Simultaneous determination of creatinine and creatine in human serum by double-spike isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-mass spectrometry (GC-MS).
This work describes the first multiple spiking isotope dilution procedure for organic compounds using (13)C labeling. A double-spiking isotope dilution method capable of correcting and quantifying the creatine-creatinine interconversion occurring during the analytical determination of both compounds in human serum is presented. The determination of serum creatinine may be affected by the interconversion between creatine and creatinine during sample preparation or by inefficient chemical separation of those compounds by solid phase extraction (SPE). The methodology is based on the use differently labeled (13)C analogues ((13)C1-creatinine and (13)C2-creatine), the measurement of the isotopic distribution of creatine and creatinine by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the application of multiple linear regression. Five different lyophilized serum-based controls and two certified human serum reference materials (ERM-DA252a and ERM-DA253a) were analyzed to evaluate the accuracy and precision of the proposed double-spike LC-MS/MS method. The methodology was applied to study the creatine-creatinine interconversion during LC-MS/MS and gas chromatography-mass spectrometry (GC-MS) analyses and the separation efficiency of the SPE step required in the traditional gas chromatography-isotope dilution mass spectrometry (GC-IDMS) reference methods employed for the determination of serum creatinine. The analysis of real serum samples by GC-MS showed that creatine-creatinine separation by SPE can be a nonquantitative step that may induce creatinine overestimations up to 28% in samples containing high amounts of creatine. Also, a detectable conversion of creatine into creatinine was observed during sample preparation for LC-MS/MS. The developed double-spike LC-MS/MS improves the current state of the art for the determination of creatinine in human serum by isotope dilution mass spectrometry (IDMS), because corrections are made for all the possible errors derived from the sample preparation step.
†Department of Physical and Analytical Chemistry, Faculty of Chemistry, University of Oviedo, Julián Clavería 8, 33006 Oviedo, Spain.
Mario
M
Fernández-Fernández
†Department of Physical and Analytical Chemistry, Faculty of Chemistry, University of Oviedo, Julián Clavería 8, 33006 Oviedo, Spain.
Pablo
P
Rodríguez-González
‡Clinical Biochemistry Service, Central University Hospital of Asturias, 33011 Oviedo, Spain.
M Elena
ME
Añón Álvarez
§Department of Organic and Inorganic Chemistry, Faculty of Chemistry, University of Oviedo, 33006 Oviedo, Spain.
Felix
F
Rodríguez
‡Clinical Biochemistry Service, Central University Hospital of Asturias, 33011 Oviedo, Spain.
Francisco V Álvarez
FV
Menéndez
†Department of Physical and Analytical Chemistry, Faculty of Chemistry, University of Oviedo, Julián Clavería 8, 33006 Oviedo, Spain.
J Ignacio
JI
García Alonso
D002247:Carbon Isotopes; D002853:Chromatography, Liquid; D003401:Creatine / Q000097:blood* / Q000737:chemistry; D003404:Creatinine / Q000097:blood* / Q000737:chemistry; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D007201:Indicator Dilution Techniques; D015394:Molecular Structure; D053719:Tandem Mass Spectrometry
D002247:Carbon Isotopes; D003404:Creatinine; D003401:Creatine