Quantitative determination of guanidinoacetate and creatine in dried blood spot by flow injection analysis-electrospray tandem mass spectrometry.
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 micromol/l of blood and 0.30 micromol/l of blood for Cr and GAA, respectively, and the response was linear over the range 0.25-12.5 micromol/l of blood for GAA and 3.57-624.7 micromol/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.
Carducci
Claudia
C
Dipartimento di Medicina Sperimentale e Patologia, Università di Roma La Sapienza, Viale del Policlinico 155, 00161 Roma, Italy. claudia.carducci@uniroma1.it
Santagata
Silvia
S
Leuzzi
Vincenzo
V
Carducci
Carla
C
Artiola
Cristiana
C
Giovanniello
Teresa
T
Battini
Roberta
R
Antonozzi
Italo
I
D000579:Amidinotransferases / Q000172:deficiency; D002648:Child; D002675:Child, Preschool; D002851:Chromatography, High Pressure Liquid; D003401:Creatine / Q000097:blood* / Q000592:standards; D005998:Glycine / Q000031:analogs & derivatives* / Q000097:blood / Q000592:standards; D050937:Guanidinoacetate N-Methyltransferase / Q000172:deficiency; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D009422:Nervous System Diseases / Q000097:blood / Q000201:enzymology; D012016:Reference Values; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods*
C494506:GAMT protein, human; D050937:Guanidinoacetate N-Methyltransferase; D000579:Amidinotransferases; C020681:glycine amidinotransferase; C004946:glycocyamine; D003401:Creatine; D005998:Glycine