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Background: Selpercatinib, a selective RET kinase inhibitor, is approved for treating various cancers with RET gene mutations such as RET-rearranged thyroid cancer and non-small cell lung cancer. The presence of process-related and degradation impurities in its active pharmaceutical ingredient (API) can significantly affect its safety and effectiveness. However, research on detecting these impurities is limited. Methods: This study developed and systematically validated a High-Performance Liquid Chromatography (HPLC) method for identifying selpercatinib and its related impurities. The method utilized a 4.6 mm × 250 mm chromatographic column with 5 μm particles, employing a flow rate of 1.0 mL/min, a detection wavelength of 235 nm, an injection volume of 10 μL, and a column temperature of 35°C. Mobile phase A was composed of a 9:1 ratio of water to acetonitrile, with the aqueous component adjusted to pH 2.5 and containing 2 mM potassium dihydrogen phosphate (KH2PO4) and 0.4% triethylamine. Mobile phase B was pure acetonitrile. The gradient elution program was as follows: 0–2 min, 5%B; 2–15 min, 5% to 15%B; 15–30 min, 15% to 35%B; 30–35 min, 35% to 45%B; 35–36 min, 45% to 5%B; 36–45 min, 5%B. Results: The chromatographic method established in this study was validated according to the ICH Q2 (R1) guidelines. The developed HPLC method demonstrated excellent specificity, sensitivity, stability, linearity, precision, accuracy, and robustness. It efficiently separated the impurities present in selpercatinib, thereby confirming the method's efficacy in ensuring the purity and quality of the drug. Conclusion: The chromatographic method established in this study can be used for the detection of selpercatinib and its impurities, providing significant reference value for the quality research of selpercatinib bulk drug and its preparations, and ensuring the safety of medication for patients. [ABSTRACT FROM AUTHOR]

Background/Objectives: Reference materials are essential for ensuring the accuracy and traceability of measurements in the quality control of medicinal products. This study explores new principles for the preparation of impure materials of active pharmaceutical substances, focusing on 1-(3-benzoylphenyl)ethanone ketoprofen impurity A (European Pharmacopoeia) as the reference material. Methods: The reference material was synthesised from commercially available acetanilide and benzoyl chloride. The obtained product was purified using preparative chromatography and characterised by infrared spectroscopy (IR), 1H and 13C nuclear magnetic resonance (NMR), and mass spectrometry. The structure was verified using primary research methods to confirm its identity as the target product. Results: The characterisation confirmed the structure and purity of 1-(3-benzoylphenyl)ethanone, achieving a purity of 99.86%, meeting regulatory documentation requirements. The synthesised product was demonstrated to be identical to the target compound and suitable for use as a reference material. Conclusions: The developed method provides a robust approach for the preparation and characterisation of 1-(3-benzoylphenyl)ethanone, enabling its use as a certified reference material in the quality control of medicinal products. This approach ensures compliance with regulatory standards and enhances the reliability of pharmaceutical quality assurance practices. [ABSTRACT FROM AUTHOR]

Background: Ponatinib, a third-generation tyrosine kinase inhibitor, is employed in the management of adult chronic myeloid leukemia. Nevertheless, the presence of process impurities and degradation impurities linked to ponatinib may potentially influence its effectiveness and safety. Therefore, the objective of this research was to establish a robust liquid chromatography method and systematically validate it for the detection of substances related to ponatinib. Methods: The separation of ponatinib and its impurities was conducted using an Agilent 5HC-C18 chromatographic column (4.6 mm × 250 mm, 5 μm). The mobile phase A comprised a mixture of water and acetonitrile in a 9:1 ratio, with an aqueous solution of pH 2.4 containing 2 mM potassium dihydrogen phosphate and 0.4% triethylamine. Mobile phase B, consisting of acetonitrile, was eluted in a gradient fashion. The flow rate was set at 1.0 mL/min, detection wavelength at 250 nm, column temperature at 40°C, and injection volume at 10 μL. Results: The method demonstrated high specificity, sensitivity, solution stability, linearity, precision, accuracy, and robustness. Additionally, this research unveiled a novel compound, imp-B, generated via the oxidative degradation of ponatinib. The molecular structure of the newly discovered product was elucidated through the utilization of nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS). Conclusion: In conclusion, the chromatographic method developed in this study has the potential to be utilized for the detection of ponatinib and its impurities, thereby offering significant insights for quality assessment in ponatinib research. [ABSTRACT FROM AUTHOR]

Background: Pralsetinib, a targeted inhibitor of the RET enzyme, plays a critical role in the treatment of adult patients with locally advanced or metastatic non-small cell lung cancer (NSCLC) characterized by RET gene fusion mutations following platinum-based chemotherapy. Nevertheless, impurities resulting from the manufacturing and degradation of pralsetinib have the potential to impact its therapeutic effectiveness and safety profile. Methods: To address this issue, a liquid chromatography method was developed and validated for the specific identification of pralsetinib and its related impurities. The separation of pralsetinib and its related impurities was achieved via a Waters X Bridge Ci8 column with dimensions of 4.6 mm x 250 mm and a particle size of 5 ^m. Mobile phase A was composed of 20 mmol/L potassium dihydrogen phosphate (KH2PO4) and acetonitrile (ACN) at a volume ratio of 19:1, while mobile phase B consisted solely of ACN, utilizing a gradient elution technique. Detection was performed at a wavelength of 260 nm, with an injection volume of 10 ^L and a flow rate of 1.0 mL/min. Results: The chromatographic method established in this study was validated according to the ICH Q2 (R1) guidelines. The method demonstrated excellent linearity over a specific concentration range (imp-A: 0.035-10.21 ^ig/mL; imp-B: 0.09-10.16 ^g/mL; imp-C: 0.15-10.19 μg/mL; pralsetinib: 0.04-10.32 ^g/mL). Additionally, the method possesses high sensitivity, with detection limits for impurities A, B, C, and pralsetinib of 0.01, 0.03, 0.015, and 0.013 ^g/mL, respectively, and quantification limits of 0.035, 0.09, 0.05, and 0.04 ^ig/mL, respectively. In terms of specificity, stability, repeatability, accuracy, and robustness, the method met the validation acceptance criteria. Overall, the chromatographic technique established in this study can effectively separate pralsetinib and its impurities, providing reliable assurance for the accurate detection and quantification of impurities. Conclusion: The chromatographic method developed in this study can be utilized for the detection of pralsetinib and its impurities, offering a crucial reference for research on the quality of pralsetinib. [ABSTRACT FROM AUTHOR]

Currently, the therapeutic potential of complex biomolecules such as peptides has significantly enhanced the demand for analytical method development and validation for their quality control. Peptides are more complex to analyze than small molecules because of their larger size and unique physical–chemical properties, which create different challenges for analytical method development. Reversed‐phase high‐performance liquid chromatography (RP‐HPLC) with ultraviolet or mass spectrometry is the most widely used technique for analyzing peptides and related substances. In the present study, a decapeptide and its six related possible impurities were synthesized in‐house and identified. A stability‐indicating RP‐HPLC method has been developed for the estimation of the decapeptide and its related substances. The developed method was validated according to the International Conference on Harmonization guidelines for specificity, linearity, limit of detection, quantification, accuracy, precision, and robustness. The stability‐indicating capability of the proposed method was studied under the stress conditions of acid, base, oxidative, thermal, humidity, and photolytic degradation. The decapeptide peak and its degradant were found to be homogeneous and pure in the studied stress trials, with mass balance for all types of degradation exceeding 95%. It confirms the reliability and appropriateness of determining the integrity and purity of the decapeptide and its related substances. [ABSTRACT FROM AUTHOR]

Apalutamide is an anti-cancer agent used for the management of prostate cancer. A new stability indicating RP-HPLC method (Gradient mode) has been developed for the estimation of Apalutamide and its related substances using Waters Alliance system (Model no. 2996 and 2695) with Inertsil ODS-3 (250 x 4.6 mm, 5μm) column (PDA detector) was used for the present study. A mixture of Ammonium phosphate buffer solution and Acetonitrile (30: 70, v/v) was used as the mobile phase for the chromatographic study (Flow rate: 1.0 mL/min; Detection wavelength: 243 nm). Stress degradation studies were performed and the method was validated as per ICH guidelines. [ABSTRACT FROM AUTHOR]

To determine the paclitaxel-related substances in a paclitaxel injection (albumin-bound), it is necessary to remove human blood albumin from the sample via solid-phase extraction and subsequently perform high-performance liquid chromatography analysis. The pre-processing operation is complicated, the duration of analysis is long, and much human power and material resources are needed in the sample testing process. Thus, the purpose of this study was to develop a quick two-dimensional liquid chromatography method that requires less consumables and possesses high sensitivity. Samples were directly injected into one-dimensional solid-phase extraction columns to remove human blood albumin. Paclitaxel and its related substances in the solid-phase extraction columns were then eluted into two-dimensional analytical columns through valve switching technology. The sample pretreatment step can be omitted in this method, which allows for the analysis of paclitaxel and its related substances to be completed more quickly, within only 15 min, under the developed two-dimensional elution conditions. The established method had good linearity (R2 = 0.9994), limit of detection (0.25 μg·mL−1), limit of quantification (0.5 μg·mL−1), precision (RSD = 0.5%), and accuracy (102.53–107.97%). [ABSTRACT FROM AUTHOR]

The retention behaviour of scopolamine (hyoscine) and its related compounds (norhyoscine, atropine, homatropine, and noratropine) was investigated on the silica-based HPLC stationary phase. The retention of investigated tropane alkaloids was interpreted by using the Soczewiński-Wachtmeister equation. A high correlation between the retention parameter (log k) and lipophilicity (log P) (R = 0.9923) confirms the significant influence of hydrophobic interactions on the retention behaviour of the aforementioned compounds. It was found that by increasing the acetonitrile fraction, a decrease in retention of the more polar epoxide derivatives (scopolamine, norhyoscine) and an increase in retention of the more lipophilic derivatives (atropine, noratropine, homatropine) is obtained. The best separation of the tropane alkaloids was achieved by a simple procedure that involved a mobile phase composed of acetonitrile and 40 mM ammonium acetate/0.05% TEA, pH 6.5; 50:50 v/v. Selected conditions were assumed for the determination of scopolamine hydrochloride in the eye drops (Scopolamini hydrobromidum 0.25%). The method was validated and it was found as selective, sensitive, precise, accurate, and robust for the further qualitative analysis of the scopolamine-related compounds. [ABSTRACT FROM AUTHOR]

This study aims to develop a chromatographic analytical method, for separation, identification and quantification of degradation products, in veterinary finished product with two active substances enrofloxacin and bromhexine hydrochloride. Analytical method used an Agilent Eclipse Plus C18 column (150 × 4.6 mm, 5 µm particle size) and a mobile phase A consisted of 2.5% (v/v) phosphoric acid adjusted to pH 2.3 with triethylamine and mobile phase B was acetonitrile. Elution was in a gradient mode with total chromatographic time being 21 min. Detection was performed at 248 nm for bromhexine hydrochloride and at 272 nm for enrofloxacin. The method was developed to assure the separation of impurities of enrofloxacin and bromhexine hydrochloride. Specificity in relation with degradation products revealed up to 20 impurities for enrofloxacin and 7 impurities related with bromhexine hydrochloride. The spectra of impurities were chosen among the compounds found in forced degradation studies. Method validation was performed according to VICH GL 2—validation of analytical procedures and included selectivity/specificity, linearity, accuracy, precision, limit of quantification, limit of detection, robustness, and system suitability. Linearity was between 0.015 and 0.06 mg/mL for enrofloxacin and 0.001125–0.005625 mg/mL for bromhexine hydrochloride. Limit of quantification was 0.00292 mg/mL for enrofloxacin and 0.001103 mg/mL for bromhexine hydrochloride. These limits assured method performance, because they are under the reporting threshold of 0.3% as stated in VICH GL11 Impurities in new veterinary medicinal products which is 0.30%. Recovery was calculated on three concentrations for every compound and was 102.99% for enrofloxacin and 102.91% for bromhexine hydrochloride. In terms of intermediate precision, a relative maximum deviation of 2.50% was obtained for area and retention time using two analysts in two different days of application. [ABSTRACT FROM AUTHOR]

A new ultra‐high‐performance liquid chromatography method was developed using quality‐by‐design principles for quantifying trace‐level impurities of ibrutinib. The method utilized an ACQUITY UPLC BEH C18 column with a mobile phase consisting of equal parts of 0.02 M formic acid in water and 0.02 M formic acid in acetonitrile. The critical method parameters, including mobile phase pH, column temperature, and flow rate, were optimized using the design of experiments. Statistical analysis revealed the impact of these parameters on critical quality attributes. Perturbation and response surface plots illustrated the individual and interactive effects of the parameters. The optimal parameter levels were determined to be pH, 2.5; column temperature, 28°C; and flow rate, 0.55 mL/min. Confirmation experiments demonstrated the method's robustness, with the separation of impurities and unknown degradation products within a 5‐min runtime. The optimized ultra‐performance liquid chromatography method was validated according to ICH guidelines. The method exhibited linear response within the range of 0.025–100 μg/mL for ibrutinib and 0.0187–0.225 μg/mL for impurities (r2 > 0.9995), with limits of detection/limits of quantification of 0.01/0.025 and 0.015/0.0187 for ibrutinib and four impurities, respectively. Recoveries for the drug and impurities ranged from 92.69 to 102.7%, and precision was below 2% and 8% relative standard deviation for ibrutinib and impurities, respectively. [ABSTRACT FROM AUTHOR]

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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.

Doxycycline hyclate (DOXH) is very important to treat acne and prevent infections caused by bacteria, so quantifying doxycycline hyclate and its related substances is crucial. In this work, All analytes of doxycycline and its related substances, including methacycline, 4‐epi doxycycline, 6‐epi doxycycline, and 2‐acetyl‐2‐carbamoyl doxycycline, were successfully separated at low concentrations in a single run utilizing the suggested isocratic high‐performance liquid chromatography method with a mobile phase comprising water:acetonitrile:perchloric acid (75:25:0.2, v/v), Phenomenex Luna C18 column (50 × 4.6 mm, 3 μm i.d.) maintained at 30°C with a flow rate of 0.7 mL/min, injection volume of 25 μL, and photodiode array detector at 269 nm. DOXH and its related substances were linear in the range of 0.25–50, 0.1–30, 0.2–35, 0.5–50, and 0.6–20 μg/mL, respectively, with correlation coefficients greater than 0.999 and good accuracy results between 98% and 102%. The method has been successfully used to determine the drug's comparative study in both generic and reference products, demonstrating the similarity between the two products. According to the International Council for Harmonization requirements, the proposed approach was validated. Additionally, the technique has effectively studied in vitro dissolution, Six Sigma, and content uniformity. [ABSTRACT FROM AUTHOR]

In total, three related substances (RS) associated with sotalol hydrochloride (STHCl) were herein identified with a novel gradient high-performance liquid chromatography (HPLC) protocol. Further characterization of these substances was then performed via liquid chromatography–mass spectroscopy (LC–MS/MS) and nuclear magnetic resonance (NMR) approaches. For these analyses, commercial STHCl samples were used for quantitative HPLC studies and the degradation of STHCl under acidic (1M HCl), alkaline (1M NaOH), oxidative (30% H2O2), photolytic (4500 Lx), and thermal stress conditions (100 °C) was assessed. This approach revealed this drug to be resistant to acidic, alkaline, and high-temperature conditions, whereas it was susceptible to light and oxidation as confirmed through long-term experiments. The putative mechanisms governing RS formation were also explored, revealing that RS3 was derived from the manufacturing process, whereas RS2 was generated via oxidation and RS1 was generated in response to light exposure. The cytotoxicity of these RS compounds was then assessed using MTT assays and acute toxicity test. Overall, this study provides details regarding the characterization, isolation, quantification, and toxicological evaluation of STHCl and associated RS compounds together with details regarding the precise, specific, and reliable novel HPLC technique, thus providing the requisite information necessary to ensure STHCl purity and safety. [ABSTRACT FROM AUTHOR]

A simple, quick, and economical reverse-phase high-performance liquid chromatography (RP-HPLC) method for the quantitative determination of related substance in milrinone API was developed and validated. The method has shown adequate separation of milrinone and eight kinds of related substance. Chromatographic separation was achieved on an Agilent ZORBAX Rx-C8 (250 × 4.6 mm, 5 µm) column at wavelength of 220 nm, using a mobile phase acetonitrile: phosphate buffer (85:15) in an isocratic elution mode at a flow rate of 1.0 mL min−1. The method was validated in terms of system suitability, specificity, linearity, range, accuracy, intermediate precision, and robustness. A linear relationship between peak area and concentration of milrinone and its related substances was observed in a level of 10–300% (determination coefficient, r2 ≥ 0.998), respectively. The method showed acceptable levels of precision (%RSD ≤ 2), accuracy (> 96% recovery), robustness (< 10% content difference), and stability (> 96% recovery) over varied environment and laboratory conditions. The validation results suggested that the developed method is sensitive enough and repeatable and could be used for qualitative and quantitative assessment of eight kinds of related substance in milrinone. As a significant part of drug development process, stress testing is also performed to identify the degradation products and validate the stability-indicating power of our analytical methods, in which milrinone is subjected to acidic, base, oxidation, thermal and photolytic stress environment. To sum up, the proposed method is suitable for purpose in quality-control laboratories for quantitative analysis of the drugs individually, as it is simple and rapid with good precision and accuracy. [ABSTRACT FROM AUTHOR]

This current analysis demonstrates the development of the related substances (RS) method for separating and quantifying BSL and its related substances from plasma using the UPLC technique. The solid phase extraction (SPE) method was employed to extract the belumosudil (BSL) and related impurities from plasma. This SPE method has been shown to have high recovery values for BSL and its related impurities. The method employed a column from Waters, specifically the X-Bridge BEH C18 150x4.6mm, 3.5µm, with a mobile phase flow rate (0.8 mL/min) and a UV-wavelength (250 nm). In this method, BSL and its impurities show good peak shapes and high resolutions. This developed method shows high sensitivity, accuracy, and precision values. While performing the stability studies, the drug was stable and it showed less than 4.53% degradation. Six different concentration solutions (BSL (25-250µg/mL) and impurities (3-18µg/mL)) were prepared for constructing linearity plots. The obtained linearity plot shows the accepted correlation coefficient (R2) value (=0.999). The Limit of detection (LOD) and Limit of quantification (LOQ) were determined using the signal-to-noise (S/N) ratio method. This UPLC method was validated according to ICH guidelines. [ABSTRACT FROM AUTHOR]

Analytical Quality by Design principles using the design of experiments were applied for the development of a capillary electrophoresis method for the determination of enantiomeric purity and chemically related impurities of tamsulosin. From initial scouting experiments, a dual cyclodextrin (CD) system composed of sulfated β‐CD and carboxymethyl‐α‐CD was selected as the chiral selector. A fractional factorial resolution V+ design was used for the identification of the critical process parameters, while a face‐centered central composite design and Monte Carlo simulations were employed for final optimization and defining the design space of the method. The experimental conditions of the working point were: 30 mM sodium phosphate buffer, pH 3.0, containing 40 mg/mL sulfated β‐CD and 7 mg/mL carboxymethyl‐α‐CD, capillary temperature 18°C, applied voltage ‐23 kV. Following the assessment of robustness by applying a Plackett‐Burman design, the method was validated according to the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use guideline Q2(R1). The method allowed the quantification of the chiral impurity and three other related impurities at the 0.1 % level with acceptable accuracy and precision. [ABSTRACT FROM AUTHOR]

to determine the structure of the unknown impurity generated during the preparation of sodium sivelestat, two new compounds, benzyl 2-(2-aminoacetamido)benzoylglycinate and benzyl (2-aminobenzoyl)glycylglycinate, were prepared, and the chemical structures were confirmed by 1H NMR, 13C NMR and MS data. The unknown impurity produced during the preparation of sodium sivelestat was finally identified as benzyl (2-aminobenzoyl)glycylglycinate, which laid the foundation for quality research on sodium sivelestat. [ABSTRACT FROM AUTHOR]

Chromatographic methods for the analysis of antibiotic degradation products are widely used to evaluate the quality of medicines. Natural multicomponent antibiotics, such as capreomycin, are the most challenging compounds in terms of developing analytical procedures for related substances. Capreomycin sulfate monographs of the leading pharmacopoeias do not contain specifications for related substances. The key requirement concerns the sum of the main components of capreomycin calculated by normalising the peak areas in the test solution chromatogram. Therefore, it is important to develop an analytical procedure for determining not only the main components but also related substances of capreomycin.The aim of the study was to develop an analytical procedure for determining both the main components (IA, IB, IIA, and IIB) and related substances of capreomycin by ion-pair ultra-high-performance liquid chromatography (UHPLC).Materials and methods. This study examined capreomycin sulfate powder, an active pharmaceutical ingredient (API). Capreomycin sulfate solutions were analysed after artificial degradation (alkaline or acid hydrolysis) to demonstrate the resolution, selectivity, and efficiency of the experimental chromatographic system. The authors used an Agilent 1100 liquid chromatography instrument (Agilent Technologies) and chromatographic columns: Kinetex C18, YMC-Triart С18, ACQUITY UPLC BEH C18, ACQUITY UPLC BEH C8, ACQUITY UPLC BEH Phenyl, and ACQUITY UPLC CSH C18 (experimental procedure) or Acclaim C18, Zorbax SB-C18, and XBridge BEH130 C18 (The International Pharmacopoeia procedure).Results. In contrast to pharmacopoeial procedures, which evaluate only the component composition, the experimental procedure under the selected chromatography conditions can determine both the component composition and related substances of capreomycin. This advantage results from substituting a column packed with 1.7 µm particles for a 5 µm column required for pharmacopoeial procedures. The experimental procedure remains suitable for liquid chromatography instruments with a pressure limit of no more than 400 bar in the gradient elution mode with two mobile phases. According to the efficiency and selectivity evaluation, ACQUITY UPLC BEH C18 columns (150 × 2.1 mm, 1.7 µm) provide optimal peak resolution for capreomycin isoforms and related substances after artificial degradation of capreomycin.Conclusions. This experimental procedure based on ion-pair UHPLC may be used in the production and stability testing of capreomycin medicines to evaluate the API quality by the content of its main components and related substances.

Mechanistic modeling is useful for predicting and modulating selectivity even in early chromatographic method development. This approach is also in accordance with current analytical quality using design principles and is highly welcomed by the authorities. The aim of this study was to investigate the separation behavior of two different types of chiral stationary phases (CSPs) for the separation of ezetimibe and its related substances using the mechanistic retention modeling approach offered by the Drylab software (version 4.5) package. Based on the obtained results, both CSPs presented with chemoselectivity towards the impurities of ezetimibe. The cyclodextrin-based CSP displayed a higher separation capacity and was able to separate seven related substances from the active pharmaceutical ingredient, while the cellulose-based column enabled the baseline resolution of six impurities from ezetimibe. Generally, the accuracy of predicted retention times was lower for the polysaccharide CSP, which could indicate the presence of additional secondary interactions between the analytes and the CSP. It was also demonstrated that the combination of mechanistic modeling and an experimental design approach can be applied to method development on CSPs in reverse-phase mode. The applicability of the methods was tested on spiked artificial placebo samples, while intraday and long-term (2 years) method repeatability was also challenged through comparing the obtained retention times and resolution values. The results indicated the excellent robustness of the selected setpoints. Overall, our findings indicate that the chiral columns could offer orthogonal selectivity to traditional reverse-phase columns for the separation of structurally similar compounds. [ABSTRACT FROM AUTHOR]

The primary objective of the research work is to develop an effective, sensitive, economical and simple reverse phase HPLC method for quantification of Sarecycline and its impurities in Sarecycline parenteral dosage form. The separation was achieved by using a stationary phase water X-Bridge shield RP18 (150 x 4.6 mm, 3.5µ) and the mobile phase consists of ammonium acetate buffer and acetonitrile in the proportion of gradient elution. The flow rate was 1.0 mL/min. Sarecycline was detected by using UV detector at the wavelength of 240 nm. The column temperature was maintained at 40°C and sample cooler temperature was maintained at 5°C, injection volume 10µL, run time was 45 minutes. The developed method was validated for various parameters as per ICH guidelines like accuracy, precision, linearity, specificity, solution stability. [ABSTRACT FROM AUTHOR]

In this research study, we developed a new rapid and simple HPLC quantitative approach for comprehensively measuring related substance impurities (LNGN N formyl; LNGN acetamide; LNGN N Boc; LNGN amino) in Linagliptin. The related substance impurities were studied by PDA detection at 225 nm after being separated on the Kromasil C18 column stationary phase. The isocratic mode elution and a flow velocity of 1.0 mL/min were performed with the 0.1% phosphoric acid having pH 2.5 and acetonitrile. The proposed approach was appropriately verified in agreement with the "International Conference on Harmonization". Linearity was demonstrated for values ranging from LOQ quantity ppm to 150% quantity ppm, with variance coefficients 0.99. The precision varied from 0.128 to 0.969% RSD and accuracy varied from 99.13% to 101.76% for four related substance impurities. The proposed approach was appropriately implemented in the quality control analysis of the linagliptin API. Stress testing on the linagliptin API is also part of this investigation. One degradant with a 10.273 min Rt was found during oxidative stress testing on linagliptin. Using LC-MS this peak was discovered to be an adduct. [ABSTRACT FROM AUTHOR]

Objective To establish a quality control method for detecting impurities in chloral hydrate raw materials, improve the quality standards and control limits of raw materials. Methods The determination methods of chloroform and halogenated carboxylic acid in chloral hydrate were established to monitor the change of impurities in chloral hydrate through stability. Results The research and establishment of chloroform and halogenated carboxylic acid methods met the requirements of relevant regulations for analytical methodology verification, which could accurately detect four impurities in raw materials and preparations by one method. Conclusion The study provides technical support for the improvement and optimization of the quality standards of chloral hydrate and preparations. It is very necessary to implement the impurity monitoring in preparation research and production process by the chloral hydrate impurity detection and the stability comparison of this product at high temperature and light, which could largely promote the safety of medication. [ABSTRACT FROM AUTHOR]

This study deals with designing and validating an HPLC procedure for the determination of favipiravir and ten favipiravir-related impurities simultaneously in favipiravir bulk forms. The ten favipiravir related substances investigated herein included 6-Bromo-3-hydroxy-amide; 6-Fluoro-3-hydroxy-nitrile; 6-Chloro-3-hydroxy-amide; Favipiravir acid; 3-Hydroxy amide; 3,6-Difluoro amide; 3,6-Dichloro amide; 6-Chloro-3-hydroxy-nitrile; 3,6-Difluoro nitrile; and 3,6-Dichloro nitrile. Column Inert Sustain C18 is used to analyze favipiravir and favipiravirrelated impurities. Mobile phases employed included 20 mM KH2PO4 (system A) and 20 mM KH2PO4: Acetonitrile combined in 700:300 (vol/vol) parts (system B,) and given to column through gradient elution. The system was examined in accordance with the ICH prerequisite and was capable of providing accurate (96.4-114.8%) and precise (0.1-0.4%) quantitative results. The peak purities measures for favipiravir and its ten impurities revealed specificity. Linearity was evidenced from the LOQ amount level to 120% amount of the specification level (0.0015 mg/mL) with R² measures > 0.999 for favipiravir and its ten related impurities. This method concluded it was beneficial for the evaluation expected. [ABSTRACT FROM AUTHOR]

A related‐substances method was developed for the anticancer drug formulation apalutamide 60 mg tablets and validated using a liquid chromatography gradient elution method. All of the impurities and degradants were separated using the Luna Omega 5 μm Polar C18, (250 × 4.6) mm HPLC column with a 1.0 ml min−1 flow rate. The detection was done at 225 nm by injecting the 10 μl of injection volume, controlling the sample temperature at 10°C and maintaining the column compartment temperature at 30°C. The total run time was 85 min. A 0.01 m disodium phosphate dihydrate pH 4.20 ± 0.05 buffer mixed with acetonitrile in the ratio of 73:27 (v/v) was used as mobile phase A. Mobile phase B consisted of water and acetonitrile in the ratio 30:70 (v/v). The proposed method was validated as per the current regulatory guidelines. The method precisions (RSD) at 100% specification level were 1.41, 1.74, 1.84, and 1.66% for the four impurities. The accuracy results were obtained between 96.0 and 106.3% for the limit of quantitation to the 150% level. The standard and sample solutions stability were established for 44 h at 10°C. The correlation coefficient (r) value was >0.999 for all four impurities, indicating good linearity between the concentration and peak response: 0.9999, 0.9999, 0.9999 and 1.0000. These results show the method's linearity. The three filter compatibility was proved and it was concluded that 0.45 μm Nylon, PTFE and PVDF filters are suitable. The robustness of the method was established by varying the conditions. The method specificity was proved and the forced degradation data reveal the method's stability‐indicating nature. [ABSTRACT FROM AUTHOR]

Related substances, such as process-related substances and degradation products, may affect the efficacy of drugs and cause adverse reactions. Therefore, identifying and controlling them is of the importance. A rapid ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF–MS) method has been developed for the separation and characterization of related substances in candesartan cilexetil tablets. After optimization, the method validation was followed according to ICH guidelines. The developed UHPLC method showed adequate specificity, sensitivity, accuracy, linearity, precision, stability and robustness for validation of analytical procedures. Commercial candesartan cilexetil tablets was subjected to stress testing (60 ℃, 90% RH and 4500 lx for ten days) and forced degradation studies (acidic, alkaline, oxidative and photolytic degradation conditions). A total of eleven related substances were detected and characterized. Among them, four related substances have not been reported in the literature yet, and one of them (RS7) was confirmed as candesartan cilexetil methoxy analog by reference substance. In addition, plausible mechanisms for the formation of these related substances are discussed. This study provides a useful reference for the quality control of candesartan cilexetil tablets. [ABSTRACT FROM AUTHOR]

Trametinib is used to treat patients with unresectable or metastatic melanoma with BRAF V600E or V600K mutations. Two process impurities, namely 5-(2-fluoro-4-iodo phenylamino)-1-(3-aminophenyl)-3-cyclopropyl-6,8-dimethylpyrido[4,3-d]pyrimidine-2,4,7 (1H,3H,6H)-trione hydrochloride (2) and 4-((3-acetamidophenyl)amino)-N-cyclopropyl-2-((2-fluoro-4-iodophenyl)amino)-1,5-dimethyl-6-oxo-1,6-dihydropyridine-3-carboxamide (3), are observed during the laboratory optimization of trametinib. These impurities have a definite impact on the quality of the drug product. In this context, the identification, synthesis, origin and the control of these potential impurities are described. This study will be helpful to the generic pharmaceutical industry for obtaining chemically pure trametinib. [ABSTRACT FROM AUTHOR]

Literature about sterilization of pharmaceutical substances is limited. The aim of this study was to evaluate the effect of nitrogen dioxide (NO 2) sterilization, a new emerging technology, on five different ophthalmic active pharmaceutical ingredients, i.e., tetracycline hydrochloride, aciclovir, dexamethasone, methylprednisolone, and triamcinolone. The NO 2 process concentrations tested were 5, 10, and 20 mg/L. The applied temperature was 21 °C and the relative humidity 30 %. The process cycle consisted of two pulses with a dwell time of 10 min each. Non-processed samples were used as a blank. The effect of the sterilization method was assessed by high performance liquid chromatography coupled to an ultraviolet/visible detector, used for the quantitative analysis of the degradation products and the relative content of the evaluated ophthalmic medicines. For tetracycline hydrochloride and aciclovir, an increase of impurities was observed by increasing the NO 2 concentration. The maximum permissible NO 2 concentrations were estimated to be 10 mg/L and 2.5 mg/L, respectively, considering the requirement for the impurities to be within the limit stated in the European Pharmacopoeia (Ph. Eur.). For both compounds, samples subjected to 20 mg/L NO 2 demonstrated a significant difference in content compared to the non-processed sample. For methylprednisolone, dexamethasone, and triamcinolone, impurities complied with the limits of the Ph. Eur. for each NO 2 concentration and relative contents were not significantly affected. Sterilization of tetracycline hydrochloride and aciclovir with NO 2 is not recommended due to extensive degradation. NO 2 sterilization of methylprednisolone, dexamethasone, and triamcinolone could find its application within the aseptic processing procedure of related pharmaceuticals. • Effect of the gas sterilant nitrogen dioxide studied on 5 ophthalmic drugs. • Content and impurity profile determined with HPLC. • Degradation of tetracycline hydrochloride and aciclovir upon nitrogen dioxide treatment. • No pronounced effect on methylprednisolone, dexamethasone and triamcinolone. [ABSTRACT FROM AUTHOR]

This article described the development and validation of a method for spiramycin related substances based on hybrid particle column. The chromatographic conditions were as follows: water - 0.2 mol/L dipotassium hydrogen phosphate (the pH value adjusted to 9.5 using a 1 mol/L KOH solution) - acetonitrile - methanol (10: 60: 28.5: 1.5, v / v / v / v) as mobile phase A, water - 0.2 mol/L dipotassium hydrogen phosphate (pH 9.5) - acetonitrile - methanol (10: 30: 57: 3, v / v / v / v) as mobile phase B and gradient elution was performed. Compared with previous analytical methods, this method has strong specificity, excellent sensitivity and stability, which could be used for the daily testing of related substances of spiramycin. Furthermore, impurities above 0.1 % were characterized using two-dimensional liquid chromatography coupled with quadrupole time-of-flight mass spectrometer (2D LC-QTOF-MS/MS) and there were 6 impurities reported for the first time. • Optimized HPLC-UV method was developed for impurity analysis of spiramycin samples. • 2D LC-QTOF-MS/MS method was developed for the characterization of impurities in spiramycin. • 6 new impurities were reported for the first time. [ABSTRACT FROM AUTHOR]

Introduction: The main objective of this work was to analysis seven drug related impurities by High Performance Liquid Chromatography (HPLC) in six samples of Metronidazole API, collected from six pharmaceutical industries installed in Algeria. Materials and methods: For the organic related-impurities analysis, a liquid chromatography apparatus HPLC-UV device equipped with an automatic injector and UV/Vis detector and a column (C18), deactivated for the bases, post-grafted (5 µm) and dimensions (w: 0.25 m, Ø: 4.6 mm) were used. Each sample of Metronidazole API was processed according to the related substances procedures of the European Pharmacopoeia (Eur pH), 8th edition. Results: The HPLC related-impurities analysis showed that the M1, M2, M3, M4 and M6 samples had an individual content of impurity A or any other unspecified impurity within the required standards and a total of all impurities present meeting the standard. M5 sample had a high content of unspecified impurity 0.170% compared to the general acceptance criterion, and a total of impurities 0.187% meeting the standard. This can be explained either by the degradation of the sample which may be due to poor storage conditions or the batch from which this sample comes was not well purified during the synthesis route. Conclusion: Seven drug related impurities were analyzed in six samples of Metronidazole API by HPLC. The impurity A and the unspecified impurities were precisely determined in the different samples of Metronidazole analyzed. All samples had an individual content and a total of all impurities present meeting the standard except M5 sample had a high content of unspecified impurity 0.170% and a total of impurities 0.187% meeting the standard.

Abstract The study aimed to establish a high-performance liquid chromatography (HPLC) method for the quantitative analysis of the related substances of bedaquiline fumarate. Nuclear magnetic resonance and mass spectrometry were used for characterization and assay. A chromatographic method was used for separation. The conditions used were: gradient elution system composed of methanol 0.01mol/L KH2PO4 and 0.01 mol/L K2HPO4 (pH = 4.1) with a flow rate of 1 mL/min, at 224 nm as the detection wavelength. In this study, three degradation products of bedaquiline fumarate have been disclosed for the first time. The related impurities and degradation products of the drug were well separated. The method provided linear responses within the concentration range, which varied from 0.20 to 10.08 μg/mL with limits of detection of 0.10 μg/mL and limits of quantification of 0.20 μg/mL. The mean percent recovery varied between 91.64 and 105.89%. The method was validated for other parameters such as specificity, stability, and robustness. This method was validated and worked well for the impurity studies and quality control analysis of the laboratory-prepared samples of bedaquiline fumarate.

Objectives: Olmesartan medoxomil (OLM) and metoprolol succinate (MPS) in fixed-dose combination (FDC) tablet formulation prescribed extensively. Stability indicating (SI) method for impurities and related substance (RS) test quantitates the amount of these analytes in formulation; the manuscript presents SI/RS-ultra-high performance liquid chromatography-photodiode array (UHPLC-PDA) method for OLM and MPS and their impurities. Materials and Methods: Well-resolved separation of all analytes was achieved with gradient elution on a Shimadzu on Shimpack GIST-C18 (100 mm x 2.1 mm, 2 µm) column maintained at 25°C. Mobile phase-A consist of 0.1% orthophosphoric acid in water and mobile phase-B was acetonitrile at a flow rate of 0.4 mL/min, data integrated at 225 nm and 16 min of short runtime for satisfactory elution of all peaks. Results: The proposed SI/RS-UHPLC-PDA method was developed and validated as per International Conference on Harmonisation (ICH) of Technical Requirements guidelines. The system suitability test complied by all eluted peaks of the interest with acceptable linearity, recovery, and precision. Specificity, robustness, and method sensitivity parameters were determined; all the parameters were found to be within the limits. All the impurities and stress-degraded peaks were well resolved. Conclusion: The proposed method was found to be simple, fast, linear, and accurate. Further, the method is precise, robust, and specific; suitable for routine IPQC during active pharmaceutical ingredient manufacturing, stability and impurity profiling studies of the titled bulk analytes. Furthermore, the method can be extended to assess the levels of impurities formed during life cycle of new FDCs of titled analytes. [ABSTRACT FROM AUTHOR]

Ezetimibe is an azetidinone derivative and a substituted β‐lactam, it is a hypolipidemic agent, and a drug substance that lowers the cholesterol levels in our body. The present work describes the origin, synthesis, and characterization of three impurities of ezetimibe, which are also positional isomers and/or related substances of ezetimibe. viz: o‐Fluoro benzene isomer, m‐Fluoro aniline analog, and des‐fluoro aniline analogs of ezetimibe. [ABSTRACT FROM AUTHOR]

Simple, accurate, precise and stable indicating HPLC methods were developed and validated to determine both related substances and the assay content of Remogliflozin etabonate (RME). The separation was established under optimized chromatographic condition using Waters e2695 HPLC coupled to Ultraviolet detector (UV) at 228 nm with Inertsil ODS-3V C18 column (250 mm x 4.6 mm, 5 μm), injection volume 10 μl for related substances and 20 μl for assay content determination. The mobile phase consisted of 10 mM KH2PO4 buffer with ortho-phosphoric acid pH 2.5. Gradient elution was maintained at a flow rate of 1.0 ml/min using 45°C column oven temperature for determination of the related substances and isocratic elution was maintained at a flow rate of 1.2 ml/min for assay content. The retention time for RME-01, RME-02, RME-03 and RME-04, RME-05 and RME were about 41.74 min, 11.42 min, 20.38 min, 37.16 min, 49.93 min, 25.05 min respectively. The linearity was found to be in the concentration range of 0.312-2.341 μg/ mL for RME-01, 0.306-2.292 μg/ mL for RME-02, 0.306-2.297 μg/ ml for RME-03 and 0.294-2.203 μg/mL for RME-04. The RME was subjected to acid, base, oxidation, and thermal degradation stress conditions. The method was validated according to the ICH guidelines for specificity, precision, linearity, accuracy and robustness and the values were found to be within the limits. [ABSTRACT FROM AUTHOR]

Abstract Background Sodium Valproate is the sodium salt of valproic acid (VPA). Valproic acid is mainly used for the treatment of epilepsy. The specific aim of the study is to develop and validate an optimized method for the determination of six related substances such as N,N-dimethyl valpronamide, valeric acid, 2-methyl valeric acid, 2-ethyl valeric acid, 2-isopropyl valeric acid and 2-n-butyl valeric acid in Sodium Valproate Oral Solution by Gas Chromatography. Chromatographic separations of these six related substances were achieved on DB-FFAP fused silica capillary column (30 m × 0.53 mm) bonded with a 0.5-µm layer of macrogol 20,000 2-nitroterephthalate materials used as stationary phase. The six related impurities were extracted using heptane and monitored by Gas Chromatography coupled with flame ionization detector. The performance of the developed method was assessed by evaluating system suitability, method precision, specificity, linearity and range, ruggedness, accuracy, robustness. Results The correlation coefficient was within the acceptance criteria in the range of 0.9998. The evaluated concentrations for Sodium Valproate were in the ranges of 5.05–25.27 ppm. The average recovery values were in the range of 92.4–100.4%. Solution Stability experiments were performed to evaluate the degradation behavior of SVS. Conclusion A novel, precise and sensitive GC method was developed, validated and optimized for the determination of six related substances in sodium Valproate oral solution. The results obtained from the validation experiments demonstrated that the method is accurate, precise, linear, specific, sensitive and robust. Hence, the proposed method can be an alternative method, for the determination of related substances in sodium valproate oral solution drug substance.

Torasemide, a pyridine-3-sulfonylurea derivative, is a high-efficiency loop diuretic. During the process development of torasemide, five process-related substances, which have been specified in the pharmacopeia, would be produced. In this study, all these related substances, including compounds A–E, were synthesized via simple procedures and subsequently characterized by 1H nuclear magnetic resonance (NMR), 13C NMR, and mass spectrometry. Particularly, a simple synthetic method for compound A has not been found in previous literature. It is worth noting that other related substances could be prepared from compound B in one or two steps. The availability of these related substances could allow for quality control in the process of torasemide.

[Display omitted] • Eltrombopag olamine was subjected to forced degradation and UPLC-ESI-MS studies were performed for major degradation products. • Known drug related substances (RS) were searched from the literature. • m / z values for major forced degradation products were correlated with known RS. • RS were subjected to Tanimoto similarity, Toxicity - ADME evaluations, and docking. • Justified entry of six RS as potential TPO receptor agonists for drug discovery. The recent pandemic has highlighted the impact of diseases on global health and the economy. The rapid discovery of new hit molecules remains a tough challenge. Pharmaceutical impurity profiling can be linked to drug discovery through the identification of new hits from compounds identified during the analytical profiling. The present study demonstrates this linkage through the extension of the impurity (forced degradation) profiling of eltrombopag (ELT) olamine, a thrombopoietin (TPO) receptor agonist. The drug was exposed to standard degradation and the degradation products were primarily resolved and identified by UPLC-ESI-MS. This led to the identification of five forced degradation products (FDP). Thirty-three other known related substances (RS) of ELT, identified in the literature, were also considered. Molecular similarity checks were performed using Tanimoto/Jaccard's similarity searches. A set of structurally and topologically similar molecules, including ELT and 15 RS, was established and subjected to in-silico toxicity-, absorption-, distribution-, metabolism-, and elimination (ADME) predictions. The RS, predicted with similar or lower toxicity than ELT and a comparable ADME profile, were subjected to molecular docking to trace changes in TPO receptor affinity. The results indicated that five RS had a high Jaccard's similarity with ELT and higher or comparable docking scores. These compounds, along with few other impurities were predicted to have lower toxicity, better or comparable absorption, distribution, metabolism, and also a better excretion profile than ELT. This justifies their entry as potential novel TPO receptor agonists in drug discovery. [ABSTRACT FROM AUTHOR]

Budesonide is an active pharmaceutical ingredient used in various dosage forms of finished products for the treatment of asthma. During the process of drug development, unbiased analysis of related substances is of utmost significance for both pharmaceutical research and quality control purposes. In this work, the official method documented in the United States Pharmacopoeia was selected to determine the related substances of budesonide considering the pros and cons of critical chromatographic parameters, compared to the European Pharmacopoeia. In doing so, several unpredictable interference peaks, namely "ghost peaks", were observed occasionally during analysis. A strategy that integrated information derived from experimental operation inspection, liquid chromatography/mass spectrometry fingerprint analysis, and mechanism‐based stress studies was then proposed for comprehensively and quickly exploring those non‐degradable and degradable peaks. Some ghost peaks were found to originate from nylon syringe filter, illumination, and alkali borosilicate glass high‐performance liquid chromatography vials. Besides, degradation pathways under alkaline conditions were also unraveled through liquid chromatography‐mass spectrometry qualitative analysis. Overall, an optimization of the analytical methodology based on the United States Pharmacopoeia for its application in impurity analysis of budesonide and corresponding formulations was carried out with the design of experiments, by which "ghost peaks" could be suppressed or prevented. The results obtained herein are not only crucial to studies on budesonide's stability or degradation kinetics but also contribute to clarifying the impurity research of other drugs. [ABSTRACT FROM AUTHOR]

A simple and sensitive high-performance liquid chromatographic (HPLC) method is established to separate and quantify the related substance present in two alkylating agents. The use of the traditional approach of analytical validation, in practice or in the literature, is communal. However, statistical verification, that looks separately the two errors (such as absolute bias and repeatability) to make a decision, presents a risk to declare that an analytical method is valid while it is not, or equally. To minimize this peril, a new approach based on the concept of total error was proposed. In this paper, we reveal the applicability and simplicity of the both methods based on the total error approach: accuracy profile and uncertainty profile. Proposed study demonstrated by validation case of a liquid chromatographic LC method for the quantification of related compounds present in two alkylating agents. Both methods showed good linearity response (> 0.995) with repeatability (%relative standard deviation less than 2%) and accuracy (94 to 106%). Accuracy profile was found within the range of ±10% and risk profile ±5% between the two series respectively. The excellence of the total error approach was presented since it enables successfully to validate the analytical procedure as well the calculation of the measurement uncertainty at each concentration level. [ABSTRACT FROM AUTHOR]

A simple, rapid, and robust reverse phase HPLC method was developed and validated for the determination of related substances for Dolutegravir dispersible tablets 10mg. The primary goal of this research is to develop and validate a new RP-HPLC method for validating the amount of Impurity B (degradation impurity) as a related substance following USP guidelines. Dolutegravir and its impurities were separated using chromatographic conditions on a Phenyl-Hexyl (250 × 4.6 mm), 5µ column with a 45 % buffer (sodium dihydrogen phosphate dihydrate and EDTA): 49 % methanol: 6 % acetonitrile mixture and a pH of 2.5 ± 0.05 adjusted with orthophosphoric acid. The flow rate in isocratic elution mode was 1.2 mL/min. The column temperature was kept constant at 35°C, and the eluted compounds were measured at a wavelength of 258 nm using the PDA detector. According to USP guidelines, the developed method was tested and found to be stability-indicating, specific, rugged, precise, linear, accurate, and robust, with a high resolution and shorter retention time. The system suitability and other validation parameters were found to be within the limits. The method was sensitive because the LOD and LOQ demonstrate its sensitivity. The linearity curves for Dolutegravir and Impurity B were found to be linear, with a correlation coefficient of at least 0.997. The average percentage of impurities recovered ranged between 80 % to 120 %. As a result, the proposed method was found to be good and accurate for the quantitative determination of related substances associated with Dolutegravir dispersible tablets 10mg. [ABSTRACT FROM AUTHOR]

In this project, we have developed a reversed phase liquid chromatography method for separation and determination of lenalidomide (LENA) and related substances by using C-8 (250×4.6 mm ID, 5 μm) HPCL column. The mobile phases A and B were phosphate buffer at Ph=3.30, and (methanol:acetonitrile)(1:5 V/V), respectively. The column oven temperature was 25°C, the wavelength was 220nm, and the injection volume was 20 µl. The degradation studies using basic, acidic, oxidation, and thermal stress, were performed. In addition, in the basic stress, a significant degradation for LENA, was observed.Also, the results showed that the resolutions of the peaks for fresh, acid stress, and thermal stress were considerably high. For example, in the case of thermal shock, the resolution of each peak to the next, was 3.6, 3.2, 5.3, and 4.7. Thus, it indicates that the method is suitable at least in view of separation and resolution for the peaks produced by thermal shock.

Compound 2β-carbomethoxy-3β-(4-chlorophenyl)tropane (β-CCT) is a key intermediate for the synthesis of some clinical dopamine transporter (DAT) imaging agents. Potential impurities from synthesis process of β-CCT and degradation during storage might have detrimental effect on the final imaging agents. Thus, it is necessary to guarantee the quality of β-CCT. In this study, a rapid, sensitive and accurate high-performance liquid chromatography (HPLC) method was developed and validated for the analysis of β-CCT and its related substances. The chromatographic separation was achieved on a reverse-phase phenomenex™ Gemini C18 column with an isocratic mobile phase consisted of methanol, water and TFA (30:70:0.1 v/v/v). The flow rate was 1.0 mL/min at 30 °C and samples were monitored at 220 nm. The method was validated concerning system suitability, linearity, accuracy, precision, specificity, robustness and stability. The limit of detection (LOD) and the limit of quantification (LOQ) of β-CCT were 0.5 and 1.5 μg/mL, respectively. The linearity range of β-CCT was 1.5–450 μg/mL with a good linear correlation coefficient (R2 = 0.9999) between the peak response and concentration. Specificity investigation through forced degradation experiments displayed that β-CCT was stable in acidic, thermal and photolytic degradation conditions, but significantly unstable in alkaline and oxidative conditions. With the developed chromatographic method, possible impurity α-CCT from synthetic process and potential degradation products could be well separated from β-CCT. Good recovery and precision were manifested in the assay method. These results indicated that the present method would be suitable for not only the quality assurance of β-CCT in regular production sample assays but also the monitoring and determination of its related substances.

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 µm); Zorbax SB C18 (150 x 4.6 mm, 3.5 µm); Acquity UPLC BEH C18 (50 x 2.1 mm, 1.7 µm), and Xterra MS C18 (100 x 4.6 mm, 3.5 µm). 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 µm, 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. [ABSTRACT FROM AUTHOR]