Reversed-phase High-performance Liquid Chromatographic Method Research Articles

Advancements in RP-HPLC Method Development and Validation: A Comprehensive Review

Reverse-phase high-performance liquid chromatography (RP-HPLC) has emerged as a cornerstone technique in analytical chemistry, offering high sensitivity, selectivity, and efficiency in separating and quantifying a wide range of compounds. This comprehensive review explores recent advancements in RP-HPLC method development and validation, focusing on key strategies, challenges, and emerging trends. The review encompasses a detailed examination of critical parameters such as column selection, mobile phase optimization, detection techniques, and validation protocols. Furthermore, it discusses innovative approaches and technologies that enhance the robustness, speed, and accuracy of RP-HPLC analyses. Through a thorough analysis of current literature and case studies, this review provides valuable insights for researchers and practitioners engaged in method development and validation in the field of chromatographyies.

RP-HPLC METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS ESTIMATION OF CURCUMIN AND RESVERATROL IN NANO-MICELLE: DUAL DRUG DUAL FORM SIMULTANEOUS ESTIMATION

Objective: To develop a reverse-phase high-performance liquid chromatography (RP-HPLC) method for simultaneous estimation of conjugated form of Curcumin (CCMN) and free form of Resveratrol (RSV) in nano-micelle. Methods: The conjugation of lipophilic CCMN and hydrophilic Chitosan (CHT) through succinyl linker produce amphipathic molecule that can self-assemble into RSV solution to form micelle. Here RSV exists in micelle core as free form and CCMN with micelle backbone as conjugated form. So it required to estimate conjugated drug and free drug simultaneously from nano-micelle. We developed a RP-HPLC method, utilized C18 column, follow flow rate of mobile phase 1.0 ml/min, which consist of acetonitrile with water (0.5% Ortho Phosphoric acid, pH 4.6) in the ratio of 1:1 for 20 min. Injection volume was 10μl and column temperature 25 ℃. Isosbestic detection of both drugs was at 254 nm. Results: The retention time of RSV and CCMN were at 8.15 min and 11.41 min respectively, completely distinguished sharp peak of CCMN and RSV developed with resolution 7.360±0.117, wide range of linearity with correlation coefficient value (R2) of CCMN and RSV were 0.99987 and 0.99992 respectively and recovery value of CCMN and RSV were 100.041±0.22 % and 100.041±0.21 % respectively. The RSD (relative standard deviation) for accuracy, precision and robustness of the method was found to be less than 2%. Conclusion: The develop method for simultaneous estimation of conjugated CCMN and free form of RSV in the nano-micelle formulation was consider to be accurate, precise, robust and sensitive.

Development of a RP-HPLC method for simultaneous determination of atenolol, metoprolol tartrate and phenol red for in-situ rat intestinal perfusion studies

Single-pass intestinal perfusion (SPIP) method is a widely used experimental model to determine the intestinal permeability of drugs. These studies are performed in the presence of a reference standard (metoprolol, MT) and a zero permeability marker (phenol red, PR). Therefore, it is important to develop a validated method for simultaneous determination of the investigated compound along with MT and PR. The aim of this study was to develop a reversed phase high-performance liquid chromatography (RP-HPLC) method with UV-detection for the simultaneous determination of atenolol (ATN), MT, and PR in the perfusion medium used in SPIP experiments. Separation of compounds were performed using an InertSustain C18 (250 × 4.6 mm, 5 µm) HPLC column at 35 °C. The mobile phase was a mixture of acetonitrile and phosphate buffer (pH 7.0, 12.5 mM) in gradient elution, and was delivered at a flow rate of 1 mL/min. The acetonitrile ratio of the mobile phase increased linearly from 10 to 35 % over 15 min. The injection volume was 20 µL, and ATN, MT and PR were detected at 224 nm. The retention times under optimum HPLC conditions were 5.028 min, 12.401 min and 13.507 min for ATN, MT and PR, respectively. The developed RP-HPLC method was validated for selectivity, specificity, calibration curve and range, accuracy and precision, carry-over effect, stability, reinjection reproducibility, recovery and robustness. The method was linear for ATN (0.76–50 μg/mL), MT (1.14–50 μg/mL) and PR (0.47–20 μg/mL) with determination coefficients of 0.9999, 0.9994 and 0.9998, respectively. The results obtained for all validation parameters of the developed RP-HPLC method met the required limits of the ICH M10 Guideline.

A stability-indicating method development and validation for the determination of related substances in novel synthetic decapeptide by HPLC.

In the present scenario, peptide is an emerging field of research having vast therapeutic applications. Diverse impurities may rise from various stages of the synthesis process and storage of the peptides. Because these contaminants may have an impact on the therapeutic safety and effectiveness of peptides in their approaching applications, they must be identified and carefully monitored. Considering the pharmaceutical importance of the extent of peptides, we were motivated to synthesize a decapeptide and establish a novel gradient reversed-phase high-performance liquid chromatography (RP-HPLC) method for its analysis along with efficient separation of its six related impurities. Different buffers, organic modifiers, and columns were used in the tests for good separation of these impurities. To establish a stability-indicating method, a stress study was also conducted. The International Conference on Harmonization (ICH) guidelines have been followed for validation of the developed analytical method. The validated method revealed sufficient accuracy, specificity, linearity, robustness, precision, and high sensitivity for its intended use. The proposed method could be appropriate for routine analysis and stability assessment of the decapeptide, which might be useful for further scientific investigation.

A greener RP-HPLC method for quaternary estimation of caffeine, paracetamol, levocetirizine, and phenylephrine acquiring AQbD with stability studies

Abstract The main objective of this article was to develop method and validate quaternary estimation of caffeine (CFE), paracetamol (PCM), levocetirizine (LEV), and phenylephrine (PHE) and to conduct degradation experiments using reverse-phase high-performance liquid chromatography (RP-HPLC). This was the first innovative approach to this drug combination, combining analytical quality by design with green analytical chemistry. This method was developed using HPLC Agilent 1220 Infinity II, a binary solvent delivery pump, an automatic sampling device injector, and a photodiode array detector. Agilent Inertsil ODS 3 (250 mm × 4.6 mm, 5 μm) was used for separation. The mobile phase is composed of ethanol and 10 mM phosphate buffer (pH 3). To adjust the pH, 1% of orthophosphoric acid was used. The flow rate was set to 0.8 ml·min−1, the injection volume was 10 μl, and the detecting wavelength was 220 nm. The analytes were eluted via gradient elution. The retention time for PCM was 3.5 min, CFE was 8.1 min, PHE was 15.9 min, and LEV was 20.5 min. Green evaluation tools used in this research include Green Analytical Procedure Index, analytical eco-scale, analytical greenness, analytical method greenness score, and carbon footprint analysis. The developed method was greener than the previously reported method, as per the results of the greenness evaluation tools.

Development and characterisation of buccal dispersible film, syrup and effervescent granules of extemporaneous antigout remedy

Homemade medicinal remedies used to treat both acute and chronic ailments for centuries need to be transformed into modern evidence-based dosage forms. Therefore, the present study aimed to develop buccal dispersible film, syrup and effervescent granules of an extemporaneous antigout homemade remedy. The dosage forms were prepared using holistic remedy (HR) as an active entity and excipients masking disagreeable features. The dosage forms and HR were subjected to physicochemical evaluation, and standardised by a reversed-phase HPLC method using chlorogenic acid, caffeic acid, vanillin, and ferulic acid as analytical standards. The standardised dosage forms were investigated for antigout activity using an appropriate model. The dosage forms were found to be physically elegant and phytochemically aligned with HR. The developed dosage forms contained the selected markers and possessed antigout activity. Hence, these standardised and evidence-based dosage forms may have wider acceptance in the market compared to HR.

Rapid Development and Validation of Atoltivimab, Maftivimab and Odesivimab in Pharmaceutical Dosage form by using the RP-HPLC Method

Background: The reverse-phase high-performance liquid chromatography (RP-HPLC) method was developed for the quantitative measurement of monoclonal antibodies (Maftivimab, Atoltivimab, and Odesivimab) in the pharmaceutical dosage form. The Food and Drug Administration (FDA) has approved these monoclonal antibodies for the treatment of Zaire ebolavirus infection in adults. Methods: Maftivimab, Atoltivimab, and Odesivimab were separated chromatographically on the Waters Alliance-e2695 platform using the Luna Phenyl Hexyl (250 x 4.6 mm, 5 μm) column and a mobile phase made up of Acetonitrile (ACN) and ortho-phosphoric acid (OPA) buffer in a ratio of 70:30 (v/v). Results: The flow rate was 1.0 ml/min, and a photodiode array (PDA) detector operating at room temperature was used to measure absorption at 282 nm. For Maftivimab, Atoltivimab, and Odesivimab, the theoretical plates were not less than 2000, and the tailing factor shouldn't be greater than 2, accordingly. All measurements have a constant relative standard deviation of peak areas that is less than 2.0. Conclusion: The suggested procedure was approved following the International Conference on Harmonisation (ICH) recommendations. When used for the quantitative analysis of Maftivimab, Atoltivimab, and Odesivimab, the approach was found to be straightforward, affordable, appropriate, exact, accurate, and robust.

Novel RP-HPLC method for estimation of a newly developed combination of tizanidine and etoricoxib in rat plasma: Eight criteria for greens evaluation.

A novel environmentally friendly reversed-phase high-performance liquid chromatography (RP-HPLC) method has been effectively validated for simultaneously measuring a prospective conjunction of tizanidine (TIZ) and etoricoxib (ETC), the combined medicine, in rat plasma. The technique employs diclofenac potassium as the internal standard, guaranteeing dependable and precise outcomes. This study aimed to assess the impact of the suggested combination therapy on treating inflammation resulting from rheumatoid arthritis (RA) in a rat model. The procedure was performed using an Agilent series 1200 model HPLC apparatus. The chromatographic conditions consist of isocratic elution mode, C18 column with dimensions of 150 mm × 4.6 mm × 5 µm, flow rate of 1.5 mL/min, wavelength of 230 nm, temperature of 50°C, and injection volume of 10 µL. The elution was performed using a mobile phase consisting of a phosphate buffer with a pH of 3.5 and acetonitrile in a ratio of 80:20 v/v. Calibration curves were conducted for TIZ and ETC within the 1-50 µg/mL range, demonstrating linear trends with R2 values over 0.999. The effectiveness and eco-friendliness of the proposed method were evaluated using eight separate environmentally conscious metrics. The addition of TIZ and ETC to arthritic rodents amplified these effects significantly. Furthermore, TIZ and ETC significantly reduced serum levels in arthritic rodents, and safety investigations revealed normal complete blood count, liver, and renal functions. TIZ and ETC appear to have antiarthritic, anti-inflammatory, and safe combinations, making them viable future treatment options for RA that are also safe and efficacious. Following validation by United States Food and Drug Administration(US-FDA) rules, all goods met the criteria.

Comparative evaluation of liquid-liquid extraction and nanosorbent extraction for HPLC-PDA analysis of cabazitaxel from rat plasma

A precise, sensitive, accurate, and validated reverse-phase high-performance liquid chromatography (RP-HPLC) method with a bioanalytical approach was utilized to analyze Cabazitaxel (CBZ) in rat plasma. Comparative research on extraction recoveries was performed between traditional liquid-liquid extraction (LLE) and synthesized graphene oxide (GO) based magnetic solid phase extraction (GO@MSPE). The superparamagnetic hybrid nanosorbent was synthesized using the combination of iron oxide and GO and subsequently applied for extraction and bioanalytical quantification of CBZ from plasma by (HPLC-PDA) analysis. Fourier- transform infrared spectroscopy (FT-IR), particle size, scanning electron microscopy (SEM), and x-ray diffraction (XRD) analysis were employed in the characterization of synthesized GO@MSPE nanosorbent. The investigation was accomplished using a shim pack C18 column (150 mm×4.6 mm, 5 µm) with a binary gradient mobile phase consisting of formic acid: acetonitrile: water (0.1:75:25, v/v/v) at a 0.8 mL/min flow rate, and a λmax of 229 nm. The limits of detection (LOD) and quantitation (LOQ) have been determined to be 50 and 100 ng/mL for both LLE and SPE techniques. The linearity range of the approach encompassed from 100 to 5000 ng/mL and was found to be linear (coefficient of determination > 0.99) for CBZ. The proposed method showed extraction recovery of 76.8–88.4% for the synthesized GO@MSPE and 69.3–77.4% for LLE, suggesting that the proposed bioanalytical approach was robust and qualified for all validation parameters within the acceptable criteria. Furthermore, the developed hybrid GO@MSPE nanosorbent with the help of the proposed RP-HPLC method, showed a significant potential for the extraction of CBZ in bioanalysis.

Therapeutic drug monitoring of six contraindicated/co-administered drugs by simple and green RP-HPLC-PDA; application to spiked human plasma

Therapeutic drug monitoring is an important clinical testing of the drugs to monitor their concentrations in plasma in order to guarantee their optimal impact, and to avoid any side effects resulting from drug-drug interactions. A green reversed-phase high-performance liquid chromatographic method using a photodiode array detector (RP-HPLC-PDA) was developed for the simultaneous determination of three carbapenem antibiotics (Imipenem, ertapenem, and meropenem) with the co-formulated drug (cilastatin) and contraindicated drugs (probenecid and warfarin) in spiked human plasma. The separation was achieved at 25 °C using a gradient elution of a mixture of mobile phase A: methanol and mobile phase B: phosphate buffer (pH 3.0). The photodiode array detector was adjusted at 220 nm. Bioanalytical method validation was carried out as per the FDA guidelines, and the method showed good linearity ranges for the six drugs that included their Cmax levels along with low limits of quantification. Based on the results, the method was found to be accurate and precise; with high % recovery and good % RSD, respectively. The method was successfully applied to spiked human plasma, signifying a good potential to be implemented in future TDM studies of these drugs when co-administered together.

Validation of High-Performance Liquid Chromatography Method for the Determination of Doxorubicin in Proliposomal Drug Delivery System Formulation

The objective of this study develop a novel proliposome formulation containing Doxorubicin (Dox) and was to validate sensitive and selective reversed-phase high-performance liquid chromatographic (HPLC) method for the evaluation of Dox concentrations of proliposome formulation. The samples were chromatographed on C18 column (Zorbax Eclipse Plus 5µm 4.6 x 250 mm) using a mobile phase with Sodium Lauryl Sulphate solution:Acetonitrile (%50:%50) and detected 254 nm. Linearity was confirmed in the concentration range 10.0–75.0 µg/mL. Specificity, linearity, working range, LOD, LOQ, accuracy, precision, robustness and system suitability studies were done from HPLC validation parameters. Liposome formulation containing Dox was developed by pH gradient method then proliposome formulation was developed with lyophilisation technique. The developed HPLC method, the encapsulation capacity (EE%) was found to be 90%±0.5 and the drug loading capacity (DL%) was found to be 100.0%±0.3. In addition, in vitro release studies and stability study results were evaluated with validated HPLC method. It was observed that developed Dox-proliposome formulation increased Dox release at pH 5.5, pH 6.5 and pH 7.5 by 23.9%, 30.2% and 14.8%, respectively, compared to commercial product. The result of F2 test performed in pH 7.5 media was 51.4%. According to the results of the physicochemical tests performed within the stability studies, it was observed that there was no significant change at the end of 12 months. These results show that the HPLC method developed, and validation study performed are important and applicable in the development, characterization, in vitro release and stability studies of the novel proliposome formulation.

Validation of HPLC-DAD Based Stability Indicating Assay Method for Ornidazole in Periodontal Polymeric Hydrogel: Robustness Study using Quality by Design (QbD) Approach

In the present study, a gradient reversed-phase high-performance liquid chromatography (RP-HPLC) method has been designed and validated to quantify Ornidazole (OZ) in the marketed formulation (oral gel) with the application of QbD. The proposed HPLC method utilizes an RP C18 column (4.6 x 250 mm, 5 μm). The mobile phase contained HPLC grade Solvent A (water) and Solvent B (acetonitrile) and ran at a flow rate of 1 mL/min in a gradient mode. The linearity was plotted in an OZ range of 1–12 μg/mL and the correlation coefficient was found to be 0.9998. The limit of quantification, as well as the limit of detection, has been recorded as 0.70 μg/mL and 0.23 μg/mL. Inter- and intra-day precision (RSD) percentages were reported respectively within a range of 0.262–0.589 and 0.179–0.879. At 80, 100, and 120 percent different OZ levels, the average accuracy was 99.55, 99.58, and 99.92, respectively. The result of the study indicates that the OZ gel estimate HPLC method has good sensitivity, reproducibility, accuracy, and precision. This method may be used for the estimation of OZ in a gel formulation in quality control laboratories.

The Quality by design Approach for Analytical Method Development and Validation of the RP-HPLC Method for Estimation of Quercetin in Pure, Marketed, and Cream Formulation

ABSTRACT: A simple, precise reverse phase high performance liquid chromatographic (RP-HPLC) method has been developed and validated for the flavonoid quercetin, isolated from Tridax procumbens L. The method was validated by using Phenomenex C18 (250 x 4.6mm i.d) Column. A simple, cost effective mobile phase consisting of (ACN and 10 m/moL Phosphate buffer as mobile phase in proportion of 50:50 v/v ) pH 3, 1.0 ml/min Flow rate at 370nm by using UV Visible detector. The retention time of Quercetin was found to be 3.392 minutes. The Quercetin linearity range was found to be 05 to 25 μg/mL. The accuracy and precision of commercially available preparations and in-house cream formulations were investigated using a one-way ANNOVA test. The Percentage recovery of both formulations was found to be 99.83%, 99.88%, 99.82% and 98.92%, 98.18%, 98.86%. Robustness of analytical method was studied by using 2/3 full factorial design by using Design expert software. The Percentage assay and % RSD of marketed capsule and in house cream was found to be 98.38 %, 97.40% and 0.05273, 0.02053 respectively. The Limit of Quantitation and Limit of detection were found to be 0.9053μg/mL and 2.5435μg/mL respectively. The development method of quercetin is simple, accurate, precise, sensitive, and robust

Validated Stability Indicating RP-HPLC Method for the Quantification of Process Related Impurities of Solifenacin and Mirabegron in Pharmaceutical Formulations

To assess solifenacin (SOL) and mirabegron (MER) simultaneously, a verified reverse-phase high-performance liquid chromatography (RP-HPLC) method has been developed to indicate stability. The method was thoroughly evaluated and found to meet satisfactory criteria for precision, linearity, accuracy, limits on detection, and robustness, limits on quantitation. The quantitation wavelength of 231 nm was determined. Linearity was successfully demonstrated across concentration ranges of 5 to 25 μg/mL of solifenacin and 50 to 250 μg/mL of mirabegron. RPHPLC separations was conducted employing a Phenomenex L. C18 column measuring 250 x 4.6 mm and containing particles as small as 5 μm. The methanol and phosphate buffer (pH 7) were combined in a volumetric ratio of 25:75 to create the mobile phase. The separation is accomplished at a 0.7 mL per minute flow rate. Time spent in retention for mirabegron and solifenacin had been established at 5.521 and 9.161 minutes, respectively. Forced degradation studies validated the stability-indicating character of the approach, which included hydrolysis under acidic and basic conditions, exposure to H2O2, thermal degradation, and photodegradation. Mirabegron and solifenacin exhibited 10 to 20% degradation under the specified conditions. Importantly, the process evaluated the two prescription drugs in detail with all degradation products generated during the forced degradation experiments. This developed method is characterized as straightforward, specific, and cost-effective, making them suitable of the simultaneous estimate of mirabegron with solifenacin in tabs dose forms.

Determination of Enantiomer in Tofacitinib Citrate Using Reversed-Phase Chiral High-Performance Liquid Chromatography

Tofacitinib citrate (RR-isomer) is a janus kinase (JAK) inhibitor approved for the treatment of rheumatoid arthritis (RA) in adults who have had an inadequate response or intolerance to methotrexate. The presence of the enantiomer of tofacitinib citrate (SS-isomer) is monitored for quality control as a possible impurity in the final product. In this study, a reversed-phase high-performance liquid chromatography (RP-HPLC) method based on a chiral recognition mechanism for the separation of tofacitinib citrate and its enantiomer was established based on the principles of green analytical chemistry. A CHIRALPAK IH column was used with a mobile phase of ammonium acetate buffer (pH 8.0) and acetonitrile in a gradient elution at a detection wavelength of 285 nm. The calibration curve exhibited excellent linearity over the range of 0.1002–20.04 μg/mL (r = 0.9999). The average recovery of the enantiomer was 98.6% with a relative standard deviation (RSD) of 0.7%. The limit of detection (LOD) and the limit of quantitation (LOQ) were 0.04 and 0.1 μg/mL, respectively. This RP-HPLC method was suitable for detecting the enantiomers of tofacitinib citrate in tablets. Furthermore, the method proved to be environmentally friendly based on the evaluation by Analytical Eco-Scale, Analytical GREEnness (AGREE) and Green Analytical Procedure Index (GAPI).

Development and Validation of a New Reversed Phase HPLC Method for the Quantitation of Azithromycin and Rifampicin in a Capsule Formulation.

This research aimed to develop a new method for simultaneously estimating the presence of azithromycin (AZT) and rifampicin (RIF) in a capsule formulation using reverse-phase high-performance liquid chromatography. The developed method utilized a Gemini column with a 60:40% v/v acetonitrile and potassium dihydrogen phosphate mobile phase, a flow rate of 1mL/min, and an injection volume of 20μL. The detection wavelengths of 210 and 254nm for AZT and RIF, respectively, were used. Validation ensures specificity with a peak purity index > 0.99999 for AZT and >0.99995 for RIF, affirming unambiguous analyte detection. The system suitability test, within acceptable limits, validates method reliability. Linearity calibration curves (R2 = 0.998) cover a 25-150% target concentration range. Accuracy studies employing the standard addition method yield recovery values between 96.6 and 103.9% for both drugs, confirming method accuracy. Precision studies reveal % relative standard deviation values consistently below 2%, highlighting reproducibility. Robustness testing supports method reliability under varying conditions. Application to a pharmaceutical capsule formulation demonstrates the method's practicality, accurately quantifying AZT (98.30%) and RIF (99.37%). This study provides a validated analytical approach for simultaneous quantification in commercial pharmaceutical products containing both drugs, enhancing pharmaceutical quality control for critical antibiotics in complex formulations.

A Novel Stability-Indicating RP-HPLC Method for the Simultaneous Estimation and In Vitro and In vivo Evaluation: Curcumin and Naringin Co-amorphous System

Curcumin (CUR) is a phytochemical widely used in food industries, cosmetics, and in the treatment of various ailments. It is a polyphenol derived from turmeric and is often considered the golden spice. CUR has a low solubility of less than 1 µg/ml and poor oral bioavailability which can be improved by co-amorphization with naringin (NRG). Analytical method to simultaneously quantify CUR and NRG is not reported in literature. This study aimed to develop a stability-indicating reverse phase HPLC method in gradient mode to simultaneously quantify CUR and NRG in co-amorphous system. The co-amorphous system of CUR and NRG in molar ratios 1:1 and 1:2 was prepared by quench cooling technique. The separation was attained on a Genesis C18, (4.6 mm × 150 mm, 4 µm) column with the mobile phase comprising of methanol and a 0.1% acetate buffer pH 3.8 at a single wavelength, 289 nm. CUR and NRG eluted at 5.1 and 11.1 min, respectively. For both the molecules, the linearity range was 0.125–16 µg/ml with LOD and LOQ of 0.063 and 0.125 µg/ml. The method developed was validated as per International Conference on Harmonization (ICH) guidelines for linearity, accuracy, precision, and robustness. The method was used to estimate CUR and NRG content in co-amorphous mixture and for in vitro evaluation.Graphical

Implementing analytical quality by design in reversed phase-high performance liquid chromatography for simultaneous estimation of teriflunomide and quercetin: Applicability in dual drug loaded topical microemulsion

The current study reports the development of a novel, robust, and sensitive reversed-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous estimation of teriflunomide (TFD) and quercetin (QCN) in dual drug-loaded microemulsion. TFD and QCN have been reported to exhibit anti-inflammatory effects by targeting various inflammatory mediators involved in the pathogenesis of rheumatoid arthritis (RA). This analytical method employs a risk-based approach and follows the principles of analytical quality by design (AQbD). The study involved preliminary screening trials and systematic risk analysis to identify critical method attributes, that significantly impact the critical quality attributes (CQAs). For the optimization of method,a face centered central composite design was utilized, and 16 experimental runs were performed using design expert software version 7.0.0. Chromatographic conditions were carefully optimized using AQbD, falling within the design space and consisting of ammonium acetate buffer (pH 3.5) and acetonitrile (60:40, % v/v) with a flow rate of 1.0 mL/min. An analytical Hibar Lichospher 100 RP-18e column (250 × 4.6 mm, 5 µm) was employed, with detection wavelength of 280 nm and 367 nm for TFD and QCN, respectively. The optimized method underwent validation according to ICH guidelines and demonstrating applicability for evaluating topical microemulsion.

A SIMPLE RP-HPLC METHOD DEVELOPMENT AND VERIFICATION FOR THE QUANTITATIVE ESTIMATION OF SODIUM BUTYRATE IN TABLETS

Objective: To develop a simple, accurate, precise, and linear reverse-phase high-performance liquid chromatographic (RP-HPLC) method and verify the quantitative estimation (assay) of sodium butyrate in tablets. Methods: The optimized RP-HPLC method uses a reverse phase stationary phase which is YMC Triart C18 column, having specifications of 250×4.6 mm; 5 μm, a mobile phase composition of pH 8.0 sodium dihydrogen phosphate buffer and acetonitrile in the proportion of 92:8 v/v, flow rate of 0.5 ml/min, injection volume of 10 μL, and detection wavelength of 210 nm using a UV/PDA detector. Results: The developed method gave sodium butyrate eluting at about 6 min. Sodium butyrate exhibited linearity in the range of 119.56–1195.6 μg/ ml. The precision was exemplified by a relative standard deviation of 0.30%. The percentage of individual recovery was found to be in the range of 97.0 and 103.0 during accuracy studies. Conclusion: A simple, specific, accurate, precise, and linear RP-HPLC method was developed and verified for the quantitative estimation (assay) of sodium butyrate in tablets and hence this method can be explored for the analysis of sodium butyrate in tablets in various pharmaceutical industries.

Ecofriendly bioanalytical validated RP-HPLC method for simultaneous determination of COVID-19 co-prescribed drugs employing quality by design and green chemistry

Molnupiravir, dexamethasone, ertapenem, and remdesivir are among the most frequently co-prescribed drugs used for COVID-19 virus treatment. For the first time, a simple isocratic reversed phase-high performance liquid chromatographic (RP-HPLC) method employing a photodiode array detector has been developed and validated for their simultaneous determination in spiked human plasma using rivaroxaban as an internal standard by the aid of quality by design and in accordance with the criteria of green chemistry. The separation was carried out utilizing a BDS Hypersil ® C18 column (300 × 4.6 mm), 5 μm particle size as the stationary phase using mobile phase consisted of methanol: acidified water; adjusted at pH 4.0 with 0.1 M orthophosphoric acid; with ratio (70:30, v:v) delivered at flow rate 0.6 mL/min, temperature adjusted at 20 °C, photodiode array detector maintained at 240 nm. The method was validated according to bioanalytical FDA guidelines with respect to linearity, accuracy, precision, selectivity, recovery, and stability. The method was found to be linear in the range of 4.00–120.00 μg/mL for molnupiravir, 10.00–140.00 μg/mL for dexamethasone, 10.00–160.00 μg/mL for ertapenem and 4.00–120.00 μg/mL for remdesivir. It is worth mentioning that the previously mentioned ranges covered the Cmax of the drugs. Finally, the green analytical score has been evaluated, and it was found to be an excellent green analytical method based on analytical eco-scale system penalty points. Moreover, it was assessed by analytical GREEnness metric approach and green analytical procedure index tool.