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Classical homocystinuria is an inborn amino acid metabolism disorder resulting from mutations in the Cystathionine-β-Synthase (CBS) gene. These mutations lead to elevated homocysteine and methionine levels and reduced cysteine levels in the blood. Typically, diagnosis occurs after patients display symptoms, and various lab methods confirm it. DNA sequencing is the best option for early detection of genetic variants in asymptomatic suspected individuals. Unfortunately, its high cost can hinder its use, especially in low-income countries like Pakistan. Aim of this study was to devise a robust low-cost diagnostic/screening assay based on Tetra-ARMS-PCR for five prevalent genetic variants found in Pakistani classical homocystinuria patients. In the current study, T-ARMS-PCR assays were developed for five mutations (c.975G > C, c.770C > T, c.752T > C, c.1039 + 1G > T, c.451 + 1GG > TA), which were characterized previously in classical homocystinuria patients. These low-cost T-ARMS-PCR assays were then used to screen the affected individuals and their family members to identify their genotypes for pathogenic variations in the asymptomatic patients and carriers in their respective families. The outcomes were entirely consistent with those obtained from Sanger DNA sequencing, confirming the sensitivity, specificity, and reliability of the T-ARMS-PCR assay for detecting CBS mutations. T-ARMS-PCR has wide applications for low-income countries for the screening and early diagnosis of asymptomatic patients and carriers in the homocystinuria affected families as well as other inherited diseases. [ABSTRACT FROM AUTHOR]

Nuclear magnetic resonance (NMR) is a rapid and accurate analytical tool for qualification and quantification. The capacity of NMR of being quantitative can also justify the calibration of other analytical methods. In pharmaceutical domain, quantitative NMR (qNMR) can be applied in the identification and quantification of drug simultaneously. The early drug development stage requires a minimum sample for analysis. Thus, priority should be given to utilize this technique to attain results with least investment, rapid analysis time and minimum sample consumption. This technique is a significant phenomenon to identify impurities, drug substance, residual solvents of in-process control (IPC) samples and characterizing the formulations. From an analyst's perspective, qNMR proved to be a routine practice in pharmaceutical industry to qualify any drug product. The absolute and relative methods offer great help in quantifying the component of interest in the process control samples and finished products. This review highlights the evolution of NMR application in the pharmaceutical industry, where determining the purity of drug substance, drug product and establishing the identity of impurities and its level are the challenging aspects. NMR in medicinal field emerging as a numero uno for Covid-19 severity detection and its dire consequences, accelerated vaccine development and the mapping of SAR-COV-2 RNA and proteins via chemical shift assignments. [ABSTRACT FROM AUTHOR]

For cilazapril (CLZ), analytical methods based on donor–acceptor phenomenon that are simple, rapid with broad linear dynamic range for the quantification of drug are not available in the literature. Considering the requirement for the methods, in this study, two economic, potent analytical methods based on the complexation of CLZ with π-acceptors, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) and 2,5-dichloro-3,6-dihydroxy-p-benzoquinone (CA) were developed, validated, and studied spectrophotometrically. Various analytical data were discussed. The effects of experimental variables were optimized from the results of in silico technique, i.e. Box–Behnken design under response surface methodology. Linear dynamic range was significantly good in the range of 6–60 µg mL−1 and 20–260 µg mL−1 for DDQ and CA methods. Moreover, molecular docking studies corroborated the experimental results. Further, the methods were supplemented by the pharmaceutical and biological application for the quantitative assay of CLZ. Collectively, the results of the reported method of the analysis suggest that the developed approach is simple, sensitive, accurate and precise. [ABSTRACT FROM AUTHOR]