Simultaneous determination of guanidinoacetate, creatine and creatinine in urine and plasma by un-derivatized liquid chromatography-tandem mass spectrometry

Abstract

Background: Creatine plays an important role in the storage and transmission of phosphate-bound energy. The cerebral creatine deficiency syndromes (CCDS) comprise three inherited defects in creatine biosynthesis and transport. They are characterized by mental retardation, speech and language delay and epilepsy. All three disorders cause low-creatine signal on brain magnetic resonance spectroscopy (MRS); however, MRS may not be readily available and even when it is, biochemical tests are required to determine the underlying disorder. Methods: Analysis was performed by liquid chromatography-tandem mass spectrometry in positive ionization mode. Samples were analysed underivatized using a rapid 'dilute and shoot' approach. Chromatographic separation of the three compounds was achieved. Stable isotope internal standards were used for quantification. Results: Creatine, creatinine and guanidinoacetate were measured with a 2.5 minute run time. For guanidinoacetate, the standard curve was linear to at least 5000 mmol/L and for creatine and creatinine it was linear to at least 25 mmol/L. The lower limit of quantitation was 0.4 mmol/L for creatine and guanidinoacetate and 0.8 mmol/L for creatinine. Recoveries ranged from 86% to 106% for the three analytes. Intra-and inter-assay variation for each analyte was ,10% in both urine and plasma. Conclusion: A tandem mass spectrometric method has been developed and validated for the underivatized determination of guanidinoacetate, creatine and creatinine in human urine and plasma. Minimal sample preparation coupled with a rapid run time make the method applicable to the routine screening of patients with suspected CCDS.

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

“…New method described in this paper has a satisfactory intra-day precision 3.39% for creatine and 6.01% for GAA and inter-day precision 4.99% and 7.66% respectively, similar to those described by R.S. Carling et al [15] by un-derivatized LC-MS/MS, that were 2.7% and 6.4% for intra-day precision but new data are slight better for inter-day assay precision that were 6.2% and 9.5% respectively for creatine and GAA; LOD and LLOQ were 0.1 mol/L and 0.4 mol/L for both creatine and GAA, higher compared with the new method: LOD 0.005 mol/L for creatine, 0.002 mol/L for GAA, LLOQ 0.05 mol/L for creatine and 0.02 mol/L GAA. Probably, new better data are due to the chromatographic separation of two additional interfering peaks presenting to the same GAA MRM transition at different retention times that could interfere with the GAA (Fig.…”

“…In recent years, introduction in the laboratory of liquid chromatography coupled with mass spectrometry, have greatly increased the diagnosis of creatine primary defects that were not previously easily diagnosed with traditional analytical techniques. Several methods for creatine and GAA determination have been described, including stable isotope dilution gas chromatography-mass spec- trometry (GC-MS) [12,13], and HPLC [6] and LC-MS/MS [14][15][16] but these methods reach less sensitivity and specificity. All these GC-MS methods require a long time for several steps of extraction and derivatization.…”

“…Moreover, Carling et al [15] mentioned that the validity of existing reference ranges quoted in literature is questionable by the relatively small numbers of subjects used to determine them and not always specified modality storage. Specification of storage modality of samples analyzed is very important since it is known that creatine, over time and changing pH of medium, varies its concentration transforming itself into creatinine [15,17].…”

“…Moreover, in those cases in which the concentrations of GAA and creatine are very low, as for AGAT deficiency, is necessary a high sensitive method, enough to safely detect values below the normal range. Previous methods [12][13][14][15][16] are useful for great increasing but not for a decrease of two metabolites. Bodamer et al [14] using the same metabolites derivatization procedures did not report LLOQ for creatine neither for GAA but only high limits of detection, especially for GAA, 1.2 mol/L.…”

Laboratory diagnosis of creatine deficiency syndromes: a technical standard and guideline of the American College of Medical Genetics and Genomics

“…LC-MS/MS-based measurement of creatine and guanidinoacetate can be performed with 55,56 or without derivatization. 56,57 Although slightly less sensitive, the underivatized method allows for more rapid preparation of urine and plasma samples, and simultaneous detection of creatinine. Samples are first combined with a stable isotope-labeled internal standard; for plasma, this may be prepared in an organic solvent (e.g., acetonitrile) to facilitate deproteinization, or an organic solvent may be added sequentially after the internal standards.…”