Isolation and Structural Elucidation of Impurity in Sulfonamide Intermediate of Glyburide

A reproducible isolation method by Reverse Phase (RP) preparative HPLC technique for the isolation of one crucial impurity at 1.65 RRT (Relative Retention Time) in sulfonamide stage of Glyburide API (Active Pharmaceuticals Ingredient) was developed. Preparative chromatography was done on Luna C8, 10µm (250 mm x 21.2mm) preparative HPLC column with acetonitrile: water in 70:30 % v/v proportion as a mobile phase and 8 ml/min as a flow rate. This impurity was detected at 300 nm UV-wavelength maximum. This impurity was isolated from synthesized crude impurity of sulfonamide stage of Glyburide substance by preparative HPLC by injecting 50 mg/ml concentration over 5 ml fixed loop. Isolated impurity was elucidated as N-methyl impurity of sulfonamide intermediate of Glyburide API by means of chromatographic and spectral data. Structural elucidation carried out by spectral data was reviewed. This impurity was analyzed by reverse phase HPLC for purity analysis. A Inertsil C8 (250 x 4.6) mm, 5µ particle size was employed for separation. The mobile phase consisted of Water: Acetonitrile: Methanol in the ratio of 60:15:25 % v/v. The flow rate was set at 1 ml/min. Detection was carried out at 300 nm. 10µL of 2 mg/ml concentration of sample in methanol was injected. The column oven temperature was at 25°C.

Molecular characterization and genetic diversity studies of Indian soybean (Glycine max (L.) Merr.) cultivars using SSR markers

Background The genetic base of soybean cultivars in India has been reported to be extremely narrow, due to repeated use of few selected and elite genotypes as parents in the breeding programmes. This ultimately led to the reduction of genetic variability among existing soybean cultivars and stagnation in crop yield. Thus in order to enhance production and productivity of soybean, broadening of genetic base and exploring untapped valuable genetic diversity has become quite indispensable. This could be successfully accomplished through molecular characterization of soybean genotypes using various DNA based markers. Hence, an attempt was made to study the molecular divergence and relatedness among 29 genotypes of soybean using SSR markers. Methods and results A total of 35 SSR primers were deployed to study the genetic divergence among 29 genotypes of soybean. Among them, 14 primer pairs were found to be polymorphic producing a total of 34 polymorphic alleles; and the allele number for each locus ranged from two to four with an average of 2.43 alleles per primer pair. Polymorphic information content (PIC) values of SSRs ranged from 0.064 to 0.689 with an average of 0.331. The dendrogram constructed based on dissimilarity indices clustered the 29 genotypes into two major groups and four sub-groups. Similarly, principal coordinate analysis grouped the genotypes into four major groups that exactly corresponded to the clustering of genotypes among four sub-groups of dendrogram. Besides, the study has reported eight unique and two rare alleles that could be potentially utilized for genetic purity analysis and cultivar identification in soybean. Conclusion In the present investigation, two major clusters were reported and grouping of large number of genotypes in each cluster indicated high degree of genetic resemblance and narrow genetic base among the genotypes used in the study. With respect to the primers used in the study, the values of PIC and other related parameters revealed that the selected SSR markers are moderately informative and could be potentially utilized for diversity analysis of soybean. The clustering pattern of dendrogram constructed based on SSR loci profile displayed good agreement with the cultivar’s pedigree information. High level of genetic similarity observed among the genotypes from the present study necessitates the inclusion of wild relatives, land races and traditional cultivars in future soybean breeding programmes to widen the crop gene pool. Thus, hybridization among diverse gene pool could result in more heterotic combinations ultimately enhancing genetic gain, crop yield and resistance to various stress factors.

Best Practices for Authentication of Cell Lines to Ensure Data Reproducibility and Integrity

Cell line misidentification and contamination are major contributors to the reproducibility crisis in academic research. Authentication of cell lines provides assurances of the data generated; however, commercially available cells are often not subjected to rigorous identification testing. In this study, commercially available cell lines underwent testing to confirm cell identity and purity. The methods reported here outline the best practices for cell line authentication. Briefly, a commercially available primary rabbit aortic endothelial cell line was purchased for the intent of producing target proteins necessary for generating species-specific recombinant antibodies. These rabbit-specific antibodies would then be utilized for the development of in-house enzyme-linked immunosorbent assays (ELISA) to evaluate blood-based biomarkers of vascular injury after total-body irradiation. To authenticate the cell line, cell identity and purity were determined by single tandem repeat (STR) testing, flow cytometry, polymerase chain reaction (PCR), and cytochrome c oxidase subunit 1 (CO1) DNA Barcoding in-house and/or through commercial vendors. Fresh cells obtained from a New Zealand White rabbit (Charles River, Wilmington, DE) were used as a positive control. The results of STR and flow cytometry analyses indicated the cells were not contaminated with human or mouse cells, and that the cells were not of endothelial origin. PCR demonstrated that cells were also not of rabbit origin, which was further confirmed by a third-party vendor. An unopened vial of cells was submitted to another vendor for CO1 DNA Barcoding analysis, which identified the cells as being purely of bovine origin. Results revealed that despite purchase through a commercial vendor, the cell line marketed as primary rabbit aortic endothelial cells were of bovine origin. Purity analysis found cells were misidentified rather than contaminated. Further investigation to determine the cell type was not performed. The most cost-effective and efficient methodology for confirming cell line identity was found to be CO1 DNA Barcoding performed by a commercial vendor.

Quality of forage seeds sold in the state of Mato Grosso

The acquisition of high-quality seeds is essential to successfully implant areas with forage. However, to be commercialized, they must meet the minimum quality standards established by law. Inspections are necessary to guarantee the quality and identity of the commercialized seed. Thus, the objective was to evaluate the quality of tropical forage seed species collected in 2018, in the state of Mato Grosso. The survey was based on data from officers’ samples, in commercial establishments and fine consumers, collected during inspections conducted by the Agricultural Defense Institute of the State of Mato Grosso - INDEA-MT and subsequently analyzed by the Guilherme de Abreu Lima Seed Analysis Laboratory (LASGAL). Purity analysis, determination of other seeds by number (DOSN), germination test and tetrazolium test were carried out. The study showed that 75% of the analyzed samples reached the minimum requirements for commercialization in terms of physical purity; 95% of the samples were within the standard for physiological quality determined by law; and 91.9% did not exceed the maximum limits regarding the number of undesirable seeds present in a sample. In general, 36.1% of the total samples analyzed did not conform in some way to quality standards, therefore were not appropriate for commercialization.

Rapid quantification of Psilocybin with reversed-phase HPLC and single-wavelength detection

The alkaloid psilocybin (4-phosphoryloxy-N,N-dimethyltryptamine) and the neurologically active psilocin (4-hydroxy-N,N-dimethyltryptamine) are the foremost compounds of pharmaceutical interest in Psilocybe mushrooms. As these compounds are infrequently analyzed in analytical labs, validated methods for rapid purity analysis are lacking. Newfound therapeutic use has invigorated academic and commercial interests in the molecules and new methods of production and available products are expanding. As a result, high-throughput methods of analysis for psilocybin must be improved to promptly determine chemical differences between mushroom genera or other sources of psilocybin and psilocin, as well as refined product purity. To address this, we developed an inexpensive HPLC technique for the efficient quantification of psilocybin and psilocin by using readily available equipment and dilute reagents. Aqueous ammonium formate (0.143 mM) was found to be preferable over techniques with much higher buffer concentrations or stronger acids for controlling psilocybin Zwitterion resolution. The chromatographic run time satisfied high-throughput analytical requirements with an efficient total runtime under 2 minutes. A standard octadecyl silica (C18) column provided excellent resolution between psilocybin and psilocin signals. The quality of the method was validated using certified analytical reference standards and was found to be accurate (3.5% bias, Psilocybin), reliable (0.32% RSD), and efficient (Psilocybin k’ = 1.78).

Phenological, Morphological and Yield based Characterization of Chickpea (Cicer arietinum L.) Germplasm Lines

Background: The characterization of chickpea genetic resources is a vital step to explore genetic variability in breeding programs. In the present study, we characterized 90 germplasm lines of desi chickpea collected across the Indian chickpea growing region. The identified trait-specific germplasm lines will be used as a valuable genetic resource for the chickpea improvement programme. Methods: An experiment was conducted in augmented design to characterize germplasm lines of chickpea for 13 qualitative and 17 quantitative agro-morphological traits under Bundelkhand agro-climatic conditions. Result: Ample variation was observed for qualitative and quantitative traits. The study revealed that the presences of high variability in qualitative and quantitative traits are useful in the identification of plant genotype for a specific trait, genetic purity analysis, germplasm conservation and also possible use of potential genotype in the breeding program.