Synthesis and Trace-Level Quantification of Mutagenic and Cohort-of-Concern Ciprofloxacin Nitroso Drug Substance-Related Impurities (NDSRIs) and Other Nitroso Impurities Using UPLC-ESI-MS/MS-Method Optimization Using I-Optimal Mixture Design.

Globally, the pharmaceutical industry has been facing challenges from nitroso drug substance-related impurities (NDSRIs). In the current study, we synthesized and developed a rapid new UPLC-MS/MS method for the trace-level quantification of ciprofloxacin NDSRIs and a couple of N-nitroso impurities simultaneously. (Q)-SAR methodology was employed to assess and categorize the genotoxicity of all ciprofloxacin N-nitroso impurities. The projected results were positive, and the cohort of concern (CoC) for all three N-nitroso impurities indicates potential genotoxicity. AQbD-driven I-optimal mixture design was used to optimize the mixture of solvents in the method. The chromatographic resolution was accomplished using an Agilent Poroshell 120 Aq-C18 column (150 mm × 4.6 mm, 2.7 µm) in isocratic elution mode with 0.1% formic acid in a mixture of water, acetonitrile, and methanol in the ratio of 475:500:25 v/v/v at a flow rate of 0.5 mL/min. Quantification was carried out using triple quadrupole mass detection with electrospray ionization (ESI) in a multiple reaction monitoring technique. The finalized method was validated successfully, affording ICH guidelines. All N-nitroso impurities revealed excellent linearity over the concentration range of 0.00125-0.0250 ppm. The Pearson correlation coefficient of each N-nitroso impurity was >0.999. The method accuracy recoveries ranged from 93.98 to 108.08% for the aforementioned N-nitrosamine impurities. Furthermore, the method was effectively applied to quantify N-nitrosamine impurities simultaneously in commercially available formulated samples, with its efficiency recurring at trace levels. Thus, the current method is capable of determining the trace levels of three N-nitroso ciprofloxacin impurities simultaneously from the marketed tablet dosage forms for commercial release and stability testing.

A sensitive ultra-performance liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of assay and trace-level genotoxic tosylate analogs (methyl and ethyl) in empagliflozin and its tablet dosage forms.

This study performed the simultaneous quantification of assay and two alkyl sulfonate (tosylate) analogs of empagliflozin (EGZ), specifically methyl 4-methyl benzene sulfonate (MMBS) and ethyl 4-methyl benzene sulfonate (EMBS) in EGZ, and its finished dosage form using an accurate and sensitive ultra-performance liquid chromatography-mass spectrometry method. The separation was achieved on a Waters Acquity BEH Shield RP18 (100 × 2.1 mm, 1.7 µm) column in gradient elution mode with 0.1% formic acid and acetonitrile as the mobile phases and a flow rate of 0.5 mL/min. For simultaneous quantification, the multiple reaction monitoring technique was utilized. The procedure was successfully validated in accordance with the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines. The peak areas of both impurities, along with their concentrations, exhibited a good relationship with Pearson's correlation coefficient (R), which was >0.999 in the range of 0.3-6 ppm with an EGZ concentration of 2 mg/mL. The percentage recoveries from the limit of quantitation (LOQ) to 200% to the specification level were in the range of 94.82%-102.92%, whereas the percentage relative standard deviation (%RSD) was <2. Therefore, this method is rapid and accurate to quantify MMBS, EMBS, and EGZ assay simultaneously from the marketed tablet dosage forms of EGZ for commercial release and stability sample testing.

A sensitive UPLC-MS/MS method for the simultaneous assay and trace level genotoxic impurities quantification of SARS-CoV-2 inhibitor-Molnupiravir in its pure and formulation dosage forms using fractional factorial design.

Two potential genotoxic impurities were identified (PGTIs)-viz. 4-amino-1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one (PGTI-1), and 1-(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2,4(1H,3H)-one (PGTI-II) in the Molnupiravir (MOPR) synthetic routes. COVID-19 disease was treated with MOPR when mild to moderate symptoms occurred. Two (Q)-SAR methods were used to assess the genotoxicity, and projected results were positive and categorized into Class-3 for both PGTIs. A simple, accurate and highly sensitive ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method was optimized for the simultaneous quantification of the assay, and these impurities in MOPR drug substance and formulation dosage form. The multiple reaction monitoring (MRM) technique was utilized for the quantification. Prior to the validation study, the UPLC-MS method conditions were optimised using fractional factorial design (FrFD). The optimized Critical Method Parameters (CMPs) include the percentage of Acetonitrile in MP B, Concentration of Formic acid in MP A, Cone Voltage, Capillary Voltage, Collision gas flow and Desolvation temperature were determined from the numerical optimization to be 12.50 %, 0.13 %, 13.6 V, 2.6 kV, 850 L/hr and 375 °C, respectively. The optimized chromatographic separation achieved on Waters Acquity HSS T3 C18 column (100 mm × 2.1 mm, 1.8 µm) in a gradient elution mode with 0.13% formic acid in water and acetonitrile as mobile phases, column temperature kept at 35 °C and flow rate at 0.5 mL/min. The method was successfully validated as per ICH guidelines, and demonstrated excellent linearity over the concentration range of 0.5-10 ppm for both PGTIs. The Pearson correlation coefficient of each impurity and MOPR was found to be higher than 0.999, and the recoveries were in between the range of 94.62 to 104.05% for both PGTIs and 99.10 to 100.25% for MOPR. It is also feasible to utilise this rapid method to quantify MOPR accurately in biological samples.

A comparative pharmacological and phytochemical analysis of in vivo & in vitro propagated Crotalaria species

OBJECTIVE@#To compare the efficacy for phytochemical, antibacterial and antioxidant activities of petroleum ether, chloroform, ethanol, and aqueous extracts of in vitro propagated plants and field grown plants of Crotalaria sps., for against five human pathogens.@*METHODS@#The preliminary phytochemistry, antimicrobial and antioxidant activities were evaluated using disc diffusion and DPPH radical scavenging methods.@*RESULTS@#The ethanolic extract of in vitro raised Crotalaria retusa (C. retusa) was effective on tested microorganisms and optimal ZOI values of 38 mm was obtained against Pseudomonas aeruginosa (P. aeruginosa). The optimal concentration (IC50) required for 50% inhibition of the DPPH radical scavenging was 57.6 μ g/mL obtained for ethanolic extract of in vitro propagated C. retusa. The in vitro propagated C. retusa has significant pharmacological activities while the Crotalaria prostrate (C. prostrate) and Crotalaria medicaginea (C. medicaginea) has low pharmacological activites. It was cleared that ethanolic extract of in vitro regenerated plants was most effective.@*CONCLUSIONS@#These findings indicate compounds isolated from ethanolic extracts of Crotalaria sps., possesses pharmacological properties and potential to develop natural compounds based pharmaceutical products. The IC(50) values for ethanolic extracts of Crotalaria sps. was evaluated through the Linear regression analysis (R(2) ≤ 1).

A comparative pharmacological and phytochemical analysis of in vivo & in vitro propagated Crotalaria species

Objective To compare the efficacy for phytochemical, antibacterial and antioxidant activities of petroleum ether, chloroform, ethanol, and aqueous extracts of in vitro propagated plants and field grown plants of Crotalaria sps., for against five human pathogens. Methods The preliminary phytochemistry, antimicrobial and antioxidant activities were evaluated using disc diffusion and DPPH radical scavenging methods. Results The ethanolic extract of in vitro raised Crotalaria retusa (C. retusa) was effective on tested microorganisms and optimal ZOI values of 38 mm was obtained against Pseudomonas aeruginosa (P. aeruginosa). The optimal concentration (IC50) required for 50% inhibition of the DPPH radical scavenging was 57.6 μ g/mL obtained for ethanolic extract of in vitro propagated C. retusa. The in vitro propagated C. retusa has significant pharmacological activities while the Crotalaria prostrate (C. prostrate) and Crotalaria medicaginea (C. medicaginea) has low pharmacological activites. It was cleared that ethanolic extract of in vitro regenerated plants was most effective. Conclusions These findings indicate compounds isolated from ethanolic extracts of Crotalaria sps., possesses pharmacological properties and potential to develop natural compounds based pharmaceutical products. The IC

Interaction of bovine serum albumin with dipolar molecules: fluorescence and molecular docking studies.

Interaction of bovine serum albumin (BSA) with two series of dipolar molecules having both rigid and flexible structures has been studied by monitoring the spectral and temporal behavior of the intramolecular charge transfer fluorescence of the systems. The binding sites of the molecular systems in BSA have been located with the help of docking studies. Three different sites of varying hydrophobicity have been identified where these molecules are located. Binding in the hydrophobic domains of BSA leads to a blue shift of the fluorescence spectra and an enhancement of fluorescence intensity and lifetime. This enhancement is found to be the largest for flexible systems in which internal motion serves as a nonradiative decay route. In the BSA-bound condition, some of the dipolar molecules exhibit not-so-common "dip-rise-dip" time-resolved fluorescence anisotropy profiles. It is shown that a large difference of the fluorescence lifetimes of the protein-bound and unbound molecules is one of the factors that contributes to this kind of anisotropy profiles. As internal motion is often responsible for the short fluorescence lifetime of the flexible dipolar molecules, a large increase in the fluorescence lifetime of these systems occurs if binding to BSA leads to disruption/prevention of this motion. It thus appears that it might be possible to obtain information on the prevention/disruption of nonradiative pathway on protein binding from the anisotropy profiles of the kind discussed above. However, since the present study reveals cases where a large change in fluorescence lifetime also occurs due to other reasons, one needs to be careful prior to making any conclusion.