Implementing tandem mass spectrometry as a routine tool for characterizing the complete purine and pyrimidine metabolic profile in urine samples.
Purines and pyrimidines are the basic constituents of DNA and RNA and constitute the basis of at least 50 other important compounds that serve equally vital but separate roles as integral components of intracellular mononucleotide pools. They maintain the supply of these basic components to the different nucleotide pools through an extremely efficient mechanism involving the degradation and recycling of the daily waste products of normal cell turnover. We have developed an LC-MS/MS diagnostic and routine monitoring method for known defects due to both purine and pyrimidine metabolism in a single analysis. Precision tests were made by spiking several urine samples with different creatinine concentrations. For nonspiked low-creatinine urine, intraday precision was in the range of 0.1-9.8% and interday precision was between 1.6 and 14.1%. For nonspiked high-creatinine urine, intraday precision was in the range 0.5-17.2% and interday precision was between 1.5 and 29%. Limit-of-detection (LOD) was in the range 0.1-10 micromol/l and limit-of-quantification (LOQ) in the range of 0.2-15 micromol/l. The current 'dilute and shoot' approach monitors many metabolites, and utilizes a reverse phase chromatographic analysis with a detection requiring 17 min of analysis time. Tandem mass spectrometry and isotope dilution technique enable the accurate quantitation of more than 30 metabolites in one analysis.
la Marca
Giancarlo
G
Meyer Children's Hospital, Metabolic Unit, Dept of Paediatrics, University of Florence, Via Luca Giordano 13, 50132, Florence, Italy. g.lamarca@meyer.it
Casetta
Bruno
B
Malvagia
Sabrina
S
Pasquini
Elisabetta
E
Innocenti
Marzia
M
Donati
Maria Alice
MA
Zammarchi
Enrico
E
D000293:Adolescent; D000328:Adult; D002138:Calibration; D002675:Child, Preschool; D005260:Female; D006801:Humans; D007231:Infant, Newborn; D008297:Male; D011687:Purines / Q000378:metabolism* / Q000652:urine*; D011743:Pyrimidines / Q000378:metabolism* / Q000652:urine*; D012015:Reference Standards; D053719:Tandem Mass Spectrometry / Q000379:methods*
D011687:Purines; D011743:Pyrimidines; C030986:pyrimidine; C030985:purine