Simultaneous determination of N(1)-methylnicotinamide, L-carnitine, and creatinine in human plasma and urine by liquid chromatography with mass spectrometry for assessing the activities of multiple renal cationic transporters.

Abstract

Organic cation transporters are responsible for the disposition of various endogenous and therapeutic agents in humans; thus, there is a great need for the development of a simple assay for simultaneous assessment of the activities of multiple transporters. Using liquid chromatography-mass spectrometry (LC/MS), we developed an assay that allows for simultaneous quantitation of plasma and urinary levels of N(1)-methylnicotinamide (a substrate of hOCT2/hMATEs), L-carnitine (a substrate of hOCTN2), and creatinine (an indicator of glomerular filtration). Samples were diluted with ultrapure water, deproteinized with trichloroacetic acid, filtered, and then injected on a cation exchange column. The analytes were separated with a gradient LC technique and detected by MS. The total assay time was less than 8 min. The lower detection limits for N(1)-methylnicotinamide, L-carnitine, and creatinine were 2, 10, and 24 ng/mL, respectively. Recovery of the analytes was almost complete. A preliminary clinical study conducted in 25 healthy subjects revealed that the mean±SD for the renal clearance (CLR) of N(1)-methylnicotinamide (272.7±81.0 mL/min) far exceeded the glomerular filtration rate (116.3±19.6 mL/min), indicating the involvement of active tubular secretion, while the mean CLR of clearance of L-carnitine was close to nil (1.5±1.4 mL/min), indicating almost complete tubular reabsorption. The present method is potentially useful for clinical studies on the genetic control of cationic transporter activities and the transporter-mediated drug interactions.