Recent advancements in disulfide bridge characterization: Insights from mass spectrometry.

RATIONALE: Disulfide bridges (DSB) play an important role in stabilizing three-dimensional structures of biopharmaceuticals, single purified proteins, and various cyclic peptide drugs that contain disulfide in their structures. Incorrect cross-linking known as DSB scrambling results in misfolded structures that can be inactive, immunogenic, and susceptible to aggregation. Very few articles have been published on the experimental annotation of DSBs in proteins and cyclic peptide drugs. Accurate characterization of the disulfide bond is essential for understanding protein confirmation. METHODS: Characterizing DSBs using mass spectrometry (MS) involves the chemical and enzymatic digestion of samples to obtain smaller peptide fragments, in both reduced and nonreduced forms. Subsequently, these samples are analyzed using MS to locate the DSB, either through interpretation or by employing various software tools. RESULTS: The main challenge in DSB analysis methods using sample preparation is to obtain a sample solution in which nonnative DSBs are not formed due to high pH, temperature, and presence of free sulfhydryl groups. Formation of nonnative DSBs can lead to erroneous annotation of disulfide bond. Sample preparation techniques, fragmentation methods for DSB analysis, and contemporary approaches for DSB mapping using this fragmentation were discussed. CONCLUSIONS: This review presents the latest advancement in MS-based characterization; also a critical perspective is presented for further annotation of DSBs using MS, primarily for single purified proteins or peptides that are densely connected and rich in cysteine. Despite significant breakthroughs resulting from advancements in MS, the analysis of disulfide bonds is not straightforward; it necessitates expertise in sample preparation and interpretation.

LC/Q-TOF-MS-based structural characterization of enasidenib degradation products and establishment of a stability-indicating assay method: Insights into chemical stability.

RATIONALE: Enasidenib (EDB) is an orally active selective mutant isocitrate dehydrogenase-2 enzyme inhibitor approved by the U.S. Food and Drug Administration to treat acute myeloid leukemia. It lacks a reported forced degradation study and a stability-indicating assay method (SIAM). This study addresses this gap by establishing a degradation profile in accordance with the International Council for Harmonisation Q1A and Q1B (R2) guidelines and developing a validated SIAM for EDB. METHODS: EDB was exposed to forced degradation under various conditions (hydrolytic, photolytic, oxidative, and thermal stress). Degradation samples were analyzed using high-performance liquid chromatography on an Agilent ZORBAX Eclipse Plus C18 column with a mobile phase consisting of 0.1% formic acid in Milli-Q water and acetonitrile at a flow rate of 1 mL/min and detection at 270 nm. Liquid chromatography-quadrupole time-of-flight-high-resolution mass spectrometry (LC/Q-TOF HRMS) was used for the identification and characterization of degradation products. Nitrosamine risk assessment was conducted using a modified nitrosation assay procedure (NAP) test due to the presence of a secondary amine group in the drug, which is liable to forming nitrosamine drug substance-related impurities (NDSRI). RESULTS: The drug exhibited significant degradation under acidic, basic, photolytic, and oxidative conditions in the solution state. A total of nine degradation products (DP) were formed (DP-I, DP-III, and DP-IV: acidic conditions; DP-I and DP-III: basic conditions; DP-II, DP-V, DP-VI, and DP-VII: oxidative stress; and DP-VII, DP-VIII, and DP-IX: photolytic conditions), which were separated and identified using reversed-phase high-performance liquid chromatography and characterized using liquid chromatography-tandem mass spectrometry. The mechanism behind the formation of EDB degradation products has been discussed, and this study was the first to develop a degradation pathway for EDB. In addition, the possibilities of NDSRI formation for EDB were studied using a modified NAP test, which can contribute to the risk assessment of the drug. CONCLUSIONS: Forced degradation studies were conducted by establishing a SIAM for EDB. All the degradation products were characterized by mass spectral data obtained using LC/Q-TOF-HRMS.

A comprehensive study on the identification and characterization of major degradation products of synthetic liraglutide using liquid chromatography-high resolution mass spectrometry.

Liraglutide (LGT) is a synthetic glucagon-like peptide-1 analogue mainly used for the treatment of type-2 diabetes or obesity. Comprehensive stability testing is essential in the development and routine quality control of synthetic therapeutic peptide pharmaceuticals. The GLP-1 peptide drugs are usually formulated in aqueous-base solution, which can generate stability issues during manufacturing, storage or shipment. The current study endeavors to observe the chemical stability behavior of LGT by exposing the drug substance to oxidative and hydrolytic stress conditions. A simple liquid chromatography (LC) method was developed where sufficient resolution between LGT and the generated degradation products was achieved. In total, 19 degradation products (DPs) were separated under acidic, basic and oxidative stress conditions. Using LC-HRMS, MS/MS studies, the generated degradation products were identified and characterized. The mechanistic fragmentation pathway for all generated DPs were established and the plausible chemical structure for the identified DPs was predicted based on MS/MS data. The results strongly suggest that LGT is highly susceptible to degrade under oxidative and hydrolytic conditions. Furthermore, this study provides insights into the hydrolytic and oxidative stability of LGT, which can be implied during generic and novel formulation drug development and discovery in synthesizing relatively stable GLP-1 analogues.

A comprehensive study on the identification and characterization of degradation products of lipoglycopeptide Dalbavancin using LC and LC-HRMS/MS.

Dalbavancin is the second-generation approved semisynthetic lipoglycopeptide by the United States Food and Drug Administration (USFDA) for the treatment of acute bacterial skin and skin-structure infections. Unlike other lipoglycopeptides, the stability behavior of Dalbavancin was least explored, which is a prerequisite. The current study endeavors to elucidate the oxidative and hydrolytic stability behavior of Dalbavancin by exposing the drug to oxidative, acidic, and basic stress conditions. A simple liquid chromatography (LC) method was developed, where significant resolution between Dalbavancin, its homologs, and the generated degradation products was achieved. Seven degradation products were identified under acidic, basic, and oxidative stress conditions. Using liquid chromatography and high-resolution mass spectrometry (LC-HRMS), MS/MS studies, the generated degradation products were identified and characterized. Formation of isomeric degradation products was identified especially upon exposure to basic stress conditions. The mechanistic fragmentation pathway for the seven degradation products was established, and the chemical structure for the identified degradation products was elucidated. The results strongly suggest that Dalbavancin is highly susceptible to degradation under oxidative and hydrolytic stress conditions. This study provides insights into the hydrolytic and oxidative stability of Dalbavancin, which can be employed during drug development and discovery in synthesizing relatively stable analogs.

Enantiomeric purity of synthetic therapeutic peptides: A review.

Synthetic therapeutic peptides are a complex and popular class of pharmaceuticals. In recent years, peptides with proven therapeutic activity have gained significant interest in the market. The determination of synthetic peptide enantiomeric purity plays a critical role in the evaluation of the quality of the medicine. Since racemization is one of the most common side reactions occurring in AAs or peptides, enantiomeric impurities such as D-isomers can form during the peptide synthesis or can be introduced from the starting materials (e.g., AAs). The therapeutic effect of a synthetic or semi-synthetic bioactive peptide molecule depends on its AA enantiomeric purity and secondary/tertiary structure. Therefore, the enantiomeric purity determination for synthetic peptides is supportive for interpreting unwanted therapeutic effects and determining the quality of synthetic peptide therapeutics. However, enantiomeric purity analysis encounters formidable analytical challenges during chromatographic separation, as D/L isomers have identical physical-chemical properties except stereochemical configuration. To ensure peptides AA stereochemical configuration whether in the free or bound state, sensitive and reproducible quantitative analytical method is mandatory. In this regard, numerous analytical techniques were emerged for the quantification of D-isomeric impurities in synthetic peptides, but still, very few reports are available in the literature. Thus, the purpose of this paper is to provide an overview of the importance, regulatory requirements, and various analytical methods used for peptide enantiomeric purity determination. In addition, we discussed the available literature in terms of enantiomeric impurity detection, common hydrolysis procedural aspects, and different analytical strategies used for sample preparation.

Size-exclusion LC-UV/HRMS based method for the analysis of aggregates in synthetic GLP-1 analog liraglutide and evaluation of excipient impact on aggregation.

Peptide aggregation is one of the key challenges associated with the development of therapeutic peptides. Peptide and protein aggregates are considered as one of the most important critical quality attributes (CQA). Therapeutic liraglutide (LGT) is proteinaceous in nature, and aggregation can be triggered by various environmental stress condition. Therefore, it is essential to separate and identify aggregation states of such drugs. In this study, we have established size exclusion chromatography-liquid chromatography-ultraviolet/high resolution mass spectrometry (SEC-LC-UV/HRMS) method to separate and identify the stress induced LGT aggregates. LGT samples were subjected to photolytic, thermal, freeze thaw and shaking stress conditions. Additionally, LGT solution was incubated with surfactant and excipient that are commonly used in peptide formulation, to evaluate their impact on aggregation level and physicochemical stability over time. The developed SEC method was also validated for specificity, accuracy, precision and linearity. The results of this study will be useful for investigators to monitor LGT aggregates during product development.

Development of stability-indicating assay method and liquid chromatography-quadrupole-time-of-flight mass spectrometry-based structural characterization of the forced degradation products of alpelisib.

The US Food and Drug Administration and the European Medicines Agency approved alpelisib in 2019 for the treatment of metastatic breast cancer. A thorough literature review revealed that a stability-indicating analytical method (SIAM) is not available for the quantification of alpelisib and its degradation products (DPs). In this study, per the comprehensive stress study recommended by the International Council for Harmonisation (ICH), alpelisib was exposed to hydrolysis, oxidation, photolysis, and thermal stress. Degradation of the drug was observed under hydrolysis, oxidative, and photolysis conditions, whereas the drug was stable under thermal stress condition. We developed a SIAM for the separation of alpelisib and its major DPs that were formed under different stress conditions. The validation of the developed method was performed per ICH Q2(R1) guidelines. Five DPs were identified and characterized. Structure elucidation of all DPs was performed with the modern characterization tool of liquid chromatography-quadrupole time-of-flight mass spectrometer (LC-Q-TOF-MS/MS). The degradation pathway of the drug and its mechanisms were outlined, and in silico toxicity prediction was performed using the ProTox-II tool.

Identification and structural characterization of major stress degradation products of halcinonide by liquid chromatography-high-resolution mass spectrometry.

Halcinonide is a topical corticosteroid approved by the US Food and Drug Administration (FDA), known for its anti-inflammatory and antipruritic properties. The therapeutic benefits of halcinonide have rendered it an effective treatment regimen for various dermatological conditions such as psoriasis, dermatitis, and eczema. However, stability of the drug substance is a prerequisite in determining the therapeutic efficacy and plays a crucial role during formulation development and long-term storage. As corticosteroids are highly susceptible to degradation, the current study aims to expose halcinonide to different stress conditions and understand its stability behavior. An HPLC method was developed for the separation of halcinonide and its degradation products. Separation was accomplished in gradient mode using an Eclipse Plus C18 column (250 × 4.5 mm, 5 µm) with ammonium formate (10 mM, pH 6.5) and acetonitrile as the mobile phases. LC-Q-TOF/MS/MS studies were conducted on halcinonide, which led to the identification of degraded products using optimized mass parameters. A potential mechanistic degradation pathway for halcinonide, along with the major identified degradation products has been established. The chromatographic method that was developed has been validated in compliance with the Q2(R1) guideline of the International Council for Harmonization. ProTox-II was used to perform in silico toxicity studies in order to evaluate the toxicity potential of both halcinonide and the identified degradation products.

Cutting-edge strategies and critical advancements in characterization and quantification of metabolites concerning translational metabolomics.

Despite remarkable progress in drug discovery strategies, significant challenges are still remaining in translating new insights into clinical applications. Scientists are devising creative approaches to bridge the gap between scientific and translational research. Metabolomics is a unique field among other omics techniques for identifying novel metabolites and biomarkers. Fortunately, characterization and quantification of metabolites are becoming faster due to the progress in the field of orthogonal analytical techniques. This review detailed the advancement in the progress of sample preparation, and data processing techniques including data mining tools, database, and their quality control (QC). Advances in data processing tools make it easier to acquire unbiased data that includes a diverse set of metabolites. In addition, novel breakthroughs including, miniaturization as well as their integration with other devices, metabolite array technology, and crystalline sponge-based method have led to faster, more efficient, cost-effective, and holistic metabolomic analysis. The use of cutting-edge techniques to identify the human metabolite, including biomarkers has proven to be advantageous in terms of early disease identification, tracking the progression of illness, and possibility of personalized treatments. This review addressed the constraints of current metabolomics research, which are impeding the facilitation of translation of research from bench to bedside. Nevertheless, the possible way out from such constraints and future direction of translational metabolomics has been conferred.

Metagenomic views on taxonomic and functional profiles of the Himalayan Tsomgo cold lake and unveiling its deterzome potential.

Cold habitat is considered a potential source for detergent industry enzymes. This study aims at the metagenomic investigation of Tsomgo lake for taxonomic and functional annotation, unveiling the deterzome potential of the residing microbiota at this site. The present investigation revealed molecular profiling of microbial community structure and functional potential of the high-altitude Tsomgo lake samples of two different temperatures, harvested during March and August. Bacteria were found to be the most dominant phyla, with traces of genomic pieces of evidence belonging to archaea, viruses, and eukaryotes. Proteobacteria and Actinobacteria were noted to be the most abundant bacterial phyla in the cold lake. In-depth metagenomic investigation of the cold aquatic habitat revealed novel genes encoding detergent enzymes, amylase, protease, and lipase. Further, metagenome-assembled genomes (MAGs) belonging to the psychrophilic bacterium, Arthrobacter alpinus, were constructed from the metagenomic data. The annotation depicted the presence of detergent enzymes and genes for low-temperature adaptation in Arthrobacter alpinus. Psychrophilic microbial isolates were screened for lipase, protease, and amylase activities to further strengthen the metagenomic findings. A novel strain of Acinetobacter sp. was identified with the dual enzymatic activity of protease and amylase. The bacterial isolates exhibited hydrolyzing activity at low temperatures. This metagenomic study divulged novel genomic resources for detergent industry enzymes, and the bacterial isolates secreting cold-active amylase, lipase, and protease enzymes. The findings manifest that Tsomgo lake is a potential bioresource of cold-active enzymes, vital for various industrial applications.

Synthetic pharmaceutical peptides characterization by chromatography principles and method development.

As per the United States Food and Drug Administration, any polymer/chain composed of 40 or fewer amino acids is called a peptide, where more than 40 amino acids are considered proteins. On many occasions, there is a change in the source of manufacturing of the peptide active pharmaceutical ingredient, where one has to prove the sameness of that product with the existing formulation by considering several aspects like the presence of impurities/degradation products, the extent of aggregations, and so forth. For the same, several chromatographic characterization techniques such as reversed-phase high-performance liquid chromatography-ultraviolet/high-resolution mass spectrometry, supercritical fluid chromatography, size-exclusion chromatography, ion-exchange chromatography, and so forth, are widely used in the pharmaceutical industry. It is well known that the method development of peptide molecules is often challenging as many variables are to be kept in mind which can affect the separation, recovery, and stability of the molecule. The present review focuses on the basics of peptide degradation and method development by using various chromatographic techniques for characterization. It also covers a deep insight of method development parameters and variables to be considered which might directly or indirectly affect the chromatographic separation and recovery and also provides a guide on the selection of chromatographic parameters.

Molecular dissemination of emerging antibiotic, biocide, and metal co-resistomes in the Himalayan hot springs.

Growing resistance among microbial communities against antimicrobial compounds, especially antibiotics, is a significant threat to living beings. With increasing antibiotic resistance in human pathogens, it is necessary to examine the habitats having community interests. In the present study, a metagenomic approach has been employed to understand the causes, dissemination, and effects of antibiotic, metal, and biocide resistomes on the microbial ecology of three hot springs, Borong, Lingdem, and Yumthang, located at different altitudes of the Sikkim Himalaya. The taxonomic assessment of these hot springs depicted the predominance of mesophilic organisms, mainly belonging to the phylum Proteobacteria. The enriched microbial metabolism assosiated with energy, cellular processes, adaptation to diverse environments, and defence were deciphered in the metagenomes. The genes representing resistance to semisynthetic antibiotics, e.g., aminoglycosides, fluoroquinolones, fosfomycin, vancomycin, trimethoprim, tetracycline, streptomycin, beta-lactams, multidrug resistance, and biocides such as triclosan, hydrogen peroxide, acriflavin, were abundantly present. Various genes attributing resistance to copper, arsenic, iron, and mercury in metal resistome were detected. Relative abundance, correlation, and genome mapping of metagenome-assembled genomes indicated the co-evolution of antibiotic and metal resistance in predicted novel species belonging to Vogesella, Thiobacillus, and Tepidimona genera. The metagenomic findings were further validated with isolation of microbial cultures, exhibiting resistance against antibiotics and heavy metals, from the hot spring water samples. The study furthers our understanding about the molecular basis of co-resistomes in the ceological niches and their possible impact on the environment.

Metagenomics revealing molecular profiling of community structure and metabolic pathways in natural hot springs of the Sikkim Himalaya.

BACKGROUND: Himalaya is an ecologically pristine environment. The geo-tectonic activities have shaped various environmental niches with diverse microbial populations throughout the Himalayan biosphere region. Albeit, limited information is available in terms of molecular insights into the microbiome, including the uncultured microbes, of the Himalayan habitat. Hence, a vast majority of genomic resources are still under-explored from this region. Metagenome analysis has simplified the extensive in-depth exploration of diverse habitats. In the present study, the culture-independent whole metagenome sequencing methodology was employed for microbial diversity exploration and identification of genes involved in various metabolic pathways in two geothermal springs located at different altitudes in the Sikkim Himalaya. RESULTS: The two hot springs, Polok and Reshi, have distinct abiotic conditions. The average temperature of Polok and Reshi was recorded to be 62 °C and 43 °C, respectively. Both the aquatic habitats have alkaline geochemistry with pH in the range of 7-8. Community profile analysis revealed genomic evidence of plentiful bacteria, with a minute fraction of the archaeal population in hot water reservoirs of Polok and Reshi hot spring. Mesophilic microbes belonging to Proteobacteria and Firmicutes phyla were predominant at both the sites. Polok exhibited an extravagant representation of Chloroflexi, Deinococcus-Thermus, Aquificae, and Thermotogae. Metabolic potential analysis depicted orthologous genes associated with sulfur, nitrogen, and methane metabolism, contributed by the microflora in the hydrothermal system. The genomic information of many novel carbohydrate-transforming enzymes was deciphered in the metagenomic description. Further, the genomic capacity of antimicrobial biomolecules and antibiotic resistance were discerned. CONCLUSION: The study provided comprehensive molecular information about the microbial treasury as well as the metabolic features of the two geothermal sites. The thermal aquatic niches were found a potential bioresource of biocatalyst systems for biomass-processing. Overall, this study provides the whole metagenome based insights into the taxonomic and functional profiles of Polok and Reshi hot springs of the Sikkim Himalaya. The study generated a wealth of genomic data that can be explored for the discovery and characterization of novel genes encoding proteins of industrial importance.

Development and validation of stability-indicating assay method and identification of force degradation products of glucagon-like peptide-1 synthetic analog Exenatide using liquid chromatography coupled with Orbitrap mass spectrometer.

Exenatide is a synthetic glucagon-like peptide 1 analog, widely used in the management of type 2 diabetes mellitus. The stability of pharmaceutical products is significantly impacted by various environmental stress conditions. The present study reports the development of a validated reverse-phase high-performance liquid chromatography (RP-HPLC) stability-indicating method for the identification of force degradation products (DPs) of synthetic glucagon-like peptide-1 analog Exenatide using UHPLC-Orbitrap fusionTM mass spectrometer. Force degradation studies were performed by subjecting Exenatide to various stress conditions, such as hydrolytic, oxidative, photolytic and thermal to investigate the stability indicating ability of the method. Significant degradation was observed during acidic, oxidative, photolytic and thermal stress conditions. Exenatide and its major DPs identification and characterization were demonstrated by employing LC-HRMS and MS/MS method. In total, five major stress DPs were characterized, and their fragmentation pathway was proposed using MS/MS studies. Finally, the proposed RP-HPLC method was validated as per ICH guidance.

Biochemical characterization of a novel acid-active endopolygalacturonase for pectin depolymerization, pectic-oligomer production, and fruit juice clarification.

Endopolygalacturonases are crucial pectinases known for their efficient and sustainable pectin depolymerization activities. The present study identified a novel gene encoding endopolygalacturonase from an acidic mine tailing metagenome. The putative gene showed a maximum identity of 67.55 % with an uncharacterized peptide sequence from Flavobacterium fluvii. The gene was cloned and expressed in a heterologous host, E. coli. Biochemical characterization of the novel endopolygalacturonase enzyme variant (EPHM) showed maximum activity at 60 °C and at 5.0 pH, while retaining 50 % activity under the temperature and pH range of 20 °C to 70 °C for 6 h, and 3.0 to 10.0 for 3 h, respectively. The enzyme exhibited tolerance to different metal ions. EPHM was characterized for the depolymerization of methylated pectin into pectic oligosaccharides. Further, its utility was established for fruit juice clarification, as endorsed by high transmittance, significant viscosity reduction, and release of reducing sugars in the treated fruit juice samples.

Combatting synthetic dye toxicity through exploring the potential of lignin peroxidase from Pseudomonas fluorescence LiP RL5.

Untreated release of toxic synthetic and colorful dyes is a serious threat to the environment. Every year, several thousand gallons of dyes are being disposed into the water resources without any sustainable detoxification. The accumulation of hazardous dyes in the environment poses a severe threat to the human health, flora, fauna, and microflora. Therefore, in the present study, a lignin peroxidase enzyme from Pseudomonas fluorescence LiP-RL5 has been employed for the maximal detoxification of selected commercially used dyes. The enzyme production from the microorganism was enhanced ~ 20 folds using statistical optimization tool, response surface methodology. Four different combinations (pH, production time, seed age, and inoculum size) were found to be crucial for the higher production of LiP. The crude enzyme showed decolorization action on commonly used commercial dyes such as Crystal violet, Congo red, Malachite green, and Coomassie brilliant blue. Successful toxicity mitigation of these dyes culminated in the improved seed germination in three plant species, Vigna radiate (20-60%), Cicer arietinum (20-40%), and Phaseolus vulgaris (10-25%). The LiP treated dyes also exhibit reduced bactericidal effects against four common resident microbial species, Escherichia coli (2-10 mm), Bacillus sp. (4-8 mm), Pseudomonas sp. (2-8 mm), and Lactobacillus sp. (2-10 mm). Therefore, apart from the tremendous industrial applications, the LiP from Pseudomonas fluorescence LiP-RL5 could be a potential biocatalyst for the detoxification of synthetic dyes.

Drug-Leachable Interaction Product Evaluation in Prefilled Syringe of Ganirelix Acetate Injection.

The concept of extractables and leachable has introduced a new era for identifying potential impurities in drug products. Pharmaceutical packaging materials encompass a variety of polymers due to their appealing properties for storing the drug product. However, numerous chemical species may leach into the drug product from these polymers, posing significant health hazards in the public domain. Identifying such leachable is crucial for assessing safety and addressing toxicological concerns. Acrylic acids are commonly used materials for adhering needles to the barrel in pre-filled syringes. In this study, we identified acrylic acid leachable impurities in ganirelix drug products available in the market using mass spectrometry as an analytical technique. These impurities leached into the drug product during storage conditions. Characterization of the impurities was carried out through data obtained from HRMS and MS/MS analysis, revealing them as polyacrylic acid-ganirelix adduct impurities. This study offers valuable insights into identifying leachable and susceptible sites, providing a foundation for potential modifications in similar classes of drug products, thereby enhancing their safety and efficacy.

Development of an effective cleaning technique and ancillary analytical method for estimation of residues of selected kinase inhibitors from stainless steel and glass surfaces by swab sampling.

Importance of cleaning validation in the pharmaceutical industry cannot be overstated. It is essential for preventing cross-contamination, ensuring product quality & safety, and upholding regulatory standards. The present study involved development of an effective cleaning method for five selected kinase inhibitors binimetinib (BMT), selumetinib (SMT), brigatinib (BGT), capmatinib (CPT), and baricitinib (BRT). For checking the effectiveness of the developed cleaning technique, a sensitive and specific RP-HPLC based analytical method employing a diode array detector has been established to quantitate drug residue on glass and stainless steel surfaces. A reproducible swab sampling protocol utilizing TX714A Alpha swabs wetted with an extracting solvent has been developed to collect representative samples from both surfaces. Chromatographic separation of selected kinase inhibitors was achieved in gradient mode using an Agilent Zorbax eclipsed C18 column with acetonitrile and 10 mM ammonium formate as the mobile phase. The analytes were chromatographically separated in a 12 min run time. The mean swab recovery for each drug from glass and stainless steel surfaces exceeded 90%. Cleaning with IPA (70%) and acetone (70%) effectively removed residues for all five drugs. A solution comprising 10 mM SDS with 20% IPA demonstrated good efficacy in cleaning residues of BGT, BRT, and CPT, but exhibited lower efficacy for SMT and BMT.

Echocardiographic predictors and associated outcomes of multiple vegetations in infective endocarditis: A pilot study.

BACKGROUND: Infective endocarditis (IE) is a life-threatening infection with an annual mortality of 40%. Embolic events reported in up to 80% of patients. Vegetations of > 10 mm size are associated with increased embolic events and poor prognosis. There is a paucity of literature on the association of multiple vegetations with outcome. AIM: To study the echocardiographic (ECHO) features and outcomes associated with the presence of multiple vegetations. METHODS: In this retrospective, single-center, cohort study patients diagnosed with IE were recruited from June 2017 to June 2019. A total of 84 patients were diagnosed to have IE, of whom 67 with vegetation were identified. Baseline demographic, clinical, laboratory, and ECHO parameters were reviewed. Outcomes that were studied included recurrent admission, embolic phenomenon, and mortality. RESULTS: Twenty-three (34%) patients were noted to have multiple vegetations, 13 (56.5%) were male and 10 (43.5%) were female. The mean age of these patients was 50. Eight (35%) had a prior episode of IE. ECHO features of moderate to severe valvular regurgitation [odds ratio (OR) = 4], presence of pacemaker lead (OR = 4.8), impaired left ventricle (LV) relaxation (OR = 4), and elevated pulmonary artery systolic pressure (PASP) (OR = 2.2) are associated with higher odds of multiple vegetations. Of these moderate to severe valvular regurgitation (P = 0.028), pacemaker lead (P = 0.039) and impaired relaxation (P = 0.028) were statistically significant. These patients were noted to have an increased association of recurrent admissions (OR = 3.6), recurrent bacteremia (OR = 2.4), embolic phenomenon (OR = 2.5), intensive care unit stay (OR = 2.8), hypotension (OR = 2.1), surgical intervention (OR = 2.8) and device removal (OR = 4.8). Of this device removal (P = 0.039) and recurrent admissions (P = 0.017) were statistically significant. CONCLUSION: This study highlights the associations of ECHO predictors and outcomes in patients with IE having multiple vegetations. ECHO features of moderate to severe regurgitation, presence of pacemaker lead, impaired LV relaxation, and elevated PASP and outcomes including recurrent admissions and device removal were found to be associated with multiple vegetations.

A novel validated ultra-performance liquid chromatography Method for separation of eszopiclone impurities and its degradants in drug products.

A selective, specific, and sensitive ultra-performance LC (UPLC) method was developed for determination of eszopiclone and its degradation products. The chromatographic separation was performed with a Waters ACQUITY UPLC system and BEH C18 column using gradient elution with mobile phases A and B. Mobile phase A was 0.01 M phosphate buffer with 0.2% (w/v) 1-octane sulfonic acid sodium salt as an ion pair reagent, adjusted pH 2.2 with orthophosphoric acid-acetonitrile (85 + 15, v/v). Mobile phase B was pH 2.2 buffer-acetonitrile (20 + 80, v/v). UV detection was performed at 303 nm. Eszopiclone and its impurities were chromatographed with a total run time of 13 min. A calibration study showed that the response for each of the impurities A, B, C, and D was linear between concentrations of 0.02 and 7.2 microg/mL (r2 > or = 0.999). The method was validated over this range for precision, intermediate precision, accuracy, linearity, and specificity. For the precision study, RSD of each impurity was <5% (n = 6). The method was found to be precise, accurate, linear, and specific. The proposed method was successfully used for determination of eszopiclone impurities in pharmaceutical preparations.