Simultaneous determination of N1-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

• An assay of cationic transporter substrates in humans is reported.
• The assay allows for simultaneous measurement of plasma or urine N1-methylnicotinamide, l-carnitine, and creatinine.
• Multiple renal cationic transporter activities in humans can be assessed simultaneously.

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 N1-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 N1-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 N1-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.