Determination Of 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.

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

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

Fast and efficient related substance determination of Rilpivirine and Dolutegravir in combined HIV therapy with RP-HPLC

The abstract discusses the development and validation of a Related Substance RP-HPLC method for the simultaneous estimation of Rilpivirine and Dolutegravir in a combined pharmaceutical dosage form, following their FDA approval for HIV treatment. The developed method utilized a simple and economical approach, employing an LC-20 AT C18 column with a Water:Methanol (70:30) mobile phase at a flow rate of 1 ml/min, and detection at 258 nm. Retention times for Rilpivirine and Dolutegravir were determined to be 3.82 min and 7.51 min, respectively, while Rilpivirine and Dolutegravir impurity retention times were found to be 4.32 min and 7.12 min, respectively. The method demonstrated excellent linearity for Rilpivirine impurity (5.0-15.0 μg/ml) and Dolutegravir impurity (7.5-22.5 μg/ml). Limits of detection (LOD) were 0.079μg/ml and 0.145μg/ml for Rilpivirine and Dolutegravir impurities, while limits of quantification (LOQ) were 0.241μg/mL and 0.441μg/mL, respectively. The proposed method was successfully applied for the simultaneous estimation of both drugs and their related impurities in a commercial combined dosage form.

"Zelena" RP-HPLC metoda za određivanje profila nečistoća amlodipina u tabletama

Increased awareness of nature preservation has encouraged the introduction of the green analytical chemistry (GAC) practice concepts concerning several important aspects, including sustainable development, environmental impact, and minimum waste. The aim of this research was to contribute to the implementation of this approach for the pharmaceutical industry while retaining the crucial aspects and strict requirements of quality control of medicines. Therefore, an ethanolbased, green and robust high-performance liquid chromatography (HPLC) method for the determination of related substances of amlodipine (AML) in film-coated tablets was developed and optimized using the Design of Experiments (DoE). The chromatographic separation was performed on an RP-select B column (250 x 4.0 mm, 5 mm), using a mixture of 0.04 M sodium dihydrogen phosphate monohydrate (pH 4.0) and ethanol (60:40 % v/v) as a mobile phase. The optimized conditions provided the separation of two specified impurities (impurity D and impurity F). The selectivity of the method was confirmed using forced degradation studies. The Analytical Eco-scale approach and AGREE metrics confirmed that the method conforms to the GAC principles. The validated method was successfully applied for the determination of related substances in three samples from the market, demonstrating the applicability of the method in routine analysis.

Green RP-HPLC methods for assay and related substances in rivaroxaban tablets

AbstractIn this study, two different ethanol-based RP-HPLC methods for assay and quantification of rivaroxaban related substances in tablets were developed, based on green analytical chemistry (GAC) principles, using the design of experiments approach. The chromatographic separation was performed on X-Bridge C18 column (250 × 4.6 mm, 5 µm particle size), using isocratic elution with ethanol : water (35:65, % v/v) for the assay and gradient elution with ethanol/water mobile phase, for related substances, with a flow rate of 1.0 mL min−1. The gradient method was optimized for the separation of three specified impurities (impurity G, impurity H, and impurity 14) and the selectivity was further confirmed using forced degradation studies. Both methods were validated in accordance with ICH guidelines. The robustness of the methods was confirmed with the Central Composite Face Design of Experiments. Analytical Eco-scale approach and AGREE metrics confirmed that both methods are in accordance with the GAC principles. The proposed ethanol-based RP-HPLC methods were applied for assay and determination of related substances in rivaroxaban 10 mg tablets obtained from three different manufacturers available on the Macedonian market.

A Stability Indicating Reverse Phase High Performance Liquid Chromatography Method for Related Substances of Sofosbuvir in Tablet Dosage Form

Sofosbuvir is an antiviral drug that is used to treat hepatitis C. A present investigation deals with simple, sensitive, rapid, precise and accurate reverse phase high-performance chromatography (RP-HPLC) method developed and validated for related substances of sofosbuvir in tablet dosage form. The chromatographic separation was achieved on a Kromasil 100 C18 (250 × 4.6 mm, 5 μ) column. A sofosbuvir and its impurities were extracted by composed mixture of Mobile Phase A: buffer solution: acetonitrile (97.5:2.5% v/v) and Mobile Phase B: acetonitrile, isopropyl alcohol, methanol and purified water (60:20:10:10 % v/v/v/v) using with flow rate was 1-mL/min, column temperature was 25°C, injection volume was 10 μL, Vial thermostat temperature was 10°C. The UV detection was carried out at 263 nm. The retention time of sofosbuvir, methyl ester and ethyl ester were 54.28, 36.31 and 43.77, respectively. The method shows linearity with correlation coefficient of sofosbuvir and its impurity was 0.999 over the 0.5–7.5 ppm range. The average recovery was found to be 90.2–113.9%. The LoD and LoQ for sofosbuvir and its impurities were found to be 0.1 and 0.5 μg/mL, respectively. The method was validated as per ICH guidelines. The developed method was precise, accurate, novel and detectable towards sofosbuvir and its impurity. This method is efficient in separating the sofosbuvir and its impurity. Hence, the proposed method can be utilized for the determination of related substances in routine analysis in quality control department of pharmaceutical Industry.

Recommendations for the Contents of a Product Specification File: Related Substances (HPLC)

This article provides recommendations for documenting an analytical procedure for the determination of related substances by high-performance liquid chromatography in a product specification file (PSF). The article contains a description of specific aspects and a template for the typical contents and layout of the PSF section dedicated to related substances. A unified approach to the PSF content and layout will ensure error-free testing, reliable results, and streamlined regulatory assessments.

Determination of Valbenazine related substances by liquid chromatography method complying with the current regulatory guidelines. Method robustness evaluation by Design of Experiments software

AbstractA simple, specific, rapid, sensitive reversed‐phase high‐performance liquid chromatography method was developed and validated for the determination of Valbenazine, impurities, and its degradation products. The proposed high‐performance liquid chromatography method showed optimum separation in 60 min using potassium dihydrogen phosphate buffer and ACN in the ratio of 90:10 (V/V) as mobile phase‐A whereas ACN and methanol in the ratio of 60:40 (V/V) as mobile phase‐B. YMC‐Pack ODS‐AQ (150 × 4.6 mm, 3.0 μm), column used along with security guard cartridge C18, (4.0 × 3.0 mm) at 1.1 ml/min flow. The injection volume was 10 μl and detection was carried out at 285 nm. The drug was degraded under different stress conditions and the method was validated as per International Conference on Harmonization guidelines on the basis of accuracy, precision, linearity, and robustness. A degradation study indicates that Valbenazine is prone to oxidative degradation. The method was linear over the concentration range of limit of quantitation to 200% for impurities and drug substances. The method was found robust and was studied by the design of experiments. The proposed method is applicable for the determination of the stability of Valibenazine and was successfully used in the quality control of bulk drug manufacturing and pharmaceuticals.

Development and Validation of Stability indicating HPLC method for determination of related substances and assay of Monobenzone drug substance

Monobenzone, a mono benzyl ether of hydroquinone, is used as a topical drug for skin depigmentation. It increases the excretion of melanin from melanocytes. A novel stability-indicating high performance liquid chromatographic approach has been established in the current study to identify related compounds and assay of Monobenzone. The Agilent 1260 system was used for the HPLC analysis, and a gradient method using 0.1% orthophosphoric acid in water and acetonitrile as the eluent was used to separate Monobenzone from known contaminants and degradation products on a Zorbax SB-phenyl (250 mm x 4.6 mm) 5 μm column. The flow rate was set at 0.8 mL/min, the column oven temperature was 30 °C, and the detector wavelength was maintained at 215 nm. The relative retention times for Hydroquinone and Monobenzone dimer were around 0.35 and 1.86 respectively, while the Monobenzone retention time was approximately 10.4 min. The total run time was 30 min. For all known analytes spanning the concentration range from LOQ to 200% of the specification level, the calibration plot revealed a linear relationship (minimum r=0.999). All known analytes' LOD and LOQ were found to be within 0.06–0.10 μg/mL and 0.15–0.24 μg/mL, respectively. A recovery study determined the accuracy of the proposed method. For Hydroquinone and Monobenzone dimer, mean recovery was found to be in the range of 95.86%–99.89% and 93.02%–99.84% respectively. The repeatability study showed that the method is precise enough within acceptable limits. Studies on solution stability and robustness produced results that fell within acceptable bounds. The proposed method shows excellent linearity, accuracy, precision, specificity, robustness, LOD, LOQ and system suitability findings. Additionally, the forced degradation study showed that the approach was stability indicating in nature.

Determination of Related Substances in Promethazine Hydrochloride and Dextromethorphan Hydrobromide in Oral Solution by RP-HPLC Method.

The goal of this study is to provide a single, widely applicable high-performance liquid chromatographic (HPLC) technique for the determination of related substances in multicomponent oral solution of promethazine hydrochloride and dextromethorphan hydrobromide. For the assessment of impurities of promethazine hydrochloride and dextromethorphan hydrobromide in oral solution, a unique, sensitive, quick, stability-indicating gradient HPLC technique has been created. For chromatographic separation, an Agilent Eclipse XDB-C18, 250mm × 4.6mm, 5μm column was used with a buffered mobile phase consisting of a mixture of potassium dihydrogen phosphate pH3.0:acetonitrile (80:20) v/v as mobile phase A and potassium dihydrogen phosphate pH3.0:acetonitrile:methanol (10:10:80) v/v/v as mobile phase B. The separation was performed at a flow rate of 1.2mL/min and a detection wavelength of 224nm. The temperature of the column oven was regulated at 40°C. With good sensitivity and resolution, all compounds were effectively separated on a reverse-phase HPLC column. Acid, base, photolytic, thermal, oxidative and humidity stress conditions significantly degraded dextromethorphan hydrobromide and promethazine hydrochloride. The developed technique was validated according to the criteria of the International Conference on Harmonization for all validation parameters such as specificity, accuracy, linearity, precision, the limit of detection, the limit of quantitation and robustness.

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.

Stability Indicating RP-HPLC Method for Quantitative Estimation of Levetiracetam and its Impurities in Pharmaceutical Dosage Form

Objectives: A simple, precise, accurate, robust and selective stability-indicating reverse phase high performance liquid chromatographic method for the separation and quantification of Levetiracetam and its impurities in Levetiracetam liquid dosage formulations. Materials and Methods: The analysis of improved RP-HPLC method for the separation and quantification of Levetiracetam and its impurities are described. Samples are analysed by means of reverse phase (RP-HPLC) using an Inertsil ODS-3V, 150 x 4.6 mm, 3μm and the mobile phase consists of two channels A and B. Channel-A: pH 5.50 phosphate buffer : acetonitrile (950:50 v/v) and channel-B: acetonitrile: water (90:10 v/v). The flow rate is 1.0 ml/min. The column temperature was maintained at 40°C and sample temperature was maintained at 25°C, injection volume 10μL and wavelength fixed at 205 nm. Results: For selectivity, the chromatograms were recorded for standard and sample solutions of Levetiracetam and its related substances. Selectivity studies reveal that the peak is well separated from each other. Therefore the method is selective for the determination of related substances in Levetiracetam. There is no interference of diluent and placebo at Levetiracetam and impurities peaks. The elution order and the retention times of impurities and Levetiracetam obtained from individual standard preparations and mixed standard preparations are comparable. The limit of detection (LOD) and limit of quantitation (LOQ) for Levetiracetam standard 0.0023% and 0.0070%, impurity-A 0.0049% and 0.0147%, impurity-C 0.0024% and 0.0074% and Levetiracetam RC-A 0.0091% and 0.0277% respectively. The linearity results for Levetiracetam and all the 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 Levetiracetam and its impurities found to be 1.000, 0.9999, 1.000 and 0.9994 respectively. The accuracy studies were shown as %recovery for Levetiracetam and its 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. For precision studies six replicate injections were performed. %RSD was determined from the peak areas of Levetiracetam and its impurities. The acceptance limit should be not more than 10, and the results were found to be within the acceptance limits. Conclusion: The developed LC method was validated with respect to specificity, precision, linearity, ruggedness and robustness. Validation study compared as per ICH guideline.

Determination and quantification of related substances and degradation products in bictegravir by full factorial design evaluated HPLC and mass spectrometry.

Determining and quantifying novel impurities and degraded impurities of a drug product is always a continuous challenge to enhancing the drug quality for patients' safety. Herein, our work deals with (i) developing a rapid, accurate, and reliable high-performance liquid chromatographic validation method to quantify the bictegravir drug (integrase inhibitors of antiretroviral drugs) and its novel related impurities at low levels, and (ii) the liquid chromatography-mass spectrometry (LC-MS) method to identify degraded impurities. Separation of bictegravir acid (impurity-I) and methyl bictegravir (impurity-II) impurities which are identified by LC-MS in the bictegravir drug was executed by developing a method and the same method performance evaluated by using full factorial design. This developed analytical technique gave a well-separated peak of bictegravir and related analytes such as bictegravir acid (impurity-I) and methyl bictegravir (impurity-II), adequate with the peak properties as per USP guidelines. The method's sensitivity and linearity are demonstrated by its detection and quantification limits at low levels with a correlation coefficient of 0.998. The method's repeatability, specificity, and accuracy suggest that this developed technique is a reliable determination strategy for the bictegravir drug substance and its related impurities (impurity-I and impurity-II) in a simple, feasible, and affordable way.

A new validated RP-HPLC-UV DAD method for assay of bisoprolol fumarate and related substances in tablets

The research focus of this work was development and validation of an efficient analytical method that can be used for separation and determination of related substances of bisoprolol in finished drug product using reversed-phase HPLC with UV-DAD. In a previous systematic study of various stationary phases and elution conditions it was found that several octadecylsilane phases can be successfully employed for separation of the critical pairs of bisoprolol and its related degradation products. Namely, method development led to conclusions that satisfactory resolution and peak shapes were obtained with gradient elution with water with 0.2% perchloric acid and acetonitrile and the reversed-phase columns: Hypersil 3 BDS C18 (100 x 4 mm, 3 m); Zorbax SB C18 (150 x 4.6 mm, 3.5 m); Acquity UPLC BEH C18 (50 x 2.1 mm, 1.7 m), and Xterra MS C18 (100 x 4.6 mm, 3.5 m). Since regular quality control includes the parameter related substances, which is of great importance for the overall quality of dosage forms, this study was oriented towards widening the available validated analytical methods for determination of this parameter. In this work, the results from the validation of the method for determination of related substances of bisoprolol using Zorbax SB C18 150 x 4.6 mm, 3.5 m, are presented. Validation parameters that were tested (linearity, accuracy, precision, selectivity) confirmed that the method is suitable for its intendance and it was used for characterization of the samples from a forced degradation study of bisoprolol tablets. Keywords: Bisoprolol, RP-HPLC-UV DAD, validation, related substances

Development and Validation of New Methods for Quality Control of the Antiviral Substance FAVIPIRAVIR

The introduction into the production of medicinal substances involves the creation of a pharmaceutical dossier, an important component of which is data on pharmaceutical development and quality control methods. The physico-chemical properties of the antiviral substance of favipiravir were investigated, and some basic sections of the analytical documentation were developed. With the help of a complex of physical, chemical, and physico-chemical methods of analysis, the "Identification" test was developed, and the structure of favipiravir was proven. Methods for the determination of related substances by the HPLC with UV detection and the assay of favipiravir by the acid-base titration method, suitable for substance quality control, have been developed. Relative retention times and correction factors for calculating their content are established for all impurities. The methods were validated on the parameters of specificity, accuracy, correctness, and linearity in the studied range of concentrations.

Development and validation method for the determination of related substances in nimesulide granules for oral suspension

One of the key stages of pharmaceutical development is the development of analytical methods for quality control of medicines. The critical quality indicator is the content of related impurities, which may increase during shelf life of the product due to the degradation of the active substance, which in turn affects the quality of the product. The aim of the work is to develop and validate the method for the determination of related impurities in nimesulide granules for oral suspension by HPLC method; to set the specification of finished product for related impurities. Materials and methods. Sample of finish product Nimesulide, granules for oral suspension, 100 mg/2 g, it was manufactured by JSC “Farmak”. The study was performed using Agilent 1260 liquid chromatograph with UV detector. 2-phenoxyaniline from Sigma-Aldrich, reference standards of nimesulide impurity D EP CRS and LGC, Nimesulide for peak identification EP CRS were used for preparation of standard impurity solutions. Acetonitrile and ammonium dihydrogen phosphate from Sigma-Aldrich were used for chromatographic analysis. Results. When developing method, the concentrations of solutions, the method of dissolving granules, filters were selected. The chromatographic column was selected, the requirements for the chromatographic system suitability were set. The effect of placebo components has been studied and considered. During the validation the characteristics were studied: specificity, linearity, LOD, LOQ, precision, accuracy, robustness. The limits of the impurity content were established in accordance with the requirements of ICH Q3B guideline, taking into account the data of stress studies, stability and impurities of the substance. Conclusions. The analytical method for the determination of related substances in finish product nimesulide granules for oral suspension was developed. The validation results showed that the method is suitable for analysis. The specification limits of finished product for related impurities were set.

Determination of related substances in egg yolk lecithin by HPLC-CAD and characterization of its profiling by HPLC-Q-TOF-MS

A high-performance liquid chromatography (HPLC) method has been developed for the determination of related substances in egg yolk lecithin. Chromatographic separation was achieved using a gradient elution on a Waters Xbridge HILIC column maintained at 35 ℃. Mobile phase A was composed of water-acetonitrile (80:20, v/v, containing 5 mM ammonium acetate), and mobile phase B was composed of acetonitrile. Analytes were monitored by a charged aerosol detector (CAD) at 50 ℃. The novel HPLC-CAD method was selective and sensitive for the determination of related substances in egg yolk lecithin in its commercial bulk batches. It was also successfully validated by the International Council for Harmonisation (ICH) guidelines. The method will be a renewal of an old Chinese Pharmacopoeia method (2020 edition). Moreover, quadrupole time-of-flight mass spectrometry (Q-TOF-MS) was integrated with HPLC to investigate phospholipid species in egg yolk lecithin. This work provides comprehensive composition profiles of egg yolk lecithin, thereby accelerating the quality control, development, and application of egg yolk lecithin.

Simultaneous Determination of Esafoxolaner, Eprinomectin, Praziquantel, BHT and Their Related Substances in a Topical Finished Product by a Single Reversed-Phase High-Performance Liquid Chromatography Method.

The topical product with three active pharmaceutical ingredients (APIs), namely, esafoxolaner, eprinomectin and praziquantel has demonstrated its efficacy in the treatment of cats with mixed infections with ectoparasites and nematodes and cestodes. A reversed-phase high-performance liquid chromatography (RP-HPLC) method has been developed and validated for assay and determination of related substances peaks of three APIs including the assay of antioxidant butylated hydroxytoluene (BHT) in the finished product. Analytes were separated on a short Zorbax SB-C18 column (50 × 4.6mm I.D., 5μm particle size, pore size: 80Å) with gradient elution at 40 °C column temperature. Analytes were detected at 245nm for praziquantel, esafoxolaner, eprinomectin and their degradation products. BHT and eprinomectin degradation product 8a-oxo-B1a were detected at 280nm. All analytes of interest were adequately separated within 40min. The assay for praziquantel, esafoxolaner, eprinomectin and BHT was conducted against their corresponding external reference standards. The related substances peaks of each API were determined by peak area and relative response factor against total peak area of their corresponding API peak in sample solution. This method has been demonstrated to be accurate, robust, specific and stability indicating.

Development and validation of stability-indicating -HPLC method for the determination of related substances in novel nitroimidazole antituberculosis drug pretomanid: Robustness study by Design-Expert and application to stability studies.

This study developed a stability-indicating reversed-phase-HPLC method for the determination of related substances of the novel antituberculosis drug pretomanid. Critical quality attributes were evaluated and established for the robust method conditions by using quality-by-design based design of experiments. Forced degradation studies were carried out under acidic, basic, thermal, oxidative, and photolytic stress conditions. Pretomanid degraded when treated under basic conditions, whereas no significant degradation was observed under other stress conditions. The impurities were separated using a Bakerbond C18 column (150 × 4.6mm, 3μm) with the mobile phases 0.1% of orthophosphoric acid and acetonitrile in a time gradient mode. The HPLC method was validated according to the ICH (International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use) tripartite guidelines. Limit of detection and limit of quantitation of pretomanid and all its impurities were 0.1 and 0.4μg/mL, respectively. The method was found to be linear with a correlation coefficient >0.99, precision (% relative standard deviation < 5.0), robustness, and accuracy (% recovery: 85-115%). Stability studies were evaluated according to the ICH-Q1A, and the drug was were found to be stable in all storage conditions.

Stability-indicating HPLC method for the determination of related substances in lansoprazole sulphide

Symmetric peak shape was on a C18 stationary phase with the dimensions of 250 mm column length, 4.6 mm as internal diameter, 5 microns particles with an economical and straightforward mass-compatible mobile phase combination of formic acid and acetonitrile delivered in gradient mode at a flow rate of 1.0 mL/min at 260 nm. The resolution between Lansoprazole sulphide (LAN30) and its impurities (LAN30-I &amp; LAN30-II) in the developed method was more than 2.0, indicating a significant separation. Regression analysis shows a correlation coefficient greater than 0.999 for Lansoprazole sulphide and its related substances. The detection limits of Lansoprazole sulphide and its impurities are 0.003%, 0.01%, and 0.01%; the quantitation limits are 0.01%, 0.03%, and 0.03% for LAN30, LAN30-I, and LAN30-II. This method indicates that the recovery at different levels is 95 to 105% accurate. The test solution was stable in the diluent for 48 h at refrigerator temperature and subjected to stress conditions. The mass balance was close to 99.5%.