Development of a Hydrophilic Liquid Interaction Chromatography−High-Performance Liquid Chromatography−Tandem Mass Spectrometry Based Stable Isotope Dilution Analysis and Pharmacokinetic Studies on Bioactive Pyridines in Human Plasma and Urine after Coffee Consumption

摘要

The paper reports on the development of an accuratenhydrophilic liquid interaction chromatography tandemnmass spectrometry (HILIC-MS/MS) based stable isotopendilution analysis for the simultaneous quantitation of thenfood-derived bioactive pyridines trigonelline, nicotinicnacid, nicotinamide, and N-methylpyridinium, as well asntheir key metabolites nicotinamide-N-oxide, N-methylnicotinamide,nN-methyl-2-pyridone-5-carboxamide,N-methyl-n4-pyridone-5-carboxamide, and N-methyl-2-pyridone-5-ncarboxylic acid in human plasma and urine. Precision ofnthe stable isotope dilution analysis (SIDA) was 1.9% andn11.9% relative standard deviation (n ) 6), and accuracynwas between 92.4% and 113.0%. The lower limit ofnquantitation (LLOQ) was 50 fmol (10 pmol/mL) injectednonto the column for all analytes with the exception ofnN-methyl-2-pyridone-5-carboxylic acid and N-methyl-2-npyridone-5-carboxamide, for which an LLOQ of 100 fmoln(20 pmol/mL) was found. The method was applied tonmonitor the plasma appearance and urinary excretion andnto determine pharmacokinetic parameters of the bioactivenpyridines as well as their metabolites in a clinical humannintervention study with healthy volunteers (six women,nseven men) after oral administration of 350 mL of anstandard coffee beverage. Trigonelline plasma levelsnincreased from 160 nmol/L to maximum concentrationsnof 5479 (males) or 6547 nmol/L (females), and Nmethylpyridiniumnplasma levels raised from virtuallyncomplete absence to maximum values of 777 (females)nor 804 nmol/L (males) within 2-3 and 1-2 h after coffeenconsumption, respectively. The high plasma levels ofnN-methylpyridinium found after coffee consumption clearlyndemonstrate for the first time that this cation is enteringnthe vascular system, which is the prerequisite for biologicalnin vivo effects claimed for that compound. In contrast,nthe coffee intervention did not significantly influence thenplasma concentrations of N-methyl-2-pyridone-5-carboxamidenandN-methyl-4-pyridone-5-carboxamide, the majornniacin metabolites. Within 8 h after coffee intervention,nan urinary excretion of 57.4 ( 6.9% of trigonelline andn69.1 ( 6.2% of N-methylpyridinium was found for thenmale volunteers, whereas females excreted slightly lessnwith 46.2 ( 7.4% and 61.9 ( 12.2% of these pyridines.