The influence of citrate, EDTA, and heparin anticoagulants to human plasma LC–MS lipidomic profiling

Lipid profiling of human plasma by liquid chromatography-electrospray ionization coupled to mass spectrometry (LC–ESI-MS) is being used to identify biomarkers of health, disease, and treatment efficacy. However, there is no consensus on the choice of anticoagulant to perform and compare lipidomic measurements. This study assessed the effect of the anticoagulants citrate, EDTA, and heparin, on eight synthetic and 80 plasma lipids, and compared lipidomic data among anticoagulants. Lipid extraction was affected distinctively by the anticoagulant of choice likely due to the different physico-chemical properties among anticoagulants. Peak areas of seventy endogenous lipids showed significant differences between citrate–heparin and EDTA–heparin comparisons similar to those observed for synthetic lipids. Only ten endogenous lipid species showed comparable peak areas among the three anticoagulants. Correction by a structurally related internal standard only partly eliminated differences among anticoagulants (ANOVA, P value <0.001). However, comparisons among anticoagulants were possible for most endogenous lipids after correction of peak areas by the sum of areas of its lipid class. Our observations indicate that the choice of anticoagulant distinctively impact the peak response of most lipid species by LC–ESI-MS. Lipidomic data from plasma obtained with different anticoagulants should address differences in matrix effects and extraction procedures since ion strength, plasma pH, and different physicochemical properties among anticoagulants influence lipid extraction and LC–ESI-MS analysis.

[1]  A. Zwinderman,et al.  Lipidomic approach to evaluate rosuvastatin and atorvastatin at various dosages: investigating differential effects among statins , 2008, Current medical research and opinion.

[2]  D. Goodier,et al.  Effect of in vitro sodium citrate anticoagulant on results of arterial blood gas analysis. , 1986, Journal of clinical pathology.

[3]  Dietrich A. Volmer,et al.  Ion suppression: A major concern in mass spectrometry , 2006 .

[4]  David S. Wishart,et al.  MetaboAnalyst: a web server for metabolomic data analysis and interpretation , 2009, Nucleic Acids Res..

[5]  B. Matuszewski.  Standard line slopes as a measure of a relative matrix effect in quantitative HPLC-MS bioanalysis. , 2006, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[6]  J. T. Lumeij,et al.  Avian Clinical Biochemistry , 1997 .

[7]  Daniel S Waterman,et al.  The influence of EDTA and citrate anticoagulant addition to human plasma on information recovery from NMR-based metabolic profiling studies. , 2010, Molecular bioSystems.

[8]  Stefan R. Bornstein,et al.  Top-Down Lipidomics Reveals Ether Lipid Deficiency in Blood Plasma of Hypertensive Patients , 2009, PloS one.

[9]  Edward A Dennis,et al.  The human plasma lipidome. , 2011, The New England journal of medicine.

[10]  T. Hankemeier,et al.  RPLC-ion-trap-FTMS method for lipid profiling of plasma: method validation and application to p53 mutant mouse model. , 2008, Journal of proteome research.

[11]  P. Taylor.  Matrix effects: the Achilles heel of quantitative high-performance liquid chromatography-electrospray-tandem mass spectrometry. , 2005, Clinical biochemistry.

[12]  Walter A. Korfmacher,et al.  Investigation of matrix effects in bioanalytical high-performance liquid chromatography/tandem mass spectrometric assays: application to drug discovery. , 2003, Rapid communications in mass spectrometry : RCM.

[13]  W. J. Dyer,et al.  A rapid method of total lipid extraction and purification. , 1959, Canadian journal of biochemistry and physiology.

[14]  J. Livesey,et al.  Effect of anticoagulants and storage temperatures on stability of plasma and serum hormones. , 2001, Clinical biochemistry.

[15]  S. Lehotay,et al.  Combination of analyte protectants to overcome matrix effects in routine GC analysis of pesticide residues in food matrixes. , 2005, Analytical chemistry.

[16]  Florian Kronenberg,et al.  Differences between Human Plasma and Serum Metabolite Profiles , 2011, PloS one.

[17]  I. Johnstone.  The importance of accurate citrate to blood ratios in the collection of canine blood for hemostatic testing. , 1993, The Canadian veterinary journal = La revue veterinaire canadienne.

[18]  Xianlin Han,et al.  Lipidomics at the interface of structure and function in systems biology. , 2011, Chemistry & biology.

[19]  A. Smilde,et al.  A lipidomic analysis approach to evaluate the response to cholesterol-lowering food intake , 2011, Metabolomics.

[20]  C. Gelfand,et al.  Minimizing preanalytical variation of plasma samples by proper blood collection and handling. , 2011, Methods in molecular biology.

[21]  J. M. Christensen,et al.  Ibuprofen piconol hydrolysis in vitro in plasma, whole blood, and serum using different anticoagulants. , 1991, Journal of pharmaceutical sciences.

[22]  Vinod P. Shah,et al.  Quantitative Bioanalytical Methods Validation and Implementation: Best Practices for Chromatographic and Ligand Binding Assays , 2007 .

[23]  H. Karnes,et al.  Assessment of matrix effects and determination of niacin in human plasma using liquid-liquid extraction and liquid chromatography-tandem mass spectrometry. , 2008, Biomedical chromatography : BMC.