (RSD) values of 0.07 and 0.09, respectively, in accuracy study. The intraday and interday precision was deemed adequate with a % RSD fewer than 2. Further, the limit of detection (LOD) and limit of quantization (LOQ) value for berberine and withanolide were found to be 0.12 mg/mL and 0.35mg/mL and 0.07, 0.20 mg/mL, respectively. In conclusion, the proposed method was validated in accordance with the International Conference on Harmonization (ICH) guidelines. The developed method can be employed to determine how much berberine and withanolide are present in pharmaceutical preparations.

Stability indicating reversed-phase-high-performance liquid chromatography method development and validation for pyridostigmine bromide and sodium benzoate in oral solution.

The present study focuses on the development of a simple, rapid, specific, and stability-indicating HPLC method for the simultaneous analysis of pyridostigmine bromide (PGB) and sodium benzoate (SBN) in oral liquid dosage forms. Analytical techniques should enhance sensitivity and specificity for the estimation of pharmaceutical drug products. Stress studies were conducted under various International Conference on Harmonization (ICH) conditions for evaluation. The further optimized HPLC method was validated in accordance with the current ICH guidelines. Chromatographic separation was accomplished using a mobile phase consisting of a 950:50 v/v ratio of perchloric acid buffer and acetonitrile as mobile phase-A, and 100% acetonitrile as mobile phase-B. The flow rate is 1.0 mL/min, and the injection volume is 20 μL. Detection of components was carried out at 220 nm for PGB and 228 nm for SBN. The validated HPLC method demonstrated high specificity, with linearity ranging between 24 and 72 μg/mL for PGB and 5.2-15.6 μg/mL for SBN. The correlation coefficient for both drugs exceeded 0.999. The method demonstrated high accuracy, exceeding 97%. In stress studies, PGB was found to be sensitive to alkaline stress conditions. The results reveal the successful applicability of the current method for the estimation of PGB and SBN in its marketed formulation, which can be reasonably inferred to assess other formulation systems.

Development and Validation of RP-HPLC method for the estimation of Plecanatide from Bulk and Pharmaceutical Dosage form

A novel, simple, efficient, rapid, and precise reverse-phase high-performance liquid chromatography (RP-HPLC) method was developed for the estimation of Plecanatide in the bulk and pharmaceutical dosage form. The currently developed method was subsequently validated according to ICH guidelines in terms of linearity, accuracy, precision, the limit of detection, the limit of quantification, robustness, etc. The separation of the selected drugs was optimized after several trials including change of mobile phase and its composition, stationary phase, flow rate, column temperature, etc. The separation was performed by using an Develosil ODS HG-5 and UV absorption was measured at 246 nm. Ortho phosphoric acid, Triethylamine buffer : Methanol [520 : 480] was selected as the mobile phase at a flow rate of 1 mL/min. As per International Conference on Harmonization (ICH) Q2 R1 guidelines, several validation parameters were evaluated which include specificity, linearity, precision, accuracy, the limit of detection (LOD), and the limit of quantitation (LOQ). The acceptable degree of linearity range was found to be 10 to 60 µg/mL. The percent recovery was found to be 98.55 to 101.88%. Hence, the proposed method is simple, selective, and specifically meets the requirements of ICH guidelines for the validation of the analytical method.

An Analytical Method for Determining N-Nitrosodimethylamine and N-Nitrosodiethylamine Contamination in Irbesartan, Olmesartan and Metformin by UPLC-APCI-MS/MS in Tablet Dosage Form.

N-nitrosamine pollutants are probable carcinogens. Regulatory agencies declared their presence in the drugs unsafe for human consumption and demanded their recall. Using ultra-performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry (UPLC-APCI-MS/MS) in tablet dosage form based on International Conference on Harmonization (ICH) tripartite guideline criteria, we aim to develop and test a new approach for identifying and validating nitrosamine-contaminants, N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) in irbesartan, olmesartan and metformin. The column was Phenomenex Luna-C18, 100 × 3.0mm and 3.0μm. A mobile gradient phase of formic acid in either water or methanol separated the impurities. NDMA and NDEA had retention times of 0.85 and 2.55min, respectively. The detector's linearity was established at concentrations ranging from 0.6 to 100ng/mL. R2 for NDMA and NDEA were 0.9996 and 0.9998, respectively, with a linear response function established at 0.6-100ng/mL. Limit of detection and limit of quantification for NDMA and NDEA were 0.35, 0.29 and 0.55, 0.37ng/mL, respectively. On average, recovery rates for NDMA and NDEA ranged from 96.0 to 98.4 and 96.2 to 98.0%, respectively. The relative standard deviation for NDMA and NDEA was 3.46 and 2.69, respectively. According to the ICH guidelines, the developed method was quick, sensitive and valid. The pharmaceutical formulations of irbesartan, olmesartan and metformin may be regularly examined using the approach provided here.

LC-MS/MS Method Development and Validation for Determination of Favipiravir Pure and Tablet Dosage Forms.

Analytical method development and validation for determination of favipiravir (FVPR) in pure and tablet dosage forms by liquid chromatography with tandem mass spectrometry/mass spectrometry (LC-MS/MS) technique. A simple LC-MS/MS method was developed for determination of a new antiviral drug, FVPR in pharmaceutical formulations. The stationary phase employed was a Shim pack GISS, C18 (100 mm × 2.1 mm, 1.9 μm) column and mobile phase used in pump A was 10.0 mM ammonium acetate and in pump B methanol was used. The gradient program was used with fixed mobile phase flow rate at 0.4 mL min-1. Total run time was 5.0 min. The proposed method was validated according to International Conference on Harmonization (ICH) guidelines. The established method found better outcomes. The linearity graph was found in the range of 50-200 μg/mL and the correlation coefficient value (R2) obtained was found to be 1.0. The limit of detection (LOD) and limit of quantification (LOQ) were 4.044 μg/mL and 12.253 μg/mL, respectively. Tremendous recovery outcomes were observed and found to be 101%, 99.0%, and 99.5% for FVPR at 150% upper, 100% middle, and 50% lower concentrations, respectively. All outcomes obtained comply with ICH guidelines. The developed method was simple, unique, accurate, robust, precise, and reproducible for determination of FVPR in tablet formulation. The method is novel and could be adopted in formulation industry.

Development and Validation of a Spectrofluorimetric Method for the Quantification of Capecitabine in Bulk and Tablets.

A new, simple, and affordable spectrofluorimetric method was established for quantification of capecitabine in bulk and in marketed formulations. Native fluorescence of capecitabine in 0.1% (w/v) cetrimide was measured at 386 nm after excitation at 313 nm. A linear relationship between fluorescence intensity and capecitabine concentration was noticed in 0.2-1.0 μg/mL range. The method was supported by checking several validation parameters as stated using International Conference on Harmonization (ICH) guidelines. The limit of detection (LOD) and quantification (LOQ) values (0.032 and 0.096 μg/mL, respectively) and results of validation parameters demonstrated that the method procedure were sensitive, accurate, precise, and reproducible (% relative standard deviation <2.0). The percentage assay in commercial formulation was found to be 99.2, which agrees with ICH guidelines. Due to the above findings, developed method can be successfully adopted for routine analysis of capecitabine in pharmaceutical dosage forms.

Charge transfer based green spectrophotometric method to determine remogliflozin etabonate applying response surface methodology supported with computational studies in pharmaceutical formulations

Spectrophotometric and computational methods were recognized to determine remogliflozin etabonate (RGE) in pharmaceutical formulations. Remogliflozin’s charge-transfer interaction is significant in pursuing its receptor-effective mechanisms. It helps develop a trustworthy charge-transfer-based spectrophotometric evaluation for remogliflozin with 2, 3-dichloro 5, 6-dicyano 1, 4-benzoquinone (DDQ) and 7,7,8,8-tetracyanoquinodimethane (TCNQ) in its pharmaceutical formulation. The spectrophotometric studies were performed at 445 and 750 nm against the reagent reference for the RGE–DDQ and RGE–TCNQ complexes. The optimization was accomplished by occupying the response surface methodology (RSM) combined with Box-Behnken design (BBD) which included three independent parameters: RGE concentration, DDQ concentration and reaction time. The energy differences associated with the charge transfer band formation were investigated at room temperature. The molar absorptivity and formation constant were attained by employing the Benesi-Hildebrand plot. The method was validated per the International Conference on Harmonization (ICH) guidelines. The current investigation’s linearity was within the range of 10–25 and 5–20 μg/mL, respectively, for the RGE–DDQ and RGE–TCNQ complexes. The formation constant (K) and Gibbs free energy (ΔG°) were found to be 4.58 × 101, 4.55 × 102 L mol−1 and – 2.13 × 103, – 3.6 × 103 k J mol−1, respectively. The results were reproducible with the analysis completed with pharmaceutical dosage samples. The proposed method's greenness profile was determined by applying the Eco-scale protocol. In the forced degradation findings, the degradation amount was within 5–20% as per the ICH guidelines. The extracted RGE was characterized using FTIR spectroscopy and supported with simulated vibrational IR, NMR and UV-visible spectroscopy. The electronic and spectral properties of RGE were examined, and the findings will be useful for further research on RGE.

Development and Validation of Robust, Highly Sensitive and Stability-Indicating RP-HPLC Method for Estimation of Deferasirox and its Degradation Products.

A rapid, simple and highly sensitive stability-indicating reverse-phase high-performance liquid chromatographic technique, coupled with a photodiode array detector, was developed and validated for the estimation of Deferasirox (DFS). The chromatographic separation was achieved using a C-18 (250 × 4.6 mm, 5μm) stationary phase and a mobile phase composed of 0.1% orthophosphoric acid and acetonitrile at a flow rate of 1mL/min. The detection was carried out at a wavelength of 245nm with a constant injection volume of 10μL throughout the analysis. With an R2 value of 0.9996, the calibration curve was determined to be linear over an appropriate concentration range of 50-500ng/mL. According to the International Conference on Harmonization (ICH) Q1 (R2) guideline, DFS was evaluated under stress conditions that included hydrolytic (acid, alkali and neutral), oxidative and thermal degradation. The findings demonstrated that significant degradation was observed in acidic degradation conditions, whereas drug substance was found to be stable when exposed to neutral, basic, oxidative and thermal degradation. The developed method was validated as per ICH guidelines. The developed method was employed successfully to estimate the amount of DFS in bulk and pharmaceutical formulation.

An eco-friendly matrix-augmented fluorescence spectroscopic approach for the analysis of mitoxantrone, an oncogenic therapy; application to the dosage form and biological matrices.

Mitoxantrone (MXN) is a synthetic anthracenedione oncogenic therapy. It is often prescribed as an anticancer agent to manage a variety of cancers. A green, fast, and easy fluorimetric technique for the assay of MXN as a topoisomerase type II enzyme suppressor. An investigation of MXN's fluorescence behavior in various media and solvents constituted the basis for this new technique. Methanol was shown to enhance the intrinsic fluorescence considerably. After excitation at 610 nm, the highest fluorescence intensity was found at 675 nm. Various experimental parameters, such as media, solvents, and pH levels, were tested and adjusted. ICH (International Conference on Harmonization) guidelines were followed when validating procedures. It was possible to achieve linearity in the 0.02-1.50 μg ml-1 with the method. The sensitivity (in terms of limit of detection and limit of quantification) was 0.003 and 0.008 μg ml-1 , indicating low toxicity. As a result, the current technology has a remarkable recovery for detecting residues in diverse bodily fluids. Also, the quantum yield was estimated for the designed system. Finally, the method was rated by eco-scale scoring.

A validated eco-friendly, operation-safe, and cost-effective RP-HPLC method for the determination of dodine in their pesticide dosage forms using ethanol:water as mobile phase

ABSTRACT A validated simple reverse-phase high-performance liquid chromatographic – diode array detection (HPLC–DAD) method was optimised for the analysis of dodine in their pesticide dosage forms by replacing more hazardous acetonitrile and methanol with safe ethanol. The chromatographic analysis was achieved on the Waters, Symmetry C8 column (150 mm × 4.6 mm; 5 µm) using an eco-friendly isocratic eluent; ethanol: water (containing 0.1% v/v trifluoroacetic acid) (50:50, v/v) as the mobile phase with the flow rate of 1.0 mL/minute. The detector wavelength was set at 205 nm. The method was validated as per the SANCO (Directorate-General Health and Consumer Protection) and ICH (International conference on harmonisation) guidelines. The calibration curve showed a good correlation coefficient (0.9998) over the concentration range of 20.04 to 180.34 mg/L. The method was highly precise and robust with a relative standard deviation value of less than 2%. The limit of detection (LOD) and limit of quantification (LOQ) was between 0.80 and 2.40 mg/L, respectively. The extraction recovery of the analyte ranged from 99.38% to 102.00%. Assessment results proved that our analytical method is considered an eco-friendly alternative to conventionally reported methods regarding usage of safe solvents and chemicals, a minimal amount of waste production, and a short time of analysis. Accordingly, the proposed method could be readily applied for quality control purposes as an eco-friendly, simple, and efficient analytical tool.

DEVELOPMENT AND VALIDATION OF THE RP-HPLC METHOD FOR ESTIMATION OF CILNIDIPINE IN RAT PLASMA

Objective: A simple, precise, accurate, robust, and reliable RP-HPLC technique for detecting cilnidipine in Wistar rat plasma was developed and validated. Methods: The chromatographic separation was performed using the C-18 reverse phase column Oyster BDS Premium C18 column (4.6 × 150 mm, 5 µm particle size). The flow rate was set to 1.2 ml/min, and the chromatogram was recorded at 240 nm. The proposed method was validated in compliance with the International Conference on Harmonization (ICH) guidelines. Results: The calibration curves plotted were found to be linear over the concentration range of 40-200 ng/ml in Wistar rat plasma, with a regression coefficient of R2&gt;0.999, suggesting that the linearity was within limits. The LOD and LOQ of the present method were found to be 2.184 and 4.518 ng/ml, respectively. Drug extraction recoveries from Wistar rat plasma were found to be &gt;98%. Conclusion: Using Wistar rat plasma, a simple, fast, specific, accurate, and precise analytical approach was developed and validated. The technique was validated according to the ICH guidelines. The obtained results show that the suggested technique can be simply and effectively utilized for routine cilnidipine analysis in Wistar rat plasma.

Identification and toxicity profiling of degradation impurities of riociguat using tandem mass spectrometry and in-silico studies

In the current scenario, the safety aspect related to impurities and their permissible level in the formulation has gained worldwide importance. The ICH (International Conference on Harmonization) guidelines and the official books have addressed this issue critically as it has tremendous regulatory implications. The present research work addresses the above-mentioned issue for the drug riociguat and toxicity profiling of its degradation impurities. As per ICH Q1A (R2) guideline, the drug was subjected to various stress conditions wherein the drug generated three unknown degradants from the four that were detected. The degradants were separated and characterized using LC and LC-QTOF/MS studies. Better separation was achieved on Agilent's Zorbax XDB column (150 mm × 4.6 mm, 5 µm) using 10 mM ammonium formate (pH adjusted to 3.7 with formic acid) and acetonitrile in the ratio of 72:28 (v/v). The flow rate was 1.0 mL/min and detection was carried out by using UV detector at 322 nm. The chemical structures of all four degradants and drug were subjected for toxicity prediction using in-silico ADMET tools and their relative affinities towards the receptor was compared with riociguat by using molecular docking. The degradants were identified as 3-(diaziridin-3-yl)-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b] pyridine (DP1), methyl (4,6-diamino-2-(3H-pyrazolo[3,4-b]pyridin-3-yl)pyrimidin-5-yl)(methyl)carbamate(DP2), 2-(1-(2-fluorobenzyl)-1H-pyrazolo [3,4-b]pyridin-3-yl)pyrimidine-4,5,6-triamine(DP3) and 6-amino-2-(1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)-7-methyl-7,9-dihydro-8H-purin-8-one (DP4).

Characterization of Oxidative Degradation Product of Canagliflozin by LC-MS/MS

Prior knowledge of chemical stability of drugs directs path for right selection of dosage form, excipients, storage conditions and packaging material. Literature survey revealed that, there are analytical methods reported for quantification and stability indication of Canagliflozin in bulk and formulation. But there is not much information available about the degradation products generated under different stability conditions. With this background, characterization of oxidative degradation product of Canagliflozin was successfully carried out by Liquid Chromatography-Mass Spectrometry (LC-MS/MS) studies. Degradation product was generated by forced degradation, according to International Conference on Harmonization (ICH) guidelines. Degradation product was separated from Canagliflozin by validated reverse phase (RP)-HPLC method using C18 column and Acetonitrile: Water pH 3.0 adjusted with 0.1% formic acid (70: 30, v/v) as mobile phase at a flow rate of 1mL/min. The developed RP-HPLC method was validated for different parameters as per ICH guidelines. The method was found to be linear in a range of 25-225 μg/mL. The developed method was found to be specific, accurate, precise, sensitive and robust. The marketed tablet formulation was analyzed by the developed method and the percent drug content was found to be 100.09 ± 1.96 % w/w. Separated degradation product was characterized by LC-MS/MS studies. From LC-MS/MS data probable structure of the degradation product was interpreted and the mechanism of degradation was proposed. The probable structure of degradation product was proposed as2-(4-Fluorophenyl)-5-({2-methyl-5-[3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]phenyl}methyl) thiophene-1-one. The mechanism of degradation was proposed by S-oxidation of thiophene ring to form thiophene oxide. This information will help synthetic chemists to design a synthesis scheme for the oxidative degradation product, which can be used as a reference standard for impurity profiling. It is also suggested to protect CN from oxidative conditions for improved stability.

A NOVEL VALIDATED STABILITY INDICATING ANALYTICAL METHOD FOR QUANTIFICATION OF EMPAGLIFLOZIN IN BULK AND MARKETED FORMULATION BY RP-HPLC APPLYING EXPERIMENTAL DESIGN APPROACH

A stability indicating reversed-phase high-performance liquid-chromatographic method for analysis of empagliflozin was developed and validated as per the ICH guidelines. Statistical design of experiment was applied for optimization, where independent variables used were methanol proportions in mobile phase and flow rate. Experiment was carried out on an analytical reversed phase column Cosmosil C18 (250 × 4.6 mm, 5 µm). Based on the results obtained from these studies, suitable mobile phase with appropriate composition was selected and utilized for method development applying DoE approach. The mobile phase used was methanol: water (85:15 V/V). The flow rate was set at 0.8 mL min-1 and UV detection was carried out at 225 nm. The retention time of empagliflozin was found to be 4.259 min. The lower solvent consumption along with the short analytical run time (≤05 minute) provides a cost effective and environment friendly chromatographic procedure. The measured signal was shown to be precise, accurate and linear over the concentration range tested (10-50 µg mL-1) with a correlation coefficient of 0.9999. Thus, the proposed methodology is rapid, selective and requires simple sample preparation steps and represents a good procedure for analysis of empagliflozin. Central Composite Design (CCD) was used for method development of empagliflozin. Two factors were selected with eight center points and response of empagliflozin was measured in terms of retention time which dependent on two factors namely, methanol content in mobile phase and flow rate. CCD was effective means in optimization of HPLC for analysis of empagliflozin in pharmaceutical formulation. The stability of the drug was examined over different stress conditions as per International Conference on Harmonization (ICH) guidelines. Results obtained from the force degradation studies indicated that the developed method is appropriate for stability studies.

Development and validation of novel rapid stability indicating mass compatible UPLC method for the simultaneous estimation of assay and related substances of oxytocin bulk and assay of injectable dosage form

Developed and validated a simple, rapid, new mass compatible reverse phase ultra-performance liquid chromatography (UPLC) method for the simultaneous estimation of Assay and related substance of Oxytocin in bulk and extending the same method to injectable dosage form for the estimation of Assay. The developed method is capable of separating all the potential process and degradation impurities within 15 minutes. Chromatography was carried out using Waters Acquity CSH C18 column (50 mm length, 2.1 mm internal diameter and 1.7μ particle size) with gradient elution. 0.1% Trifluoroacetic acid used as Mobile phase A and mixture of 0.1% Trifluoroacetic acid and Acetonitrile in the ratio of 50:50 v/v was used as Mobile phase B. The flow rate of the mobile phase was set at 0.4 mL/min and the column temperature was maintained at 35°C. The detection was carried out using UV detector at 220 nm. The Retention time of Oxytocin is about 3 minutes and all the potential impurities are eluted within 10 minutes. Method validation was performed according to ICH (International Conference on Harmonization) guidelines. The validated method is simple, rapid, mass compatible, economical, specific, accurate, linear, precise, rugged, sensitive, robust and stability indicating.

A Stability Indicating RP-HPLC Method Validation for Simultaneous Estimation of Metformin HCl, Dapagliflozin and Saxagliptin in Pharmaceutical Dosage Form

Aims: Metformin HCl, Dapagliflozin and Saxagliptin is a new drug combination for the treatment of Diabetes Mellitus which is one of the oldest and lethal diseases of mankind. Aim of the research work was to develop and validate novel, rapid, sensitive, specific, robust stability indicating analytical method for simultaneous estimation of: Metformin HCl, Dapagliflozin and Saxagliptin in pharmaceutical dosage form as fixed dose formulation.&#x0D; Study Design: Method development and validation was performed as recommended in ICH guideline “Validation of analytical procedures: Test and Methodology Q2 (R1)”.&#x0D; Methodology: Method develop with chromatographic parameters as C18 column (250mm×4.6 mm, 5mm particle size), HPLC system with PDA detector and mobile phase contained a mixture of Phosphate Buffer pH 3.5 and Acetonitrile (80:20 v/v) + 1 ml triethylamine per 100 ml mobile phase. The flow rate was set to 1 ml/min with responses measured at 265 nm, injection volume was 20 µl, and run time of 15 mins.&#x0D; Results: The retention time of Metformin HCl, Dapagliflozin and Saxagliptin was 5.8 min, 6.8 mins and 8.4 min respectively with resolution of 3.5 between Metformin HCl and Dapagliflozin and 4.5 between Dapagliflozin and Saxagliptin. Linearity was established in the range of 250-1500 µg/ml for Metformin HCl, 1.25-7.5 µg/ml for Dapagliflozin and Saxagliptin with correlation coefficients more than 0.999. The percentage recoveries were between 98.39-101.66 for Metformin HCl, 99.01-101.77 for Dapagliflozin and 98.88-101.87 for Saxagliptin Validation parameters were evaluated according to the International Conference on Harmonization (ICH) Q2 R1 guidelines. The forced degradation studies were performed by using HCl, NaOH, H2O2, thermal and UV Radiation. The developed method was successfully applied for the quantification and hyphenated instrumental analysis.&#x0D; Conclusion: Significance of developed method is that it can be utilize for routine or unknown sample analysis of assay of Metformin HCl, Dapagliflozin and Saxagliptin in pharmaceutical dosage form developed by various Pharmaceutical Industry.