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

Background: Guarudinoacetate (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 mu mol/l of blood and 0.30 mu mol/l of blood for Cr and GAA, respectively, and the response was linear over the range 0.25-12.5 mu mol/l of blood for GAA and 3.57-624.7 mu mol/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. (C) 2005 Elsevier B.V All rights reserved