HPLC Method For Simultaneous Estimation Research Articles

Development and Validation of Reverse Phase HPLC Method for Simultaneous Estimation of Allopurinol and Lesinurad in its API and Pharmaceutical Dosage Form

A new, reliable, and validated reverse phase-high-performance liquid chromatography (HPLC) method was developed to quantify the amount of allopurinol and lesinurad simultaneously in solid (tablet) dosage form. A clear chromatographic division was attained on inertsil ODS (4.6 x 250 mm, 5 mm) column, and a mixture of 0.1% trifluoroacetic acid and methanol in the ratio of 40:60 v/v was used as mobile phase. The rate of flow was set at 1 mL/min, and UV detection was achieved at λmax of 255 nm. Injection volume was set to 20 μL. The correlation coefficient of 0.999 was established, and the accurateness was found to be 100.69 and 100.49 for both the drugs, respectively. Therefore, the developed method was simple, specific, precise, and stable. Hence, the method can be employed to estimate the said drugs in other pharmaceutical formulations.

DEVELOPMENT, VALIDATION OF RP-HPLC METHOD FOR THE SIMULTANEOUS ESTIMATION OF TENOFOVIR DISPROXIL FUMARATE, EMTRICITABINE AND RILPIVIRINE HYDROCHLORIDE IN BULK, FORMULATION AND USED IN NANOSUSPENSION

The present research work illustrates the development and validation of RP HPLC method for simultaneous estimation of tenofovir disproxil fumarate, emtricitabine and rilpivirine hydrochloride in bulk and formulated in a pharmaceutical dosage form as a nanosupension. Antiretroviral drug treatment is the primary line of therapy for treating HIV. The multicomponent system formulated as a nanosuspension evidenced increased hydrophilicity, potency and decreased side effects. The separation was carried out by using efficient BDS hypersil C18 HPLC column with empower software. Combination method of Precipitation—ultrasonic homogenization was used for the preparation of the nanosuspension. The mobile phase used was methanol, water, acetonitrile (80:13.4:6.6) v/v and flow rate 1mL /min. The developed method was thus validated as per ICH guidelines for various parameters whose results advocated the reliability of the method. The results for parameters viz. retention times of tenofovir disproxil fumarate, emtricitabine and rilpivirine were 3.09 min, 2.78 min and 3.68 min, linearity range was between 7.5-90, 5-60, 0.625-7.5µg/mL, respectively. Thus the new RP-HPLC method is optimum, reliable and can be used for the simultaneous estimation of tenofovir disproxil fumarate, emtricitabine and rilpivirine hydrochloride.

Stability-indicating HPLC method for simultaneous quantification of 14 impurities in excedrin tablet formulations and identification of new impurity by LC-MS in accelerated stability studies.

We developed novel stability-indicating HPLC method for simultaneous estimation of 14 impurities in excedrin tablet, a formulation with a combination of acetaminophen, aspirin, and caffeine. In addition, a new impurity that was generated through degradation of aspirin at high temperatures during the accelerated stability conditions was positively identified and confirmed, using liquid chromatography-mass spectrometry technique. The HPLC method was optimized using the Inertsustain C18 , 250 × 4.6 mm, 5.0 μm column, employing simple gradient method. Forced degradation studies were performed under acidic, basic, oxidative and thermal conditions to prove the scope and stability-indicating the nature of the method. The optimized method was validated as per the International Conference on Harmonization guidelines. The HPLC method showed linearity from LOQ concentration to 21 μg mL-1 . Precision and intermediate precision values were <5% RSD. The validated HPLC method is currently applied for the routine testing of excedrin tablet formulations in quality control laboratories.

Quality by design based development and validation of HPLC method for simultaneous estimation of paclitaxel and vinorelbine tartrate in dual drug loaded liposomes

This paper describes the development of HPLC method for simultaneous estimation of paclitaxel and vinorelbine tartrate loaded in dual drug liposomes, using quality by design (QbD) approach. The main objective was to identify the robust chromatographic conditions where an adequate separation of the components with quality peaks, within acceptable run time can be achieved. Based on this objective, Target analytical profile (TAP) was defined and systematic risk analysis was carried out to identify critical method attributes (CMA) having impact on critical quality attributes (CQA). % Organic phase, pH, and ammonium acetate concentration in the aqueous phase were identified as CMA. Box-Behnken design was employed to establish the quantitative relationship between CMA and CQA which was further utilized to generate analytical design space and to develop a control strategy. The effective chromatographic separation was accomplished using C18 (4.6 mm, 100 mm, dp 5 μ) column, and mobile phase consisting of acetonitrile – aqueous phase (30 mM ammonium acetate adjusted to pH 3.2 using ortho phosphoric acid) (55:45, v/v) at ambient temperature and at flow rate of 1 mL/min. The elution was monitored at 249 nm using an ultraviolet detector. This developed HPLC method was validated using ICH guidelines. The method has been successfully used for quality analysis of development batches of dual drug liposomes and stability samples and will be applicable throughout the life cycle of the product.

A validated stability-indicating HPLC method for simultaneous estimation of resveratrol and piperine in cubosome and human plasma

Resveratrol and piperine are proven for their therapeutic benefits to treat various diseases. Due to their synergistic actions and combined drug delivery application, a rapid and specific RP-HPLC method was developed and validated as per ICH guidelines, by using an isosbestic point. The chromatographic separation was performed with Luna 5 μ 100 Å C-18(2) HPLC column by using acetonitrile (ACN): phosphate buffer (0.01% orthophosphoric acid) (55:45) as mobile phase, at 1 mL/min of flow rate and 330 nm. The developed method was found to be linear over the concentration range of 0.25–8 μg/mL with correlation coefficient value >0.999. The developed method was accurate (percent recovery 98.06–101.74%), precise (percent relative standard deviation <2.0%), and robust. The limit of detection and limit of quantification for resveratrol were found to be 0.02 and 0.08 μg/mL, respectively and 0.04 and 0.11 μg/mL, for piperine, respectively. The developed method was also validated in human plasma as per ICH guidelines. Moreover, stress degradation studies of both phytoconstituents were studied and the relevancy of the developed method was analyzed on cubosome nanoformulation. A good separation of drug peaks was observed in the presence of the degradation products. This method could thus be used for regular in vitro and in vivo estimation of piperine and resveratrol.

Stability Indicating HPLC Method for Simultaneous Estimation of Nicotinamide and Salicylic Acid

The purpose of the present study was to develop stability indicating HPLC method for simultaneous estimation of Nicotinamide and Salicylic acid and to validate developed method in accordance with ICH guidelines. Stability indicating reverse phase high performance liquid chromatography method was developed for Nicotinamide and Salicylic acid. Various trials with aqueous phase and organic phase were performed and mobile phase was optimized. The separation was achieved using Chromatopak C-18 (250mm x 4.6 mm, 5 µm) column, mobile phase containing Methanol and Water (0.1% TEA and 0.15 gm Hexane sulphonic acid and pH 3.0 adjusted with Glacial acetic acid) in the ratio of (40:60 v/v), at a flow rate of 1.0 ml/min. The retention time for Nicotinamide and Salicylic acid was 4.343 min and 17.673 min respectively. Forced degradation study was performed by applying stress conditions like acid hydrolysis, alkali hydrolysis, and neutral hydrolysis, and oxidation, thermal and photolytic degradation. %degradation was calculated with respect to drugs and specificity.LC method was validated by ICH Q2 (R1) guideline. The linearity was observed in the concentration range of 225-315 μg/ml for Nicotinamide and 75-105 μg/ml for Salicylic acid. The correlation coefficient was found to be 0.9971 and 0.9985 for Nicotinamide and Salicylic acid respectively. Degradation in all conditions was found to be 10 -20%. The method specifically estimates both the drugs in the presence of all the degradant generated during forced degradation study. The developed method was specific and precise and can be used for simultaneous estimation of Nicotinamide and Salicylic acid.

Chromatographic Analysis of Famotidine, Paracetamol and Ibuprofen from Tablet Formulation

Aim of the present work was to develop Reversed phase HPLC method for simultaneous estimation of famotidine (FAM), paracetamol (PCT) and ibuprofen (IBU)in bulk drug and tablet formulation. Separation was carried out on Nucleosil C-18 (15cm, 4.6 mm I.D × 250 mm) using a mobile phase methanol and water (ophosphoric acid, 0.05%) (83: 17, v/v) at a flow rate of 0.7 ml/min. The detection wavelength was set at 270 nm. Retention time of FAM, PCT and IBU were found to be 1.81, 2.58 and 8.58 min respectively. Linear relationship was found in the concentration range of 0.5–3 μg/ml for FAM (r2 = 0.999), 5–25 μg/ml for PCT (r2 = 0.999) and 5–30 μg/ml for IBU (r2 = 0.999). The % purity was found to be 98.40±1.49, 99.86±0.27 and 100.31±0.76, for FAM, PCT and IBU, respectively. The proposed method was validated as per ICH Q2 (R1) guidelines. The mean % recoveries were found be 100.90±1.99, 99.96±0.97 and 100.51±1.72 for FAM, PCT and IBU, respectively.

Development and Validation of HPLC method for simultaneous estimation of Rifampicin and Ofloxacin using experimental design

ABSTRACTA accurate, precise and robust isocratic HPLC method has been developed and validated for simultaneous determination of Rifampicin and Ofloxacin. The chromatographic separation was carried out on Kinetex C18, 100 A Phenomenex column with a mixture of 0.03M Potassium dihydrogen phosphate buffer pH 3.0: Acetonitrile (55:45) as mobile phase at 230 nm. The retention times were 2.91 and 4.87 min for Ofloxacin and Rifampicin, respectively. Calibration plots were linear over the concentration range 1–5 and 2–10 µg/ml for Rifampicin and Ofloxacin, respectively. The method was validated for linearity, sensitivity accuracy, precision, and robustness. Percent recoveries were found to be close to 100% with low variability. Fractional factorial design with four factors was chosen for robustness testing. The volume of acetonitrile and flow rate showed significant effect on retention factor of both the drugs and asymmetry factor of ofloxacin. The method may be adopted for routine analysis at industry.

Validated RP – HPLC Method for Simultaneous Estimation of Dapagliflozin and Saxagliptin Hydrochloride in Tablet Dosage Form

Validated RP – HPLC Method for Simultaneous Estimation of Dapagliflozin and Saxagliptin Hydrochloride in Tablet Dosage Form

Development and Validation of Stability-indicating HPLC method for simultaneous estimation of Tenofovir, Emtricitabine and Efavirenz in fixed dose combination drug product

A novel simultaneous determination of stability indicating method was developed for three combination drug product, the commercially available Atripla tablets were used for development, commercially available tablet combination of the Emtricitabine, Efavirenz, and Tenofovir. The developed novel isocratic HPLC method able to separate the three compounds in shorter runtime than reported methods and offering minimum usage of chemical reagents. The separations was achieved on Kromosil C18 100 x 4.6, 3.5μ Column, Mobile phase 0.1M ortho phosphoric acid buffer and Acetonitrile in 60: 40 v/v ratio, at flow rate of 0.5 mL/min and detection wavelength at 265 nm. The drug product was subjected to various stress conditions as per ICH stress conditions. The developed method was validated with respect to specificity, linearity, accuracy, precision, ruggedness and robustness as per ICH guidelines. The developed method transferred to Quality control for routine testing and stability studies.

A validated HPLC method for simultaneous estimation of Melatonin and Octyl Methoxycinnamate in combined pharmaceutical applications

A simple, fast and precise reverse phase high performance liquid chromatographic method has been developed for the simultaneous determination of Melatonin and Octyl Methoxycinnamate. Melatonin has become an attractive substrate in sunscreen formulations because of its high antioxidant and photo-protection properties. Octyl methoxycinnamate is one of the chemical UV filter that can be found most of the sunscreen formulations up to 7.5 % according Food and Drug Administration. The aim of the present study was to develop and validate a High-Performance Liquid Chromatography method for the determination of Melatonin and Octyl Methoxycinnamate in combined pharmaceutical or cosmetic applications. As a model of combined pharmaceutical applications, a microemulsion consisting of Melatonin and Octyl Methoxycinnamate was also prepared and characterized in terms of droplet size, pH and viscosity. The separation was performed with a Waters XTerra RP C18 (5 μm, 4.6 x 150 mm). All HPLC assays were performed with 10 μl injection volume, mobile phase consisting of acetonitrile and water, using gradient elution starting at 20% and ending at 90% of acetonitrile with a flow rate of 1.5 ml min-1.The eluent was monitored with UV detection at 222 nm for Melatonin and 306 nm for Octyl Methoxycinnamate. The method was validated according to ICH guidelines. Validation parameters were specificity, accuracy, precision (repeatability and reproducibility), linearity, limit of detection (LOD) and limit of quantification (LOQ). Analytical method development results indicated that the LOD values were 0.132 and 0.049 μg/ml; LOQ values were 0.4 and 0.15 μg/ml and assay exhibited a linear range of 0.5- 60 μg/ml for Melatonin and Octyl Methoxycinnamate, respectively.

Development and Validation of Stability Indicating HPLC Method for the Simultaneous Estimation of LevocloperastineFendizoate and Chlorpheniramine Maleate in Pharmaceutical Dosage Form

The aim of the present study was to develop and validate stability indicating HPLC method for simultaneous estimation of LevocloperastineFendizoate (LCP) and Chlorpheniramine Maleate (CPM). HPLC method for simultaneous analysis of both drugs was developed and validated according to ICH guideline. Efficient chromatographic separation was achieved on ODS column C18 (250 mm × 4.6 mm, 5 μm) using the optimized mobile phase. Stability indicating assay method was carried out by different stress degradation conditions. In HPLC method, the Retention time for LCP and CPM was 3.173 min and 5.060 min using optimized mobile phase Phosphate buffer (pH-3.5): Methanol (60:40 %v/v) and UV detection at 273 nm. The degradation of LCP, CPM and Formulation was shown to be highest in alkaline condition. Linearity was observed in concentration range of 20-80 μg/ml for LCP and 4-16 μg/ml for CPM. The correlation coefficient of LCP and CPM were respectively 0.9992 and 0.9994. All validation parameters were within the acceptable range. The LOD and LOQ values for HPLC method were found to be 0.146 μg/ml and 0.444 μg/ml for LCP and 0.0113 μg/ml and 0.0344 μg/ml for CPM respectively. The Method validation parameters showed %RSD value less than 2.

DEVELOPMENT AND VALIDATION OF STABILITY INDICATING CHROMATOGRAPHIC METHOD FOR SIMULTANEOUS ESTIMATION OF SACUBITRIL AND VALSARTAN IN PHARMACEUTICAL DOSAGE FORM

Objective: This study aims to develop and validate a stability indicating HPLC method for simultaneous estimation of sacubitril and valsartan in pharmaceutical dosage form.Methods: Sacubitril and valsartan separation were achieved by LC-20 AT C18 (250 mm x 4.6 mm) column and buffer (potassium phosphate, pH 3.0): methanol (50:50) as mobile phase, at a flow rate of 1 ml/min (millilitre per minute). Detection was carried out at 224 nm (nanometer). The different HPLC experimental parameters were optimized and the method was validated according to the standard guideline. Forced degradation experiments were carried out by exposing sacubitril and valsartan standard and sample for thermal, photolytic, oxidative and acid-base hydrolytic stress conditions.Results: Retention time of sacubitril and valsartan were found to be 4.170 min (minute) and 6.530 min (minute) respectively. The method has been validated for linearity, accuracy, precision, LOD, and LOQ. Linearity observed for sacubitril is 12.25-36.75 μg/ml (microgram per milliliter) and for valsartan is 12.75-38.25 μg/ml (microgram per milliliter). The results showed that sacubitril and valsartan and the other degradation products were fully resolved and thus the proposed method is stability-indicating.Conclusion: The proposed HPLC method was found to be simple, specific, precise, accurate, rapid and economical for simultaneous estimation of valsartan and sacubitril in bulk and tablet dosage form. Thus the validated economical method was applied for forced degradation study of sacubitril and valsartan tablet.

Development and Validation of HPLC Method for Simultaneous Estimation of Famotidine, Paracetamol and Diclofenac in their Raw Materials and Pharmaceutical Formulation

A simple, sensitive and rapid HPLC method has been developed for simultaneous estimation of a ternary mixture of famotidine, paracetamol and diclofenac in their raw materials and pharmaceutical formulations. The analysis was performed on C8 column (250 mm × 4.6 mm i.d., 5-μm particle size) with mobile phase consisted of methanol and 0.01 M sodium dihydrogen phosphate (80:20, v/v) with apparent pH 3.5. The UV detector was operated at 280 nm and the effluents were pumped with flow rate of 1.0 mL/min using felodipine as internal standard. The run time under the optimum chromatographic conditions is less than 6 min. Linearity, accuracy and precision were found to be acceptable over the concentration range of 5-100 μg/mL, 10-300 μg/mL and 5-100 μg/mL for famotidine, paracetamol and diclofenac respectively. The sensitivity of the method allows the determination of the studied drugs with limit of quantification of 3.22 μg/mL, 8.93 μg/mL and 4.16 μg/mL for famotidine, paracetamol and diclofenac respectively. The high sensitivity and the simplicity of the proposed method allow the successful determination of the studied drugs in their combined tablets as well as the determination of famotidine in its dosage forms and in its combined tablets with ibuprofen. Moreover, the proposed method was fully validated according to ICH guidelines.

Application of hplc method for simultaneous estimation of rutin and myricetin from micro and nano powders of leaves of syzygium jambos (l.) Alston

Application of hplc method for simultaneous estimation of rutin and myricetin from micro and nano powders of leaves of syzygium jambos (l.) Alston

Targeting kynurenine pathway in olfactory bulbectomised mice: Inflammatory and neurodegerative pathway of depression

Aims and objectivesThe aim of study was to evaluate the pharmacotherapeutic efficacy of NDGA in experimental paradigm of depression i.e. olfactory bulbectomy (OB) specifically targeting kynurenine pathway.Materials and methodDepression like behaviours was induced in OB mice and evaluated by assessment of various behavioural (olfactory deficit test, forced swim test, splash test, open field test, sucrose preference test), biochemical (catalase, reduced glutathione, SOD, nitrite, MAO-A, MDA, corticosterone), inflammatory cytokines (TNF- α, IL-1β, IL-6, IFN-γ) levels and alterations in delta sleep was recorded using EEG. Kynurenine pathway metabolites were determined in plasma and brain using HPLC method. After 14 days post-surgery, olfactory bulbectomized (OBX) mice were administered nordihydroguaiaretic acid (5 mg/kg, 10 mg/kg and 25 mg/kg) daily i.p.ResultsWe have developed a new HPLC method for simultaneous estimation of monoamines and kynurenine pathway metabolites in plasma and brain samples of mice. Chronic treatment with nordihydroguaiaretic acid significantly restored all behavioural, biochemical and neurochemical alterations in OBX mice and increase in quinolinic acid and decrease in kynurenic acid point out the neurodegeneration hypothesis of depression.ConclusionNordihydroguaiaretic acid showed potent neuropharmacotherapeutic effect in OBX mice by virtue of its strong anti-oxidant, anti-inflammatory, anti-stress and by restoring quinolinic acid levels.

Development and Validation of Stability-indicating HPLC Method for Simultaneous Estimation of Cefixime and Linezolid.

A stability-indicating reverse phase high performance liquid chromatography method was developed and validated for cefixime and linezolid. The wavelength selected for quantitation was 276 nm. The method has been validated for linearity, accuracy, precision, robustness, limit of detection and limit of quantitation. Linearity was observed in the concentration range of 2-12 μg/ml for cefixime and 6-36 μg/ml for linezolid. For RP-HPLC, the separation was achieved by Phenomenex Luna C18 (250×4.6 mm) 5 μm column using phosphate buffer (pH 7):methanol (60:40 v/v) as mobile phase with flow rate 1 ml/min. The retention time of cefixime and linezolid were found to be 3.127 min and 11.986 min, respectively. During force degradation, drug product was exposed to hydrolysis (acid and base hydrolysis), H2O2, thermal degradation and photo degradation. The % degradation was found to be 10 to 20% for both cefixime and linezolid in the given condition. The method specifically estimates both the drugs in presence of all the degradants generated during forced degradation study. The developed methods were simple, specific and economic, which can be used for simultaneous estimation of cefixime and linezolid in tablet dosage form.

A gradient based facile HPLC method for simultaneous estimation of antioxidants extracted from tea powder.

A new simple, rapid and precise RP-HPLC method was developed for the extraction and quantitative estimation of caffeine (C), (-)-epigallocatechin gallate (EGCG), (+)-catechin(Ct), (-)-epicatechin(EC), and (-)-epicatechin gallate (ECG) (collectively named as Tea Powder Bioactives TPBAs) extracted from tea powder using different ratios of ethanol: water. The simultaneous determination of TPBAs was performed using the UV spectrophotometric method which employs the absorbance at 205nm (λmax of caffeine and polyphenols). This method is a gradient based HPLC method with a flow rate of 0.8mL/min using Inertsil ODS 100×4.6mm, 3μm column with methanol and ammonium dihydrogen phosphate (pH-2.8) as mobile phase. The method was validated in terms of specificity, precision, linearity, accuracy, limit of quantification (LOQ), and limit of detection (LOD). The linearity of the proposed method was investigated for concentration ranging between 0.5-60μg/mL with regression co-efficient, R(2)=0.999-1.0. This method estimates all the TPBAs simultaneously with enhanced precision and linearity as per the ICH guidelines. Also, to confirm the individual TPBA, the antioxidant property of the each TPBA was analyzed which was commensurate with that of the previous reports.

Uncovering interaction by QbD in Optimization of Stability-indicating HPLC Method for Simultaneous Estimation of Low Dose Aspirin and Lansoprazole

In the present study a QbD acquiescent method utilizing design of experiment and method operable design regions methodology was evaluated for optimization of chomatographic condition for separation of ten degradation products along with peaks of aspirin and lansoprazole. Box-Behnken design was exploited to evaluate the main and interaction effects of the selected critical process parameters on the critical quality attributes, viz resolution between thee peak pairs and retention time of last eluting peak. The optimal separation was predicted at pH 3.6, with a gradient starting at 20% of acetonitrile and initial hold time of 10 min. The results of experimental methods show excellent agreement with predicted results, highlighting the importance of design of experiments in method optimization. The selected working condition was then fully validated according to ICH guidelines for linearity, range, accuracy, precision and robustness.

Development and Validation of New HPLC Method for Simultaneous Estimation of L-Lysine Hydrochloride and L-Carnitine-L-Tartrate in Pharmaceutical Dosage Form.

A new analytical method is developed and validates for simultaneous estimation of L-lysine hydrochloride and L-carnitine-L-tartrate in single pharmaceutical dosage form by means of high resolution HPLC. The chromatographic procedure was carried out using C18-5 μm (4.6 mm × 250 mm), column. Optimally suited mobile phase was made to run in an isocratic elution mode whose composition was 10 mM of potassium dihydrogen phosphate adjusted with triethylamine to a pH of 7.5 and pumped at a flow rate of 0.50 ml/min through chromatographic system. The detector's wavelength was 214 nm. The accuracy of the analytical method was determined keeping into account the recovery of analytes, which in this case remains well within the accepted standards that is 95-105%. Detection limit for L-lysine hydrochloride and L-carnitine-L-tartrate are 1.47 μg/ml and 0.85 μg/ml while quantitation limit for L-lysine hydrochloride and L-carnitine-L-tartrate is 4.41 and 2.55 μg/ml, respectively. All the statistical results were validated performing precision, accuracy, linearity, specificity studies for analytes concentration from 70-130%. The outcome of results confirmed that the method was in consonance with the acceptance criteria.