Reversed-phase High-performance Liquid Chromatographic Method Research Articles

"Development and Validation of an Isocratic Reverse-Phase High-Performance Liquid Chromatographic Method for the Simultaneous Determination of Desloratadine and Montelukast sodium in Bulk Drug, Pharmaceutical Dosage Forms, and Human Plasma.”

"Development and Validation of an Isocratic Reverse-Phase High-Performance Liquid Chromatographic Method for the Simultaneous Determination of Desloratadine and Montelukast sodium in Bulk Drug, Pharmaceutical Dosage Forms, and Human Plasma.”

Method Development and Validation of Gallic Acid in Liquid Dosage Form by Using RP-HPLC Method.

A simple, rapid, precise, sensitive, and reproducible reverse-phase high-performance liquid chromatography (RP-HPLC) method has been developed for the quantitative analysis of gallic acid in the pharmaceutical dosage form. Chromatographic separation of gallic acid was achieved on Waters Alliance-e 2695, by using Waters X-Terra RP-18 (150×4.6mm, 3.5μ) column and the mobile phase containing 0.1% formic acid and ACN in the ratio of 70:30% v/v. The flow rate was 1.0mL/min; detection was carried out by absorption at 275 nm using a photodiode array detector at ambient temperature. The number of theoretical plates and tailing factor for gallic acid was NLT 2000 and should not be more than 2 respectively. Percentage relative standard deviation of peak areas of all measurements is always less than 2.0. The proposed method was validated according to ICH guidelines. The method was found to be simple, economical, suitable, precise, accurate, and robust method for quantitative analysis of gallic acid.

Applicability of an HPLC method for analysis of alcoholic and glycolic Brazilian green-propolis extracts

Brazilian green propolis has been widely used in food and pharmaceutical products due to its valuable source of phenolic compounds and versatile biological activities. The development and validation of analytical methods are extremely useful for the characterization and quality control of products containing propolis. Therefore, the aim of this study was to optimize, validate and investigate the applicability of a reversed-phase HPLC method for analysis of different types of Brazilian green propolis extracts (glycolic and ethanolic). The method showed to be selective for the propolis phenolic markers. The analysis of variance and residues demonstrated that the method had significant linear regression, without lack of fit. It was also a precise, accurate and robust method, which was of utmost importance to analyze both glycolic and ethanolic extracts and at different concentrations. Moreover, as these products can display most complex matrices to analyze, a valid HPLC method can also prove to be specific and versatile.

A stability-indicating reverse phase-HPLC method development and validation for newly approved drug, Belzutifan in bulk and pharmaceutical dosage forms

A stability-indicating reverse phase-HPLC method development and validation for newly approved drug, Belzutifan in bulk and pharmaceutical dosage forms

Preparation and evaluation of lipid matrix microencapsulation for drug delivery of azilsartan kamedoxomil

The aim of the work is to consolidate azilsartan-kamedoxomil (AZM) into lipid matrix controlled-release microparticles to enhance its permeability because AZM belongs to Biopharmaceutical classification (BCS) IV which characterized by poor permeability and to protect AZM from light and humidity and execute a prolonged release profile.
 Materials and methods. A reversed-phase HPLC method was created and validated to estimate the drug. AZM microparticles formulations were invented using melt dispersion technique and waxy materials such as carnuba wax, beeswax, stearic acid in the ratio of waxy-substance: drug ranging from 0.25: 1 to 1:1 and stabilizer namely; tween 80 and Poloxamer 407 in ratio of stabilizer: drug ranging from 0.5:1 to 1:1. Azilsartan formulations were assessed for azilsartan-medoxomil content, loading, entrapment efficiency, the zeta potential,the particle size, the morphology by scanning electronic microscopy (SEM), and in-vitro release profile.
 Results. Zeta potential results for microparticle formulations using beeswax and carnuba range from -21.1 mV to -26.6 mV and -20.6 mV to -26.7 mV, respectively. This difference indicates that the azilsartan microparticles containing stearic acid are better stabilized with zeta potential of 25.3 - 29.7 mV. Furthermore, the release from azilsartan microparticle formulations containing stearic acid exceeded 80 % after 8 h and remained for 24 h while release from beeswax did not exceed 65 % after the same period and less than 60 % in case of carnuba formulations
 Conclusions. The formulation (AZSP4) exhibited the highest zeta potential and released exceeding 80 % of AZM over the course of 8 hours and remained over a day. AZSP4 microparticles formulation containing, poloxamer 407, in a 0.8:0.8:1 drug: stearic acid: poloxamer ratio proved the ability of stearic acid microencapsulation employing poloxamer as stabilizer in a certain ratio can prolong the release of AZM

Stability Indicating Isocratic HPLC Method for Bilastine and Characterization of Forced Degradation Products by LC-MS/MS

Abstract: The present study aims to develop and validate a simple, precise, accurate, stability indicating and isocratic reversephase high-performance liquid chromatography (RP-HPLC) method for estimating Bilastine in bulk and synthetic mixture. Bilastine from its degradation products were well separated and estimated on Discovery C8 column (250 mm x 4.6 mm, 5μm) using methanol: 0.1% ortho-phosphoric acid (55:45 %v/v) as a mobile phase and detection was performed at 276 nm by PDA detector. The degradation of Bilastine was studied under different ICH recommended stress conditions. The developed stability-indicating method was validated for system suitability, linearity, accuracy, precision, robustness, detection limit and quantitation as per ICH guidelines. The method was linear over 25-150 μg/ml. Mathematically computed Limit of Detection and Limit of Quantitation were found to be 0.19μg/ml and 0.57μg/ml, respectively. It was discovered that Bilastine degrades under acid and oxidation conditions. By using LC-MS/MS analysis, the structure of Bilastine breakdown products created amid acidic and oxidative settings was elucidated. A complete fragmentation pathway of Bilastine was established to elucidate the degradation products' structures using LC–MS/MS. The elemental concentrations of oxidation products and their fragmentation products were studied using the acquired mass values. The overall data was utilized to describe the degradation of products and their fragmentation process. The developed method can be used for routine analysis of Bilastine in dosage form as it can estimate of Bilastine in presence of excipients and degradation products.

Development and Validation for the Estimation of Fenofibrate in Pharmaceutical Dosage form by Reversed-phase High-performance Liquid Chromatography

Very simple, exact, precise and accurate reversed-phase high-performance liquid chromatography (RP-HPLC) methods used to estimate the amount of fenofibrate in both the bulk and tablet formulations. The reversed-mobile phase buffer and ACN in the proportion (40:60) v/v at a flow rate of 1.0 mL/min were used to create the Princeton (C18) (250 mm x 4.6 mm, 5) column. At 240 nm wavelength, the detection procedure was approved. The RT was established to be 3.905 minutes under fenofibrate’s optimum circumstances. The calibration curve had a range of 87 to 232 g/mL and a correlation coefficient of 0.9994. All-important parameters’ RSD values were below 2.0%. A 99.13 and a 100.44% recovery rate, respectively, were shown to exist. In accordance with ICH requirements, the established technique was evaluated for linearity, specificity, precision, accuracy, and system applicability. The feasibility and repeatability of the suggested technique for determining the amount of the commercially available dosage forms of fenofibrate in tablet and bulk form were also demonstrated.

Efficient Separation of C-Tetramethylcalix[4]resorcinarene Conformers by Means of Reversed-Phase Solid-Phase Extraction.

A reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed to study the conformer formation generated during the reaction for obtaining C-tetramethylcalix[4]resorcinarene. The chromatographic method was used to design a strategy for purifying the reaction products, using solid-phase extraction columns (RP-SPE) and gradient elution. The chromatographic profiles of the cyclocondensation reaction between resorcinol and acetaldehyde show the presence of three products under the different reaction and precipitation conditions studied. Using RP-SPE, it was possible to enrich the products, which were later characterized by means of RP-HPLC and 1H nuclear magnetic resonance (NMR). This investigation explored and established a new method for RP-HPLC analysis and RP-SPE separation of conformational isomers obtained in the formation reaction of C-tetramethylcalix[4]resorcinarene.

Определение статинов методом обращенно-фазовой ВЭЖХ в подвижных фазах с разными вариантами модифицирования

The chromatographic behavior of atorvastatin, rosuvastatin and simvastatin in the presence of the main modifiers of the mobile phase: elution strength and buffer solution, has been studied by means of the reverse-phase high-performance liquid chromatography (HPLC) method. Optimal modification conditions have been found and their application for the determination of statins under study in the pharmaceutical preparations Atorvastatin-OBL, Liprimar and Tulip has been shown. The Sr value is 0.03. The relative determination error does not exceed 7%.

Development and validation of a stability-indicating, single HPLC method for sacubitril-valsartan and their stereoisomers and identification of forced degradation products using LC-MS/MS.

The aim of this research work was to develop and validate a stability-indicating, single reversed-phase HPLC method for the separation of five impurities, including enantiomers, diastereomers, and degradation products in sacubitril-valsartan tablets. The method was developed using a Chiralcel OJ-RH column (150 × 4.6mm, 5μm) at 45°C with a gradient program of (T/%B) 0.01/25, 10.0/25, 25/38, 37.0/45, 39.0/25, and 45.0/25 at a flow rate of 0.8ml/min. Mobile phase A consisted of 1ml of trifluoroacetic acid in 1000 ml of Milli-Q water. Mobile phase B consisted of 1ml of trifluoroacetic acid in a mixture of acetonitrile and methanol in the ratio of 950:50 (v/v). Sacubitril, valsartan, and their five impurities were monitored at 254 nm. Degradation was not observed when sacubitril-valsartan was subjected to heat, light, hydrolytic, and oxidation conditions. In acid degradation study (1N HCl/60°C/2h) impurity 1 (m/z 383.44) was formed, and in base degradation study (0.1N NaOH/40°C/1h) impurities 1 and 5 (m/z 265.35) were formed; both impurities were confirmed using LC-MS. The degradation products, enantiomers, and diastereomers were well separated from sacubitril and valsartan, proving the stability-indicating power of the method. The developed method was validated per the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidelines. The inter- and intra-day percentage relative standard deviation for sacubitril, valsartan, and their five impurities was less than 5.2%, recovery of the five impurities was between 93 and 105%, and linearity was ≥0.999. The limit of detection was 0.030-0.048 μg/ml, and the limit of quantification was 0.100-0.160 μg/ml.

Determination of Firocoxib and Its Related Substances in Bulk Drug Substance Batches of Firocoxib by a High-Speed Reversed-Phase HPLC Method With a Short Fused-Core Biphenyl Column.

Firocoxib is a nonsteroidal anti-inflammatory drug. It provides control of postoperative pain and inflammation associated with soft tissue and orthopedic surgery in dogs, and control of pain and inflammation associated with osteoarthritis in horses. A high-speed stability-indicating reversed-phase high-performance liquid chromatography method was developed to determine firocoxib and its related substances in bulk batches of firocoxib drug substance. Firocoxib was dissolved in neat acetonitrile (ACN) and analyzed on a short HALO (fused-core) biphenyl column (30 × 4.6mm i.d., 2.7-μm particle size) at flow rate of 2.5mL/min. Column temperature was maintained at 50°C. Mobile phase A is composed of 0.1% of H3PO4 in water and mobile phase B is composed of ACN. Analytes were detected with UV detection at 240nm and quantitated against an external reference standard. Firocoxib and its related compounds were adequately separated within 4min by a gradient elution. The method was validated for specificity, linearity, accuracy, precision and robustness according to method validation guidelines described in The International Conference on Harmonization. The validation data demonstrated that this method is sensitive, accurate, robust, specific and stability-indicating.

Development and Validation of Stability-Indicating High-Performance Liquid Chromatography Method for Estimation of Lacosamide in Bulk and Its Pharmaceutical Dosage Form.

A simple, specific, accurate and stability-indicating reversed-phase high-performance liquid chromatographic method was developed for the determination of lacosamide, using C18 column and a mobile phase composed of phosphate buffer (pH4.0):acetonitrile (40:60v/v). The retention time of lacosamide was found to be 2.7min. Linearity was established for lacosamide in the range of 10-50μg/mL. The percentage recovery of lacosamide was found to be in the range of 97.37-99.20%. The drug was subjected to acid, alkali, oxidation, dry heat and photolytic degradation. The degradation studies indicated condition was well resolved from the pure drug with significant differences in their retention time values. This method can be successfully employed for quantitative analysis of lacosamide in bulk drug and formulation.

Development and Validation of RP-HPLC Method for Simultaneous Estimation of Haloperidol and Trihexyphenidyl Hydrochloride in Tablet Dosage Form

Haloperidol is a common antipsychotic drug used to treat schizophrenia, and Trihexyphenidyl hydrochloride is an antiparkinsonian medication. The combination formulation is used to treat schizophrenia as well as depression. A simple, accurate, and precise assay as well as a rapid stability-indicating reversed-phase high performance liquid chromatography (RP-HPLC) method for the simultaneous estimation of Haloperidol (HAL) and Trihexyphenidyl hydrochloride (THP) from their combination drug product have been developed and validated. The proposed method is based on reversed-phase separation of the two drugs using a Zorbax Eclipse XDB C18 column (150 mm x 4.6 mm, 5 um) kept at 30°C. The optimal mobile phase was composed of 0.5 mL triethylamine 0.05 M phosphate buffer having pH 3.75: acetonitrile: (65:35 v/v), a flow rate of 1.2 mL.min-1, and PDA detection at 210 nm. The method was validated in accordance with ICH standards. It was found to be precise and reproducible. Linearity was achieved in the concentration ranges of 6-60 ug mL-1 for HAL and 8-80 ug mL-1 for THP, with correlation coefficients of 0.999 and 1.000, respectively. The mean percent recovery of triplicate samples at each level for both drugs was 99.10-100.7% for Haloperidol and 98.7-100.6% for Trihexyphenidyl hydrochloride. The proposed method can be used to successfully test quality control and stability samples from bulk manufacturing and pharmaceutical dosage forms.

VALIDATION, STABILITY STUDIES, AND SIMULTANEOUS ESTIMATION OF CO-ENCAPSULATED CURCUMIN, EPIGALLOCATECHIN GALLATE NANOFORMULATION BY RP-HPLC METHOD

Objective: A new reverse-phase high-performance liquid chromatography (RP-HPLC) method was developed to simultaneously determine curcumin and epigallocatechin gallate (EGCG) in novel nanoformulation. Methods: The high-performance liquid chromatography (HPLC) method was achieved by using a Thermo Scientific Hypersil Base Deactivated Silica (BDS) C18 column (25 cm X 4.6 mm, 5 µm) at 35 °C column oven temperature. The chromatographic procedure was performed with a mobile phase of acetonitrile and 0.025 M (pH 4.0) potassium dihydrogen phosphate (KH2PO4) buffer by gradient mode of elution. The injection volume was 20 µl, and the flow rate was 1.5 ml/min, with ultraviolet (UV) detection using a diode array detector (DAD) at a 268 nm isosbestic wavelength. Results: Drug entrapment efficiency studies were performed with co-encapsulated EGCG and curcumin nanoformulation, which were found to be 94.35 % and 95.12 %, respectively. This shows that the developed method is highly effective. EGCG and curcumin were eluted at 3.9 min and 10.7 min, respectively. The linearity range was 25-175 µg/ml for EGCG and 12.5-100 µg/ml for curcumin. The correlation coefficient was 0.991 for EGCG and 0.999 for curcumin from the linearity curve, which indicates that the method can produce good sensitivity. Forced degradation studies were conducted in acidic, basic, oxidative, thermal, photolytic, and UV stress conditions, where all the degradation peaks were monitored. Conclusion: The developed method was linear, simple, rapid, robust, and precise. It could be used to quantify EGCG and curcumin simultaneously in various nanoformulations for in vivo and in vitro applications.

QUALITY CONTROL ASSAY OF TWO ANTIHYPERTENSIVE REPRESENTATIVES USING RP-HPLC BASED METHODOLOGY: STRESS ASSESSMENT ON ANTIHYPERTENSIVE REPRESENTATIVES

Objective: A quick simultaneous separation and quality control assay of two antihypertensive representatives, Azilsartan (AZIL) and Cilnidipine (CLIN) in bulk and tablet formulation was developed and validated using a Reverse phase (RP) HPLC method within a run time of 10 min. Methods: All chromatographic separations of AZIL and CILN were operated on a “Supelco C18 column (250 × 4.6 mm, 5 μ)”, using a mobile phase of Na2SO4 (0.1 M, pH 4.0): methanol at 60:40 (v: v) ratio and the samples were analyzed at 239 nm. Stability assessments of AZIL and CILN were carried out as per the ICH Q1A (R2) regulation. The methodology for determining AZIL and CILN in bulk and formulations tablets was verified by adhering to International Conference on Harmonization (ICH) recommendations. Results: Retention times of AZIL and CILN samples were 4.023 and 5.732 min, respectively, indicating a quick elution time. Over the tested range of 20–60 µg/ml for AZIL and 5–15 µg/ml for CILN determination, calibration curves have displayed linearity and satisfactory results. LOD of AZIL and CILN are 0.083 µg/ml and 0.056 µg/ml, respectively. The approach suggested herein has satisfactory precision (RSD: 0.1013% for AZIL and 0.4944% for CILN) and accuracy (recovery: 99.20 to 100.34 % for AZIL and 100.17 to 101.59 % for CILN). Furthermore, the approach has also been shown to be effective in detecting degradants of AZIL and CILN and resolving them with high resolution. Conclusion: This approach is shown to be acceptable for the accurate quality control assay of two antihypertensive representatives, AZIL and CLIN in both bulk and tablet formulation.

Determination of Genotoxic Impurities N-nitrosamine's in Efavirenz Drug Substance Using Rp-Hplc Technique

Aim: The main purpose of the research is to develop a effective, sensitive, economical and simple reverse phase HPLC method for the determination of genotoxic N-Nitrosamine's impurities in Efavirenz drug substance.
 Study Design: HPLC based quantification studies
 Place and Duration of Study: Department of Chemistry, Acharya Nagarjuna University, Guntur, Andhra Pradesh between April 2022 and August 2022.
 Methodology: Determination of genotoxic N-Nitrosamine's in Efavirenz drug substance. N-Nitrosamine's impurities were determined by RP-HPLC method using Zorbax SB C18 (150x4.6mm, 3.5µ) column as stationary phase. Column temperature maintained 25°C, injection volume 20 µL, flow rate 1.0 mL/min and sample cooler temperature 5°C and run time was 15 minutes. The mixture of 0.1% formic acid buffer and methanol in the ratio of 50:950 (v/v) was used as mobile phase. wavelength 240 nm. respectively.
 Results: There is no interference of blank at N-Nitrosamine's impurities peaks. The elution order and the retention times of impurities and Efavirenz obtained from individual standard preparations and mixed standard preparations are comparable.
 The limit of detection (LOD) and limit of quantitation (LOQ) for NDMA 0.03 & 0.09 µg/mL, NDEA 0.02 & 0.06 µg/mL and NDIPA 0.03 & 0.09 µg/mL respectively.
 The linearity results for N-Nitrosamine's impurities in the specified concentration range are found satisfactory, with a correlation coefficient greater than 0.99.Calibration curve was plotted and correlation co-efficient for impurities found to be 1.000, 1.000 and 1.000 respectively.
 The accuracy studies were shown as %recovery for N-Nitrosamine's impurities at specification level. The limit of % recovered shown is in the range of 80 and 120% and the results obtained were found to be within the limits. Hence the method was found to be accurate.
 The technique has been validated in accordance with ICH rules, and all validation parameters meet the acceptance criteria of the ICH Q2 specification.
 Conclusion: According to ICH guidelines, an RP-HPLC method that can assess genotoxic Nitrosoamines in Efavirenz at trace level concentration has been established. The method's specificity, precision, linearity, and accuracy ensured its efficacy. As a result, the technique is appropriate for the goals it was designed to achieve and can be successfully used for release testing of efavirenz into the market.

Formulation, Characterization, and Evaluation of Eudragit-Coated Saxagliptin Nanoparticles Using 3 Factorial Design Modules.

Background and Introduction: Saxagliptin is a hypoglycemic drug that acts as a dipeptidyl peptidase-4 (DPP-4) inhibitor and is preferably used in the treatment of Type 2 Diabetes Mellitus (T2DM). It is safe and tolerable; however, the major disadvantage associated with it is its low bioavailability. Aim: The present research aimed to enhance the bioavailability of the drug by enteric coating with a polymer that controls the rate of drug delivery, and it was prepared as Solid Lipid Nanoparticles (SLNs). Methodology: In the current study, various SLN formulations were developed using a central composite design (CCD) module using Design Expert-11 software. A modified solvent injection technique was used to prepare Saxagliptin nanoparticles coated with Eudragit RS100. The CCD was used to determine the independent variables and their effect on dependent variables at varied levels. Evaluation studies such as particle size analysis, Zeta potential, polydispersity index (PDI), drug loading, entrapment efficiency, in-vitro drug release studies, and in vivo pharmacokinetic studies were performed for the optimized SLN formulation. The reversed-phase HPLC method was developed and validated for the estimation of the pharmacokinetic parameters of the pure drug and prepared SLNs. Results: The effect of independent variables (A1: amount of lipid, A2: amount of polymer, A3: surfactant concentration, and A4: homogenization speed) on dependent variables (R1: particle size, and R2: entrapment efficiency) was established in great detail. Observed responses of the prepared and optimized Saxagliptin SLN were close to the predicted values by the CCD. The prepared SLNs depicted particle sizes in the range of 212-442 nm. The particle size analysis results showed that an increase in the lipid concentration led to an increase in particle size. The developed bioanalytical method was noted to be very specific and robust. The method accuracy varied from 99.16% to 101.95% for intraday, and 96.08% to 103.12% for inter day operation at low (5 mcg/mL), moderate (10 mcg/mL), and higher (15 mcg/mL) drug concentrations. The observed Zeta potential values for the prepared SLNs were in the range of -41.09 ± 0.11 to 30.86 ± 0.63 mV suggesting quite good stability of the SLNs without any aggregation. Moreover, the polydispersity indices were in the range of 0.26 ± 0.051 to 0.45 ± 0.017, indicative of uniformity of sizes among the prepared SLNs. In vivo study outcomes proved that Saxagliptin oral bioavailability significantly enhanced in male Albino Wistar Rats via SLN formulation and Eudragit RS100 coating approach. Conclusions: The developed and optimized Saxagliptin SLNs revealed enhanced Saxagliptin bioavailability in comparison to the native drug. Thus, this formulation strategy can be of great importance and can be implied as a promising approach to enhance the Saxagliptin bioavailability for facilitated T2DM therapy.

Characterization of Degradation Products of Lifitegrast by Mass Spectrometry: Development and Validation of a Stability-indicating Reversed Phase HPLC Method

Lifitegrast API was subjected to forced degradation studies under various conditions of hydrolysis (acidic, alkaline, and neutral/water), oxidation, photolysis, and thermal as prescribed by International Conference on Harmonisation guideline Q1A (R2). A short and simple reversed phase HPLC method was developed. The method was developed on a Shimadzu Ultra C18, (100 x 2.1) mm, 3.0 μm column. The gradient method consisted of 0.1% orthophosphoric acid as mobile phase A and mixture of acetonitrile and methanol in the ratio of 50:50 (v/v) as mobile phase B. The flow rate of the mobile phase was 0.8 mL/min. The developed method was validated using ICH Q2 (R1) guidelines. The parameters considered for method validation were solution stability, specificity, DL/QL, linearity, accuracy, precision and robustness. The drug showed significant degradation in acidic and alkaline conditions while slight degradation was observed in oxidative condition. The drug was found stable in water, photolytic and thermal conditions. The characterization of three major degradation products (DP1, DP2, and DP3) was done with LC-Q-TOF-MS/MS in combination with accurate mass measurements. The most probable mechanisms for the formation of DPs have been proposed on the basis fragmentation pattern.

Enantioselective Green HPLC Method for Simultaneous Determination of Enantiomer, and Potential Impurities in Apremilast Drug Substance

A single reversed-phase HPLC method was developed for the quantification of seven impurities of Apremilast and its enantiomer in the drug substance. An immobilized chiral stationary phase with a chiral selector “tris (3,5-dimethyl phenyl carbamate) derivative of amylose-Chiralpak IA-3 (250 mm × 4.6 mm, 3 μm) was employed to achieve the desired separation. A mobile phase consists of buffer (0.01M NH4HCO3, PH 8.0) and acetonitrile in the ratio 50:50 (v/v) and is pumped at a flow rate of 0.4 mL/min with an isocratic elution mode. The column oven temperature is set at 25°C, and the chromatographic output is monitored at 225 nm with a total run time of 45 min. A test concentration of 500 µg/mL of Apremilast in the mobile phase is used with an injection volume of 10 µL. Induced degradation studies were carried out to study the intrinsic chemical behavior of the drug. The degradation sample solutions were utilized to demonstrate the stability-indicating nature of the developed analytical method. The obtained mass number [M+H]+ for the primary degradation product formed in acid hydrolysis was identified as 418.36 and in base hydrolysis 478.12. The two impurities were identified as impurity-5(Deacetylated impurity), and impurity-2(open ring acid impurity), respectively, and the degradation pathways were established. Following ICH Q2 and USP<1225>guidelines, complete method validation was carried out. The RSD for the drug and impurities in interday and intraday studies are less than 4.0%, and the recoveries for the impurities are between 96.1-102.1% and linearity r ≥ 0.9997. LOQ results for the drug and impurities are between 0.052 µg/mL and 0.107 µg/mL, and LOD results are between 0.016 µg/mL and 0.032 µg/mL. The greenness of the method was evaluated by using an analytical eco scale, GAPI, and AGREE, and it was found that the method is green.

Development and Validation of a Reversed-Phase HPLC Method with UV Detection for the Determination of L-Dopa in Vicia faba L. Broad Beans.

L-Dopa (LD), a substance used medically in the treatment of Parkinson's disease, is found in several natural products, such as Vicia faba L., also known as broad beans. Due to its low chemical stability, LD analysis in plant matrices requires an appropriate optimization of the chosen analytical method to obtain reliable results. This work proposes an HPLC-UV method, validated according to EURACHEM guidelines as regards linearity, limits of detection and quantification, precision, accuracy, and matrix effect. The LD extraction was studied by evaluating its aqueous stability over 3 months. The best chromatographic conditions were found by systematically testing several C18 stationary phases and acidic mobile phases. In addition, the assessment of the best storage treatment of Vicia faba L. broad beans able to preserve a high LD content was performed. The best LD determination conditions include sun-drying storage, extraction in HCl 0.1 M, chromatographic separation with a Discovery C18 column, 250 × 4.6 mm, 5 µm particle size, and 99% formic acid 0.2% v/v and 1% methanol as the mobile phase. The optimized method proposed here overcomes the problems linked to LD stability and separation, thus contributing to the improvement of its analytical determination.