International Conference On Harmonization Guidelines Research Articles

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.
 Study Design: Method development and validation was performed as recommended in ICH guideline “Validation of analytical procedures: Test and Methodology Q2 (R1)”.
 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.
 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.
 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.

A Stability Indicating RP-HPLC Method Validation for Simultaneous estimation of Metformin HCl and Canagliflozin in Pharmaceutical Dosage Form

Aims: Canagliflozin and Metformin HCl is a new drug combination for the treatment of Diabetes Mellitus which is one of the oldest and lethal diseases of the mankind. Aim of the research work was to develop and validate novel, rapid, sensitive, specific, robust stability indicating analytical method for the simultaneous estimation of Canagliflozin and Metformin HCl in the pharmaceutical dosage form as fixed dose formulation.
 Study Design: Method development and validation was performed as recommended in ICH guideline “Validation of analytical procedures: Test and Methodology Q2(R1)”.
 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 5.0 and Acetonitrile (60:40 v/v). The flow rate was set to 1ml/min with responses measured at 290 nm, injection volume was 20 µl, and run time of 15 mins.
 Results: The retention time of Metformin Hydrochloride and Canagliflozin was 5.4 min and 7.6 min respectively with resolution of 7.0. Linearity was established in the range of 10-30 µg/ml for Metformin Hydrochloride and 0.5-1.5 µg/ml for Canagliflozin with correlation coefficients more than 0.999. The percentage recoveries were between (98.62-101.22%) and (98.68-101.27%) for Metformin Hydrochloride and Canagliflozin respectively. 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.
 Conclusion: Significance of developed method is that it can be utilize for routine or unknown sample analysis of assay of Metformin HCl and Canagliflozin in pharmaceutical dosage form developed by various Pharmaceutical Industry.

Isolation and Analytical Method Validation for Phytocomponents of Aqueous Leaf Extracts from Vaccinium bracteatum Thunb. in Korea

In this study, major phytochemical compounds of Vaccinium bracteatum Thunb. (VB) aqueous leaf extract were isolated and analyzed using a HPLC-based method, followed by method validation in accordance with the International Conference on Harmonisation (ICH) guidelines for drug development. Five major compounds were isolated in VB extract. Apart from vaccinoside, which had been the only compound isolated in VB extract to date, vanillic acid and protocatechuic acid were isolated for the first time. Isolation of orientin and isoorientin in the VB extract helped validate the reverse-phase analytical method. A new simple and rapid high-performance liquid chromatography (HPLC)-based method was developed for the validation of orientin and isoorientin in VB extract and was determinated according to the ICH guidelines. The analytical method was validated through a Waters Alliance HPLC System containing an e2695 separation module and a 2998 photodiode array (PDA) detector. The VB extract and solutions of orientin and isoorientin were analyzed using a reverse-phase Eclipse XDB-C18 column (4.6 × 250 mm ID, 5 µm, Waters), which was maintained at 30 °C. A mobile phase of methanol and 0.01% formic acid in water was used at a flow rate of 1.0 mL/min to achieve gradient elution. The linearity of the orientin and isoorientin was excellent results (R2 ≥ 0.9999) in the concentration range of 1.0–50.0 μg/mL. Precision values ranged 98.55–101.70% and 98.70–101.18%, respectively. The intra-day and inter-day relative standard deviation (RSD) values of the orientin and isoorientin were all <2.0%. The average recoveries of orientin ranged 98.30–101.57%, whereas isoorientin ranged 97.81–102.14% with RSD values <2.0%. Quantitative analysis found that VB extract contained 2.90 mg/g of orientin and 3.45 mg/g of isoorientin.

A New Stability-indicating Chiral RP-HPLC Method for the Determination of Degradation Products in Meclizine Hydrochloride

Background:An enantiomeric separation of the stability-indicating high-performance liquid chromatographic method was developed and validated for the analysis of Meclizine enantiomers. The degradation behavior of Meclizine Hydrochloride was investigated under different stress conditions recommended by the International Conference on Harmonization (ICH). Methods:Enantiomeric resolution of the drug and complete separation from its degradation products were successfully achieved on a Phenomenex® lux cellulose 1 C18 (250 mm × 4.6 mm i.d, 5 μm particle size) column, using UV detector at a wavelength of 230 nm, with a mobile phase consisting of acetonitrile, 20mM ammonium bicarbonate at the ratio of 75:25 (v/v), and a flow rate of 1 mL/min. The drug was subjected to alkaline, acidic, neutral, oxidative and photolytic conditions in order to mimic stress conditions. Result:The degradation products were well resolved from main peak and proving the stability-indicating power of the method. The developed method provided linear responses within the concentration range 1-5 µg/mL, and regression analysis showed a correlation coefficient value (r2) of 0.999. The HPLC method was validated as per ICH guidelines with respect to specificity, precision, linearity and robustness. Limit of detection (LOD) and limit of quantification (LOQ) were found to be 0.25 µg/mL and 1.00 µg/mL respectively. Conclusion: The method provides good sensitivity and excellent precision and reproducibility. The method was highly selective, where degradation products and co formulated compounds did not interfere. The proposed method was successfully applied in pharmaceutical preparations.

Method Development and Validation of Polmacoxib in Capsule Dosage Form by RP-HPLC

A simple and sensitive RP-HPLC method developed for estimation of Polmacoxib in bulk by using a high performance liquid chromatography with PDA detector using Phenomenex luna C18 column (250mmx4.6mm), 5 µm; mobile phase comprises of Water : ACN as (1:1) at the flow rate 1.0 ml/min and the wavelength of detection 238 nm and eluted at 8.12 minutes. The proposed method is validated for specificity, Linearity, Precision, accuracy, ruggedness, and Robustness. All the parameters were found within the acceptable limits. RP-HPLC method was a simple, reliable economic and acceptable and it confirmed that method is suitable for the intended use for routine quality control and assay of drugs. This method is successfully applied for the determination of commercial dosage form capsule preparation. This method is validated as per ICH (International conference on harmonization) Guidelines.
 Keywords: Polmacoxib, RP-HPLC, ICH, PDA Detector, Precision

Theophylline Determination in Pharmaceuticals Using a Novel High-performance Liquid Chromatographic Process

Objective: The current study aims to find a suitable, accurate, and faster RP-HPLC technique for the determination of theophylline, which could then be validated in accordance with the International Conference on Harmonization (ICH) guidelines. The Aim of this Study: The aim of this study was to develop an efficient, accurate, and faster RP-HPLC method for determining theophylline, which was then validated using the International Conference on Harmonization (ICH) guidelines. Methods: In the HPLC analysis, the Waters 2695 was used. The drug was isolated better using an Ion Pac zorbax 300-SCX Agilent Column, 5 m, 4.6 250 mm, with a liquid phase of Orthophosphoric acid (0.1 percent Orthophosphoric acid in HPLC acetonitrile and Methanol in the ratio of 50:50 v/v at a flow rate of 1ml/min, with discovery at 280 nm using a PDA detector. Results: Theophylline's preservation time was discovered to be 3.747 0.127 min. In the 5-25 mg/l range, the procedure was found to be linear, with a parallel coefficient (R2) of 0.9998. The LOD and LOQ of the system were determined to be (0.99 and 3) g/ml, respectively. The technique and system precisions were predicted using, and the outcomes were determined as percent RSD principles, which were noticed to be within the strict limitations. Theophylline recovery was detected to be in the 99-100 percent range, confirming the method's precision. Conclusion: Using basic ICH guidelines, the suggested RP-HPLC process was validated. The following methodology can be used successfully and easily for routine diagnostic analysis.

RP-HPLC Method Development and Validation of Tapentadol Hydrochloride in Bulk and Pharmaceutical Formulations

Objective: Tapentadol Hydrochloride was approved (November 2008) by the United States Food and Drug Administration for the relief of moderate to severe pain. It is an opioid analgesic, acts by dual mechanism as opioid receptor agonist and norepinephrine reuptake inhibitor. The present research work was aimed to develop an accurate, precise, and rapid RP-HPLC method and subsequently validates the method according to the International Conference on Harmonization (ICH) guidelines for the determination of Tapentadol Hydrochloride.
 Methods: Tapentadol Hydrochloride was analyzed by using High-Performance Liquid Chromatography. Better separation of the drug was achieved by using a Symmetry C18 column (150x4.6mm, 3.5μm) with the mobile phase consisted of a mixture of Orthophosphoric acid (0.1% of Orthophosphoric acid in HPLC water) and acetonitrile in the ratio of 30:70 v/v at a flow rate of 1 ml/min, and the detection was at the wavelength of 219nm using a PDA detector.
 Results: The retention time of Tapentadol Hydrochloride was found to be 3.747 ± 0.127 min. The method was found to be linear in the range of 10-200 ug/ml with a correlation coefficient (r2) of 0.9991. The LOD and LOQ of the method were calculated to be 0.1 and 1μg/ml respectively. The method precision and system precision was estimated and the results were calculated as % RSD values, which were found to be within the limits. Recovery of Tapentadol Hydrochloride was found to be 100.1%, which confirms the efficiency of the method.
 Conclusion: The developed RP-HPLC method was validated using standard ICH guidelines. The developed method can be used for the analysis of both tapentadol hydrochloride bulk and formulations.

Design of Experiment (DoE)-Approach Based RP-HPLC Analytical Method Development and Validation for Estimation of Efavirenz in Bulk and Formulations

Present study reports design of experiment (DoE) based development and validation of a simple, rapid and sensitive reversed-phase high-performance liquid chromatography (RP-HPLC) method for estimation of efavirenz (EFZ), a non-nucleotide reverse transcriptase inhibitor (NNRTs), used in the treatment of acquired immunodeficiency syndrome (AIDS). Plackett-Burman design was explored to screen the critical method variables (CMVs) for the RP-HPLC method. A response surface Box-Behnken design was employed to optimize the screened CMVs which affect the analytical responses (ARs) of RP-HPLC method. Using the optimized CMVs the HPLC method was developed and validated according to International Conference on Harmonization (ICH) guidelines. EFZ in marketed formulation was estimated using the validated method. Acetonitrile proportion, pH of the phosphate buffer and mobile phase flow rate were the CMVs and retention time and number of theoretical plates were the ARs for the study. The optimized chromatographic parameters were acetonitrile proportion in mobile phase: 51.17%v/v, pH of phosphate buffer: 4.04 and flow rate: 1.25mL/min. Use of these optimized parameters resulted in retention time of 11.031min and 9,498.787 number of theoretical plates as ARs of the HPLC method. The method was further validated in harmony with current ICH guidelines Q2 (R1). The method was capable of the successful estimation of EFZ in marketed formulation. The study depicts successful development and validation of a simple RP-HPLC method of EFZ using DoE approach.

A reversed phase HPLC method for the quantification of HIV gp145 glycoprotein levels from cell culture supernatants

A reversed phase high performance liquid chromatography (RP-HPLC) method was developed for the quantitative determination of recombinant HIV-1 gp145 produced in CHO-K1 cells, as measured directly from culture supernatants. Samples were diluted in 50% D-PBS and 50% PowerCHO-2 (PC2) spent medium, and resolved on a Zorbax 300SB-C8 Rapid Resolution (2.1 × 50 mm, 3.5 µm) column, fitted with a C8 guard column (Zorbax 300SB-C8, 2.1 × 12.5 mm, 5 µm), using 0.1% TFA and 2% n-propanol in LC-MS water as mobile phase A and 0.1% TFA, 70% isopropanol, and 20% acetonitrile in LC-MS water as mobile phase B. The column temperature was 80 °C, the flow rate was 0.4 mL/min and the absorbance was monitored at 280 nm. The procedures and capabilities of the method were evaluated against the criteria for linearity, limit of detection (LOD), accuracy, repeatability, and robustness as defined by the International Conference on Harmonization (ICH) 2005 Q2(R1) guidelines. Two different variants of the HIV-1 envelope protein (Env), CO6980v0c22 gp145 and SF162 gp140, were analyzed and their retention times were found to be different. The method showed good linearity (R2 = 0.9996), a lower LOD of 2.4 µg/mL, and an average recovery of 101%. The analysis includes measurements of accuracy, inter-user precision, and robustness. Overall, we present a RP-HPLC method that could be applied for the quantitation of cell culture titers for this and other variants of HIV Env following ICH guidelines.

Development and Validation of a Headspace Gas Chromatographic (HS-GC) Method for Determination of Residual Solvents in Nitazoxanide API

Controlling residual solvents in the drug substances or active pharmaceutical ingredients (API) is mandatory to the specified limits as per the International Conference on Harmonisation (ICH) Q3C guidelines. Residual solvents in pharmaceuticals are mostly determined by Gas Chromatography with Headspace. A simple and sensitive headspace gas chromatographic (HS-GC) method has been developed for the determination of Acetone, Dichloromethane and Cyclohexane in Nitazoxanide API. The separation of analytes was achieved with DB – 624 (30 m length, 0.53 mm inner diameter and 3.0 μm in film thickness) capillary column. Dimethyl formamide was used as a diluent. Nitrogen was used as carrier gas with 3.0 mL/minutes and Flame ionisation detector (FID) for detecting analytes. The oven temperature was set at 60°C for 5 minutes at initial and programmed at a rate of 20°C per minute to a final temperature of 240°C for 2 minutes. Run time was 16 minutes, and total GC cycle time was 25 minutes. The spilt ratio used as 1:20 to get optimum peak response. The developed method was validated as per the ICH guidelines for specificity, accuracy, precision, linearity, range, the limit of detection, the limit of quantification and robustness. The results of validation were indicated no interference, good recoveries, precise, linear, rugged and robust method, suitable for the determination of residual solvents in Nitazoxanide API for research and routine quality control laboratory.

A new gas chromatographic method development and validation for the simultaneous determination of ibuprofen and caffeine in bulk and pharmaceutical dosage form

BackgroundIbuprofen is a nonsteroidal anti-inflammatory drug (NSAID) that has analgesic, anti-inflammatory, and antipyretic properties. Caffeine is one of the most common adjuvant analgesic drugs which is combined with ibuprofen in commercially available formulations. Combining analgesics offers the possibility of increasing effectiveness without increasing dose and therefore risk. Prescribing ibuprofen and caffeine together is common in clinical practice. This is the first work reporting a new and validated gas chromatographic method for the simultaneous determination of ibuprofen and caffeine in bulk and pharmaceutical dosage form. The separation was performed on a TRB-17 column (30.00 m in length, 0.25-mm ID, and 0.25-μm df). Detection was carried out using a flame ionization detector (FID). Methyl paraben was used as an internal standard. The injection volume was 1 μL with 1:50 split ratio using nitrogen as a carrier gas at a flow rate of 1 mL/min. The oven temperature was programmed at 150 °C for 0.5 min, with a rise of 10 °C/min up to 250 °C. The injector temperature was 280 °C, and the detector temperature was 300 °C. The validation of the method including linearity, range, detection limit (DL), quantitation limit (QL), accuracy, precision, specificity, system suitability, and robustness was carried out utilizing International Conference on Harmonization (ICH) guidelines.ResultsThe retention times of methyl paraben, ibuprofen, and caffeine were 1.687, 2.594, and 4.031 min, respectively. The method was linear in the range of 1000–7000 μg/mL for ibuprofen and 162.5–1137.5 μg/mL for caffeine with a correlation coefficient of 0.9999 for both drugs. The DL was found to be 131.68 μg/mL and 15.74 μg/mL for ibuprofen and caffeine, respectively, whereas QL was found to be 399.02 μg/mL for ibuprofen and 47.68 μg/mL for caffeine. The accuracy of the method was validated by mean percentage recovery, which was found to be in the acceptable range. The precision study results of the new method were less than the maximum allowable limit percentage of relative standard deviation %RSD ≤ 2.0.The specificity was evaluated by the standard edition method, and the results of the recovery data showed that excipients do not affect the accuracy of the proposed method. The results of system suitability and robustness tests were also within the acceptable limits.ConclusionThe first reported method for simultaneous determination of ibuprofen and caffeine by gas chromatography in bulk and combined dosage form was carried out in this work. The developed method gave a good separation of the drugs and internal standard. The analytical performance of the method was statistically validated as per ICH guidelines, and satisfactory results were obtained. The proposed method can be easily adopted for the routine analysis of ibuprofen and caffeine.

Univariate and Chemometrics-Assisted Spectrophotometric Methods for Determination of Flibanserin in a Recently Released Dosage Form

Five simple, selective, and rapid spectrophotometric methods are developed for the determination of flibanserin (FL) in the presence of its oxidative degradation products (ODPs). Stress studies are performed according to the International Conference on Harmonization (ICH) guidelines to assess the behavior of FL against oxidative, thermal, and acidic conditions. FL was stable against thermal and acidic conditions, while it was susceptible to oxidative degradation. Three of the spectrophotometric methods were univariate methods, namely, third derivative (D3), ratio difference of ratio spectra (RD), and first derivative ratio of spectra (D1R), while the other two methods were multivariate methods, namely, partial least square (PLS) and principal components regression (PCR). No preliminary separation steps were required in these methods. The chemical structures of the ODPs were confirmed using mass spectrometry and 1H NMR spectroscopy. The proposed methods were developed and validated according to the ICH guidelines. The linearity, accuracy, and precision were determined, and the selectivity was assessed by analyzing synthetic laboratory mixtures containing different ratios of FL and its ODPs. Statistical comparisons were applied to the five spectrophotometric methods, and no significant differences were found. The validated methods could be applied for routine testing and quality control.

VALIDATION OF THE QUALITATIVE DETERMINATION OF HPLC METODS FOR SUCROSE AND PEG-2000-DSPE IN A LIPOSOMAL FORM OF THE PHOTOSENSITIZER LIPOPHTHALOCYAN

Objective: This study was undertaken with the aim of the validate the simple isocratic metods high-performance liquid chromatographic (HPLC) for the estimation of PEG-2000-DSPE and sucrose in liposomal medicinal formulations of the phthalocyanine photosensitizer Lipophthalocyan.
 Methods: HPLC quantification was carried out by using of YMC-Pack Polyamine II column. The mobile phase (for sucrose: acetonitrile: water: ethyl acetate in the ratio of 450: 200: 20; for PEG-2000-DSPE: water in the ratio 10: 90) was pumped at a flow rate of 1 ml/min. Following the guidelines of the International Conference on Harmonization (ICH), the methods was validated for various analytical parameters like specificity, linearity, detection limit, quantitative limit, correctness, and accuracy.
 Results: The obtained results of the analysis were validated statistically. The correlation coefficient for the linearity was 0.999292, for sucrose, and 0.997650 for PEG-2000-DSPE. The methods can be assessed as correct, as the results obtained are close to the true value and the confidence interval for both methods include 100%. The coefficients of variation in both methods in determining the accuracy were less than 3%.
 Conclusion: The proposed HPLC methods were validated according to the ICH guidelines and results and statistical parameters demonstrated that the developed methods are sensitive, precise, reliable and simple for the estimation of PEG-2000-DSPE and sucrose in Lipophthalocyan.

A new validated stability-indicating RP-HPLC method for simultaneous quantification of dolutegravir and lamivudine in bulk and pharmaceutical dosage form

BackgroundA fresh selective, rapid, accurate, precise and RP-HPLC stability-indicating method was developed and validated for the quantitative simultaneous determination of dolutegravir and lamivudine in the bulk as well as pharmaceutical dosage form. A chromatographic separation was done by using Inertsil ODS 3V (250 × 4.6 mm, 5 μm) column and mobile phase composed of phosphate buffer, pH 3.0:acetonitrile:methanol (50:20:30% v/v/v) with flow rate of 1.0 mL/min, and the detection of eluents was carried out at a wavelength of 257 nm utilizing a PDA detector. The drugs, dolutegravir and lamivudine, were subjected to varied conditions like base hydrolysis, acid hydrolysis, oxidation, thermal, photochemical and UV. The suggested method was analysed statistically and validated to fulfil requirements of International Conference on Harmonisation (ICH) and the validation covered accuracy, precision, linearity, limit of detection (LOD), limit of quantification (LOQ), robustness, ruggedness and specificity.ResultsThe retention time of dolutegravir and lamivudine were observed to be 6.36 and 2.16 min, respectively. The method was found to be linear within the range of 14.98 to 91.25 μg/mL for lamivudine and 2.54 to 15.35 μg/mL for dolutegravir. The percentage recoveries (accuracy) for dolutegravir and lamivudine were in the range of 98.35 to 102.14% and 98.01 to 101.5%. The computed relative variance (%RSD) was within the suitable criterion of less than 2.0.ConclusionThe suggested method was set to be precise as well as stability-indicating since no interfering degradant peaks and excipients were evident. All the peaks of degradation were successfully resolved by the use of the developed analytical method with altered retention times. Results obtained were analysed statistically and found to be acceptable in line with the ICH guidelines. Hence, such method is often employed successfully for routine analysis of active analytes in the bulk as well as pharmaceutical dosage form. It is going to be extended to review for its estimation in plasma and other biological fluids and may even be employed for quality control stability sample estimation and in cleaning method analysis during cleaning validation.

Three Spectrophotometric Methods for Quantitative Analysis of Duloxetine in Presence of its Toxic Impurity: 1-Naphthol.

Duloxetine hydrochloride (DUL) is a drug used to treat depression and anxiety. 1-Naphthol is a potential toxic impurity of DUL, as it causes hepatotoxicity in humans, and it is harmful to aquatic life. Three simple, selective, rapid, accurate and precise methods were developed and validated according to International Conference on Harmonization (ICH) guidelines with respect to linearity, accuracy, precision and selectivity for analysis of duloxetine hydrochloride (DUL) in the presence of its potential toxic impurity 1-Naphthol in different laboratory-prepared mixtures and pharmaceutical formulations. Method (A) is the first derivative of the ratio spectra spectrophotometric (1DD) method which allows determination of DUL at 251 nm and 1-Naphthol at 305.2 nm without interference from each other. Method B (dual wavelength) means that two different wavelengths were chosen to each drug, where the absorbance difference at these two wavelengths is equal to zero to the second drug. The chosen two wavelengths for DUL were 221.4 nm and 235.6, where the absorbance difference for 1-naphthol at these two wavelengths was equal to zero. While the chosen wavelengths for 1-naphthol were 247.8 nm and 297 nm, where the absorbance difference for DUL at these two wavelengths was equal to zero. Method (C) is the mean centering of ratio spectra spectrophotometric (MCR) method, which depends on measuring the mean centered values of ratio spectra of both DUL and 1-Naphthol at 226 nm. These methods were validated and agreed with the requirements of ICH guidelines with respect to linearity, accuracy, precision and selectivity. The results indicate the ability of developed methods to be used for routine quality control analysis of DUL in bulk and pharmaceutical formulations in the presence of its potential impurity 1-Naphthol.

ANALYTICAL METHOD DEVELOPMENT AND METHOD VALIDATION FOR DETERMINATION ASSAY AND CONTENT UNIFORMITY OF LEVONORGESTREL BY REVERSED-PHASE HIGHPERFORMANCE LIQUID CHROMATOGRAPHY

Object: The main objective of the complete study is to develop a new method and also to validate the developed method for the determination of Assay and Content Uniformity of Levonorgestrel by reverse-phase high performance liquid chromatography (RP-HPLC).
 Methods: RP-HPLC method was developed for simultaneous estimation of levonorgestrel using Hypersil ODS, 125 mm×4.6 mm×5 μm C8 column with a mixture of water, and acetonitrile solution with a ratio of 50:50 as a mobile phase at a flow rate of 1.3 mL/min with a detection of quantification wavelength of 242 nm. Method was selected after calculating system suitability and validated as per International Conference on Harmonization (ICH) guidelines.
 Results: The developed analytical method parameters found within the limits as given in ICH and USP Guidelines and the total chromatographic analysis time per sample was 8 min with Levonorgestrel Eluting with retention time of 4.479, 4.479, and 4.467 min, respectively. The validated HPLC method was successfully applied for the determination of dissolution of levonorgestrel tablets.
 Conclusion: The method is simple, precise, specific, and accurate. The newly developed method can be used for routine analysis of Levonorgestrel in tablet dosage form.

A Simple, Specific, Mass Compatible and Validated Gas Chromatographic Method for the Estimation of Piperidine-3-amine Content in Linagliptin Finished and Stability Samples without Derivatization

A new simple, selective, highly sensitive, specific (stability indicating), robust, rugged and mass compatible gas chromatographic method and sample with direct injection-mode was developed for the quantitative determination of amino-3-piperidine (3-aminopiperidine, 3-AMP) in linagliptin. As the CAD-LC method did not proved for specificity and pre-derivitizations are challenging task for quality control (QC), a simple GC method has been developed. Compared to LC-CAD method, which is not proved for specificity and pre-column derivitization methods having the limitations to analyze the reaction monitoring in-process samples and degradation samples, the present method is selective, simple, cost effective, QC friendly, widely available GC technique with direct injection and high in sensitivity. Also this method is mass compatible, specificity proved by forced degradation, method was validated as per ICH guidelines. Mass balance was proved by analyzing the stressed samples for net degradation by HPLC and assay by HPLC methods. This GC method also provides an advantage to analyze the in-process samples to monitor the progress of the synthetic process, where the reaction monitoring samples are unpurified or non-isolated samples and contains many process related impurities (reference) and solvents, which will have interference with 3-AMP in LC-CAD and pre-column derivitization methods. This method involves simple sample preparation process and direct injection with GC-FID technique. Hence, this method can be used to analyze the finished product samples, degradation samples, stability samples and reaction monitoring samples. The method was developed with widely available GC column (diphenyl dimethyl polysiloxane as stationary phase, 30 m length, 0.53 mm internal diameter & 5.0 μm thickness), helium as carries gas, FID detector set at 240 ºC, column oven starts at 200 ºC and starts increases after 2 min with 20 ºC/min and holds up to 11 min, which will ensure the column bake. The solvents used for the process were well separated from 3-AMP peak. Mass balance was reported > 99%. The limit of quantification and limit of detection values for 3-AMP were 0.002% (0.4 μg/mL) and 0.007% (1.4 μg/mL) of targeted concentration (20 mg/mL), respectively. The method was precise at LOQ and at specification level with %RSD values 2.8 and 4.7, respectively. Linearity was established in the range of 0.0014 mg/mL (LOQ) to 0.045 mg/mL for 3-AMP with correlation coefficient (r2 > 0.9995). The percentage recoveries were obtained between 99.9% and 104.4%. The range of the method was established from LOQ (0.0014 mg/mL) to 150% (0.045 mg/mL) of the specification targeted limit of 0.15% (0.03 mg/mL). The standard and spiked sample solutions were studied up to 2 days and are stable at room temperature. The forced degradation studies were performed by using HCl, NaOH, H2O2 thermal, UV radiation and water. A mild degradation bout 0.25% was observed in base degradation condition, which was confirmed with mass number by GC-MS analysis. Validation parameters were evaluated according to the International Conference on Harmonization (ICH) Q2 guidelines.

Stability-indicating HPLC-DAD method for the simultaneous determination of fluoroquinolone in combination with a non-steroidal anti-inflammatory drug in pharmaceutical formulation

We developed and validated a stability-indicating assay method for the simultaneous determination of enrofloxacin and piroxicam in combination and in the presence of degradation products. Reversephase high-performance liquid chromatography analyses were carried out on a Vertisep C18 column and acetonitrile-water (48:52 v/v, pH 3.0) mobile phase with a 1.00 mL min−1 flow rate. The efficient chromatographic separation of these drugs and their forced degradation products was achieved in less than 5 min with a peak purity match factor higher than 950. The method used showed linearity in the concentration ranges of 0.25 to 16.0 μg mL−1 for enrofloxacin (r = 0.9997) and 0.125 to 8.0 μg mL−1 for piroxicam (r = 0.9999) as well as precision (relative standard deviation lower than 2%), accuracy (mean recovery 100 ± 2%), and robustness, according to ICH (International Conference on Harmonization) and AOAC (Association of Official Analytical Chemists) guidelines. This method can simultaneously determine the combination of these drugs in a veterinary formulation and separate the drug peaks from their forced degradation products. Additionally, its optimized chromatographic conditions can contribute to the quality control of this formulation in pharmaceutical manufacturing plants and minimize waste from the organic solvent.