Simultaneous determination of creatine and guanidinoacetate in plasma by liquid chromatography–tandem mass spectrometry (LC–MS/MS)

Guanidinoacetate (GAA) and creatine are reliable biochemical markers for primary and secondary creatine defects. We describe a method by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for simultaneous determination of plasma GAA and creatine. We analyzed 283 healthy subjects from 0 to 63 years old to obtain age-related control values.Plasma samples were extracted with acetonitrile containing 13C2-GAA and d3-creatine. Samples were analyzed by LC-MS/MS in positive ionisation mode, after derivatization to butyl-esters. Optimal chromatographic separation was achieved using a column Supelcosil™ LC-4.6mm with isocratic elution in 5min.Run time was 5min. Standard curves were linear from 0.05 to 200μmol/L for creatine and from 0.02 to 40μmol/L for GAA. Limit of detection (LOD) and limit of quantitation (LOQ) were respectively 0.005 and 0.05μmol/L for creatine; LOD and LOQ were 0.002 and 0.02μmol/L respectively for GAA. Intra and inter-assay CVs for creatine and GAA were <8%. Recovery experiments adding 50 and 100μmol/L creatine and 10 and 20μmol/L GAA were 102.1% and 101.2%, for creatine; 102.95% and 96.45% for GAA. The method was applied to 283 plasma controls from healthy subjects to obtain control values in three specific age ranges: 0-12, 13-20, >20 years old.A rapid and high sensitive LC-MS/MS method was developed and validated for determination of creatine and GAA in plasma and it could also be applied to other biological materials, such as CFS and urines. This method is useful for diagnoses of primary and also for secondary creatine defects that may occur in inherited metabolic diseases in which precursors of creatine biosynthesis are involved.