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Simultaneous quantification of 12 different nucleotides and nucleosides released from renal epithelium and in human urine samples using ion-pair reversed-phase HPLC

Contreras-Sanz, Alberto, Scott-Ward, Toby S., Gill, Hardyal S., Jacoby, Jennifer C., Birch, Rebecca E., Malone-Lee, James, Taylor, Kevin M. G., Peppiatt-Wildman, Claire M., Wildman, Scott S.P. (2012) Simultaneous quantification of 12 different nucleotides and nucleosides released from renal epithelium and in human urine samples using ion-pair reversed-phase HPLC. Purinergic Signalling, 8 (4). pp. 741-751. ISSN 1573-9538. E-ISSN 1573-9546. (doi:10.1007/s11302-012-9321-8) (KAR id:53063)


Nucleotides and nucleosides are not only involved in cellular metabolism but also act extracellularly via P1 and P2 receptors, to elicit a wide variety of physiological and pathophysiological responses through paracrine and autocrine signalling pathways. For the first time, we have used an ion-pair reversed-phase high-performance liquid chromatography ultraviolet (UV)-coupled method to rapidly and simultaneously quantify 12 different nucleotides and nucleosides (adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, adenosine, uridine triphosphate, uridine diphosphate, uridine monophosphate, uridine, guanosine triphosphate, guanosine diphosphate, guanosine monophosphate, guanosine): (1) released from a mouse renal cell line (M1 cortical collecting duct) and (2) in human biological samples (i.e., urine). To facilitate analysis of urine samples, a solid-phase extraction step was incorporated (overall recovery rate ? 98 %). All samples were analyzed following injection (100 ?l) into a Synergi Polar-RP 80 Å (250 × 4.6 mm) reversed-phase column with a particle size of 10 ?m, protected with a guard column. A gradient elution profile was run with a mobile phase (phosphate buffer plus ion-pairing agent tetrabutylammonium hydrogen sulfate; pH 6) in 2-30 % acetonitrile (v/v) for 35 min (including equilibration time) at 1 ml min(-1) flow rate. Eluted compounds were detected by UV absorbance at 254 nm and quantified using standard curves for nucleotide and nucleoside mixtures of known concentration. Following validation (specificity, linearity, limits of detection and quantitation, system precision, accuracy, and intermediate precision parameters), this protocol was successfully and reproducibly used to quantify picomolar to nanomolar concentrations of nucleosides and nucleotides in isotonic and hypotonic cell buffers that transiently bathed M1 cells, and urine samples from normal subjects and overactive bladder patients.

Item Type: Article
DOI/Identification number: 10.1007/s11302-012-9321-8
Uncontrolled keywords: Nucleotide, Nucleoside, HPLC, Solid-phase extraction, Ion-pair agent, Urine samples
Subjects: R Medicine > RM Therapeutics. Pharmacology
Divisions: Divisions > Division of Natural Sciences > Medway School of Pharmacy
Depositing User: Scott S.P. Wildman
Date Deposited: 10 Dec 2015 15:23 UTC
Last Modified: 16 Nov 2021 10:22 UTC
Resource URI: (The current URI for this page, for reference purposes)

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Peppiatt-Wildman, Claire M..

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Wildman, Scott S.P..

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