Reversed-phase High-performance Liquid Chromatographic Method Research Articles

Development and validation of a high-performance liquid chromatography method for the estimation of esomeprazole in bulk and tablet dosage form

Introduction: An accurate, simple, precise, rapid, economic and reproducible reverse-phase high-performance liquid chromatography method was developed and validated for the estimation of Esomeprazole (ESO) in bulk and tablet dosage form. Method: The separation was carried out on Finepak SIL C18T-5 column (250 × 4.6 mm, 5.0 µm i. d.) using potassium dihydrogen phosphate buffer (0.025M): ACN (20:80 v/v) and at a flow rate of 1.0 mL/min. using UV detector at 302 nm with a run time of 10 min. The method was validated for accuracy for linearity, accuracy, precision, limit of detection (LOD), limit of quantification (LOQ) and robustness. Results: The standard calibration curve was linear with R2 = 0.995. LOD and LOQ obtained for esomeprazole were 0.0001 and 0.0004 µg/mL respectively. The method was found robust for possible changes. Results of analysis of other parameters were also tested and validated as per ICH guidelines and recovery studies confirmed the accuracy of the proposed method. validation studies showed that the developed HPLC method is simple, reproducible, rapid, precise and reliable. The high recovery and low relative standard deviation confirm the suitability of the developed method for the determination of esomeprazole in the tablet dosage form. Conclusion: This method may be used as a more convenient and efficient option for the analysis of esomeprazole to establish the quality of the substance during routine analysis with consistent and reproducible results.

Application of UV spectrophotometry and reversed-phase HPLC to determine the ionization constant of a new biologically active compound

Aim to determine the ionization constant (pKa) of the biologically active compound 2-ABPPC by UV spectrophotometry and high performance liquid chromatography (HPLC).
 Material and methods. The object of the study was the substance 2-ABPPC (2-amino-1-(4-bromophenyl)-5-(3,3-dimethyl-2-oxobutylidene)-4-oxo-4,5-dihydro-1H-pyrrole-3- carboxamide). The methods of UV spectrophotometry and high performance liquid chromatography were used to determine the ionization constant. The spectrophotometric analysis was carried out on a Shimadzu UV-1800 spectrophotometer. The chromatographic determination of the pKa value was carried out on an LC-20 Prominence liquid chromatograph (Shimadzu) using a Zorbax Extend-C18 reversed-phase column.
 Results. In the course of study, it was found that 2-ABPPC has one pKa value. When using two different methods to estimate the ionization constant, the comparable results were obtained: 7.64 (UV spectrophotometry method) and 7.40 (reversed phase HPLC method).

Simultaneous determination of enantiomeric and organic impurities of vildagliptin on a cellulose tris(3-chloro-4-methylphenylcarbamate) column under revered-phase conditions

A new, reversed-phase HPLC (RP-HPLC) method was developed for the simultaneous determination of the dipeptidyl-peptidase-IV-inhibitor antidiabetic drug vildagliptin (VIL) enantiomeric impurity and four other achiral related impurities. An initial screening was performed on five polysaccharide-type chiral stationary phases (Lux Amylose-1, Lux Amylose-2, Lux-Cellulose-1, Lux-Cellulose-2, Lux-Cellulose-3) in polar organic mode with methanol, ethanol, 2-propanol, or acetonitrile containing 0,1% diethylamine as mobile phase to identify the best conditions for the separation of VIL enantiomers. Lux-Cellulose-2 column was found to provide the best chiral resolution for VIL enantiomers. Further experiments were conducted using different aqueous-organic mobile phases to achieve the simultaneous chiral-achiral separation of the selected compounds. Experimental design-based optimization was performed by using a face-centered central composite design. The optimal separation conditions (Lux Cellulose-2 stationary phase, 45 °C, mobile phase consisting of methanol/water/diethylamine 80:20:0.2 (v/v/v), and 0.45 mL/min flow rate) provided baseline separation for all 6 compounds. The optimized method was validated according to the ICH guideline and proved to be reliable, specific, linear, precise, and accurate for the determination of at least 0.1% for all impurities in VIL samples. The validated method was applied for determinations from a commercially available drug formulation and proved to be suitable for routine quality control of both enantiomeric and organic impurities of VIL.

QbD-Engineered Development and Validation of a RP-HPLC Method for Simultaneous Estimation of Rutin and Ciprofloxacin HCl in Bilosomal Nanoformulation.

In the given study, a new reverse-phase high-performance liquid chromatography (RP-HPLC) method has been reported for the simultaneous estimation of ciprofloxacin hydrochloride (CPX) and rutin (RUT) using quality by design (QbD) approach. The analysis was carried out by applying the Box-Behnken design having fewer design points and less experimental runs. It relates between factors and responses and gives statistically significant values, along with enhancing the quality of the analysis. CPX and RUT were separated on the Kromasil C18 column (4.6 × 150 mm, 5 μm) using an isocratic mobile phase combination of phosphoric acid buffer (pH 3.0) and acetonitrile with the ratio of 87:13% v/v at a flow rate of 1.0 mL/min. CPX and RUT were detected at their respective wavelengths of 278 and 368 nm using a photodiode array detector. The developed method was validated according to guideline ICH Q2 R (1). The validation parameters taken were linearity, system suitability, accuracy, precision, robustness, sensitivity, and solution stability which were in the acceptable range. The findings suggest that the developed RP-HPLC method can be successfully applied to analyze novel CPX-RUT-loaded bilosomal nanoformulation prepared by thin-film hydration technique.

Regulatory Perspective Reverse Engineering Analysis of the Mast Cell Stabilizer and the Histamine Receptor Antagonist (Olopatadine HCl): Instrumental and Classical Methods for Multiple Formulations.

This study evaluates the unknown qualitative (Q1) and quantitative (Q2) formulas for nasal spray and ophthalmic solution formulations of olopatadine HCl by classical and instrumental techniques to match the generic formula with reference-listed drugs to avoid clinical study. Reverse engineering of olopatadine HCl nasal spray 0.6% and ophthalmic solution 0.1, 0.2% formulations was accurately quantified using a simple and sensitive reversed-phase high-performance liquid chromatography (HPLC) method. Both formulations possess similar components, namely ethylenediaminetetraacetic acid (EDTA), benzalkonium chloride (BKC), sodium chloride (NaCl), and dibasic sodium phosphate (DSP). These components were qualitatively and quantitatively determined using the HPLC, osmometry, and titration techniques. With derivatization techniques, EDTA, BKC, and DSP were determined by ion-interaction chromatography. NaCl in the formulation was quantified by measuring the osmolality and using the subtraction method. A titration method was also used. All the employed methods were linear, accurate, precise, and specific. The correlation coefficient was >0.999 for all components in all the methods. The recovery results ranged from 99.1 to 99.7% for EDTA, 99.1-99.4% for BKC, 99.8-100.8% for DSP, and 99.7-100.1% for NaCl. The obtained % relative standard deviation for precision was 0.9% for EDTA, 0.6% for BKC, 0.9% for DSP, and 1.34% for NaCl. The specificity of the methods in the presence of other components, diluent, and the mobile phase was confirmed, and the analytes were specific.

Analytical Quality by Design-Driven RP-HPLC Method Conditions to Concomitantly Determine Cinnarizine and Morin Hydrate in Combined Drug Solution and Dual Drug-Loaded Formulations.

The replacement of traditional oils with a camphor and menthol-based eutectic mixture is done to prepare oil-less emulsion-like dispersions for co-delivery of cinnarizine (CNZ) and morin hydrate (MH) for managing Meniére's disease (MD). Since two drugs are loaded into the dispersions, the development of a suitable reverse phase-high performance liquid chromatography (RP-HPLC) method for their simultaneous analysis becomes inevitable. By applying the analytical quality by design (AQbD) approach, the RP-HPLC method conditions were optimized for the concomitant determination of two drugs. The systematic AQbD started with identifying critical method attributes (CMA) through an Ishikawa fishbone diagram, risk estimation matrix, and risk priority number-based failure mode effect analysis followed by screening using fractional factorial design and optimization by face-centered central composite design. The concomitant determination of two drugs by the optimized RP-HPLC method condition was substantiated via specificity checking using combined drug solution, drug entrapment efficiency, and in vitro release of the two drugs from emulsion-like dispersions. The AQbD optimized RP-HPLC method conditions revealed the retention time for CNZ and MH at 5.017 and 5.323, respectively. The studied validation parameters were found within the ICH-prescribed limits. Exposing the individual drug solutions to acidic and basic hydrolytic conditions yielded extra chromatographic peaks for MH, probably due to the degradation of MH. The DEE % values of 87.40 ± 4.70 and 74.79 ± 2.94, respectively, were noticed for CNZ and MH in emulsion-like dispersions. More than 98% CNZ and MH release was occurred from emulsion-like dispersions within 30 min post-dissolution in artificial perilymph. Overall, the AQbD approach could be helpful for systematic optimization of RP-HPLC method conditions to estimate concomitantly other therapeutic moieties. The proposed article shows the successful application of AQbD for the optimization of RP-HPLC method conditions to concomitantly estimate CNZ and MH in combined drug solution and dual-drug-loaded emulsion-like dispersions.

Liquid chromatography separation and identification of new oxidative degradation impurity in edoxaban tosylate hydrate drug substance by using liquid chromatography with tandem mass spectrometry.

Edoxaban is an anti-coagulant medication and a director factor Xa inhibitor. A novel reverse phase liquid chromatography-mass spectrometry compatible method developed for separation and identification of new oxidative degradation impurities in edoxaban tosylate hydrate drug substance. The separation of three oxidative degradation impurities was achieved by using YMC Triart phenyl (250 × 4.6) mm, 5 µm column with mobile phase containing gradient elution of mobile phase-A as 10 mM ammonium acetate and mobile phase-B as acetonitrile:methanol (1:1)% (v/v). The flow rate of the mobile phase is 0.7 mL/min with a column temperature of 40 °C and detection wavelength of 290 nm. Edoxaban tosylate hydrate shows significant degradation in oxidative stress conditions and forms three oxidative degradation products. The degradation products were identified and characterized by using a high-resolution mass spectrometry quadrupole-time of flight mass detector. The three oxidative degradation impurities of Edoxaban drug substance were well resolved with each other and along with Edoxaban drug substance peak. Among the three oxidative degradation impurities di-N-oxide impurity was the new oxidative degradation impurity identified for the first time and a novel reverse-phase high-performance liquid chromatography method was developed for separation of the three oxidative degradation impurities.

RP UFLC method for estimation of valsartan chemometricaly

A simple, sensitive, rapid, precise and economical reverse phase HPLC method has been developed for the estimation of valsartan from pharmaceutical dosage forms. The method was carried out using Chromatographic conditions were established by employing a C18 analytical phenomenal (Kinetex) column (5micron -C18, 250x4.6mm) with a mobile phase of phosphate buffer (PH 3.5) and acetonitrile in a 60:40 ratio. The sample was injected at a volume of 20 microliters, and the mobile phase was degassed using a Sonica ultrasonic sonicator before being pumped into the HPLC system. The flow rate was set at 1 ml per minute, and the wave length 273 nm was chosen for detection. The column temperature was kept at 25oC respectively. The developed method was validated in terms of accuracy, precision, linearity, limit of detection, limit of quantitation and solution stability. The proposed method can be used for the estimation of these drugs in dosage forms.

A New Stability-Indicating RP-HPLC Method for Simultaneous Quantification of Diacerein and Aceclofenac in Novel Nanoemulgel Formulation and Commercial Tablet Dosage Form in the Presence of their Major Degradation Products Using DoE Approach.

The present study aimed to develop a simple, robust, sensitive and effective stability-indicating reversed-phase high-performance liquid chromatography method for simultaneous quantification of diacerein (DCN) and aceclofenac (ACE) in novel nanoemulgel formulation and commercial tablets in the presence of their main degradation product: rhein (RH) and diclofenac sodium (DLS), respectively. A fractional factorial design was used to screen the crucial independent factors, whereas a central composite design was used for the optimization of the chromatographic conditions. The separation was carried out on Phenomenex C18 column (5μm, 250 × 4.6mm), using a mobile phase consisting of phosphate buffer pH3 (0.1% v/v orthophosphoric acid) and acetonitrile (40:60v/v) at a flow rate of 1mL/min with detection at 264nm. The analytes were exposed to a variety of stress conditions, including heat, alkali, acid, oxidation, photochemical, humidity and hydrolysis. DCN, ACE, RH and DLS were found to have retention times of 4.32 ± 0.15, 5.77 ± 0.07, 8.28 ± 0.20 and 9.10 ± 0.18min, respectively. The percent recovery for all four analytes was found to be between 98 and 102, and the procedure was discovered to be linear in the range of 0.1-64μg/mL with R2 value > 0.999. The established method was validated as per ICH guidelines and successfully used to assay DCN and ACE in their combined marketed tablet dosage form and developed nanoemulgel formulation.

Advancing posaconazole quantification analysis with a new reverse-phase HPLC method in its bulk and marketed dosage form

Introduction: Posaconazole is a widely used antifungal drug, and its accurate quantification is essential for quality control and assessment of its pharmaceutical products. This study aimed to develop and validate a reverse-phase high-performance liquid chromatography (HPLC) analytical method for quantifying Posaconazole in bulk and dosage form. Methods: The HPLC method was developed and validated based on International Conference on Harmonisation (ICH) guidelines. The developed method was then applied to quantify Posaconazole in a marketed tablet formulation. The method's specificity, linearity, precision, accuracy, robustness, and stability were evaluated. Results: The developed HPLC method showed good linearity over a 2-20 μg/mL concentration range. The percentage recovery of Posaconazole from the bulk and marketed formulations was found to be 99.01% and 99.05%, respectively. The intra-day and inter-day precisions were less than 1%, and the method was stable under different conditions. The HPLC method was successfully applied to quantify Posaconazole in the marketed formulation. Conclusion: The developed and validated HPLC method is reliable and efficient for analyzing Posaconazole in bulk and dosage forms. The method's accuracy, precision, specificity, linearity, robustness, and stability demonstrate its effectiveness. The method can be used for the quality control and assessment of Posaconazole-containing pharmaceutical products.

Validated stability-indicating RP-HPLC assay method for fesoterodine fumarate in pharmaceutical products

The goal of the current study was to develop a precise, sensitive and validated reverse phase HPLC method for determination of fesoterodine fumarate (FST). The current investigation was performed for a stability indicating method in order to obtain both assay and impurity profiles of FST. All the experiment was done on HPLC Shimadzu Prominence 20A having stainless steel ACE5 C18 column with a particle size of 5µm and a dimension of 4.6 mm X 150 mm. The mobile phase was in gradient mode with mobile phase A (0.03M ammonium acetate buffer : acetonitrile : methanol (55:30:15 v/v) pH 3.8) and mobile phase B (water) . The flow rate was 1.2 mL/min and the wavelength 208 nm was selected for detection. This method was validated for linearity and range, accuracy, precision and robustness in accordance with ICH requirements. The retention time of fesoterodine was found as 11.0 min. The main degradation product 5-hydroxymethyltolterodine (5-HMT; active metabolite of FST) and the other process impurities; aldehyde, benzylated hydroxy, tolterodine ester, diester impurities were detected easily at the retention times 3.8, 13.5, 16.7, 21.5, 37.1 minutes, respectively. It was observed that the retention times of the main peak and its impurities were not affected and the selectivity of the method was proved under the applied stress conditions. With this developed method, FST and its impurities can easily be detected. It is also stable, simple, accurate, and completely validated RP-HPLC technique for the analysis of FST and related impurities that meets with ICH standards.

Extraction and Quantification of Ano Rectal Formulation by UV-LC Techniques

Suppositories very popular formulations and are currently used therapeutically all over the world. Especially in pediatrics, where they can be used for the effective lowering of high fever accomplished with seizure. The choice of a suppository as the mode of drug delivery system in all cases even in infants when oral delivery is impossible, that is an unconscious or vomiting patient, or in the case of infants. Therefore, the quality control study of this dosage form with a modern instrumental analytical technique is highly desired. In consequence of the increased application of suppositories, there is a current demand for a rapid, effective analytical methods required for routine analysis. The literature search revealed that methods for the UV and high-performance liquid chromatography (HPLC) analysis of DZP are very rare.Thereforethe aim of the present research was to develop and validate a suitable general sample preparation and chromatography method for suppositories containing DZP. The primary method (I) is based on UV Spectroscopy. UV spectra of Diazepam are recorded. The secondary method is based on the Reverse phase HPLC method was developed. The UV detector was operated at 286nm. After method development the proposed method was validated as per ICH guidelines. The developed method was accurate and precise which shows the evident from the analytical data and recovery studies. The analytical results were in good and it complies with the label claims.

New N-acyl Thiourea Derivatives: Synthesis, Standardized Quantification Method and In Vitro Evaluation of Potential Biological Activities.

New N-acyl thiourea derivatives with heterocyclic rings have been synthesized by first obtaining isothiocyanate, which further reacted with a heterocyclic amine, characterized by (FT-IR, NMR spectroscopy and FT-ICR) and tested for their in vitro antimicrobial, anti-biofilm and antioxidant activities to obtain a drug candidate in a lead-optimization process. From the tested compounds, those bearing benzothiazole (1b) and 6-methylpyridine (1d) moieties revealed anti-biofilm activity against E. coli ATCC 25922 at MBIC values of 625 µg/mL. Compound 1d exhibited the highest antioxidant capacity (~43%) in the in vitro assay using 1,1-diphenyl-2-picrylhydrazyl (DPPH). Considering the in vitro results, the highest anti-biofilm and antioxidant activities were obtained for compound 1d. Therefore, a reversed-phase high-performance liquid chromatography (RP-HPLC) method has been optimized and validated for the quantitative determination of compound 1d. The detection and quantitation limits were 0.0174 μg/mL and 0.0521 μg/mL, respectively. The R2 correlation coefficient of the LOQ and linearity curves were greater than 0.99, over the concentration range of 0.05 μg/mL-40 μg/mL. The precision and accuracy of the analytical method were within 98-102%, confirming that the method is suitable for the quantitative determination of compound 1d in routine quality control analyses. Evaluating the results, the promising potential of the new N-acyl thiourea derivatives bearing 6-methylpyridine moiety will be further investigated for developing agents with anti-biofilm and antioxidant activities.

A stability-indicating, reversed-phase HPLC method for quantification of assay and organic impurities in doxycycline hyclate bulk and parenteral dosage forms.

The aim of this study is to develop a stability-indicating, reversed-phase HPLC method for the quantification of assay and organic impurities (process and degradation) of doxycycline hyclate in a doxycycline injectable formulation. Both the active and dosage forms are officially present in the USP monograph, and assay and impurity methods are provided by separate UPLC techniques, which are highly sensitive to the flow rate and temperature, considering the quality control requirements and user-friendliness. A simple stability-indicating HPLC method with a shorter run time was developed for the simultaneous quantification of assay and impurity. The method was developed using HPLC with a gradient program and a reversed-phase Waters XBridge BEH C8 column (150 × 4.6 mm, 3.5 μm i.d.). Mobile phase A consisted of phosphate buffer (pH 8.5, 25 mM potassium phosphate, 2 mM ethylenediaminetetraacetic acid, and 0.5 ml of triethylamine). Mobile phase B consisted of methanol with a flow rate of 1.7 ml/min, a column temperature of 55°C, a UV wavelength of 270 nm, and an injection volume of 25 μl. Modern research represents a concomitant method for quantifying assay and organic impurities of doxycycline hyclate (active form) and doxycycline for injection (dosage form). The assay and impurity method were validated per United States Pharmacopeia (USP)1225 and International Conference on Harmonization (ICH)guidelines. The retention time of doxycycline and degradation impurity, 4-epidoxycycline, was about 9.8 and 6.4 min, respectively. The linearity range of doxycycline and 4-epidoxycycline was 0.5-150 and 0.5-18 μg/ml, respectively. The percentage of recovery of doxycycline and 4-epidoxycycline was 98.7-100.6% and 88.0-112.0%. Validation of the analytical method demonstrated that the method is suitable, specific, linear, accurate, precise, rugged, and stability indicating for estimating the assay, known and degraded impurities of doxycycline, and doxycycline for injection.

A stability indicating RP-HPLC method development and validation for simultaneous quantification for assay of rifampicin in pharmaceutical solid dosage form

A cost effective stability indicating reverse phase high performance liquid chromatography (RP-HPLC) for simultaneous quantification for assay of Rifampicin in pharmaceutical solid dosage form (tablets) was developed and validated. Chromatographic procedure was conducted using stationary phase packing c18 column (Luna), having 5 µm particle size with inner diameter (250 x 4.6) mm, mobile phase consists of 60% acetate buffer (pH 4.5) and 40% acetonitrile (HPLC grade of sharlau manufacturer). The detection wavelength was uv 254 nm with flow rate of 1.0 ml/min and injection volume was 10 µL with run time 10 min. Based on proposed method validation was conducted following system precision, specificity or selectivity, method precision, accuracy, linearity, robustness, solution stability, limit of quantitation (LOQ), limit of detection. It was observed the system (Waters, HPLC) was precise enough to quantify Rifampicin, no interference of blank, placebo or other known impurity at the elution zone of Rifampicin; % RSD for six sample solutions of Rifampicin was 0.9% (% average assay: 98.7%); accuracy observed 101.1% for 80% recovery, 100.4% for 100% recovery and 100.5% for 120% recovery; co-relation coefficient of linearity (r =1.000); limit of quantification (LOQ) found 0.18 ppm.

Development and validation of stability indicating RP-HPLC method for determination of aspirin and pantoprazole sodium in synthetic mixture stability indicating HPLC method

The present study describes a new accurate and precise stability indicating reverse phase HPLC method for quantitative computation of pantoprazole sodium and Aspirin from physical simulated mixture. The proposed chromatographic method employs Hypersil ODS C18 column (250 x 4.6 mm, 5μ) as the stationary phase and combination of methyl alcohol and water in ratio of 70: 30 v/v as the elution medium. Overall separation was carried out at 0.8 ml/minute flow rate and elution was monitored at 254 nm. The proposed system gave well resolved peak of Aspirin and Pantoprazole sodium with elution time of 2.32 and 5.85 minute respectively. Same system was effective in separation of active components and degradation products when the components were subjected to forced degradation as per regulatory guidelines (ICHQ1). Finally, the optimized method was successfully validated as per ICH Q2R1 guidelines and applied for quantitative analysis of both active components in synthetic mixture.

Advances, Applications, and Challenges in RP HPLC Method Development

High-performance liquid chromatography (HPLC) has become an indispensable tool for modern analytical chemistry, and reversed-phase (RP) HPLC is one of the most widely used techniques for the separation and analysis of complex mixtures. In recent years, there have been numerous advances in RP HPLC method development, including the development of new stationary phases, improved column technology, and novel optimization strategies. These advances have led to increased speed, resolution, and sensitivity, as well as improved selectivity and reduced non-specific adsorption. Moreover, RP HPLC has found applications in a wide range of industries, including pharmaceuticals, food and beverage, environmental analysis, forensic science, and biotechnology. Despite its many benefits, RP HPLC method development is not without its challenges, including issues with column stability, sample preparation, and optimization. In this review article, we provide a comprehensive overview of the principles, optimization strategies, and recent advances in RP HPLC method development. We discuss the applications of RP HPLC in various industries and identify the common challenges and emerging trends in the field. Additionally, we evaluate the impact of RP HPLC on the accuracy, sensitivity, and selectivity of analysis and its potential to improve the quality and safety of products and the environment. Overall, this review highlights the importance of RP HPLC in modern analytical chemistry and provides insights and recommendations for researchers and practitioners who are interested in this technique. By exploring the latest advances, applications, and challenges in RP HPLC method development, we hope to inspire continued innovation and progress in this important field.

Comparative Extraction and Quantifi cation of Myricetin from Leaves of Madhuca longifolia Using RP-HPLC

Background: This study’s purpose was to develop a sensitive, repeatable reverse-phase high-performance liquid chromatography (RP-HPLC) method for extracting the main fl avonoid Myricetin from the leaves of Madhuca longifolia and quantifying it. Materials and Methods: Diff erent leaves extracts were prepared using various solvent systems, including ethanol, ethanol: water (50:50), and water, through various extraction methodologies, including hot and cold maceration and soxhlet-assisted extraction, in order to optimize the best solvent system and ideal extraction methodology (SAE). Myricetin was separated by chromatography using a C18 column. Results: According to the fi ndings, the hydroalcoholic extract had the greatest content of myricetin at 2.524 ng. This study reports the presence of fl avonoid content in M. longifolia leaves extract that can be extracted using, water:ethanol and ethanol. The best solvent system was water:ethanol, and the best extraction technique was cold maceration. Conclusion: M. longifolia extracts were produced by hot and cold maceration, SAE techniques, and their fl avonoid concentrations were measured using HPLC-ESI-MS/MS. Water: Cold maceration and ethanol were shown to be promising solvent systems. While trying to extract myricetin from M. longifolia leaves, the extraction approach was more eff ective than other traditional extraction methods.

Estimation of Digoxin in Chloroform Extract of <i>Moringa concanensis</i> Leaves Using Newly Developed and Validated RP-HPLC Method

Cardiac glycosides are the secondary metabolites that are used in atrial fibrillation and atrial flutter as it binds with the sodium-potassium ATPase pump and increases the force of contraction. There are two types of cardiac glycosides i.e. cardenolides and bufadienolides. Digoxin is a cardenolide type of cardiac glycoside. Digoxin is found in chloroform extract of Moringa concanensis leaves. In the present study, the reverse-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated to determine the content of digoxin in chloroform extract of M. concanensis leaves from three different geographical sources. The chromatographic method was developed at 220 nm wavelength with an Acetonitrile: Water: 1% Orthophosphoric acid (50:50:0.1 %V/V/V) mobile phase. In this method, the digoxin shows linearity in the range of 10 – 50 μg/ml with a regression coefficient of 0.995. The Limit of detection (LOD) and Limit of quantitation (LOQ) was found to be 0.232 μg/ml and 0.703 μg/ml respectively.

Development and Validation of RP-HPLC Method for the simultaneous determination of Nirmatrelvir and Ritonavir in bulk and pharmaceutical formulation

In this study, a rapid and sensitive stability indicating reversed phase HPLC method was developed for quantitation of Nirmatrelvir and Ritonavir simultaneously in bulk and tablet formulation. Nirmatrelvir and ritonavir were separated on a Thermo C18 column with mobile phase containing 0.01M potassium dihydrogen phosphate buffer and acetonitrile (45:55, v/v). The flow rate was 1 mL/min and detection wavelength was 272 nm. Method linearity was established over a range of 75–225 μg/mL for nirmatrelvir and 50–150 μg/mL for ritonavir. Limit of quantification was 0.694μg/mL for nirmatrelvir and 0.820μg/mL for ritonavir. The recovery (%) was 99.96 to 100.45 (Nirmatrelvir) and 100.25 to 101.35 (Ritonavir). The method precisions were 0.11% (Nirmatrelvir) and 0.33% (Ritonavir). Method was suitable to assay nirmatrelvir and ritonavir in tablet formulation (Paxlovid). Stress degradation studies have shown that this method can be implemented to assay nirmatrelvir and ritonavir in the presence of its degradants.