Reversed-phase High-performance Liquid Chromatographic Method Research Articles

QbD-based RP-HPLC method development for quantitative computation of phase III composition comprising apixaban and clopidogrel

The presented work highlights the use of the quality by design methodology in accordance with International Council on Harmonization (ICH) recommendations for developing an reverse phase high performance liquid chromatographic (RP-HPLC) method for apixaban (APX) and clopidogrel (CLP). An efficient approach based on a factorial design incorporating the essential method parameters of the RP-HPLC method such as flow rate, pH, and proportion of organic phase, is presented. The optimum conditions for analysis were derived by using Design Expert software 10.0 Version, i.e., Hypersil ODS C18 column (5.0 μ, 25 cm × 4.6 mm), methanol and 0.05M potassium dihydrogen phosphate buffer (63.5:36.5%v/v, pH 3) as mobile phase, and 0.8 ml/minute as the flow rate. The derived condition gave excellent resolution between APX and CLP with Rt of 4.89 and 14.35 minutes, respectively, with the best possible system suitability parameters. The developed method presented excellent linearity in the range of 1.25–3.75 μg/ml for APX and 37.5–112.5 μg/ml for CLP at 245 nm. The optimized method was further validated in accordance with ICH guidelines on analytical method validation. Finally, the approach was successful in determining APX and CLP from a binary combination.

A SIMPLE RP-HPLC METHOD DEVELOPMENT AND VERIFICATION OF THE DISSOLUTION OF BROMELAIN-A COMPLEX MIXTURE OF PROTEOLYTIC ENZYMES, IN DELAYED-RELEASE TABLETS

Objective: To develop a simple, accurate, precise and linear Reverse Phase High-Performance Liquid Chromatographic (RP-HPLC) method and verify for the quantitative estimation (Dissolution) of Bromelain in delayed-release tablets. Methods: The optimized RP-HPLC method for both acid and buffer stage dissolutions of delayed-release tablets uses Zorbax 300 SB-C8 column (150 mm X 4.6 mm; 3.5μ), a mobile phase-A of 0.1% trifluoroacetic acid in water and a mobile phase-B of 0.1% Trifluoroacetic acid in Acetonitrile in the gradient proportion, flow rate of 1.0 ml/min, injection volume of 25 µl, detection wavelength of 280 nm using a UV/PDA detector, column temperature of 40 °C, sample tray/compartment temperature of 5 °C and a run time of 20 min. Results: The developed method gave Bromelain eluting at about 6 min. Bromelain exhibited linearity in the range 53.4-800.6 μg/ml (r2=0.99992). The precision is exemplified by relative standard deviation of 1.3 and 2.3% for acid and buffer stages, respectively. Percentage recovery of the drug was found to be between 90.0 and 110.0 during accuracy studies. Conclusion: A simple, accurate, precise, and linear RP-HPLC method was developed and verified for the quantitative estimation (Dissolution) of Bromelain in tablets and hence this method can be explored for the analysis of Bromelain in tablets in various pharmaceutical industries.

Quantification of rifampicin loaded into inhaled polymeric nanoparticles by reversed phase high-performance liquid chromatography in pulmonary nonphagocytic cellular uptake.

Rifampicin is an antibiotic effective against both Gram-negative and Gram-positive bacteria and is commonly used as a first-line treatment for tuberculosis caused by Mycobacterium tuberculosis. In this study, a reversed-phase high-performance liquid chromatography method was developed and validated to assess rifampicin, either free or combined with ascorbic acid, loaded into chitosan/Tween 80-coated alginate nanoparticles. The method utilized a reversed-phase C18 Restek column with specific chromatographic conditions: a mobile phase of 60 : 40 ratios of methanol/buffer phosphate (pH 7.0), at a flow rate of 0.8 mL min-1, and an injection volume of 15 μL. rifampicin and the internal standard (rifamycin) had retention times of 4.0 and 2.5 min, respectively, and were detected at 334 nm. The method demonstrated the stability of stored samples after freezing-thawing cycles and specificity for rifampicin, even in the presence of degradation products from stress conditions. The high-performance liquid chromatography method was found to be specific, precise, robust, and sensitive. Results indicated that rifampicin accumulation and uptake kinetics varied based on cell type, formulation (free or loaded in nanoparticles), rifampicin concentration, and incubation time. Confocal fluorescence microscopy images supported these findings, showing isothiocyanate fluorescein nanoparticles distribution in different intracellular regions depending on the cell type used. The societal impact of this research lies in its potential to advance the treatment of respiratory infectious diseases, such as tuberculosis, through the development of more effective and specific drug delivery methods. By optimizing the way drugs, particularly rifampicin in this case, interact with lung cells, we aim to achieve greater treatment efficacy and alleviate the overall burden of disease. Furthermore, our study offers novel insights into the intracellular behavior of rifampin from polymeric nanoparticles, paving the way for personalized medicine approaches in the treatment of respiratory infections. This dual focus on social impact and innovation underscores our commitment to improving global health outcomes and addressing pressing public health challenges.

Inflammatory-stimuli-responsive turn-on NIR fluorogenic theranostic prodrug: adjuvant delivery of diclofenac and hydrogen sulfide attenuates acute inflammatory disorders.

Prolonged use of very commonly prescribed non-steroidal anti-inflammatory drugs (NSAIDs) is often associated with undesired side effects, including gastrointestinal ulcers due to the non-selective inhibition of cyclooxygenases. We describe the development of an inflammatory-stimuli-responsive turn-on fluorogenic theranostic prodrug DCF-HS for adjuvant drug delivery. Upon activation by reactive oxygen species (ROS), the prodrug releases diclofenac DCF (active drug) and the NIR fluorophore DCI-NH2 along with carbonyl sulfide (COS). The second activation of COS by the enzyme carbonic anhydrase (CA) generates hydrogen sulfide (H2S). The prodrug was conveniently synthesized using multi-step organic synthesis. The UV-Vis and fluorescence studies revealed the selective reactivity of DCF-HS towards ROS such as H2O2 in the aqueous phase and the desired uncaging of the drug DCF with turn-on NIR fluorescent reporter under physiological conditions. Furthermore, the release of fluorophore DCI-NH2 and drug DCF was confirmed using the reverse phase HPLC method. Compatibility of prodrug activation was studied next in the cellular medium. The prodrug DCF-HS was non-toxic in a representative cancer cell line (HeLa) and a macrophage cell line (RAW 264.7) up to 100 μM concentration, indicating its biocompatibility. The intracellular ROS-mediated activation of the prodrug with the release of NIR dye DCI-NH2 and H2S was investigated in HeLa cells using the H2S-selective probe WSP2. The anti-inflammatory activity of the active drug DCF from the prodrug DCF-HS was studied in the lipopolysaccharide (LPS)-induced macrophage cell line and compared to that of the parent drug DCF using western blot analysis and it was found that the active drug resulted in pronounced inhibition of COX-2 in a dose-dependent manner. Finally, the anti-inflammatory potential of the prodrug and the turn-on fluorescence were validated in the inflammation-induced Wister rat models.

VALIDATED STABILITY INDICATING RP-HPLC METHOD FOR QUANTIFICATION OF SPARSENTAN IN PHARMACEUTICAL DOSAGE FORM: A 2023 USFDAAPPROVED DRUG

The quantitative analysis of Sparsentan in the pharmaceutical dosage form has been accomplished through the development and validation of a simple rapid, precise, and sensitive reverse-phase high-performance liquid chromatography (RP-HPLC) method. Using a Zorbax SB 18 (150 x 4.6 mm, 3.5 µm) column and a mobile phase containing acetonitrile and potassium dihydrogen phosphate pH-3.0 adjusted with diluted orthophosphoric acid in a ratio of 40:60% v/v, Sparsentan was separated chromatographically using a Waters Alliance-e2695. The flow rate was 1.0 mL/min, and a photodiode array detector (PDA) operating at room temperature was used for absorption detection at 287 nm. For Sparsentan, the theoretical plate count was greater than 2000 and the tailing factor was less than 2, respectively. All measurements have a relative standard deviation of less than 2.0 for their peak areas. The suggested approach was verified in compliance with ICH Q2(R1) standards. The approach was determined to be an easy, affordable, appropriate, robust, precise, and accurate way for the quantitative study of Sparsentan.

Box-Behnken guided development of an ecofriendly RP-HPLC analytical method for simultaneous quantification of pantoprazole sodium and piperine co-loaded mucoadhesive GRDDS formulation for H. pylori eradication

Helicobacter pylori (H. pylori) infection is the leading cause of chronic peptic ulcer disease worldwide. Many treatment options are available to treat H. pylori infection. However, the eradication is still a challenge due to the poor bioavailability of the currently available formulations. To improve the efficacy of therapy, novel formulations are necessary. Proton pump inhibitors (PPIs) are already a part of the treatment regimen with antibiotics. P-glycoprotein (P-gp) inhibitor was reported to have increased the efficacy of antibiotic treatment in H. pylori infections. To make available the P-gp inhibitor and PPI on the intestinal mucosal surface we have formulated a gastroretentive drug delivery system (GRDDS) as an adjuvant therapy with antibiotics. The objective of this study is to simultaneously estimate pantoprazole (PAN) and piperine (PIP) by reverse-phase (RP) high-performance liquid chromatography (HPLC) method from the chitosan-based sodium alginate mucoadhesive beads utilizing the design of experiments (DOEs) methodology. The HPLC settings were optimized using DOEs software. The final optimized HPLC method used a hyperclone Octadecylsilane C18 column as the stationary phase and methanol: ammonium acetate at pH 4.5 (70:30 v/v) as the mobile phase. The flow rate was 0.9 ml/minute. The validation of the developed RP-HPLC method was done as per the International Conference on Harmonization (ICH) Q2(R1) guideline. The method was linear from 0.5 to 20 μg/ml for both PAN and PIP with an R2 value of 0.999 and 0.999, respectively. The validated RP-HPLC method showed specificity for both drugs despite interference from degradation products and other GRDDS excipients. The entrapment efficiency of the final formulation was determined to be 80%–85% for PAN and 60%–67% for PIP. The novelty and merit of the DOE-based method development are that it reduces the number of trials, thereby reducing reagent wastage, and is environmentally friendly suggested by the Green Analytical Procedure Index (GAPI) tool scoring six green, six yellow, and three red.

Analytical method development and validation of antifungal drugs in updated ointment formulation using UV spectroscopy and RP-HPLC

A precise, simple, and validated Reverse-phase high-performance liquid chromatography (RP-HPLC) method alongside spectrophotometric analysis has been established for the simultaneous quantification of clobetasol propionate, miconazole nitrate, and salicylic acid in an updated ointment formulation. The impact of organic modifiers on the retention of the target compounds was assessed. The chromatographic analysis was conducted using a simple low-pressure gradient method with UV detection at 282 nm on a C18 column (5 ?m, 4.6 ? 250 mm HSS). The mobile phase consisted of a mixture of methanol, acetonitrile, and tetra ethylamine acetate buffer at pH 4, adjusted with acetic acid in a 40:40:20 v/v ratio, at a flow rate of 1.2 mL min-1. The method demonstrated linearity in the 5-15 ?g mL-1 concentration range for clobetasol propionate, 30-90 ?g mL-1 for miconazole nitrate, and 15-45 ?g mL-1 for salicylic acid. The limits of detection and quantification were determined to be 1.49 ?g mL-1 and 4.53 ?g mL-1 for clobetasol propionate, 8.72 ?g mL-1 and 26.43 ?g mL- 1 for miconazole nitrate, and 3.37 ?g mL-1 and 10.22 ?g mL-1 for salicylic acid, respectively. The recoveries for drugs in formulation range from 95 % to 99 %.

Development and validation of a green RP-HPLC method for detection and quantitation of meropenem trihydrate in the bulk: A comparison with HPTLC method

This research introduces an eco-friendly green Reverse-Phase High Performance Liquid Chromatography (RP-HPLC) method for detecting and quantifying meropenem trihydrate. Traditional methods use buffered solutions, gradient mobile phases, and longer retention times, but this method offers a rapid RP-HPLC technique with a C8 column and isocratic mobile phase (60% methanol, 40% ultra-pure water), eliminating the need for buffers and acetonitrile. It features a short 2.1-minute retention time and a high r² value of 0.9995. It delivers accuracy (101.1-102.3%) and precision (RSD ≤ 2%), coupled with low LOD and LOQ values of 1.72 and 5.20 µg/ml. Aqueous dilution simplifies sample preparation, reducing degradation and interference. The method is compared with an HPTLC method, showing an extended linear range (6.25-200 µg/ml for HPLC, 7.81-62.5 µg/ml for TLC) and high sensitivity, making it significant for meropenem trihydrate quality control in bulk and dosage forms.

Analytical quality by design approach to develop an eco-friendly RP-HPLC method for estimation of irbesartan in chitosan polymeric nanoparticles: forced degradation studies and assessment of in vitro release mathematical modelling.

Irbesartan is an angiotensin converting enzyme blocker, primarily utilized for the management of hypertension and the mitigation of diabetic nephropathy progression. The present study introduces rapid, robust and environmentally sustainable reverse phase high performance liquid chromatography (RP-HPLC) validated under the analytical quality by design (AQbD) framework according to ICH guidelines. Utilizing a central composite design, the method's systemic optimization was achieved, ensuring reproducibility and accuracy. Chromatographic separation was accomplished utilizing an ethanol and sodium acetate buffer (60 : 40 v/v) isocratic mobile phase system on a zorbax sb C18 column, with a flow rate of at 0.6 mL min-1. Studies on forced degradation outlined stability of irbesartan and its degradation processes, enhancing our understanding of its chemical robustness under varied conditions. Complementing the green chemistry paradigm, the method's environmental impact was critically assessed, affirming its alignment with sustainability objectives. The validated method proved pivotal in determining the percent entrapment and loading efficiency of the formulated nanoparticles and holds potential for application in biological matrices. Furthermore, the encapsulation of IRB within chitosan nanoparticles was explored to assess release kinetics and enhance bioavailability. This study not only advances the analytical sciences by merging eco-friendly practices with method development but also broadens the applicative landscape of HPLC methodologies in drug delivery research.

Analytical Method Development and Validation of RP-HPLC Method for Estimation of Pazopanib Drug Sample and It’s Dosage Form

The study focuses on developing and verifying a cost-effective Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) technique for quantifying Pazopanib HCl in both bulk and tablet forms. The study aims to develop a cost-effective approach for routine quality control analysis by utilizing the simplicity and wide accessibility of HPLC. A Shimadzu C18 column (5 µm, 250 mm × 4.6 mm) was successfully used to separate Pazopanib HCl via chromatography. The mobile phase consisted of a mixture of Potassium dihydrogen phosphate in water (pH 2.9, corrected with Phosphoric Acid) and Acetonitrile in a ratio of 25:75 v/v. The isocratic elution mode was utilized with a flow rate of 1.0 mL/min, a column temperature of 25°C, and an injection volume of 20 µL. Pazopanib hydrochloride had a retention period of 2.8 minutes when measured at an isobestic wavelength of 215 nm. The described RP- HPLC technique showed exceptional specificity, accuracy, precision, linearity, and durability, rendering it a viable instrument for Pazopanib HCl tablets' regular quality control analysis. This method is both efficient in terms of analytical performance and economically profitable, making it very suitable for frequent pharmaceutical analysis.

Development and Validation of Reverse-Phase High-Performance Liquid Chromatography Method for Simultaneous Estimation of Doxorubicin and Clotrimazole.

A reverse-phase high-performance liquid chromatographic (RP-HPLC) method was developed to analyze the simultaneous estimation of doxorubicin and clotrimazole. The method was achieved by Nucleodur C18 column with dimension 250 × 4.6 mm (5 μm) using gradient elution. The mobile phase contained 0.2% formic acid (pH 3.2) and acetonitrile. The flow rate was kept at 1.0 mL/min and detection and quantitation of both drugs (doxorubicin and clotrimazole) were achieved using a photodiode array detector at 276 nm, which was the isosbestic point for both drugs. The proposed method was validated according to the current International Council for Harmonization of Technical Requirements of Pharmaceuticals for Human Use guidelines for specificity, linearity, accuracy, precision, and robustness. The developed method showed a linear response (R2 > 0.999), and was accurate (recoveries 97%-103%), precise (resolution ≤1.0%), sensitive, and specific. Thus, the developed RP-HPLC method for the simultaneous estimation of both drugs was successfully validated and can be utilized for the estimation of these drugs in the formulations being developed.

Development of Reverse Phase HPLC Method and Validation: Sofosbuvir and Velpatasvir Quantification in Bulk and Tablets

A new reverse-phase high-performance liquid chromatography (RP-HPLC) method for measuring the amount of sofosbuvir and velpatasvir in active pharmaceutical ingredients (API) and tablet forms has been developed and the method was also validated. Discovery column C18 150 mm x 4.6 mm, pore size of 5 μm was used for the analysis of drugs. Elution of drugs was achieved with 0.1% OPA Buffer: Acetonitrile mixed in the proportion of 40:60 v/v was used as mobile phase, maintaining 1.0 mL/min as flow rate, column temperature maintained at 30°C and uv detector wavelength was fixed at 272 nm. Sofosbuvir and velpatasvir were eluted at retention time (RT) of 2.183 and 3.038 minutes, respectively. Linearity parameter was performed in the levels from 25 to 150% and the square of correlation coefficient (r2) was found to be 0.9992. The %recovery obtained for sofosbuvir was 99.6 and for velpatasvir was 99.4. In precision %RSD for sofosbuvir was found to be 0.4 and for velpatasvir was 0.9. Validation was performed for the developed method and all of the validated parameter results obtained was within the ICH Q2 guidelines acceptance range. So, the developed RP-HPLC method can be used in pharmaceutical industry for quantification of sofosbuvir as well as velpatasvir at once in API and tablet dosage form.

Development and Validation of Stability Indicating RP-HPLC Method and Characterization of Degradation Products of Anti-neoplastic Agent by LCMS-MS

In present research a stability indicating reversed-phase high-performance liquid chromatography (RP-HPLC) was developed and validated for quantification of azacitidine and its degradants. The chromatographic isolation of azacitidine carried out using Phenominex C18 (150×4.6 mm, 5 μ) column, movable phase of 0.1% HCOOH in water and acetonitrile (30:70 v/v), at a flowing rate of 1-mL/min analyzed at 210 nm. The %recovery findings ranged between of 99.13–99.41%. The limit of detection (LoD), and limit of quantitation (LoQ)values were assessed from the rectilinear plot and were found to be 3.78 and 15.12 μg/mL. The regression equation of the linearity curve was found to be y = 851.76x + 310.38 and r2 value of 0.9998. The samples were subjected to stress degradation and characterized by three acid degradation products by liquid chromatography-mass spectrometry (LC-MS/MS) - DPI, DPII, and DPIII. The stationary phase and movable system attained excellent resolution as chromatographic isolation and structure identification was accomplished using LC-MS/MS fragmentation

Response Surface Methodology Assisted RP-HPLC Method for the Determination of Meropenem in Human Plasma: Application to a Pharmacokinetic Study

A simple, accurate and precise development of an reversed-phase high-performance liquid chromatography (RP-HPLC) method for the quantification of meropenem in human plasma samples for therapeutic drug monitoring (TDM) was developed and validated after optimization of various chromatographic parameters. The best recovery of meropenem was achieved through sample preparation based on a simple extraction method that included deproteinization and extraction with acetonitrile. The developed method reported acceptable values for selectivity, linearity, accuracy and precision, carry-over, dilution integrity and stability. The developed method was efficiently shown to be applicable to a real-time monitoring and pharmacokinetic study of meropenem in critically ill patients.

RP-HPLC Method for the Simultaneous Determination of Sofosbuvir and Daclatasvir in Pure and Pharmaceutical Dosage Forms

Daclatasvir and sofosbuvir in pure and medicinal dosage forms can be determined quickly and easily using a reversed-phase high-performance liquid chromatography (RP-HPLC) technique. An accurate, reproducible method was developed and validated. The mobile phase contained a mixture of 90% methanol:10% water (0.05% OPA). UV estimation was done with a flow rate of 0.7 mL/min and wavelength of 275 nm. The temperature was 30°C. Sofosbuvir stays in the body for 3.361 minutes and daclatasvir for 5.745 minutes. A quantitative study of commercial dosage forms went well with this method, which was made and tested. Sofosbuvir and daclatasvir each had a %RSD of 0.43 and 0.28, respectively. Recoveries were 97.85 and 98.52% for sofosbuvir and daclatasvir, respectively. Three methods were checked for precision, accuracy, linearity, selectivity, specificity, limit of detection (LoD), limit of quantitation (LoQ), robustness, and ruggedness according to rules of the International Council for Harmonisation (ICH).

Development of Novel RP-HPLC Method for Estimating Tepotinib in Bulk and Pharmaceutical Dosage Form

Objective: The study’s goal is to develop a highly appropriate, fast, precise, and validated reverse-phase high-performance liquid chromatography (RP-HPLC) approach that indicates stability for the estimation of tepotinib in pharmaceutical dosage form and bulk, in accordance with ICH recommendations. Method: The components were separated by chromatography using a Phenomenex Kinetex XB-C18 (150×4.6 mm, 5μ) column. The absorbance was measured at 272 nm, and the flow rate was 1.0 mL̸min. The International Council for Harmonisation (ICH) states that checks were made for linearity, precision, accuracy, system appropriateness, specificity, and protocol robustness. Results: Tepotinib was shown to have a retention period of 3.4 minutes. The results showed that the linearity ranged from 5 to 25 μg/mL (r2 = 0.9993), and the percentage mean recoveries for tepotinib’s accuracy and precision fell within the range of (%RSD˂ 2). It was discovered that the Limit of Detection (LoD), and Limit of Quantitation (LoQ) were, respectively, 0.429 and 1.3 μg/mL.

Design of experiment-oriented development and validation of novel bioanalytical reverse phase high performance liquid chromatography method of palbociclib in rat plasma and tissues, and its application in pharmacokinetic studies.

The current research involved the development and validation of an easy, cost-effective, and sensitive bioanalytical reverse-phase high-performance liquid chromatography method for the assessment of palbociclib (PAL) in rat plasma and kidney, liver, spleen and heart. A response surface methodology-based Box-Behnken design was used to optimize critical chromatographic conditions such as buffer pH, organic phase concentration and flow rate to attain good sensitivity, tailing factor and retention time. The conditions were: pH of buffer, 4.5; organic phase concentration, 40%; Shimpac column with 1.0ml/min flow rate. The responses were: tailing factor, 1.29 ± 0.03, sensitivity, 366,593 ± 8,592; and retention time, 4.5 ± 0.05 min. The samples were extracted by a protein precipitation method, and absolute recoveries were in the range of 88.99-95.08%. The linearity of the developed method was validated over the range 100-2,000 ng/ml (r2 ≥ 0.994) in all tested matrices. The developed bioanalytical method showed greater accuracy (0.98 and 4.01%) and precision (<4.88%). The method was optimized for the sensitive analysis of the PAL in rat plasma, and the kidney, liver, spleen and heart were effectively applied to pharmacokinetic studies.

Absolute Quantification of Hepatitis B Core Antigen (HBcAg) Virus-like Particles and Bound Nucleic Acids.

Effective process development towards intensified processing for gene delivery applications using Hepatitis B core Antigen (HBcAg) virus-like particles (VLPs) relies on analytical methods for the absolute quantification of HBcAg VLP proteins and bound nucleic acids. We investigated a silica spin column (SC)-based extraction procedure, including proteinase K lysis and silica chromatography, for the absolute quantification of different species of nucleic acids bound to HBcAg VLPs analyzed by dye-based fluorescence assays. This revealed load-dependent nucleic acid recoveries of the silica-SC-based extraction. We also developed a reversed-phase high-performance liquid chromatography (RP-HPLC) method to separate and quantify the HBcAg proteins and the bound nucleic acids simultaneously without prior sample treatment by dissociation reagents. The method demonstrated sufficient linearity, accuracy, and precision coefficients and is suited for determining absolute protein and nucleic acid concentrations and HBcAg protein purities at various purification stages. Both the silica-SC-based extraction and the RP-based extraction presented overcome the limitations of analytical techniques, which are restricted to relative or qualitative analyses for HBcAg VLPs with bound nucleic acids. In combination with existing analytics, the methods for an absolute quantification of HBcAg VLPs and bound nucleic acids presented here are required to evaluate downstream purification steps, such as the removal of host cell-derived nucleic acids, concurrent protein loss, and efficient loading with therapeutic nucleic acids. Hence, the methods are key for effective process development when using HBcAg VLP as potential gene delivery vehicles.

Gradient reversed-phase HPLC method for the quantitation of azelnidipine and chlorthalidone in a fixed-dose synthetic mixture

Gradient reversed-phase HPLC method for the quantitation of azelnidipine and chlorthalidone in a fixed-dose synthetic mixture

Stability indicating RP-HPLC method development and validation for estimation of Crisaborole in topical dosage form

A simple, specific, accurate, precise , rapid, robust and selective stability indicating reverse phase high performance liquid chromatography (RP-HPLC) method has been developed for assay and related substances and validated for quantification of Crisaborole with its excipients in its topical dosage form. Forced degradation study is done to determine stability of the product. In mobile phase Water: Methanol (30: 70 v/v), the maximum of Crisaborole was measured to be 241nm. The technique is highly sensitive, with linearity ranging from 5 to 25g/ml (regression equation: y = 316606x - 427583; r2 = 0.9992). For verifying and testing this approach, several parameters according to ICH recommendations and USP are used. The detection and quantitation limits were determined to be 0.1094 and 0.3316 g ml-1, respectively. The results revealed that the process is accurate, specific, and repeatable (RSD 2%), as well as easy, inexpensive, and less time consuming, and suitable for determining Crisaborole in topical dosage form. The method have been robust under various variation with flow rate, detection wavelength and column oven temperature. The drug was exposed to stress conditions. Method resolves all degraded product as compared to Crisaborole. Developed method can be used routinely for estimation of drug Crisaborole with its excipient methyl paraben in dosage form and stability sample.