Validation of an HPLC method for the determination of urinary and plasma levels of N-1-methylnicotinamide, an endogenous marker of renal cationic transport and plasma flow

N-1-Methylnicotinamide (NMN) is an endogenous cationic metabolite of nicoti namide (niacine, vitamine PP) whose renal clearance reflects both the capac ity of the renal tubular transport system to secrete organic cations and re nal plasma flow. NMN is present in human plasma and urine at the 1-117-ng m l(-1) and 0.5-25-mug ml(-1) concentration range, respectively, and its leve l depends notably on pathophysiological (age, renal or hepatic diseases) co nditions. We report the optimization and validation of an HPLC method for t he measurement of endogenous NMN in biological fluids after derivatization into a fluorescent compound. Plasma is first deproteinized with TCA 20% and the urine diluted 1:10 with HCl 10(-4) M prior to the derivatization proce dure, which includes a condensation reaction of NMN with acetophenone in Na OH at 0 degreesC, followed by dehydration in formic acid and subsequent for mation of the fluorescent 1,6-naphthyridine derivatives after heating sampl es in a boiling water bath. The synthetic homologous derivative N-1-ethylni cotinamide (NEN) reacts similarly and is added as internal standard into th e biological fluid. The reaction mixture is subjected to reverse phase high performance liquid chromatography on a Nucleosil 100-C18 column using a mo bile phase (acetonitrile 22%, triethylamine 0.5%, 0.01 M sodium heptanesulf onate adjusted to pH 3.2), delivered isocratically at a flow rate of 1 ml m in(-1). NMN and NEN are detected at 7.8 and 10 min by spectrofluorimetry wi th excitation and emission wavelengths set at 366 and 418 nm, respectively. The addition-calibration method is used with plasma and urine pools. Calib ration curves (using the internal standard method) are linear (r(2) > 0.997 ) at concentrations up to 109 ng ml(-1) and 15.7 mug ml(-1) in plasma and u rine, respectively. Both intra- and inter-assay precision of plasma control samples at 10, 50 and 90 ng ml(-1) were lower than 3.3% and concentrations not deviating more than 2.7% from their nominal values. In urine intra- an d inter-assay CVs of control samples at 1, 5 and 9 mug ml(-1) are lower tha n 8.3%, with concentrations not deviating more than -9.0 to +11.8% from the ir nominal values. This analytical method has therefore the required sensit ivity and selectivity to measure NMN in plasma and urine, enabling the non- invasive determination of the tubular secretory capacity of the kidney and the renal plasma flow. (C) 2001 Elsevier Science B.V. All rights reserved.