Correction of creatine-creatinine conversion during serum creatinine quantification by

two-dimensional liquid chromatography and double-spike isotope dilution tandem mass

spectrometry
Daniela Pineda-Cevallos, María Funes Menéndez, Adriana González-Gago, Pablo Rodríguez González, J. Ignacio García Alonso
University of Oviedo, Faculty of Chemistry, Department of Physical and Analytical Chemistry
Universidad de Oviedo

GRAPHICAL ABSTRACT BACKGROUND

 CREATININE = CALCULATION OF GFR
 ROUTINE CLINICAL LABORATORY
JAFFE-> INTERFERENCES
 IDMS-> REFERENCE METHOD
 INTERCONVERSION CREATININE-CREATINE

OBJECTIVE

Development of a candidate REFERENCE METHOD based on

2D-MHC-HPLC-ESI-MS/MS and DOBLE SPIKE isotope
dilution for SERUM creatinine quantificatrion capable of
correcting for creatinine-creatine INTERCONVERSION

ESI-MSMS
2D
MULTIPLE +
REVERSED PHASE DOUBLE SERUM
+ HEART SPIKE
CATIONIC ANALYSIS
CUTTING ISOTOPE
EXCHANGE DILUTION

METHODOLOGY: 1D VS 2D-HPLC-MS/MS TRUENESS OF THE METHODOLOGY

Creatine-specific SRM transitions 2D-HPLC-MS/MS: High resolution sampling TWO FROZEN HUMAN SERUM CERTIFIED
1D-SCX-ESI-MS/MS chromatogram of serum A 1D-C18-HPLC UV DETECTION REFERENCE MATERIAL OF CREATININE
ERM
sample ERM-DA252A (LOW LEVEL) 3.1±0.5 µg g-1 ERM
POLAR
253A
COMPOUNDS 252A
ERM-DA253A (HIGH LEVEL) 50±2 µg g-1

LOW
RETENTION
IN C18

B COLLECTION OF THREE FRACTIONS IN THE 1D CHROMATOGRAPHIC PROFILE

HIGHEST
SENSITIVIT
Y

Creatine

C
Ion suppression is barely observed
when increasing the volume CREATININE CONCENTRATION IN AGREEMENT WITH CERTIFIED VALUES NEGLIGIBLE VALUES OF INTERCONVERSION FACTORS
10 µL -> Highest sensitivity

INTRA-DAY AND INTER-DAY VARIABILITY

Important INTERFERENCES at the tr of
creatine INTRA-DAY: FOUR INDEPENDENT REPLICATES AND FIVE INJECTIONS PER SAMPLE IN THE SAME DAY
INTER-DAY: REPEATING THE EXPERIMENT ON FOUR DIFFERENT DAYS
Ionic SUPPRESSION effects due to ANALYSIS OF SERUM CONTROLS BIORAD LIQUID ASSAYED MULTIQUAL 1 AND 3 AND A POOLED SERUM
MATRIX constituents
 Sample PURIFICATION
LOW TOLERANCE of SCX column to
matrix
 MINIMIZE ionic suppression and spectral
interferences INTRA-DAY PRECISION
ACCURATE creatine
QUANTIFICATION and creatine-
 Enhancement of the SCX COLUMN LIFE 0.5-1.6% FOR CREATININE
1.3-13% FOR CREATINE
creatinine INCONVERSION  Use of MOBILE PHASES not compatible INTER-DAY PRECISION
determination for LOW LEVEL SERUM with ESI (TFA)
1.6-2.1% FOR CREATININE
simples is CHALLENGING 2.4-13% FOR CREATINE

ANALYSIS OS SERUM SAMPLE CONCLUSIONS

CONCENTRATION mg dL-1 CONCENTRATION OF CREATININE (Cn) VS CREATINE (Cr)
BIDIMENSIONAL CHROMATOGRAPHY
THREE GROUPS
N=93 1. Low concentration of Cr and Cn +
2. Low concentration of Cr and increasing DOUBLE SPIKE ISOTOPE DILUTION
CV< 2% concentration of Cn +
3. Increasing concentration of Cr and Cn TANDEM MASS SPECTROMETRY

INTERCONVERSION VALUES VS CONCENTRATION

 REFERENCE METHOD
F2 = -0.7±0.2%
F1 = 0.2±0.2%  SIMPLE SAMPLE PREPARATION
F2 = % CREATINE -> CREATININE
 FRACTION SELECTED IN REVERSED PHASE
F1 = % CREATININE -> CREATINE  MINIMIZE MATRIX EFFECTS AND ION SUPPRESSION
 VALIDATION ACCORDING TO CLSI GUIDELINES
F1 AND F2 ARE NEGLIGIBLE AND  NEGLIGIBLE CREATINE-CREATININE INTERCONVERSION
INDEPENDENT FROM MEASURED
CONCENTRATIONS OF BOTH Cr AND Cn

ACKNOWLEDGEMENTS BIBLIOGRAPHY
[1] M. Fernández-Fernández, P. Rodríguez-González, M.E. Añón Álvarez, F. Rodríguez, F.V. Álvarez
Daniela Pineda-Cevallos acknowledges the Principality of Asturias, Spain, for their financial support
Menéndez, J.I. García Alonso. Anal. Chem. 2015, 87, 3755–37631
through the Severo Ochoa scholarship ref. BP22-061. The Spanish Ministry of Science and Innovation is
[2] D. Pineda-Cevallos, M. Funes Menéndez, Adriana González-Gago, P. Rodríguez-González, J.I. García
acknowledged for the funding through Project MCIU-22-PID2021-125795NB-I00.
Alonso. Clinica Chem Acta. 2024, 554, 117778