Assessing the role of the graphene family nanomaterials (GFNs: Graphene, GO, rGO) in modifying the toxicity potential and environmental risk of flame retardant, tetrabromobisphenol-A (TBBPA) in the marine microalgae Chlorella sp.

In recent years, a growing concern has emerged regarding the environmental implications of flame retardants (FRs) like tetrabromobisphenol-A (TBBPA) and graphene family nanomaterials (GFNs), such as graphene, graphene oxide (GO), and reduced graphene oxide (rGO), on marine biota. Despite these substances' well-established individual toxicity profiles, there is a notable gap in understanding the physicochemical interactions within the binary mixtures and consequent changes in the toxicity potential. Therefore, our research focuses on elucidating the individual and combined toxicological impacts of TBBPA and GFNs on the marine alga Chlorella sp. Employing a suite of experimental methodologies, including Raman spectroscopy, contact angle measurements, electron microscopy, and chromatography, we examined the physicochemical interplay between the GFNs and TBBPA. The toxicity potentials of individual constituents and their binary combinations were assessed through growth inhibition assays, quantifying reactive oxygen species (ROS) generation and malondialdehyde (MDA) production, photosynthetic activity analyses, and various biochemical assays. The toxicity of TBBPA and graphene-based nanomaterials (GFNs) was examined individually and in combinations. Both pristine TBBPA and GFNs showed dose-dependent toxicity. While lower TBBPA concentrations exacerbated toxicity in binary mixtures, higher TBBPA levels reduced the toxic effects compared to pristine TBBPA treatments. The principal mechanism underlying toxicity was ROS generation, resulting in membrane damage and perturbation of photosynthetic parameters. Cluster heatmap and Pearson correlation were employed to assess correlations between the biological parameters. Finally, ecological risk assessment was undertaken to evaluate environmental impacts of the individual components and the mixture in the algae.

A Systematic Evaluation of Risk Predictors for COVID-19 Sequelae.

Background Multisystem involvement in coronavirus disease 2019 (COVID-19) is known since the beginning of the pandemic, and post-COVID-19 sequelae have often been reported. The term 'long Covid' encompasses these signs and symptoms. The aim of our study was to study different after-effects which patients endured within 12 months after recovery from acute COVID-19 and to study the various risk predictors. Methods This was a longitudinal observational study of a cohort of 146 patients who recovered from COVID-19 illness. Patients were enrolled during the first four weeks of the onset of their illness, and a monthly follow-up assessment was done for six months that included a detailed history of persistent or new symptoms, new illnesses diagnosed, and complete biochemical, pulmonary, cardiac, neurological and psychiatric evaluation, both objective and subjective. A final follow-up was also done at the end of one year of enrolment. Based on the patient's self-reported history and our multi-system assessment, recorded sequelae were classified according to the involved organ system. These were correlated with possible risk predictors and statistically significant associations were established. Results One hundred and twenty subjects out of 146 total subjects qualified for final analysis. Pulmonary sequelae (48/120; 40%) were the most followed by psychiatric (30/120; 25%), neurological (26/120; 21.7%), and opportunistic infections (7/120; 5.8%). A total of 39/120 (32.1%) cases complained of prolonged dyspnoea. Six out of 120 i.e. 5% of study participants had new-onset diabetes. Twenty-six out of 120 (21.7%) had radiological signs of pulmonary fibrosis. Patients with co-morbidities, older age, higher body mass index, and patients with severe disease were found to be at higher risk of developing these sequelae. Poor nutrition, female gender, and hospitalization were predictors of psychiatric sequelae. Diabetes and liberal steroid use during COVID-19 management were predictors of opportunistic fungal infections. Conclusion This study evaluated post-COVID-19 sequalae in-depth both objectively and subjectively. Some specific predictors for specific sequelae were confirmed on statistical correlation. Long-term follow-up of high-risk persons is therefore recommended after the cure of COVID-19.

Ecotoxicological significance of bio-corona formation on micro/nanoplastics in aquatic organisms.

The unsustainable manufacturing, utilization and inadequate handling of plastics have led to a surge in global plastic pollution. In recent times, there has been increasing concern about the plausible hazards associated with exposure to micro/nanoplastics (M/NPs). As aquatic systems are considered to be the likely sink for M/NPs, it is crucial to comprehend their environmental behavior. The bioavailability, toxicity and fate of M/NPs in the environment are predominantly dictated by their surface characteristics. In the aquatic environment, M/NPs are prone to be internalized by aquatic organisms. This may facilitate their interaction with a diverse array of biomolecules within the organism, resulting in the formation of a biocorona (BC). The development of BC causes modifications in the physicochemical attributes of the M/NPs including changes to their size, stability, surface charge and other properties. This review details the concept of BC formation and its underlying mechanism. It provides insight on the analytical techniques employed for characterizing BC formation and addresses the associated challenges. Further, the eco-toxicological implications of M/NPs and the role of BC in modifying their potential toxicity on aquatic organisms is specified. The impact of BC formation on the fate and transport of M/NPs is discussed. A concise outlook on the future perspectives is also presented.

Cancer nanomedicine: a review of nano-therapeutics and challenges ahead.

Cancer is known as the most dangerous disease in the world in terms of mortality and lack of effective treatment. Research on cancer treatment is still active and of great social importance. Since 1930, chemotherapeutics have been used to treat cancer. However, such conventional treatments are associated with pain, side effects, and a lack of targeting. Nanomedicines are an emerging alternative due to their targeting, bioavailability, and low toxicity. Nanoparticles target cancer cells via active and passive mechanisms. Since FDA approval for Doxil®, several nano-therapeutics have been developed, and a few have received approval for use in cancer treatment. Along with liposomes, solid lipid nanoparticles, polymeric nanoparticles, and nanoemulsions, even newer techniques involving extracellular vesicles (EVs) and thermal nanomaterials are now being researched and implemented in practice. This review highlights the evolution and current status of cancer therapy, with a focus on clinical/pre-clinical nanomedicine cancer studies. Insight is also provided into the prospects in this regard.

Preparation of Celery Essential Oil-Based Nanoemulsion by Ultrasonication and Evaluation of Its Potential Anticancer and Antibacterial Activity.

INTRODUCTION: Plants have always been a significant source of natural active components with biological properties. Celery seed oil (extracted from Apium graveolens) has several potential applications, but its therapeutic uses in the form of nanoemulsion formulation need to be investigated further in order to meet the demand in cancer treatment, and to alleviate the prevailing crisis arising from increased antimicrobial resistance. METHODS: The therapeutic potential of celery seed oil was investigated through the formulation and testing of a nanoemulsion developed with Tween 80 (a non-ionic surfactant) and the utilization of an ultrasonication technique. Anticancer and apoptotic properties of the formulation were evaluated through MTT and Annexin V-FITC assays. The clonogenic assay aided in the identification of the antiproliferative properties of the formulation on oral squamous cell carcinoma. The antimicrobial study was supported by agar well diffusion assay, membrane integrity test and scanning electron microscopy. RESULTS: Experiments identified relevant parameters, including optimal surfactant concentration and emulsification time. GC-MS analysis identified various components in the celery oil, but not their biological activities. A sonication time of 20 min resulted in a droplet diameter of 23.4 ± 1.80 nm. The IC50 concentration of the optimal nanoemulsion formulation against SAS cells was 1.4 µL/mL. At this concentration, cell proliferation was significantly reduced through inhibition of the anchorage-independent cell growth by disrupting colony formation and inducing cell death (apoptosis) of cancer cells. The nanoemulsion was also treated with a microbial suspension of S. aureus, and displayed antibacterial properties through lipid membrane fusion, causing cytoplasmic leakage as verified through agar well diffusion and membrane permeability assays. Scanning electron microscopy revealed complete distortion of the bacterial pathogen. CONCLUSION: The results in this study present celery as a possible constituent for cancer therapeutics and as a candidate for aggressive, yet safe cancer treatment. The celery-based nanoemulsion has the potential to act as a key alternative to standard antibiotic therapy.

Ultrasonic Nanoemulsification of Cuminum cyminum Essential Oil and Its Applications in Medicine.

BACKGROUND AND STUDY: Cumin seed oil (extracted from Cuminum cyminum) has many applications but conclusive evidence of its therapeutic uses has not been presented. This study has explored the anticancer and antibacterial properties of the seed oil. METHODS: The cumin nanoemulsion was prepared with Tween 80 non-ionic surfactant employing ultra-sonication technology. The anticancer activity of the nanoscale-based emulsion was evaluated through cell viability (MTT), antiproliferation evaluation through clonogenic assay, and apoptosis through Annexin V-FITC assay. Agar well diffusion was used to study the antimicrobial activity, and this was supported by membrane integrity analysis. RESULTS: A thorough study of process parameters, aimed at obtaining the optimal surface concentration and emulsification time, was completed. GC-MS data indicated cumaldehyde as a major component. The resultant droplet diameter after a sonication time of 5 min was 10.4 ± 0.5 nm. MTT assay revealed the IC50 value at 1.5 µL/mL and the early induction of apoptosis was evident. Tongue carcinoma cell line treated with cumin nanoemulsion presented a diminished colony formation. The nanoemulsion exhibited significant antibacterial activity against S. aureus. A significant cytoplasmic leakage was observed on treatment with cumin nanoemulsion. The consequences of the analysis projected cumin as a potential component for cancer therapy. CONCLUSION: This study provides definitive evidence for cumin essential oil nanoemulsion as a legitimate plant-based medicine that can bypass the drawbacks of the present aggressive treatment of cancer, can overcome the antimicrobial resistance, and can also meet all prerequisites.

A Study to Assess The Efficacy of Local Application of Oral Probiotic in Treating Recurrent Aphthous Ulcer and Oral Candidiasis.

To study the efficacy of local application of oral probiotics in improving oral health in recurrent aphthous ulcer and oral candidiasis. Eighty patients with diagnosis of recurrent aphthous ulcer and oral candidiasis were included in the study. They were divided into group A = 40 patients (patients treated with oral application of probiotic as an adjuvant) and group B = 40 patients (patients treated without probiotic). Both the groups were divided into two subgroups, group AU and group BU for recurrent aphthous ulcer and group AC and BC for oral candidiasis. Clinical signs and symptoms were assessed at the beginning of the study and at the end of the study. Pregnant or lactating women, patients with localised or systemic diseases such as Steven Johnson syndrome, ulcerative colitis, Behcet's syndrome and patients on chemotherapy or radiotherapy were excluded from the study. Bacillus Clausii, was used as a probiotic in our study. Patients in group A showed significant improvement in erythema (p = 0.001), pain reduction (p = 0.0001), decreased oral thrush (p = 0.006) and burning sensation in the mouth (p = 0.005) on day 5, whereas there was no significant difference on day 10 follow up. The study demonstrated the efficacy and rapidity of response to oral probiotic as an adjuvant in treating aphthous ulcer and oral candidiasis. Hence, oral application of probiotics can be used as an adjuvant in treating various oral pathology.

Anticancer and antibacterial effects of a clove bud essential oil-based nanoscale emulsion system.

BACKGROUND AND PURPOSE: The essential oil derived from clove buds (Syzygium aromaticum) has been used as a chemopreventive agent in Ayurvedic medicine. The antiviral, antibacterial, and anticancer properties of its chemo-skeleton have motivated this study to explore its efficacy in pharmaceutics. METHODS: Nanoscale-based emulsions were prepared by employing a spontaneous emulsification technique through self-assembly using varying concentrations of Tween 20 and Tween 80 surfactants. Their physicochemical properties and stability were studied in order to choose an optimum formulation which was clear and stable. The cytotoxicity of the stable oil-based emulsion system was evaluated using MTT assay, colony formation assay, and Annexin V-FITC assay against the thyroid cancer cell line (HTh-7). RESULTS: All three methods verified apoptosis and reduction in cancer cell proliferation, making the formulation a promising candidate as an alternative cancer drug. The oil-based emulsion system was also tested for its antibacterial properties against Staphylococcus aureus. Membrane permeability studies proved its efficacy to permeate through cell membrane, thereby increasing the leakage of cytoplasmic contents. CONCLUSION: Many current treatments for cancers are aggressive yet ineffective. This study positions the clove bud-based nanoscale emulsion as a suitable candidate for further in vivo studies and trials as a cancer drug.

Nanoemulsion of eucalyptus oil and its larvicidal activity against Culex quinquefasciatus.

Filariasis is a mosquito-borne disease that causes lymphedema and the main vector is Culex quinquefasciatus. A simple measure was taken to eradicate the vector using nanoemulsion. Eucalyptus oil nanoemulsion was formulated in various ratios comprising of eucalyptus oil, tween 80 and water by ultrasonication. The stability of nanoemulsion was observed over a period of time and 1:2 ratios of eucalyptus oil (6%) and surfactant (12%) was found to be stable. The formulated eucalyptus oil nanoemulsion was characterized by transmission electron microscopy and dynamic light scattering. The nanoemulsion droplets were found to have a Z-average diameter of 9.4 nm and were spherical in shape. The larvicidal activity of eucalyptus oil nanoemulsion and bulk emulsion was tested and compared. Our nanoemulsion showed higher activity when compared to bulk emulsion. The histopathology of larvae-treated and untreated nanoemulsion was analyzed. Furthermore, biochemical assays were carried out to examine the effect of nanoemulsion on biochemical characteristics of larvae. The treated larval homogenate showed decrease in total protein content and a significant reduction in the levels of acetylcholinesterase. The levels of acid and alkaline phosphatase also showed reduction as compared to control larval homogenate.

Ultrasonic emulsification of eucalyptus oil nanoemulsion: antibacterial activity against Staphylococcus aureus and wound healing activity in Wistar rats.

The plant derived essential oil nanoemulsion was prepared using a mixture of components containing eucalyptus oil as organic phase, water as continuous phase, and non ionic surfactant, Tween 80, as emulsifier at a particular proportion of 1:1 v/v%. The ultrasonication was applied for varied processing time from 0 to 30 min to study the effect of time on the formation of nanoemulsion and physical stability of formulation by this method. The transparency and stability of emulsion was enhanced when the sonication time was increased compared to hand blender emulsion. The most stable nanoemulsion was obtained in 30 min sonication having the mean droplet diameter of 3.8 nm. The antibacterial studies of nanoemulsion against Staphylococcus aureus by time kill analysis showed complete loss of viability within 15 min of interaction. Observations from scanning electron microscopy of treated bacterial cells confirmed the membrane damage compared to control bacteria. Furthermore, the wound healing potential and skin irritation activity of the formulated nanoemulsion in Wistar rats, suggested non-irritant and higher wound contraction rate with respect to control and neomycin treated rats. These results proposed that the formulated system could be favourable for topical application in pharmaceutical industries.

Design and formulation technique of a novel drug delivery system for azithromycin and its anti-bacterial activity against Staphylococcus aureus.

Azithromycin, an important member of the azalide subclass is effective against both Gram-positive and Gram-negative organisms. Certain physicochemical properties of the drug like poor water solubility and relatively low bioavailability of 37% due to incomplete absorption after ingestion, aroused the need for the development of a novel drug delivery system to enhance the solubilization potential and antibacterial activity against Staphylococcus aureus at a very low concentration. Cinnamon oil (Cinnamonum zeylanicum)-based microemulsion system formulated using non-ionic surfactant, Tween 20, and water was characterized. The drug-incorporated system F4 (oil to surfactant ratio of 1:4 (v/v)) showed enhanced solubilization of the drug, droplet diameter of 5-8 nm, and a good thermodynamic stability. The effect of surfactant concentration exhibited a negative correlation with droplet size diameter and turbidity and a positive correlation with stability and viscosity. The system was investigated for its antibacterial activity that demonstrated a significantly higher activity at a minimum concentration (4 µg/ml) of the novel drug-loaded system in comparison with the conventional formulation (128 µg/ml). Examination through scanning electron microscopy analysis further confirmed a considerable morphologic variation due to alteration in the membrane permeability of the microemulsion-treated system. The small droplet size of the microemulsion system and the antibacterial property of cinnamon oil, together, accounts clearly for the enhanced efficacy of the new formulated system F4 and not just azithromycin alone. Staining with acridine orange/ethidium bromide dyes as examined through fluorescence microscopy also substantiated with the results of membrane permeability of bacteria. Thus, our study discloses a potential oral drug delivery system of azithromycin with improved biocompatibility.

Cytotoxicity of TiO2 nanoparticles and their detoxification in a freshwater system.

In the current study, two aspects concerning (i) the cytotoxicity potential of TiO2 nanoparticles (NPs) toward freshwater algal isolate Scenedesmus obliquus and (ii) the potential detoxification of NPs by the microalgae were assessed under light (UV-illumination) and dark conditions at low exposure levels (≤1 µg/mL), using sterile freshwater as the test medium. The statistically significant reduction in cell viability, increase in reactive oxygen species production and membrane permeability (light vs. dark) suggested photo-induced toxicity of TiO2 NPs. The electron micrographs demonstrated adsorption of the NPs onto the cell surface and substantiated their internalization/uptake. The fluorescence micrographs and the confocal laser scanning (CLSM) images suggested the absence of a definite/intact nucleus in the light treated cells pointing toward the probable genotoxic effects of NPs. In a separate three cycle experiment, a continuous decrease in the cytotoxicity was observed, whereas, at the end of each cycle only fresh algae were added to the supernatant containing NPs from the previous cycle. The decreasing concentrations of the NPs in the subsequent cycles owing to agglomeration-sedimentation processes exacerbated by the algal interactions played a crucial role in the detoxification. In addition, the exo-polymeric substances produced by the cells could have rendered the available NPs less reactive, thereby, enhancing the detoxification effects.

Improved efficacy of fluconazole against candidiasis using bio-based microemulsion technique.

Candida albicans is a common fungal pathogen that causes systemic and superficial infections in most immunocompromised patients. Fluconazole, a synthetic triazole antifungal agent, is the most prescribed drug used in treating this pathogen. But because of its poor solubilization in water and the emergence of resistant strains against this antimycotic drug, we aimed at devising a unique microemulsion drug delivery system for fluconazole against candidiasis. A clear oil-in-water microemulsion system, consisting of clove oil as oil phase, Tween 20 as surfactant, and water as aqueous phase was developed using a ternary phase diagram. Physicochemical characterization was done to understand the internal physicochemical state. The bulk drug, fluconazole, that measured several microns in length was reduced to a 10-65 nm range with no means of high-energy methods as confirmed by transmission electron microscopy. The very small and uniform spherical structure of the drug-loaded microemulsion system could be of high impact to the biological system as the efficacy of fluconazole is greatly improved when compared with its conventional bulk form. The optimized microemulsion exhibited significantly higher antifungal activity at a minimum concentration (8 µg/ml) of fluconazole as examined by fluorescence and scanning electron microscopy. Thus, our report discloses an excellent oral drug delivery system.

Hepatoprotective activity of Musa paradisiaca on experimental animal models.

OBJECTIVE: To investigate the hepatoprotective activity of stem of Musa paradisiaca (M. paradisiaca) in CCl4 and paracetamol induced hepatotoxicity models in rats. METHODS: Hepatoprotective activity of alcoholic and aqueous extracts of stem of M. paradisiaca was demonstrated by using two experimentally induced hepatotoxicity models. RESULTS: Administration of hepatotoxins (CCl4 and paracetamol) showed significant biochemical and histological deteriorations in the liver of experimental animals. Pretreatment with alcoholic extract (500 mg/kg), more significantly and to a lesser extent the alcoholic extract (250 mg/kg) and aqueous extract (500 mg/kg), reduced the elevated levels of the serum enzymes like serum glutamic-oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), alkaline phosphatase (ALP) and bilirubin levels and alcoholic and aqueous extracts reversed the hepatic damage towards the normal, which further evidenced the hepatoprotective activity of stem of M. paradisiaca. CONCLUSIONS: The alcoholic extract at doses of 250 and 500 mg/kg, p.o. and aqueous extract at a dose of 500 mg/kg, p.o. of stem of M. paradisiaca have significant effect on the liver of CCl4 and paracetamol induced hepatotoxicity animal models.

Transplantation of hippocampal cell lines restore spatial learning in rats with ventral subicular lesions.

We have demonstrated in our previous studies that ventral subicular lesion induces neurodegeneration of the hippocampus and produces cognitive impairment in rats. In the present study, the efficacy of transplanted green fluorescent protein (GFP)-labeled hippocampal cell line (H3-GFP) cells in establishing functional recovery in ventral subicular lesioned rats has been evaluated. The survival of H3-GFP transplants and their ability to express trophic factors in vivo were also investigated. Adult male Wistar rats were subjected to selective lesioning of ventral subiculum and were transplanted with H3-GFP cells into the cornu ammonis 1 (CA1) hippocampus. The transplants settled mainly in the dentate gyrus and expressed neurotrophic factors, brain-derived neurotrophic factor (BDNF), and basic fibroblast growth factor (bFGF). The ventral subicular lesioned (VSL) rats with H3-GFP transplants showed enhanced expression of BDNF in the hippocampus and performed well in eight-arm radial maze and Morris water maze tasks. The VSL rats without hippocampal transplants continued to show cognitive impairment in task learning. The present study demonstrated the H3-GFP transplants mediated recovery of cognitive functions in VSL rats. Our study supports the notion of graft meditated host regeneration and functional recovery through trophic support, although these mechanisms require further investigation.

A comparative study of cardiac dysautonomia in autosomal dominant spinocerebellar ataxias and idiopathic sporadic ataxias.

OBJECTIVES: Comparative evaluation of cardiac dysautonomia in spinocerebellar ataxias (SCA) and idiopathic sporadic ataxias (IA) not fulfilling the criteria of multiple system atrophy. MATERIAL AND METHODS: Cardiac autonomic functions were evaluated in 14 SCA (SCA1 = 6, SCA2 = 5 and SCA3 = 3) and 10 IA patients, comparable for age, age at onset, duration and severity of illness. The results were categorized as early, definitive, or severe autonomic involvement (EI, DI and SI respectively) based on the degree of abnormalities on tests of parasympathetic and sympathetic pathways. RESULTS: Cardiac autonomic dysfunction was present in all (EI = 25.0%, DI = 41.7% and SI = 33.3%), parasympathetic dysfunction being an early feature. SI was most often present in SCA3 (100%), followed by those with SCA1 (66.7%), and SCA2 (20%) and none in IA. CONCLUSIONS: Cardiac dysautonomia was common in both SCA and IA, although the severity was greater in SCA. Among SCAs, the severity was greatest in SCA3, followed by SCA2 and least in SCA1.

(3S,4R)-4-(4-Fluoro-phen-yl)-3-(hydroxy-meth-yl)piperidinium chloride.

The title compound, C(12)H(17)FNO(+)·Cl(-), is a degradation impurity of paroxetine hydro-chloride hemihydrate (PAXIL), an anti-depressant belonging to the group of drugs called selective serotonin reuptake inhibitors (SSRIs). Similar to the paroxetine hydro-chloride salt with protonation having taken place on the basic piperidine ring, the degradation impurity also exists as the hydro-chloride salt. The cyclic six-membered piperidinium ring adopts a chair conformation with the hydroxy-methyl and 4-fluoro-phenyl groups in the equatorial positions. The ions form a tape along the b axis through charge-assisted N(+)-H⋯Cl(-) hydrogen bonds; these tapes are connected by O-H⋯Cl(-) hydrogen bonds along the a axis.

Myasthenia gravis and acetylcholine receptor antibodies: a clinico immunological correlative study on South Indian patients.

UNLABELLED: Acquired myasthenia Gravis (MG), a disorder of impaired neuromuscular transmission is recognized as an autoimmune disorder, with a majority of the patients having antibodies against acetylcholine receptor (AChR antibodies) in the serum. AIM AND OBJECTIVES: To correlate the AChR antibody status with the clinical characteristics of patients with myasthenia gravis. STUDY DESIGN: Retrospective and prospective study. MATERIALS AND METHODS: This study was carried out in patients with definite MG, attending the Neurology services of the National Institute of Mental Health and Neurosciences, Bangalore, India, during the period 1999-2003. The AChR antibody status was determined using the direct and indirect enzyme linked immunosorbent assay (ELISA) technique. RESULTS: There were 165 patients in this study (M : F :: 1.5 : 1). The overall seropositivity rate was 59.4%. Seropositive patients had higher age of onset and presentation, and more frequent occurrence of crises, both at presentation and at any time during the course. Other parameters, viz. gender of the patient, Osserman staging, thymic enlargement on CT and remission during follow-up did not differ between the two groups. CONCLUSION: This communication reports the result of AChR antibody assay in a large cohort of patients, using a simple diagnostic tool, namely direct and indirect ELISA technique. In addition, the characteristics of a large cohort of patients with seronegative myasthenia gravis are described.

Identification and characterization of potential impurities of amlodipine maleate.

Six impurities ranging from 0.43 to 1.42% in amlodipine maleate were detected by a simple isocratic reverse-phase high performance liquid chromatography (HPLC). LC-MS was performed to identify the mass of the impurities. Based on the spectral data (IR, NMR and MS), the structures of these impurities were characterized as 3-ethyl 5-methyl 4-(2-chlorophenyl)-2-[2-(1,3-dioxo-2,3-dihydro-1H-2-isoindolyl)ethoxymethyl]-6-methyl-1,4-dihydro-3,5-pyridinedicarboxylate (impurity I); 5-ethyl 3-methyl 4-(2-chlorophenyl)-2-methyl-6-[2-(2-methylcarbamoylphenyl-carboxamido)ethoxymethyl]-1,4-dihydro-3,5-pyridinedicarboxylate (impurity II); besylate salt of 3-ethyl 5-methyl 2-(2-aminoethoxymethyl)-4-(2-chlorophenyl)-6-methyl-3,5-pyridinedicarboxylate (impurity III); dimethyl 2-(2-aminoethoxymethyl)-4-(2-chlorophenyl)-6-methyl-1,4-dihydro-3,5-pyridinecarboxylate (impurity IV); 3-ethyl 5-methyl 2-(2-aminoethoxymethyl)-4-(4-chlorophenyl)-6-methyl-1,4-dihydro-3,5-pyridinedicarboxylate (impurity V); diethyl 2-(2-aminoethoxymethyl)-4-(2-chlorophenyl)-6-methyl-1,4-dihydro-3,5-pyridinedicarboxylate (impurity VI).