Simultaneous quantification of acidic and basic flupirtine metabolites by supercritical fluid chromatography according to European Medicines Agency validation.
Supercritical fluid chromatography (SFC) holds the potential to become an orthogonal method to HPLC/UHPLC in xenobiotic metabolism studies, due to its outstanding capacity to simultaneously separate highly similar (as HPLC) and physicochemically different analytes (problematic using HPLC). Paucity of guideline-conform validation, however, has been a major obstacle to clinical application of SFC, even in cases where biotransformation yields chemically dissimilar metabolites that require more than one HPLC method for comprehensive analysis. Here, a method based on supercritical fluid chromatography coupled to single quadrupole MS detection was developed to simultaneously quantify the divisive analgesic flupirtine and its acidic and basic metabolites, represented by 4-fluorohippuric acid (4-FHA) and the active metabolite D-13223 respectively, using custom-made synthetic internal standards. Experimental data on the fundamental retention mechanisms under supercritical conditions, indicating the importance of halogen and π-π-bonding for specific retention on polysaccharide-based stationary-phases, is discussed. Compared to previous HPLC methods, the novel method offers higher versatility in terms of the target metabolite range (addressing both acidic and basic metabolites within a singular method), faster analysis (7.5 min), and compliance with green chemistry principles. Validation was performed according to EMA criteria on bioanalytical method validation, demonstrating selectivity, carry-over, calibration curve parameters (LLOQ, range, and linearity), within- and between-run accuracy and precision, dilution integrity, matrix effect and stability. For proof-of-concept, the SFC method was applied to clinical samples of human urine obtained after single intravenous (100 mg), single oral (100 mg), and repeated oral administration (400 mg). Flupirtine, D-13223, and 4-FHA could be quantified, shedding light on the extent of oxidative flupirtine metabolism in humans in the context of the unresolved biotoxification that has led to the withdrawal of specific neuronal KV7 openers.
['Hofstetter|Robert K|RK|', 'Hasan|Mahmoud|M|', 'Fassauer|Georg M|GM|', 'Bock|Christian|C|', 'Surur|Abdrrahman S|AS|', 'Behnisch|Steven|S|', 'Grathwol|Christoph W|CW|', 'Potlitz|Felix|F|', 'Oergel|Tobias|T|', 'Siegmund|Werner|W|', 'Link|Andreas|A|']
D000143:Acids D000328:Adult D000631:Aminopyridines D000700:Analgesics D002138:Calibration D025924:Chromatography, Supercritical Fluid D005060:Europe D005260:Female D006801:Humans D057230:Limit of Detection D008297:Male D055442:Metabolome D015203:Reproducibility of Results D012997:Solvents D013696:Temperature D055815:Young Adult
Flupirtine Supercritical fluid chromatography Validation Metabolism Green chemistry Halogen bonds