RP-HPLC Method For Simultaneous Estimation Research Articles

A new method development and validation for simultaneous estimation of ramipril and telmisartan by RP-HPLC method in combined dosage form

The present research work was development and validation of new RP-HPLC method for simultaneous estimation of Ramipril and Telmisartan in combined dosage form. In simultaneous RP-HPLC method development, Waters 2695 Separations Module with PDA Detector and column used is C8 SB ZORBAX (150 X 4.6mm) column with 3.5-micron particle size. Injection volume of 10 µL is injected and eluted with the mobile phase selected after optimization was Phosphate buffer and Acetonitrile in the ratio of 70:30 was found to be ideal. The optimized flow rate was at 1.0 mL/min. Detection was carried out at 230 nm. This system produced symmetric peak shape, good resolution and reasonable retention times of Ramipril and Telmisartan were found to be 2.275 and 4.261 minutes respectively. The Ramipril and Telmisartan showed linearity in the range of 20-60 µg/mL and 160-480 µg/mL respectively. The %RSD values for precision was found to be within the acceptable limit, which revealed that the developed method was precise. The developed method was found to be robust and the %RSD value for percentage recovery of Ramipril and Telmisartan was found to be within the acceptance criteria. The results indicate satisfactory accuracy of method for simultaneous estimation of the Ramipril and Telmisartan.

Simultaneous Estimation of Atazanavir & Ritonavir in API & Marketed Formulations by Using RP-HPLC

Development and validation of RP-HPLC method for simultaneous estimation of Atazanavir and Ritonavir in their combined tablet dosage form. Atazanavir and Ritonavir are antiviral agents used in treatment of HIV. A simple, precise, rapid, accurate and cost-effective high- performance liquid chromatography (HPLC) method was successfully developed and validated for simultaneous estimation of Atazanavir and Ritonavir in their combined tablet dosage form. The selected mobile phase was Methanol: Phosphate Buffer in proportion 65:35 v/v respectively. The optimized columns used are C18 column, Symmetry and Zodiac column. X bridge C18 (4.6×150 mm, 5 µm) particle size was found to be ideal as it gave good peak shape and resolution at 1ml/min flow for Atazanavir and Ritonavir. In this study, the validation of Atazanavir and Ritonavir in API and marketed formulations were performed keeping in accordance with the parameters like system suitability, specificity, linearity, accuracy, precision (reproducibility & repeatability), robustness. The developed stability indicating method is capable for determination of impurities of Atazanavir and Ritonavir in combined tablet dosage form as well as individual dosage forms also. The method has been successfully validated according to ICH guidelines and the results obtained by using RP – HPLC are rapid, accurate and precise. The proposed methods were successfully applied for the analysis of synthetic mixtures and pharmaceutical formulations of Atazanavir and Ritonavir.

Development and Validation of New Stability Indicating Analytical RPHPLC Method for Simultaneous Estimation of Metformin and Alogliptin.

The current study focuses on developing and validating a reverse phase-high performance liquid chromatography (RP-HPLC) method for quantifying metformin HCL (MET) and alogliptin (ALO) in both dosage and bulk drug forms. ALO and MET in tablet form were determined simultaneously using an RP-HPLC technique. The Reverse Phase (Waters) C18 (4.6 mm x 150 mm; 5 µ) column, a 20-injection loop are the components of the gradient system for the RP manufactured by Agilent Technologies. In the procedure, a 50:50 volumetric combination of methanol and water (0.1% acetic acid) was used as the mobile phase at pH 4.5. The developed approach yielded retention times of 3.856 minutes for MET and 6.302 minutes for ALO. The reliability of the established technique was demonstrated in compliance with the International Council of Harmonization (ICH) standards. In general, all of the metrics including linearity, accuracy, range, and robustness, were found to be within the acceptable ranges. Consequently, the methodology was assessed to be simple, precise, cost-effective, and replicable. Hence, the analysis of MET and ALO in both bulk medication and formulated states can be regularly conducted utilising the procedures specified for the purpose of quality control.

Development and Validation of a Stability Indicating RP-HPLC Method for Simultaneous Estimation of Metformin, Tenegliptin, and Pioglitazone in bulk and its Pharmaceutical Dosage Form .

A straightforward, reliable, and thorough approach was created for contemporaneously estimating Teneligliptin, Metformin, and Pioglitazone in solid dosage form. It was conducted by means of Agilent C18 150x4.6mm, 5µ. The mobile phase comprising buffer, acetonitrile, and Methanol (55:35:10 v/v) passed across the column at a pace of 0.9ml/min. The procedure relied on a 0.01N K2HPO4 buffer having a pH of 3.5, which was achieved by diluting an orthophosphoric acid solution. The temperature was kept constant at 30°C—optimized wavelength for Teneligliptin. Metformin and Pioglitazone were 220.0 nm. Retention times of Teneligliptin, Metformin, and Pioglitazone were 2.970 min, 2.395 min, and 3.642 min. %RSD of a system for Teneligliptin, Metformin, and Pioglitazone were 1.3, 0.6, and 1.0, respectively. %RSD of a method for Teneligliptin, Metformin, and Pioglitazone was 0.6 for each drug. The % recovery was 100.43, 100.45, and 100.22% for Teneligliptin, Metformin, and Pioglitazone, accordingly. The regression equations are used to generate the LoD and LoQ values of Teneligliptin, Metformin, and Pioglitazone, and LoD values were 0.016 ppm, 0.26 ppm, 0.42ppm, while LOQ values of Teneligliptin, Metformin and Pioglitazone were 0.048 ppm, 0.78 ppm,1.28 ppm respectively. The regression equation of Teneligliptin was y = 56655x + 920.87. Metformin was y = 14194x + 3082.1 and of Pioglitazone was y = = 41330x + 792.61.Reduced retention durations make the new approach easy and cost-effective for use in routine industrial quality control testing.

RP-HPLC method for simultaneous estimation of trifluridine and tipiracil in pure and tablet dosage form

RP-HPLC method for simultaneous estimation of trifluridine and tipiracil in pure and tablet dosage form

DoE-Aided Optimization of RP-HPLC Method for Simultaneous Estimation of Amoxicillin and Tinidazole Loaded Mucoadhesive GRDDS Formulation for the Treatment of H. pylori

Helicobacter pylori (H. pylori) infection is one of the primary risk factors of peptic ulcer disease worldwide. Treatment of H. pylori with the conventional dosage form is often challenging due to the ineffective reach of the antibiotics to the inner layers of gastric mucosa, where the organism resides. This study developed an eco-friendly, stability-indicating RP-HPLC method to simultaneously estimate amoxicillin and tinidazole from mucoadhesive formulation targeting H. pylori infection. The mucoadhesive GRDDS formulation of antibiotics was developed with a goal of improving bioavailability at the gastric mucosa. The multivariate Box–Behnken design (BBD) was utilized to optimize chromatographic parameters. Independent variable such as ratio of mobile phase, flow rate, pH and injections volume were optimized using DoE, and analyzed using perturbation plots. A desirability of 0.981 was achieved for the optimized variables. The optimized method utilized methanol and phosphate buffer (25:75) at pH 6.3 as the mobile phase in an isocratic elution mode on a Luna ODS C18 column kept at 25 °C as the stationary phase. The method was linear from 0.25 to 20 µg/mL, for both the drugs with R2 values of 0.9993 and 0.9997 for amoxicillin and tinidazole, respectively. This validated RP-HPLC technique demonstrated selectivity in the presence of possible degradation products and excipients present in the mucoadhesive GRDDS beads. The method was used for the determination of entrapment efficiency and in vitro release profile for tinidazole and amoxicillin in the mucoadhesive GRDDS formulation.Graphical

Development and validation of RP-HPLC method for simultaneous estimation of 5-fluorouracil and silibinin in corn-derived protein-polysaccharide nanocomplex: Forced degradation and mathematical modeling of drug release

In the current work, RP-HPLC method was developed and validated for simultaneous analysis of 5-Fluorouracil (5-FU) and silibinin from corn-derived protein polysaccharide nanocomplex. The method employs a mobile phase of water (A) and acetonitrile: methanol (60:40) (B) on a Waters C18 column (4.6 x 150 mm, 5 μm), with detection wavelengths set at 266 nm and 288 nm for 5-FU and silibinin, respectively. The runtime was 15 min at a flow rate of 0.7 mL/min. The analytical method validation adhered closely to ICH guidelines, exhibiting remarkable selectivity, specificity, linearity, and precision, all falling comfortably within acceptable thresholds. The analytical method was found to be linear from 1 to 150 µg/mL with R2 of 0.9998. Forced degradation studies were carried to assess the stability of 5-FU and silibinin within the nanocomplex across diverse conditions. Additionally, nanocomplexes loaded with 5-FU and silibinin were formulated and characterized, demonstrating a particle size of 209.83 ± 3.83 nm and a zeta potential of 57.93 ± 1.06 mV. The %entrapment efficiency for 5-FU and silibinin was found to be 88.73 ± 0.37 % and 97.00 ± 1.58 % with a %drug loading of 6.73 ± 0.92 % and 2.74 ± 0.61 % respectively. The %in vitro drug release was determined using developed and validated analytical methods. Further, various release kinetic models were applied to determine the release behavior from the nanocomplex system. To conclude, we ensured the eco-friendliness of the developed analytical method by assessing its greenness through Eco-scale, the green analytical procedure index (GAPI), and the Analytical GREEnness metric (AGREE).

METHOD DEVELOPMENT AND VALIDATION OF RAPID ISOCRATIC RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF PARACETAMOL, CAFFEINE, AND PROPYPHENAZONE IN PHARMACEUTICAL FORMULATION

Objective: The objective is to develop a novel, rapid, simple, precise, accurate, and reproducible RP-HPLC method for simultaneous estimation of paracetamol, caffeine, and propyphenazone in bulk and a pharmaceutical dosage form. Method: Optimized chromatographic conditions were used for isocratic elution with Shimadzu C18 (4.6×250 mm, 5 μm), methanol, and 20mM phosphate buffer (60:40, v/v, pH 2.5) as mobile phase, flow rate 1.2 mL/min, and UV detector at λ max 272 nm. The method was validated for specificity, precision, linearity, accuracy, robustness, and solution stability as per the International Council for Harmonization (ICH) guidelines. Result: It was discovered that the retention time for paracetamol, caffeine, and propyphenazone was found to be 2.6 min, 3.0 min, and 7.5 min. The method proved to be rapid, simple, linear (R2>0.999), precise {relative standard deviation (RSD<2.0%)}, accurate (recovery 98–102%), sensitive, and robust. Conclusion: The proposed novel isocratic RP-HPLC method was found to be rapid (short run time of 10 min), highly selective, accurate, and sensitive. The method has been successfully applied to the simultaneous analysis of paracetamol, caffeine, and propyphenazone in a pharmaceutical formulation.

A RP-HPLC Method for Simultaneous Estimation of Vilanterol Trifenatate and Fluticasone Furoate in Dry Powder Inhalation Formulation

A RP-HPLC Method for Simultaneous Estimation of Vilanterol Trifenatate and Fluticasone Furoate in Dry Powder Inhalation Formulation

Analytical Method Development and Validation of Beclomethasone Dipropionate and Fusidic Acid in pure and Pharmaceutical Dosage Form (Cream) by RP-HPLC

A simple, rapid, specific, cheap, eco-friendly, and accurate RP-HPLC method for simultaneous estimation of Beclomethasone Dipropionate and Fusidic Acid in pure and combination drugs has been developed and validated. A stationary phase of BDS Hypersil C18 ((250mm x 4.6m, 5m) and a mobile phase of Acetonitrile: Methanol: Orthophosphoric acid (60:20:20v/v) were used for the separation. The flow rate was continued at 1.0 ml/min and the effluent was observed at 230nm. The designated situations were appropriate for separating Beclomethasone Dipropionate and Fusidic acid eluted with a mean retention time of 7.2 and 5.1 minutes, respectively. The planned technique was validated as per the International Council for Harmonisation recommendation. Its linearity, specificity, system suitability, accuracy and precision, limit of detection, limit of quantitation, and robustness are some of the parameters that have been validated. The concentration range produced linear calibration curves of 1-6g/ml and 2-12g/ml for Beclomethasone Dipropionate and Fusidic Acid with R2 = 0.9961 and 0.9963. Percent RSD values for both intraday and interday were determined to be less than 2.0%. Demonstrating the accuracy of the approach. The technique was proven to be reliable. A successful application of the developed RP-HPLC method for the quantification of Beclomethasone Dipropionate and Fusidic Acid in marketed semi-solid dosage form (Cream).

A Review on Method Development and Validation of RP-HPLC Method for Simultaneous Estimation of Teneligliptin, Metformin Hydrochloride and Pioglitazone Hydrochloride in Pharmaceutical Dosage Form

A Review on Method Development and Validation of RP-HPLC Method for Simultaneous Estimation of Teneligliptin, Metformin Hydrochloride and Pioglitazone Hydrochloride in Pharmaceutical Dosage Form

Development and validation of stability indicating RP-HPLC method for simultaneous estimation of silodosin and mirabegron in synthetic mixture

The study focuses to validate and develop a precise, simple and accurate stability indicating RP-HPLC method for estimation simultaneously of and silodosin and mirabegron in synthetic mixture. The chromatographic separation was achieved by using Shimpack Solar C18 column (250×4.5mm, 5μm) with acetonitrile: 5mM ammonium acetate in ratio of 90:10% v/v as a mobile phase at a constant flow rate of about 1.2mL/min. The development and validation were carried out at detection wavelength of 229nm. We developed a robust RP-HPLC method, validated for linearity, precision, accuracy, specificity, and system suitability. The method demonstrated excellent linearity with correlation coefficient value r2 was nearly 0.998 with linearity range 8-18μg/mL for Silodosin and 24-54μg/mL for mirabegron. LOD and LOQ were found to be lower; hence, the method is sensitive. Percentage recovery was obtained 99.97% and 99.99% for silodosin and mirabegron, respectively. In case of precision, robustness and repeatability, RSD was found to be less than 2. The validated and developed RP-HPLC method offers an efficient and practical approach for the simultaneous quantification of silodosin and mirabegron in pharmaceutical formulations, making it a valuable tool for quality control and pharmaceutical research.

Simultaneous estimation of emtricitabine, tenofovirdisoproxilfumarate and rilpivirine in pharmaceutical dosage form by using rp-hplc

The study describes method development and subsequent validation of RP-HPLC method for simultaneous estimation of emtricitabine, tenofovir disoproxil fumarate and rilpivirine in combined tablet dosage forms. Chromatographic separation was achieved on a hypersilBDSC18 column (250 mm x 4.6 mm, 5 µm) using a mobile phase consisting of (45:55 v/v) buffer: acetonitrile at a flow rate of 1 mL/min. The detection wavelength is 280 nm. The retention times of emtricitabine, tenofovirdisoproxil fumarate and rilpivirine were found to be 2.692, 4.402 min and 5.725 min respectively. The developed method was validated as per ICH guidelines. The developed and validated method was successfully used for the quantitative analysis of emtricitabine,tenofovirdisoproxilfumarate and rilpivirine in tablet dosage forms.

Multipurpose RP-HPLC Method for Simultaneous Estimation of Fixed-Dose Combinations of Anti-diabetic Drugs: Integrating Green, Economical, and Robust Approaches with Design of Experiments and White Analytical Chemistry

Multipurpose RP-HPLC Method for Simultaneous Estimation of Fixed-Dose Combinations of Anti-diabetic Drugs: Integrating Green, Economical, and Robust Approaches with Design of Experiments and White Analytical Chemistry

Development and Validation of an Analytical RP-HPLC Method for Simultaneous Estimation of Losartan and its Active Metabolite (EXP-3174) in Isolated Perfused Rat Liver

Background: The liver perfusion method is frequently used in drug pharmacokinetic studies and the various effects of drugs on liver tissue. The aim of this study was to establish and validate an analytical method using high-performance liquid chromatography to determine the simultaneous concentration of losartan and its active metabolite, EXP-3174, in an isolated perfused rat liver study. Method: An HPLC system with isocratic mode was used. Various chromatographic parameters were adjusted to develop and validate a method for determination of losartan and its active metabolite in liver perfusion media. Results: In this study, losartan and its active metabolite, EXP-3174, were separated using a C18 stationary phase, a mobile phase consisting of acetonitrile: phosphate buffer at a flow rate of 1 mL.min-1, and UV detection at 254 nm. Retention times for losartan and the metabolite were 10 and 16 minutes, respectively. Linearity from 25-250 ng.ml-1 was validated with acceptable accuracy and precision. The LOD and LOQ for losartan were 7.0 and 21.1 ng.ml-1, respectively. The LOD and LOQ for metabolite were 7.4 and 22.4 ng.ml-1, respectively. ChromGate® software was used to acquire and process the data. Conclusions: The optimized and validated technique was effectively used to analyze losartan and its active metabolite in isolated perfused rat liver.

A Validated RP-HPLC Method for Simultaneous Estimation of Benidipine and Telmisartan in Bulk and Tablet Dosage Form

Background: In this work, a novel, simple and accurate reverse phase high-performance liquid chromatographic method with UV detection is presented for the simultaneous estimation of Benidipineand Telmisartan in API and marketed formulations. Objective: To develop and validate this method in accordance to regulatory guidelines i.e. ICH Q2B-R1 for analytical parameters. Methods: In RP-HPLC the separation was achieved using a Xbridge C18 (250x 4.6 i.d; 5µm) column with isocratic elution using a mobile phase consisting of methanol: acetonitrile (80:20v/v/ with 0.1% v/v TEA) adjusted to pH 5.2 with glacial acetic acid at 1mL/min flow for 10min. Benidipine and Telmisartan, were detected at dual wavelength at 237 and 297nm with retention time (Rt) 6.891 and 8.997 minutes respectively. Results: Our new methods (RP-HPLC) were found to (r2>0.999) for both Benidipine and Telmisartan. The developed methods are highly selective, precise and reproducible with %RSD < 2%, Moreover the methods are accurate with recovery more than 99% in both methods. Conclusion: The RP-HPLC method results are efficient, simple and easy. These highly sensitive methods were successfully applied for assay determination of both Benidipine and Telmisartan at fixed dose combination.

Simultaneous Estimation of Eugenol and Scopoletin from the in House <i>Avipattikar Churna</i> by RP-HPLC Method and Estimation of Scopoletin from Different Extracts of <i>Jalap</i>

Background: Although standardising polyherbal medicine requires immediate attention, it is a tedious undertaking. Phytochemical profiling is a particularly useful tool for assessing the quality and effectiveness of polyherbal medicines, among various methods used for standardization. The proposal aimed to develop a precise RP-HPLC method for simultaneous estimation of eugenol and scopoletin in in-house Avipattikar churna. This method was also used to estimate scopoletin in various extracts of Ipomoea turpethum. Methods: The phytomarkers in Avipattikar churna, hydroalcoholic and alcoholic extracts of Jalap were estimated by RP-HPLC system. In this setup, RP-ODS C8 column was employed with methanol: water (30:70 v/v, 0.1% formic acid) at 1 ml/min for 0-10 minutes, and then with methanol: water (60:40 v/v) at 0.8 ml/min for 10.01-25 minutes. Detection was done at 280 nm for eugenol and 366 nm for scopoletin using a UV/VIS detector. The method was validated by performing validation parameters as per ICH guidelines. Results: The linearity of eugenol and scopoletin was performed, with correlation coefficients of 0.999 and 0.9969 respectively. In repeatability, % RSD was observed as 0.856 and 0.909 for eugenol and scopoletin correspondingly. The LOD (detection limit) of eugenol was 0.67 μg/mL and of scopoletin was 1.39 μg/mL. While LOQ (quantification limit) of eugenol was found as 2.04 μg/mL and 4.03 μg/mL for scopoletin. The % recovery was ranging from 102.96 - 100.45 % for eugenol and from 102.65 - 101.3 %w/w for scopoletin, after adding a pre-quantified amount (20 μg/mL) in the different concentrations of the standards. The eugenol and scopoletin were estimated 0.1366 %w/w and 0.0465 %w/w respectively in Avipattikar churna. The hydro alcoholic extract of Jalap showed presence of more scopoletin than in the alcoholic extract. Conclusion: The validated process is established as accurate, consistent and precise which results as a better standardization drive for Ayurvedic dosage forms.

Development and Validation of RP-HPLC Method for Simultaneous Estimation of Gallic Acid, Scopoletin, Umbelliferone, and Imperatorin in Bael avaleha

Bael avaleha is a semisolid ayurvedic formulation containing the fruit of bael, which acts as an anthelmintic to treat diarrhea, dysentery, and IBS. It strengthens the gastrointestinal tract, heals ulcers, and alleviates abdominal pain. The avleha contains excess sugar, which can result in faster absorption of active ingredients in the biological system. Researchers have begun scrutinizing ayurvedic formulations for quality and consistency, with quality testing being conducted to ensure that the quality of ayurvedic formulations adheres to ayurvedic standards. Phytochemical fingerprinting by spectroscopic and chromatographic techniques is becoming more popular in assessing the efficacy of ayurvedic products. Previous studies evaluated phenolic compounds, flavonoids, monoterpenes, and sesquiterpenes in Bael fruit using Gas Chromatography Mass Spectrometry (GC-MS), HPTLC, and HPLC methods.This study evaluated marketed bael avaleha formulations (BA I and II) for organoleptic characteristics, physical parameters, and phytoconstituents. A UV-vis spectrophotometric study was conducted for quantitative analysis of phytochemicals. The extract of bael avaleha formulations BA I and BA II were found to have a total phenolic content of 42.34 ± 0.090 mg GAE/g and 38.52 ± 0.065 mg GAE/g, a flavonoid content of 14.34 ± 0.070 mg QE/g and 12.42 ± 0.086 mg QE/g, sugar content of 69.44 ± 0.020 mg Glu/g and 67.18 ± 0.065 mg Glu/g, and a reducing sugar content of 31.34 ± 0.025 mg Glu/g and 29.08 ± 0.047 mg Glu/g, respectively. The Fourier transform infrared (FTIR) spectra of extract of bael avaleha formulations were recorded in region 4000–400 cm−1 and confirmed the presence of hydroxy group, aromatic C-H stretch, C=C and C-O group. RP-HPLC method was developed and used for the estimation of gallic acid, scopoletin, umbelliferone, and imperatorin in the extract of Bael avaleha formulations

Simultaneous Chromatographic Estimation of Vildagliptin and Dapagliflozin Using Hybrid Principles of White Analytical Chemistry and Analytical Quality by Design.

The fixed-dose combination of vildagliptin (VDG) and dapagliflozin (DGZ) is used in the treatment of type 2 diabetes mellitus. According to the literature survey, RP-HPLC and HPTLC methods have been reported for routine analysis of VDG and DGZ. These chromatographic methods have been developed using potentially neurotoxic and teratogenic solvents, which are unsafe for human and aquatic animal life and hazardous to the environment. These types of organic solvents shall be replaced or reduced during chromatographic analysis of drugs for the safety of human and aquatic animal life and the protection of the environment. The novel white analytical chemistry (WAC) approach has been introduced, which emphasizes robust, green, user-friendly, economical, and rapid analysis of drug samples. Hence, the WAC-based RP-HPLC method has been developed for the estimation of VDG and DGZ using lower toxic and economical solvents. The development of the RP-HPLC method includes the implementation of the analytical quality by design approach using principles of design of experiments to reduce organic waste generation and regulatory compliance of analytical method. The central composite design was applied for response surface modeling (RSM) and optimization of the RP-HPLC method. The method validation was carried out according to ICH Q2 (R1) guidelines. The fixed-dose combinations of VDG and DGZ were assayed, and results were found in compliance with their labeled claim. The published and proposed RP-HPLC methods were assessed for chromatographic analysis of VDG and DGZ using the Red-Green-Blue (RGB) model, AGREE calculator, Eco-Scale Assessment tool, GAPI software, and NEMI standards. The proposed method was found to be robust, green, economical, and user-friendly for chromatographic analysis of VDG and DGZ. The proposed method can be an economical and eco-friendly analytical tool in the pharmaceutical industry for quality control and routine analysis of fixed-dose combinations of VDG and DGZ. Hybrid principles of WAC and analytical quality by design to RP-HPLC method for simultaneous estimation of VDG and DGZ in their fixed-dose combinations.

A Novel RP-HPLC Method for Simultaneous Estimation of Vilanterol Trifenatate, Umeclidinium Bromide and Fluticasone Furoate in Inhalation Dry Powder Formulation.

The dry powder inhalation formulation containing vilanterol trifenatate, umeclidinium bromide and fluticasone furoate intended for the therapy of bronchospasm related to chronic obstructive pulmonary disease and bronchial asthma was selected for the development and validation of a novel, selective, accurate, precise, quick and cost-efficient reversed-phase, high-performance liquid chromatography method. Neither an official monograph nor a single method has yet been published for the simultaneous estimation of these three compounds, which makes this method novel. The stationary phase of an ACE-C18-PFP column (250mm × 4.6mm, 5μ) was used with a mobile phase of 25-mM sodium perchlorate buffer (pH2.5 adjusted with ortho-phosphoric acid) and acetonitrile (40:60% v/v) at a flow rate of 1mL/min to optimize chromatographic variables. The column temperature was kept at 40°C, and detection was at 224nm, which was the isosbestic point of these three drugs. Well-resolved good peak symmetry was obtained for all three molecules by isocratic elution in less than 10min, and the retention times of vilanterol trifenatate, umeclidinium bromide and fluticasone furoate were found to be 3.7, 5.4 and 8.3min, respectively. The proposed method was validated as per ICH Q2 (R1) guidelines, and the calibration curves were linear in concentration ranges of 5-35μg/mL for vilanterol trifenatate, 5-80μg/mL for umeclidinium bromide and 5-150μg/mL for fluticasone furoate, with mean % recoveries of 99-100%. The limits of detection and quantitation are 0.15 and 0.45μg/mL for vilanterol trifenatate, 0.58 and 1.77μg/mL for umeclidinium bromide and 0.32 and 0.96μg/mL for fluticasone furoate, respectively. Hence, the proposed RP-HPLC technique was successfully used to quantify the inhalation formulation containing all three compounds.