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

• Published in: Clinica chimica acta Vol. 364; no. 1; pp. 180 - 187
• Main Authors: Carducci, Claudia, Santagata, Silvia, Leuzzi, Vincenzo, Carducci, Carla, Artiola, Cristiana, Giovanniello, Teresa, Battini, Roberta, Antonozzi, Italo
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-02-2006
• Subjects: AGAT; Amidinotransferases - deficiency; Child; Child, Preschool; Chromatography, High Pressure Liquid; Creatine; Creatine - blood; Creatine - standards; Dried blood spot; GAMT; Glycine - analogs & derivatives; Glycine - blood; Glycine - standards; Guanidinoacetate; Guanidinoacetate N-Methyltransferase - deficiency; Humans; Infant; Infant, Newborn; Nervous System Diseases - blood; Nervous System Diseases - enzymology; Reference Values; Reproducibility of Results; Spectrometry, Mass, Electrospray Ionization - instrumentation; Spectrometry, Mass, Electrospray Ionization - methods; Tandem mass spectrometry; GAMT; Tandem mass spectrometry; GAA; Dried blood spot; AGAT; ESI-MS/MS; Creatine; Guanidinoacetate; Cr; FIA
• ISSN: 0009-8981; 1873-3492
• DOI: 10.1016/j.cca.2005.06.016

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.
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).
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.
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.