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.
Clonazepam contains one benzodiazepine ring in its chemical structure which makes it vulnerable to degradation. In this study, green analytical chemistry approach was applied in attempts for the ...development of validated stability indicating RP-HPLC method for determining clonazepam and its related substances in pharmaceutical formulation. Validation has been performed according to ICH guidelines. Assay was capable of simultaneous monitoring of the intact drug in the presence of its related substances within the same run. HPLC assay involved an ODS column and a mobile phase composed of 2% sodium dodecyl sulfate, 0.05M sodium acetate buffer pH 3.5 and isopropanol in ratio (25:55:20) at a flow rate of 1.5mL/min and detection was carried out at 254nm. HPLC method allowed good resolution between the peaks that corresponded to the active pharmaceutical ingredients and its degradation products with good linearity, precision, accuracy, specificity, LOD and LOQ. The expanded uncertainty (0.33%) of the method was also estimated from method validation data. This analytical technique is not only ecofriendly but also faster than the conventional liquid chromatographic system official in the USP-36.
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.
This article, which is partly biographical and partly scientific, summarizes a life in academic medicine. It relates my progress from benchside to bedside and then to academic and research ...administration, and concludes with the teaching of human biology to college undergraduates. My experience as an intern (anno 1953) treating a youngster in diabetic ketoacidosis underscored our ignorance of the controls in human fuel metabolism. Circulating free fatty acids were then unknown, insulin could not be measured in biologic fluids, and beta-hydroxybutyric acid, which was difficult to measure, was considered by many a metabolic poison. The central role of insulin and the metabolism of free fatty acids, glycerol, glucose, lactate, and pyruvate, combined with indirect calorimetry, needed characterization in a near-steady state, namely prolonged starvation. This is the main topic of this chapter. Due to its use by brain, D-beta-hydroxybutyric acid not only has permitted man to survive prolonged starvation, but also may have therapeutic potential owing to its greater efficiency in providing cellular energy in ischemic states such as stroke, myocardial insufficiency, neonatal stress, genetic mitochondrial problems, and physical fatigue.
The processing of tea leaves plays a crucial role in the formation of the taste of the resulting tea. In order to study the compositions of and changes in taste-related substances during the ...processing of Rizhao green tea, non-targeted metabolomics was used, based on UHPLC-Q Exactive MS. Totals of 529, 349, and 206 non-volatile metabolites were identified using three different detection modes, of which 112 secondary metabolites were significantly changed. Significant variations in secondary metabolites were observed during processing, especially during the drying stage, and the conversion intensity levels of non-volatile metabolites were consistent with the law of “Drying > Fixation > Rolling”. The DOT method was used to screen tea-quality-related compounds that contributed significantly to the taste of Rizhao green tea, including (−)-epicatechin gallate, (−)-epicatechin gallate, gallic acid, L-theanine, and L-leucine, which make important contributions to taste profiles, such as umami and bitterness. Metabolic pathway analysis revealed that purine metabolism, caffeine metabolism, and tyrosine metabolism perform key roles in the processing of Rizhao green tea in different processing stages. The results of this study provide a theoretical basis for tea processing and practical advice for the food industry.
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.
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.
The third generation of antiepileptic drugs that have been approved by international regulatory agencies between 2007 and 2018 include rufinamide, stiripentol, eslicarbazepine acetate, lacosamide, ...perampanel, brivaracetam and everolimus. As part of demonstrating their safety profile, stability indicating methods are developed to monitor these drugs and their impurities. In this context, this review describe some characteristics, impurities and the stability indicating methods used for the determination of these drugs and the presence of their related substances. Through a search in official compendia and scientific articles, fifty-six analytical methodologies were identified up to October 2020. The methodologies were developed using techniques of HPLC, UPLC, HPTLC, GC and UV/Vis spectrophotometry. A majority of the methods (∼70%) employed HPLC-UV. A number of these antiepileptic drugs were found to have had a small number of studies related to their stability and for the detection of impurities. The presentation of the current level of research on third generation antiepileptic drugs highlights the need for new stability and safety studies that are necessary to develop new pharmaceutical products containing these drugs.
Synthetic opioids such as fentanyl account for over 71,000 of the approximately 107,000 overdose deaths reported in the United States in 2021. Fentanyl remains the fourth most identified drug by ...state and local forensic laboratories, and the second most identified drug by federal laboratories. The unambiguous identification of fentanyl-related substances (FRS) is challenging due to the absence or low abundance of a molecular ion in a typical gas chromatography-mass spectrometry (GC-MS) analysis and due to a low number of fragment ions that are similar among the many potential isomers of FRS. This study describes the utility of a previously reported gas chromatography-infrared (GC-IR) library for the identification of FRS within a blind, interlaboratory study (ILS) involving seven forensic laboratories. Twenty FRS reference materials, including those with isomer pairs in the library, were selected based on either their presence in the NIST library and/or some similarity of the mass spectra information produced. The ILS participants were requested to use the Florida International University (FIU) GC-MS and GC-IR libraries supplied by FIU to search for matches to their unknown spectra generated from in-house GC-MS and GC-IR analysis. The laboratories reported improvement in the positive identification of unknown FRS from ~75% using GC-MS alone to 100% correct identification using GC-IR analysis. One laboratory participant used solid phase IR analysis, which produced spectra incompatible with the vapor phase GC-IR library to generate a good comparison spectrum. However, this improved when searched against a solid phase IR library.
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% H
O
), 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.
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