Dual-Mode Gradient HPLC and TLC Densitometry Methods for the Simultaneous Determination of Paracetamol and Methionine in the Presence of Paracetamol Impurities.

BACKGROUND: Paracetamol (PC) is one of the most widely used analgesic and antipyretic drugs and has recently been integrated into the supportive therapy of COVID-19. Pharmaceuticals containing methionine (MT) with PC may contribute to avoid hepatotoxicity and eventual PC overdose-dependent death. OBJECTIVE: The current work purposes to develop and validate two chromatographic methods for the simultaneous determination of MT and PC in the presence of two PC impurities (4-nitrophenol [NP] and 4-aminophenol [AP]). METHOD: Two chromatographic methods were established and validated according to the International Conference on Harmonization guidelines. The first one was an RP-HPLC/UV method based on applying a "dual-mode" gradient elution. The separation was realized via varying both the composition of the ternary mobile phase (acetonitrile-methanol-water) and its flow rate. This strategy enabled a relatively rapid analysis with a satisfactory resolution, although the investigated compounds exhibit a significant difference in lipophilicity. The second one relied on TLC-densitometry, where the optimum separation was realized using a quaternary mobile phase system composed of butanol-dioxane-toluene-methanol (8:2.5:3.5:0.3, by volume). Both methods were monitored at 220 nm. RESULTS: The developed methods were proven to be robust, accurate, specific, and appropriate for the routine analysis of PC in its pure form or in pharmaceutical formulations with MT in quality control laboratories. CONCLUSIONS: The corresponding methods are suitable to determine MT and PC in the presence of PC impurities. HIGHLIGHTS: The study achieves the analysis of MT and PC in the presence of PC impurities via the application of HPLC and TLC-densitometry methods.

Simultaneous Determination of Paracetamol, Propyphenazone and Caffeine in Presence of Paracetamol Impurities Using Dual-Mode Gradient HPLC and TLC Densitometry Methods.

Two chromatographic methods were validated for the determination of the widely prescribed analgesic and antipyretic drug combination of paracetamol (PC) (recently integrated into the supportive treatment of COVID-19), propyphenazone (PZ) and caffeine (CF) in the presence of two PC impurities, namely 4-aminophenol and 4-nitrophenol. A "dual-mode" gradient high-performance liquid chromatography method was developed, where the separation was achieved via "dual-mode" gradient by changing both the ternary mobile phase composition (acetonitrile: methanol: water) and the flow rate. This enables a good resolution within a relatively shorter analysis time. The analysis was realized using Zorbax Eclipse XDB column C18, 5 µm (250 × 4.6 mm) and the UV detector was set at 220 nm. The other method is a thin-layer chromatography densitometry method, where the separation was achieved using a mobile phase composed of chloroform: toluene: ethyl acetate: methanol: acetic acid (6: 6: 1: 2: 0.1, by volume). Densitometric detection was performed at 220 nm on silica gel 60 F254 plates. The developed methods were fully validated as per the ICH guidelines and proved to be accurate, robust, specific and suitable for application as purity indicating methods for routine analysis of PC in pure form or in pharmaceuticals with PZ and CF in quality control laboratories.

Validated UPLC and TLC-Densitometry Stability Indicating Methods for the Determination of Rafoxanide in the Presence of Its Degradation Products.

Two sensitive and accurate stability-indicating chromatographic methods were developed and validated for the determination of rafoxanide (RFX). Degradation products were identified by mass spectrometry and IR spectroscopy. The first is ultra-performance liquid chromatography method where separation was performed using acetonitrile:0.005 M potassium dihydrogen orthophosphate (pH 3.5) in a ratio of 80:20 by volume as a mobile phase using a Hypersil GOLD™ C8 column 1.9 mm (50 × 2.1 mm), UV detection was adjusted at 220 nm and the flow rate was 0.6 mL min-1. The other is a thin-layer chromatography-densitometry method where separation was achieved using a mobile phase composed of chloroform:ethyl acetate:toluene:ammonia (5:4:3:0.1 by volume) on silica gel 60 F254 plates, and densitometric detection was done at 280 nm. Validation was achieved as per the ICH guidelines. The proposed methods proved to be accurate, robust, specific and suitable for application as stability-indicating methods for routine analysis of RFX in quality control laboratories.

Enhancing prediction power of chemometric models through manipulation of the fed spectrophotometric data: A comparative study.

Effect of data manipulation in preprocessing step proceeding construction of chemometric models was assessed. The same set of UV spectral data was used for construction of PLS and PCR models directly and after mathematically manipulation as per well known first and second derivatives of the absorption spectra, ratio spectra and first and second derivatives of the ratio spectra spectrophotometric methods, meanwhile the optimal working wavelength ranges were carefully selected for each model and the models were constructed. Unexpectedly, number of latent variables used for models' construction varied among the different methods. The prediction power of the different models was compared using a validation set of 8 mixtures prepared as per the multilevel multifactor design and results were statistically compared using two-way ANOVA test. Root mean squares error of prediction (RMSEP) was used for further comparison of the predictability among different constructed models. Although no significant difference was found between results obtained using Partial Least Squares (PLS) and Principal Component Regression (PCR) models, however, discrepancies among results was found to be attributed to the variation in the discrimination power of adopted spectrophotometric methods on spectral data.

Increasing the binding affinity of VEGFR-2 inhibitors by extending their hydrophobic interaction with the active site: Design, synthesis and biological evaluation of 1-substituted-4-(4-methoxybenzyl)phthalazine derivatives.

A series of anilinophthalazine derivatives 4a-j was initially synthesized and tested for its VEGFR-2 inhibitory activity where it showed promising activity (IC50 = 0.636-5.76 µM). Molecular docking studies guidance was used to improve the binding affinity for series 4a-j towards VEGFR-2 active site. This improvement was achieved by increasing the hydrophobic interaction with the hydrophobic back pocket of the VEGFR-2 active site lined with the hydrophobic side chains of Ile888, Leu889, Ile892, Val898, Val899, Leu1019 and Ile1044. Increasing the hydrophobic interaction was accomplished by extending the anilinophthalazine scaffold with a substituted phenyl moiety through an uriedo linker which should give this extension the flexibility required to accommodate itself deeply into the hydrophobic back pocket. As planned, the designed uriedo-anilinophthalazines 7a-i showed superior binding affinity than their anilinophthalazine parents (IC50 = 0.083-0.473 µM). In particular, compounds 7g-i showed IC50 of 0.086, 0.083 and 0.086 µM, respectively, which are better than that of the reference drug sorafenib (IC50 = 0.09 µM).