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)

Abstract

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.

Metabolic Effects of Clenbuterol and Salbutamol on Pork Meat Studied Using Internal Extractive Electrospray Ionization Mass Spectrometry

“…A large amount of chemical components were successfully detected from pork tissues and identified based on the CID experiments, the NIST/EPI/NIH Mass Spectral Library, authentic compounds (Figure S1) and literatures data. The majority of dominant peaks in both normal pork sample and pork contaminated by clenbuterol and salbutamol at mass range of m / z 50–500 were assigned as amino acids 20, 23, 24 (e.g., m / z 118 [Valine+H] + , m / z 147 [Lysine+H] + , m / z 156 [Histidine+H] + , m / z 175 [Arginine+H] + ), sugars ( m / z 219 [Glucose+K] + ), alkaloids ( m / z 104 [Choline] + ), polyamine ( m / z 203 [Spermine+H] + ), and m / z 132 [Creatine+H] + 25 , m / z 162 [L-carnitine+H] + , m / z 170 [Vitamin B 6 +H] + , m / z 227 [Carnosine+H] + 26 . The main peaks at mass range of m / z 700–900 were identified as phosphatidylcholines (PCs) including m / z 808 [PC (36:2)+Na] + , m / z 824 [PC (36:2)+K] + , m / z 782 [PC (36:4)+H] + , m / z 780 [PC (34:2)+Na] + , m / z 796 [PC (34:2)+K] + and m / z 832 [PC (38:4)+Na] + 27–29 , etc.…”

LC-MS/MS Method for Serum Creatinine: Comparison with Enzymatic Method and Jaffe Method

“…They observed a small but systematic conversion of creatine to creatinine during the sample preparation process and found in most cases, this interconversion reaction had no influence in the final Cre results, since the creatine concentration was very small. Only when creatine has similar or even higher level than Cre, then significant bias may occur [ 27 ].…”

Development of Ratiometric Fluorescent Biosensors for the Determination of Creatine and Creatinine in Urine

“…Alternatively, a second enzyme-catalyzed hydrolysis of creatine mediated by sarcosine oxidase results in the production of hydrogen peroxide (H 2 O 2 ) as the end product, which can be detected with a chronoamperometric technique [ 7 , 8 , 9 ]. Creatine detection has been also performed with flow-injection analysis (FIA) systems combined with immobilized enzyme reactors [ 10 ] and more expensive analytical tools such as high-performance liquid chromatography [ 10 , 11 ], capillary electrophoresis [ 12 ], and mass spectrometry [ 13 ]. However, the use of creatine sensors and other analytical tools is associated with practical issues related to sensitivity, reproducibility, stability, and interferences.…”