Method For Related Substances Research Articles

New stability indicating RP-HPLC methods for the determination of related substances and assay of trametinib acetic acid: a mass balance approach.

New stability indicating related substances and assay methods for trametinib acetic acid by RP-HPLC have been developed and validated through forced degradation and mass balance studies for the first time. In related substances (RS) method, trametinib acetic acid was successfully separated from its six related substances namely, cyclopropanamide impurity, desiodo trametinib, desacetyl trametinib, trione acetamide intermediate, trione intermediate, and trione PTSA intermediate using YMC-Triart C18 (150 × 4.6mm, 3µm) column. Orthophosphoric acid (0.15%) in water was used as buffer. Gradient elution was programmed using mobile phase-A (buffer and acetonitrile mixture in 80:20 v/v ratio) and mobile phase-B (buffer and acetonitrile mixture in 20:80 v/v ratio). Acetonitrile and methanol mixture (1:1 v/v) was used as diluent. Flow rate, injection volume, column temperature, and wavelengths were kept as 0.8mL/min, 10µL, 55°C, and 240nm, respectively. Desacetyl trametinib and cyclopropanamide impurity were identified as potential degradation impurities in acid and base degradation conditions, respectively. Assay method specific to above six related substances was also developed. Assay of forced degradation samples was also determined, and the mass balance was established by adding total impurities formed in RS method to assay of trametinib acetic acid.

Understand the Stabilization Engineering of Ascorbic Acid, Mapping the Scheme for Stabilization, and Advancement.

Vitamin C is extensively used in cosmetic formulation, howbeit stability is the supreme demerit that limits its use in beautifying products. Numerous techniques are being employed to inhibit the degradation of vitamin C caused by formulation components to facilitate the use in skin rejuvenating products. Diverse materials are being exercised in formulation to stabilize the ascorbic acid and ingredients selected in this formulation composition help for stabilization. The initial stable prototype is developed and further optimization is accomplished by applying the design of experiment tools. The stable pharmaceutical formulations were evaluated for the evaluation parameters and designated as two optimized formulations. The analytical method for the assay of ascorbic acid from the United States pharmacopeia and the related substance method from European pharmacopeia has been modified to be used for cream formulation. The DoE design exhibited that the stability of formulation is impacted by citric acid and tartaric acid but not by propylene glycol and glycerin. The analysis results of topical formulations for the evaluation parameter exhibited satisfactory results. The in-vitro release study method has been developed, optimized, and validated to fit the analysis. The in-vitro studies have been performed for selected compositions and both the formulation has similar kinds of release patterns. The stability study as per ICH guidelines exhibited that the product is stable for accelerated, intermediate, and room-temperature storage conditions. The optimized formulation shows constant release and permeation of ascorbic acid through the skin. The formulation with the combinations of citric acid, tartaric acid, and tocopherol is more stable and the degradation of vitamin C has been reduced significantly. The beaucoup strategies in the unique composition help to protect the degradation by inhibiting the multitudinous degradation pathways.

Stress degradation study of Etoricoxib, isolation, and characterization of major degradation impurity by preparative high‐performance liquid chromatography, liquid chromatography‐mass spectrometry, and nuclear magnetic resonance: Validation of ultra‐performance liquid chromatography method

AbstractEtoricoxib (ETO) is a selective cyclooxygenase‐2 inhibitor, used as a nonsteroidal anti‐inflammatory drug. In this study, a detailed investigation was conducted on the degradation behavior of ETO under acidic, basic, neutral, photolytic, oxidative, and thermal degradation conditions as per the International Conference on Harmonization (ICH) guideline Q1A (R2). A gradient “ultra‐performance liquid chromatography (UPLC)” method was developed for separating the degradation products formed under various degradation conditions along with process‐related impurities. Acquity BEH phenyl column was used for the separation of analytes and 0.1% formic acid in water and 0.05% formic acid in acetonitrile:methanol (8:2 %v/v) were used as mobile phase‐A and mobile phase‐B. A major degradant namely, N‐oxide impurity was observed in oxidative degradation along with two minor degradants where N‐oxide was confirmed based on LC‐mass spectrometry (LC‐MS) studies. Further, the major degradant was enriched and isolated using preparative HPLC and the conformation of the N‐oxide impurity was established using nuclear magnetic resonance techniques including two‐dimensional studies and high‐resolution MS. In addition, the method for related substances by UPLC has been validated by considering a major degradant, namely N‐oxide along with process‐related impurities as per the ICH guidelines validation parameters.

Using the AQbD Approach, Development and Validation of a Simple, Rapid Stability Indicating Chromatographic Method for Quantification of Related Impurities of Apixaban.

Apixaban is a new oral anticoagulant that has been approved by the United States Food and Drug Administration for the prevention of stroke and other cardiovascular complications in people with non-valvular atrial fibrillation. Then, later, it was given the green light to treat deep venous thrombosis and pulmonary embolism. The biggest challenge in the development of pharmaceutical drugs is comprehending science- and risk-based techniques for developing and maintaining analytical procedures appropriate for evaluating the quality of drug substances and drug products. The improved technique provides a methodical strategy to acquire and improve understanding of an analytical method. The current study discusses the related substances method development for an apixaban drug substance employing a regulated authority refined and approved approach of the quality-by-design concept. For the quantification of impurities, and apixaban drug substance, a simple, quick and stability-indicating reverse-phase liquid chromatographic method was developed using a full factorial design. The separation between apixaban and its nine impurities was accomplished using an Zorbax Stable Bond Phenyl, 150 × 4.6mm, 5μm column. The mobile-phase components for gradient elution at a flow rate of 1.2mL/min were chosen to be a mixture of water, acetonitrile, methanol and perchloric acid in various ratios with a total run time of 15min. Chromatograms were extracted at 278nm after a 5μL solution injection. According to regulatory requirements, the developed method has been validated for its intended purpose.

Forced degradation studies and development of RP-HPLC method for related substances of Tenofovir alafenamide in tablet dosage form

The development and validation of an easy and rapid stability-indicating RP-HPLC technique for Tenofovir alafenamide and its impurities. In this newly developed method, chromatographic separation of Tenofovir alafenamide and its impurities was achieved on an Inertsil ODS-3V C18 (250 mm x 4.6 mm, 5 μ) column. The impurities were extracted by a mixture of Mobile Phase A: buffer solution: Acetonitrile: Purified water (20:02:78) and Mobile Phase B: Solvent Mixture and Purified water (75:25), The injection volume was 20 μL, the column temperature was 40°C, the flow rate was 1 mL/min, and the detection was carried out at 262 nm. The retention time of Tenofovir alafenamide, PMPA, PMPA anhydrate, Phenyl PMPA and PMPA isopropyl alaninate were 56.24, 5.69, 8.71, 25.89 and 37.13 respectively. The Correlation Coefficient within the acceptance criteria is not less than 0.999. The evaluated concentrations for Tenofovir alafenamide and its impurities were in the range of 0.5–7.5 ppm. The average recovery value was in the range of 90.2–113.9%. Tenofovir alafenamide’s LOD and LOQ were determined to be 0.1 μg/mL and 0.5 μg/mL respectively. Tenofovir alafenamide solution degradation behaviour was assessed using solution stability studies. A new, accurate, and precise method has been developed, evaluated, and elevated for the detection of impurities in Tenofovir alafenamide tablet dosage form. The results obtained from the validation study established that this method is specific, reliable, precise and effective. As a result, the proposed strategy can serve as an alternate method to determine related substances in routine analysis of tablet dosage form.

Quantitative determination of related substances for Lamotrigine extended release tablet by RP-HPLC

Lamotrigine extended release tablet dosage form LAMICTAL XR used as an anticonvulsant in the treatment of generalized tonic clonic, absence seizures and partial seizures. The objective of the present study is to develop and validate analytical method for the estimation of related substances in the LAMICTAL XR from GSK; however it is very important to have simple, sensitive, robust and validated analytical method. Hence a precise RP-HPLC analytical method developed for the determination of Related substances in LAMICTAL XR tablet dosage form with gradient elution pattern having mobile phase A as buffer pH 8.0 and mobile phase B as an Acetonitrile at 1.5 mL/min flowrate, using Hypersil BDS C18 column, ambient column temperature and PDA detector with wavelength 220 nm. The analytical method is validated as per ICH guidelines including its forced degradation studies. The method was found to be linear in the range of 0.2 ppm to 2.5 ppm with correlation coefficient 0.999. Accuracy performed at LOQ to 250% level and recovery was found to be in the range of 95% to 105%. Therefore the developed related substances method provides a safe, easy and reproducible for the stability studies and QC release testing for the estimation of related substances.

Development, stability-Indicating assessment, and evaluation of influential method conditions using a full factorial design for the determination of Nintedanib Esylate related impurities.

The design of an appropriate analytical method for assessing the quality of pharmaceuticals requires a deep understanding of science, and risk evaluation approaches are appreciated. The current study discusses how a related substance method was developed for Nintedanib esylate. The best possible separation between the critical peak pairs was achieved using an X-Select CSH Phenyl Hexyl (150 × 4.6) mm, 3.5 μm column. A mixture of water, acetonitrile, and methanol in mobile phase-A (70:20:10) and mobile phase-B (20:70:10), with 0.1% trifluoroacetic acid and 0.05% formic acid in both eluents. The set flow rate, wavelength, and injection volumes were 1.0 mL/min, 285 nm, and 5 μL, respectively, with gradient elution. The method conditions were validated as per regulatory requirements and United States Pharmacopeia general chapter <1225>. The correlation coefficient for all impurities from the linearity experiment was found to be >0.999. The % relative standard deviation from the precision experiments ranged from 0.4 to 3.6. The mean % recovery from the accuracy study ranged from 92.5 to 106.5. Demonstrated the power of the stability-indicating method through degradation studies; the active drug component is more vulnerable to oxidation than other conditions. Final method conditions were further evaluated using a full-factorial design. The robust method conditions were identified using the graphical optimization from the design space. This article is protected by copyright. All rights reserved.

Forced degradation of timolol maleate on high temperature for verification of HPLC method for related substances in Timolol eye drop 0.5%

Forced degradation of timolol maleate on high temperature for verification of HPLC method for related substances in Timolol eye drop 0.5%

Related Substances Method Development and Validation of Axitinib, Zanubrutinib and Upadacitinib Using RP-HPLC and its Degradation Products were Characterized by Using LC-MS/MS

Background: Axitinib was used to treat carcinoma of renal cells and Upadacitinib was used to treat rheumatoid arthritis and Zanubrutinib used as an anti cancer medication for mantle cell lymphoma. In the current application, Zanubrutinib, Upadacitinib and Axitinib and their associated substances will be developed and validated. Materials and Methods: The optimised method includes the Zanubrutinib, Upadacitinib and Axitinib gradient elution and associated flow rates with dimensions of 1 mL/min and phenyl column X-bridge (150 x 4.6 mm, 3.5 μ). A mobile phase was employed with 1.2 g of Hexane sulphonic acid of pH-2.5 adjusted with ortho-phosphoric acid (buffer) and acetonitrile. Zanubrutinib, Upadacitinib and Axitinib were separated from their associated substances for a total run time of 60 min. The approach developed has been validated in accordance with the ICH guidelines. Results: The test concentration LOD and LOQ were established for both drugs and for their impurities. The diagrams that were drawn were straightforward with an R2 regression coefficient &gt; 0.999. As part of the method validation, recovery, specificity, linearity, accuracy, robustness was determined and the results were found to be within an acceptable range. Conclusion: HPLC was used to validate the method in terms of accuracy, linearity, method precision, accuracy, limit of detection, limit of quantification, robustness, and degradation, and LC-MS/MS was used to characterise the degradation products. The system was validated in all of these aspects.

Circumventing glass vial and diluent effects on solution stability of small molecule analytes during analytical method development and validation

Solution stability of analytes plays an important part in qualitative analysis, especially in conducting accurate, quantitative analyses. Sample diluents and glass vials as sample containers for HPLC analyses can play a critical role and should be evaluated during chromatographic method development. We have encountered several instances during pharmaceutical development where the glass vial/diluent combination has negatively impacted method performance. One case encompasses adsorption of piperazine, a secondary amine, to non-silanized glass vials, resulting in recovery failures during analytical method transfer. Two further cases describe the propensity for peracetylated C-aryl glucosides being subject chemical transformations relating to sample diluent. The first reports transesterification with methanol-based diluents and the second describes hydrolysis with acetonitrile/water diluents mediated by the mild alkalinity of certain brands of Type I borosilicate vials. A final case explores development of a related substance method, it was found that an impurity was prone to hydrolysis and another impurity with a primary amine tended to be adsorbed on glass vials. Diluents of appropriate pH and buffer strength were strategically selected to neutralize the mild alkalinity of the glass vials as well as to mitigate adsorption of the amine analyte on glass vials. As a result, excellent sample stability and reproducibility were achieved, regardless the quality and brand of Type I glass vials used. Here we present four case studies that demonstrate how the negative impact of Type I glass vials on those susceptible analytes can be effectively eliminated by using appropriate sample diluents, which is essential to ensure accurate analytical data and provide for a smooth method validation and transfer.

Dual Organic Modifier, Solid Core Technology and Quality by Design Based Tripartite Synergistic Model for Development and Validation of Stability Indicating Method by Reverse Phase-High Performance Liquid Chromatography for Assay and Impurities of Etoricoxib Tablets

Tripartite synergistic model of solid core technology, dual organic modifiers and combined mixture design was implemented to achieve combined assay and related substances method by reverse phasehigh performance liquid chromatography with short run time, enhanced sensitivity and improved resolution between multiple impurity peaks. pH of mobile phase, ternary mobile phase composition and high performance liquid chromatography column temperature are experimented as variable parameters. Acetonitrile and isopropyl alcohol mixture was experimented as dual organic modifier. Special focus was given to detailed methodology of dealing with elution order changes by assigning negative sign for resolution. Separation of etoricoxib and related impurities was evaluated as a case study to prove this concept. The method was developed with Ascentis® Express C18, 150×4.6 mm, 2.7 μ column. Mobile phase comprised of buffer (0.1 % v/v ortho phosphoric acid, pH 3.6), acetonitrile and isopropyl alcohol (65.3:29:5.7 v/v) with a flow rate of 1.0 ml/min and ultraviolet detection at 285 nm. Forced degradation studies revealed that the method was stability indicating, suitable for both assay and impurities of drug product. The recoveries for impurities and assay were found to be in the range of 94.0 %-111.0 % and 97.9 %-101.8 %, respectively. Linearity was established for impurities and assay in the range of 0.25-2.0 μg/ml and 125-750 μg/ml, respectively. The method was validated as per international conference on harmonisation guidelines. The method can be successfully employed for determination of assay and impurities of etoricoxib in bulk drugs and formulations.

Separation and Characterization of the Related Substances of Bedaquiline Fumarate Using HPLC and Spectral Methods

The study focuses on isolating and characterizing the potential degradation products (DPs) and impurities of Bedaquiline fumarate in bulk drug form. A stability-indicating related substance method was developed and validated using high-performance liquid chromatography. Agilent Poroshell 120EC- C18 (150mm × 4.6mm, 2.7μm) column showed an optimum separation of 10 analytes. The mobile phase contained 0.05% trifluoroacetic acid, acetonitrile and methanol in a time gradient mode. Bedaquiline fumarate was susceptible to acid hydrolysis, oxidation, base hydrolysis and showed three potential DPs, including DP-1, DP-2 and Impurity-8. Degradation products, DP-1 and DP-2, were isolated and characterized by ESI-MS, 1H, NMR and 13C NMR spectroscopy. The developed method was validated according to ICH tripartite guidelines and showed adequate specificity, accuracy and linearity. The limit of detection and limit of quantitation were 0.05 and 0.15μg/mL, respectively.

A rapid RP-HPLC stability-indicating method development and validation of moxifloxacin hydrochloride-related substances in finished dosage forms.

A reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the identification and quantification of moxifloxacin hydrochloride-related substances in finished dosage forms. Chromatographic separation was achieved on an Agilent C18 column (150 × 4.6 mm, 5 μm) with the mobile phase (0.01 M potassium dihydrogen orthophosphate as buffer and methanol in the ratio of 70:30) eluted in isocratic mode. The HPLC flow rate was 1.0 mL min-1 , and peaks were monitored at 230 nm using a photodiode array (PDA) detector. The column temperature was kept constant at 30°C, and the injection volume was 10 μL. The run time of the method was 16 min. The method was validated as per the International Conference on Harmonization (ICH) guidelines. Linearity was recorded at various concentrations ranging from 0.25 to 1.5 μg mL-1 for all the moxifloxacin impurities. Linearity, regression value, recovery, %relative standard deviation (RSD) of method precision values were found within the acceptance limits. The method for related substances (RS) in moxifloxacin was found to be specific, linear, accurate, precise, rugged, and robust. The validated method was suitable for the quantification of the RSs in moxifloxacin drug products. The method was applied in quality control lab for the analysis of moxifloxacin impurities in stability analysis.

Identification and characterization of Prothionamide degradation impurities by mass spectrometry, NMR spectroscopy, and ultra high performance liquid chromatography method development

Stability-indicating and liquid chromatography-mass spectrometry compatible ultra high performance liquid chromatography method was developed for the degradation and drug substances related impurities of Prothionamide. Forced degradation of Prothionamide was carried out under acidic, basic, thermal, oxidative, and photolytic stress conditions. The impurities separation was achieved on Acquity UPLC BEH-C18 (50mm × 2.1mm, 1.7 μm) with the mobile phase of 10mm ammonium acetate pH 6.0 and Acetonitrile in a time gradient mode. Related substances by ultra-performance liquid chromatography method was validated according to ICH tripartite guidelines. Degradation products were isolated by Column chromatography and characterized by liquid chromatography-mass spectrometry, 1 H, and 13 C nuclear magnetic resonance spectroscopy. The developed related substances method showed adequate specificity, sensitivity, accuracy, linearity (0.4-1.5μg/mL), precision, and robustness in line with ICH tripartite guidelines for validation of analytical procedures. Limits of detection and quantitation were 0.1 and 0.4μg/mL, respectively, for Prothionamide and all the impurities. The method was found to be linear with a correlation coefficient>0.99, precise (%RSD<5.0), robust and accurate (%recovery 85-115%).

Related substances method development and validation of an LCMS/MS method for quantification of selexipag and its related impurities in rat plasma and its application to pharmacokinetic studies

The present application wish to seem at the event of validation of bio analytical method and pharmacokinetic study of selexipag and its related impurities in rat plasma using LC–MS/MS. The optimized method contains gradient elution of selexipag with a flow rate of 1 ml/min and X-Bridge phenyl column (150 × 4.6 mm, 3.5 µ). A buffer of 1 mL formic acid in l liter water and acetonitrile mixture is used as mobile phase. 30 min run time was used for separation of selexipag and its related impurities with Ambrisentan as internal standard and impurity-D as active metabolite. The linearity curves are linear in between the percentages of 10 to 200% of rat plasma and R2 value of each analyte was observed as 0.999. This application denotes all the parameters like precision, accuracy, recovery and stability were got the results within the limit of USFDA guidelines. This method applies effectively for the investigation of pharmacokinetic studies using rat plasma.

Identification of Tartaric Acid Adduct Impurities in Dipyridamole Capsule Formulation Related Substances Method

Two tartaric acid adduct degradation products namely dipyridamole tartaric acid monoester and dipyridamole ditartaric acid ester are observed in a dipyridamole capsule formulation. The adduct impurities are inevitable in the formulation due to the interaction of multilayers of dipyridamole on tartaric acid pellets. Present study reported a simple procedure for generating these two major adducts degradation products from a mixture of dipyridamole drug substance and tartaric acid by stress study. The obtained stress mixture was characterized by liquid chromatography-tandem mass spectrometry (LC-MS) to assure the identity of adduct degradant impurities. The obtained solid stress mixture was stable for more than one year and the prepared solution can be used as reference solution to identify both the degradants during related substance analysis. Practically, the identification of tartaric acid degradants applied to the British pharmacopeia monograph related substances method, where no mechanism for identification of these adduct impurities was described. This study establishes relative retention times for the British pharmacopeia method, which enables the chemist to monitor these two major degradants during quality control release testing and shelf life stability. The same kind of experimental approach for identifying tartaric acid adduct impurities in the British pharmacopeia method can be extended to any of the in-house laboratory-developed related substance methods.

A Validated RP-HPLC method for related substances of Dabigatran etexilate mesylate

The scope of this research work was to develop a reverse-phase liquid chromatographic method for the quantification of related impurities of dabigatran and to validate the method according to ICH guidelines. Chromatographic conditions were optimised with Poroshell SB C18, 150mm, 4.6mm, 2.7µm particle size column, mixer of Phosphate buffer with phosphoric acid in water and acetonitrile with percentage of 10:90 (v/v) as solvent-A and acetonitrile and buffer with a percentage of 70:30 (v/v) as solvent-B. Gradient compassion mode is employed for mobile phase delivery with a flow rate of 0.8 mL/min. Stationary phase was maintained at 35°C temperature and detection at 230 nm, with 10 µL of sample injection volume. Water and acetonitrile in the percentage 30:70(v/v) were used as a diluent. The developed RP-HPLC method was validated according to ICH guidelines. LOD and LOQ values for dabigatran its impurities were in the range from 33 to 55 ppm and 112 to 168 ppm correspondingly. Method validation results for all the parameters are meeting the ICH guidelines acceptance criteria for the parameters of robustness, ruggedness, linearity, reproducibility and recovery. The proposed method was found to be suitable for the quantitative determination of potential impurities in the bulk samples of Dabigatran etexilate mesylate API.

A Novel Validated Eco-friendly RP-UHPLC Method for Assay and Related Substances in Meropenem

A simple, rapid, sensitive, specific, eco-friendly and stability-indicating linear gradient liquid chromatographic method (RP-UHPLC) for simultaneous estimation of assay and its related compounds in Meropenem API samples is developed and validated. Chromatographic separation was achieved on Zorbax Eclipse plus C18, (100 x 4.6) mm, 3.5 µm RRLC short column and 10 mM potassium dihydrogen orthophosphate is used as buffer, buffer solution used as eluent A and buffer and acetonitrile combination 30: 70 v/v ratio used as eluent B and Agilent RRLC (UHPLC) system is used for analysis. The mobile phase flow rate was 1.0 ml/min, and the eluted compounds were monitored at 220 nm for related substance method and 290 nm for assay method. Excellent resolution was obtained between Meropenem and its related compounds which were eluted within 10 min. The correlation co-efficient(r) is > 0.995 for both the methods from linearity data and percentage of recovery is 98.0 to 102.0 and 80.0 to 120.0 % for assay method and for related substance method respectively. Sensitivity of the method is found to be less than 0.316 µg/ml. Peak homogeneity data for Meropenem in the chromatograms from the stressed samples were obtained by using photodiode array detector demonstrated the specificity of the method for analysis of Meropenem in presence of the degradation compounds. The performance of the method was validated according to the present ICH guidelines for specificity, limit of detection, limit of quantification, linearity, accuracy, precision, and robustness.

DEVELOPMENT AND VALIDATION OF STABILITY INDICATING RELATED SUBSTANCE METHOD FOR ACETAZOLAMIDE TABLETS

DEVELOPMENT AND VALIDATION OF STABILITY INDICATING RELATED SUBSTANCE METHOD FOR ACETAZOLAMIDE TABLETS Patel Suresh*, Dr. Omray Lavakesh, Soni Pushpendra Radharaman Institute of Pharmaceutical Sciences, Bhopal, Madhya Pradesh, India ABSTRACT Acetazolamide is the prototype of new class of antiepileptic drugs. Drugs which is carbonic anhydrase inhibitors used in mainly as an antiepileptic. The present work deals with development and validation of related substance method development through stability indicating of acetazolamide tablets, an adequate simple, precise, selective, economic High performance liquid chromatographic method has been developed then validated for identification and analysis purpose. Reversed phase chromatography was performed on a C-18 column with Phosphate buffer and acetonitrile in the ratio as mobile phase with a flow rate 1.0 ml/minute. 265nm wavelength shown adequate peak shape and height at retention time approximately 7.8 minutes. The % RSD of peak area response of acetazolamide in LOQ level concentrated shown less than 2%. The chromatographic method was validated in many parameters such as specificity, accuracy, precision, robustness, reproducibility according to ICH guidelines. Statistically found that method was qualify with all validation parameters on acetazolamide tablets. This method was suitable for routine analysis because proved that wide linearity range covered, sensitive, shorter retention time, simple mobile phase as well Keywords: Acetazolamide, RP-HPLC, Anti-epileptic drugs. Full Text Article

A NOVEL VALIDATED UHPLC METHOD FOR ESTIMATION OF ASSAY AND ITS RELATED SUBSTANCES OF TRICHOSTATIN-A

Objective: The main objective of the research work is to develop and validate a rapid UHPLC method for the estimation of assay and its related substances of Trichostatin A (TSA) in pharmaceutical samples.&#x0D; Methods: The UHPLC method developed for chromatographic separation between TSA and its related compounds on Poroshell 120 SB C18(50×4.6) mm; 2.7 µm RRLC column using Agilent RRLC (UHPLC) system with linear gradient elution.&#x0D; Results: The developed UHPLC method has shown excellent chromatographic separation between TSA and its related compounds within 12 min run time, during validation experiments, specificity study revealed that the peak threshold was more than the peak purity and no purity flag was observed. Repeatability, intra, and inter-day precision results were well within the tolerable limits. Limits of detection concentrations were found between 0.075 to 0.077 ppm and the limit of quantitation is between 0.252 to 0.258 ppm for related compounds and TSA. The related substances method recoveries were found between 80 and 120 % and assay method recovery was found between 98.0 to 102.0%.&#x0D; Conclusion: The developed method capability was proven for the assay of TSA and its related compounds in pharmaceutical samples and the method shows eco-friendlier than routine, conventional HPLC methods in terms of analysis time, cost and HPLC effluent waste.