Quantitation of adenosine, inosine and hypoxanthine in biological samples by microbore-column isocratic high-performance liquid chromatography.
This paper describes a simple and sensitive high-performance liquid chromatographic method for measuring adenosine, inosine and hypoxanthine in cell suspensions. The method involves direct injection of the filtered sample on a microbore C18 reversed-phase column with UV detection at 259 nm. The mobile phase consisted of 125 mM potassium dihydrogenphosphate, 1.0 mM tetrabutylammonium hydrogen-sulfate, 1.5% acetonitrile and 20 mM triethylamine, pH 6.5. The minimum detectable amounts (signal-to-noise ratio of 3:1) were 2.0 pmol of adenosine, 2.5 pmol inosine and 3.5 pmol of hypoxanthine. The limits of quantitation were 2.9 +/- 0.2 pmol for adenosine, 4.2 +/- 0.3 pmol for inosine and 4.9 +/- 0.4 pmol for hypoxanthine. This method was used to quantitate adenosine release by dispersed rat renal outer medullary cells (tubules) under conditions of normoxia and hypoxia.
Gayden
R H
RH
Department of Internal Medicine, University of Texas, Health Science Center, Houston 77030.
Watts
B A
BA
Beach
R E
RE
Benedict
C R
CR
D000241:Adenosine / Q000032:analysis*; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D019271:Hypoxanthine; D007042:Hypoxanthines / Q000032:analysis*; D007288:Inosine / Q000032:analysis*; D007679:Kidney Medulla / Q000737:chemistry*; D008297:Male; D010100:Oxygen / Q000737:chemistry; D051381:Rats; D011919:Rats, Inbred Strains; D013056:Spectrophotometry, Ultraviolet
D007042:Hypoxanthines; D019271:Hypoxanthine; D007288:Inosine; D000241:Adenosine; D010100:Oxygen