Validation of an Reverse phase high performance liquid chromatographyMethod for In Vitro Quantification and Degradation Analysis of Naphthol AS-E Phosphate in Bulk Drugs and Nanoparticles.

Objective: This study was designed to conduct forced degradation and validation studies for determination the benzalkonium chloride (BKC) as major homologues form in azelastine hydrochloride pharmaceutical ophthalmic formulation by a novel stability indicating reverse phase high-performance liquid chromatographic method (RP-HPLC).Methods: Forced degradation study and validation were carried out with a analytical approach of reverse phase chromatographic analysis with C18, cosmosil (250mm x 4.6mm i.d. x 5μm) column using mobile phase consisting acetonitrile – buffer of pH 5.0 with 5N NaOH (45:55 %v/v) at a flow rate of 1.5 ml per minute. Column temperature was maintained at 25°C and detection wavelength was 210nm.By using these chromatographic conditions of method, two major homologues of benzalkonium chloride were separated without any interference of drug components and exciepients.Results: Forced degradation studies were carried out as per ICH guidelines and established a stability indicating property of a method .The method was found linear in concentration range 40μg/ml to 60μg/ml (50μg/ml±20%), correlation coefficient was found 0.999. The recovery was found to be 99.2%, 100.7% and 101.4% at ±30% of target concentration. The %RSD for method precision and intermediate precision were found 0.28% and 0.30 % respectively. The method was found specific, selective, precise, accurate, linear, robust and stability indicating for quantification benzalkonium chloride in azelastine pharmaceutical ophthalmic formulation.Conclusion: The method has been found successful for determination of BKC pharmaceutical formulation and validated for all the parameters of validation and found within the acceptance criteria as per ICH guideline Q2R1.

Aim: A simple, rapid, accurate, precise and economical reverse phase high performance liquid chromatographicmethod is developed for simultaneous separation and quantification of two anti-hypertensive drugs, viz., olmesartan medoxomil and indapamide. Materials and Methods: The separation of both the drugs was achieved on ACE C 18 AR column (250 × 4.6 mm id, 5 μm particle size) column using a mobile phase of sodium perchlorate and triethylamine buffer solution (at pH 3): Acetonitrile (60:40 v/v). The flow rate was 1 ml/min and detection was done at 280 nm. Results: The retention time for indapamide was 5.3 min and for olmesartan medoxomil was 6.8 min. Olmesartan medoxomil and indapamide showed a linear response in the concentration range of 50-300 μg/ml and 3.75-22.5 μg/ml respectively. The correlation co-efficients for olmesartan medoxomil and indapamide were 0.9999 and 0.9998 respectively. The percentage recoveries obtained forolmesartan medoxomil and indapamide ranges from 99.3% to 99.8% and 99.7% to 100.9% respectively. The results of the analysis have been validated as per International conference on Harmonisation (ICH) guidelines. Validation results indicated that method shows satisfactory linearity, accuracy, precision, and ruggedness. Conclusion: The extremely low flow rate, simple mobile phase composition makes this method cost effective, rapid, and non-tedious and can also be successfully employed for simultaneous estimation of both drugs in commercial products.

A new stability indicating reverse phase high performance liquid chromatography (RP-HPLC) method for analysis of Naftopidil (NAF), both as a bulk drug and in formulation, was developed and validated. The separation was achieved using a C 18 GRACE column (250 mm × 4.6 mm i.d., 5 μm particle size) and gradient mobile phase system consisting of (A) 10mM of ammonium acetate buffer pH adjusted to 4.0 with glacial acetic acid and (B) acetonitrile. The fl ow rate was 1.0 mL/min with UV detection at 284 nm. NAF was subjected to stress conditions like hydrolysis (acid, alkali and neutral degradation), oxidation, photolytic and thermal decomposition. The linearity of the proposed method was investigated in the range of 10–150 μg/mL. Application of design of experiments for the robustness study method was carried out, where in fi ve factors were selected: pH of mobile phase, fl ow rate, strength of the buffer and column temperature. These factors were examined using JMP@ (SAS Institute) software. Key words: RP-HPLC ; Forced degradation studies ; Naftopidil ; Design of experiments

In this work, an eco-friendly simple, precise reverse phase high-performance liquid chromatography (HPLC) method has been developed and validated for Favipiravir in bulk and tablet dosage form followed by its force degradation study. The proposed method was validated to obtain official requirements including stability, accuracy, precision, linearity, robustness and selectivity as per International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) Guidelines. The estimation was developed on C (18) column reversed-phase using the mobile phase composition as methanol:water (10:90v/v). The flow rate was set as 1ml/min, and the maximum absorption was observed at 323nm using Shimadzu Photo Diode Array detector. The Favipiravir, drug showed a precise and good linearity at the concentration ranges of 10-50μg/mL. The Revearse Phase High Perforance Liquid Chromatography assay showed the highest purity ranging from 99.90 to 100.02% for Favipiravir, tablet dosage form, and 100.15% was the mean percentage purity. The percent recovery was found within the acceptance limit of (98.6-100.0%). Intra- and inter-day precision studies of the method were less than the maximum allowable limit percentage of relative standard deviation ≤ 2.0. The Favipiravir retention time was found to be 5.00min. To examine the stability of the drug, various forced degradation studies were conducted on Favipiravir Active Pharmaceutical Ingredient. The developed method was validated according to the ICH guidelines. A very quick, cost-effective, precise and accurate HPLC method for the determination of Favipiravir has been developed and validated in compliance with ICH guidance Q2.

ABSTRACTA simple and simultaneous reverse phase high-performance liquid chromatographic method was developed for the quantification of aripiprazole (ARI) and two preservatives, namely, methyl paraben and propyl paraben in ARI oral solution. The method was developed on ACE C18 (4.6 × 250 mm, 5 µm) column using gradient elution of 0.1% v/v trifluoroacetic acid in water and acetonitrile as mobile phase components. Flow rate of 1.0 mL/min and 30°C column temperature were used for the method at quantification wavelength of 254 nm. The developed method was validated in accordance with International Conference on Harmonization guideline for various parameters. Forced degradation study was conducted in acid, base, peroxide, heat, and light stress conditions. ARI was found to degrade in oxidation, acid hydrolysis, and heat while it was stable under the remaining conditions. Specificity of the method was verified using Photo Diode Array (PDA) detector by evaluating purity of peaks from degradation samples. Major degradation impurities formed during stress study were identified using liquid chromatography–mass spectrometry method. The present method was useful for determining the content of all the three main analytes present in the oral solution without interference from degradation impurities. The method was robust under the deliberately modified conditions.

Development and validation of RP-HPLC and RP-UPLC methods for quantification of parathyroid hormones (1-34) in medicinal product formulated with meta-cresol

Abiraterone acetate is a well-known anticancer drug and a steroidal derivative of progesterone for treatment of patients with hormone-refractory prostate cancer. Chemometrics-assisted reverse phase high performance liquid chromatography (RP-HPLC) development of the drug abiraterone acetate has been employed in this study using an analytical quality by design (AQbD) approach. Drug separation was performed using a Princeton Merck-Hibar Purospher STAR (C18, 250 mm × 4.6 mm) i.d., 5 μm particle size) with ultraviolet detection at 235 nm. A Box-Behnken statistical experimental design with response surface methodology was executed for method optimization and desired chromatographic separation from its formulation with a few numbers of experimental trials. The impact of three independent variables, namely, composition of the mobile phase, pH, and flow rate, on response retention time and peak area was studied by constructing an arithmetic model from these variables. Optimized experimental conditions for the proposed work include the mobile phase acetonitrile and phosphate buffer (10 mM KH2PO4) (20:80 %v/v). At the concentration range of 2-100 μg/mL, a linear calibration curve was found. Recovery was performed at three concentrations and was foun to be between 98% and 102%. The 3D response surface curves revealed that mobile phase composition and flow rate were the most substantial critical factors affecting desired responses. An attempt has been made to develop and validate an economical, precise, robust, stability-indicating AQbD-based RP-HPLC method that can be employed successfully for the routine analysis of abiraterone acetate in quality control labs.

Ranolazine is a novel anti-anginal drug used in the treatment of chronic stable angina in adults. The present study was designed with an objective to develop and validate a simple, fast, precise, selective and accurate Reverse phase high performance liquid chromatography (RP-HPLC) method for quantitative estimation of Ranolazine in bulk drugs and in selected dosage forms. The efficient chromatographic separation was achieved on a SunShell C18 column (150mm X 4.6mm, 2.6μm particle size) as stationary phase with a mobile phase consisting of buffer (0.01M Ammonium acetate pH adjusted to 5.0 with diluted acetic acid) and the methanol in the ratio of 40:60 v/v at a flow rate of 1.0ml/min and the detection wavelength was 273nm. The method was statistically validated for linearity, accuracy (recovery), precision, specificity, robustness, and forced degradation study. The method was successfully applied for analysis of a tablet formulation.

A precise and feasible reversed-phase high-performance liquid chromatographic method for the determination of an antipsychotic drug quetiapine fumarate (QTF) in pharmaceuticals and spiked human urine sample has been developed and validated. The analysis was carried out using a ODS (250 mm × 4.6 mm i.d., 5 µm particle size) chromatopack column. Mobile phase containing a mixture of acetonitrile and 0.1% phosphate buffer (pH 3.1) (40:60) was pumped at a flow rate of 1 mL/min with UV detection at 240 nm at ambient column temperature (25 oC). The method showed good linearity in the range of 0.09 – 18 µg/mL QTF with limits of detection (LOD) and quantification (LOQ) values of 0.03 and 0.09 µg/mL, respectively. The suggested method was successfully applied for the analysis of QTF in bulk drug, tablets and human urine with average recoveries of 100.06, 100.26 and 98.83%, respectively. The intra- and inter-day RSD values were less than 5%. The method is accurate, precise, sensitive and selective for routine analysis in quality control laboratories.

ABSTRACTObjective: Xanthium strumarium is a cocklebur or burweed belonging to family Asteraceae and commonly found as a weed, is widely distributed inNorth America, Brazil, China, Malasia, and hotter part of India. The herb is traditionally used mostly in treating several ailments. The present studydeals with development and validation of a reliable reverse phase high-performance liquid chromatography (RP-HPLC) method for the simultaneousestimation of Stigmasterol and β-sitosterol in the various extracts of the plant.Methods: The proposed method utilizes a Qualisil Gold C18 column (250×4.6 mm), 5 µm particle size, under isocratic elution conditions with themixture of acetonitrile:ethanol:water (85:14:1 v/v/v) at 25° as a mobile phase. An effluent flow rate of 1 ml/minute and ultraviolet detection at202 nm was used for the analysis of Stigmasterol and β-sitosterol.Results: The described method was linear in the range of range of 100-500 µg/ml and 10-500 µg/ml for stigmasterol and β-sitosterol respectively,with excellent correlation coefficients. The precision, robustness, and ruggedness values were also within the prescribed limits (<2%). The recoveryvalues were within the range, which indicates that the accuracy of the analysis was good and that the interference of the matrix with the recovery ofphytosterols was low. The phytosterols were found to be stable in a stock solution for 24 hrs (percentage relative standard deviation was below 2%)and no interfering extra peaks were observed under controlled stress conditions.Conclusion: The proposed method is simple, specific, precise, accurate, and reproducible and thus can be used as appropriate method for routineanalysis of X. strumarium phytosterols in quality control laboratories.Keywords: Xanthium strumarium, Reverse phase high-performance liquid chromatography method, Precision, Phytosterols, Quality control.

An analytical method was developed using reverse phase high performance liquid chromatograph (RP-HPLC) system and a C-18 reverse-phase column (Nucleodur C18, 250mm × 4.6mm i.d.,5μ) for simultaneous estimation of simvastatin and glimepiride. Acetonitrile and potassium dihydrogen phosphate buffer pH 4 (75:25, v/v) were used as mobile phase. The flow rate was 1 mL min−1 and the chromatogram of both drugs was detected at wavelength of 232 nm. Method was validated as per ICH Q2 (R1) guidelines. The retention times of glimepiride and simvastatin was found to be 4.726 min and 9.829 min, respectively. Both drugs have shown linearity over the concentration range 2–10μg/mL with r2 of 0.997 for GLM and 0.998 for SIM. The mean percentage recovery of both the drugs was found within 98–102% at all the levels which indicated that the method was accurate. The percentage relative standard deviation was found less than 2% which indicated that method was satisfactorily precised. The LOD and LOQ were found to be 0.24 and 0.73 for simvastatin and 0.32 and 0.96 for glimepiride. The method was found to be robust as there was no significant change in response with variation in pH, flow rate and mobile phase composition. It was concluded that the developed method has passed all the validation tests and can be successfully applied to estimate the presence of both the drugs in bulk as well as in pharmaceutical formulations.

A reversed-phase high-performance liquid chromatographic method is developed and validated for the quantitative determination of talinolol and to characterize its degradation products. A very good resolution between peaks is achieved using a C18 column at 40°C. The mobile phase comprises of a mixture of acetonitrile and potassium dihydrogen orthophosphate buffer (pH 4.4) in the ratio of 27:73 (v/v). The method is validated with respect to linearity, accuracy, precision, robustness, and forced degradation studies, which further proved the stability indicating power. During the forced degradation studies, talinolol is observed to be labile to hydrolytic stress and thermal stress (in the solution form). However, it is stable to the oxidative, photolytic, and thermal stress (in the solid form). The degraded products formed are investigated by electrospray ionization (ESI), time-of-flight mass spectrometry, nuclear magnetic resonance, and infrared spectroscopy. A possible degradation pathway is outlined based on the results. The method is found to be sensitive with a detection limit of 0.125 μg/mL and a quantitation limit of 0.378 μg/mL. The method is also demonstrated to be robust, as it is resistant to small variations of chromatographic variables such as pH, mobile phase composition, flow rate, and column temperature.

A sensitive, stability-indicating reversed-phase high-performance liquid chromatographic method is developed and validated for the quantitative determination of retapamulin in topical dosage form. The chromatographic separation is achieved by using a C18 column (XTerra RP 18 250 × 4.6 mm, 5 µm) at 30°C. The mobile phase comprises a mixture of 0.05M potassium dihydrogen phosphate buffer (pH 6.1), acetonitrile and methanol in the ratio of 35:50:15 (v/v/v). The flow rate is set at 1.0 mL/min and chromatograms are extracted at 243 nm using a photodiode array detector. The method is validated with respect to linearity, accuracy, precision, robustness and forced degradation studies, which further prove the stability-indicating supremacy of the method. During forced degradation studies, retapamulin is observed to be labile to oxidative and base hydrolysis stress and stable in thermal, photolytic and acid hydrolysis stress. The degradation products are well separated from the retapamulin peak, thus proving the stability-indicating superiority of the method. The method is found to be sensitive for retapamulin, with a detection limit of 25 ng/mL and a quantification limit of 80 ng/mL. The proposed method is found to be very sensitive and accurate for the determination of retapamulin in topical dosage form. The method is also demonstrated to be robust, because it is resistant to small variations of chromatographic variables such as pH, mobile phase composition, flow rate and column temperature.

The analysis of phenolic compounds in wines is of considerablecommercial importance, since they are known to play the major role in defyingthe sensorial characteristics of wines, such as astringency, flavor, and color,as well as in the browning process, causing product deterioration. In thispaper, an automated reversed phase high performance liquid chromatographicmethod, using a multistep binary gradient elution, is developed for the determinationof five phenolic acids: caffeic, ferulic, vanillic, salicylic, and p-hydroxy-benzoicacid. The separation method was based on mobile-phase optimization and off-linesolid-phase extraction (SPE) from wines and wine vinegar samples, using novelsorbent materials. The analytical column, an Inertsil C8, 250 ×4 mm, 5 μm, used, was operating at ambient temperature. The elution solventswere classified as A: 5–95 (v/v) CH3OH-H2Oand B: 90−10 (v/v) CH3OH - H2O,both A and B acidified with glacial CH3COOH at pH 3–4.The samples were eluted with gradient starting with 10% in B and ending to100% in B. Nicotinic acid was used as internal standard at a concentrationof 10 ng/μL The flow rate of mobile phase was 0.8 mL/min and observed inletpressure ranged from 270 to 345 kg/cm2. A diode-arraydetector monitored the effluent and chromatograms were recorded at 290 and256 nm. Comparing their retention time values and UV spectra in the 190–300nm range with authentic standards stored in a data bank, we made identificationof phenolic compounds. The statistical evaluation of the method was examined performing intra-day(n=8) repeatability and inter-day (n=8) precision assays at three concentrationlevels and was found to be satisfactory, with high accuracy and precisionresults. High percentage recoveries from wines (98.7 ± 5.9, with RSD6.0) and wine vinegar (100.8 ± 6.4, with RSD 6.4) samples were achievedusing Nexus SPE cartridges with hydrophilic and lipophilic properties andsolvent B as eluent.

Theobromine, theophylline, and caffeine retention times in a C18 HPLC column (stationary phase) were investigated as a function of mobile phase flow rate, mobile phase composition, and column temperature. When the mobile phase flow rate increased from 0.1 to 1 mL/min and the methanol concentration increased from 5 to 30%, the retention time and peak width of these three compounds were found to be reduced. While there was a small influence of increasing the mobile phase flow rate on the resolution, decreasing the methanol concentration in the mobile phase considerably reduced the resolution. In addition, the mobile phase flow rate and composition were determined to have a more significant impact than the column temperature. According to the findings, theobromine, theophylline, and caffeine were most effectively separated by liquid chromatography at a flow rate of 0.5 mL/min with a mobile phase methanol concentration of 15%.