Quantitative determination of guanidinoacetate and creatine in dried blood spot by flow injection analysis-electrospray tandem mass spectrometry

Abstract

Background Guanidinoacetate (GAA) and creatine (Cr) are reliable biochemical markers of primary creatine disorders. The aim of this study was to develop and validate a method for the determination of GAA and Cr in dried blood spot through the use of stable isotope dilution and flow injection analysis (FIA)-ESI-MS/MS. Methods Dried blood spots were extracted using methanol–water solution containing D3-Cr. After evaporation and formation of butyl esters, samples were analyzed using multiple reaction monitoring mode ( m/ z 174.2→101.1 for GAA, 188.3→90.1 for Cr and 191.3→93.1 for D3-Cr). Results The analysis was very fast (1 min). The detection limits were 0.34 μmol/l of blood and 0.30 μmol/l of blood for Cr and GAA, respectively, and the response was linear over the range 0.25–12.5 μmol/l of blood for GAA and 3.57–624.7 μmol/l of blood for Cr. Recovery range was 93–101% for Cr and 94–105% for GAA and between-run CVs were 5.3% for GAA and 4.5% for Cr. Ion suppression effect was also studied. The method was applied to spots obtained from two patients affected by GAMT deficiency, four patients affected by AGAT deficiency (including a newborn) as well as 282 healthy subjects. Conclusions The detection of GAA in dried blood spot by FIA-ESI-MS/MS is a highly reliable and high throughput method for the diagnosis of GAMT and AGAT deficiencies and a possible tool for newborn screening of both these tractable disorders.