Simultaneous Determination Of Methionine Research Articles

Peak shape enhancement using diethylamine in hydrophilic liquid interaction chromatography: Application in simultaneous determination of methionine and paracetamol

Methionine (MET) is combined with paracetamol (PAR) in a pain relief soft capsule in order to prevent the haematologic damage of paracetamol. A hydrophillic liquid chromatographic (HILIC) method was developed for simultaneous determination of PAR and MET in the combined formulation. Various analytical conditions were investigated, and the final method was chosen using silica column (150 × 4,6 mm; 5 μm), mobile phase of acetonitrile – aqueous solution of 10 mM formic acid 5 mM diethylamine (60:40, v/v), UV detection at 254 nm for PAR and 210 nm for MET. The method was validated according to ICH guidelines in terms of selectivity, linearity, accuracy, precision and robustness. The method was successfully applied for quantitation of both compounds in soft capsule preparations bought from the market. Notably, in this study, a novel approach was proposed to improve peak shape of amino acid – a problem often observed in HILIC. The addition of diethylamine to mobile phase shortened the retention time of MET and significantly improved peak shape on both silica and cyano columns, due to electrostatic interaction competition and silanol end-capping effect. The result of this research demonstrated the advantages of HILIC in simultaneous analysis of a polar compound amino acid, especially in combination with a less polar substance. The use of diethylamine as a mobile phase modifier to enhance peak shape is a new suggestion that can be used in further studies on amino acid analysis by HILIC.

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

BackgroundParacetamol is one of the most widely analgesic and antipyretic drugs recently integrated into the supportive therapy of COVID-19. The pharmaceuticals containing methionine with paracetamol may contribute to avoid hepatotoxicity and eventual paracetamol overdose-dependent death.ObjectiveThe current work purposes to develop and validate two chromatographic methods for the simultaneous determination of methionine and paracetamol in presence of two paracetamol impurities (4-nitrophenol and 4-aminophenol).MethodsTwo chromatographic methods were established and validated according to the International Conference on Harmonization guidelines. The first one was a 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.ResultsThe developed methods were proven to be robust, accurate, specific, and appropriate for the routine analysis of paracetamol in its pure form or in pharmaceutical formulations with methionine in quality control laboratories.ConclusionsThe corresponding methods are suitable to determine methionine and paracetamol in the presence of paracetamol impurities.HighlightsThe study achieves the analysis of methionine and paracetamol in the presence of paracetamol impurities via the application of HPLC and TLC- densitometry methods.

Simultaneous Determination of Methionine and Homocysteine by on-column derivatization with o-phtaldialdehyde

A fast and simple HPLC-based assay has been developed for the simultaneous determination of homocysteine (Hcy) and methionine (Met) in plasma and urine samples, utilizing as small volume of sample as 10μL. The assay uses on-column derivatization with o-phthaldialdehyde. The separation of Hcy and Met was achieved in 14min on a reversed phase C-18 column, followed by fluorescence detection (excitation at 348nm and emission at 438nm for Met; excitation at 370nm and emission at 480nm for Hcy). Linearity of the detector response was observed in the range of 2–60 μmol L−1 for Met and 2–40 μmol L−1 for Hcy. The method was successfully applied for Met and Hcy quantification in human and mouse plasma and urine samples from cystathionine β-synthase deficient and unaffected individuals.

Microwave-assisted acid digestion protocol for the determination of methionine and selenomethionine in selenium-enriched yeast by species specific isotope dilution GC-MS

A quantitative and fast microwave assisted protein digestion method is described for the simultaneous determination of methionine (Met) and selenomethionine (SeMet) in yeast. Extraction of Met and SeMet from the selenized yeast was performed in a focused microwave system using methanesulfonic acid (MSA). The effects of parameters such as extraction time, temperature, power and sample mass on the extraction efficiencies of Met and SeMet were investigated. Species specific isotope dilution (ID) calibration using 13C enriched Met and SeMet spikes was employed to obtain accurate results. Analytes were derivatized with methyl chloroformate and extracted into chloroform prior to species specific ID GC-MS analysis. Using a 20 minute extraction time at 165 °C and 6 ml of 4 M MSA was found to be efficient for both analytes based on a 50 mg sample mass. Under these conditions, concentrations of 5862 ± 32 and 3366 ± 60 μg g−1 (one standard deviation, n = 3) for Met and SeMet, respectively, were obtained in SELM-1 yeast certified reference material (CRM). The obtained results are in good agreement with the certified values of 5758 ± 277 and 3448 ± 146 μg g−1 (expanded uncertainty, k = 2). Compared to previous MSA reflux digestion, this newly proposed method offers dramatic reduction in extraction time from 8–16 hours of the conventional MSA reflux to 20 minutes by microwave extraction, significantly improving the sample throughput. Additionally, the microwave extraction is fully automated and uses 75% less reagent (MSA) than the conventional acid reflux setup. The developed method is suitable for quasi real time production monitoring of Met and SeMet in Se enriched yeast and other food products.