Impact of internal standard selection on measurement results for long chain fatty acids in blood

Abstract

Introduction: Internal standards correct for measurement variation due to sample loss. Isotope labeled analytes are ideal internal standards for the measurement of fatty acids in human plasma but are not always readily available. For this reason, quantification of multiple analytes at once is most often done using only a single or few internal standards. The magnitude of the impact this has on method accuracy and precision is not well studied for gas chromatography-mass spectrometry systems. Objective: This study aims to estimate bias and changes in uncertainty associated with using alternative fatty acid isotopologue internal standards for the estimation of similar or dissimilar long chain fatty acids. Method: Using a previously reported method for the quantification of 27 fatty acids in human plasma using 18 internal standards we obtained estimates of bias and uncertainty at up to three levels of fatty acid concentration. Results: With some notable exceptions, method accuracy remained relatively stable when using an alternative internal standard (Median Relative Absolute Percent Bias: 1.76%, Median Spike-Recovery Absolute Percent Bias: 8.82%), with larger changes in method precision (Median Increase in Variance: 141%). Additionally, the degree of difference between analyte and internal standard structure was related to the magnitude of bias and uncertainty of the measurement. Conclusion: The data presented here show that the choice of internal standard used to estimate fatty acid concentration can affect the accuracy and reliability of measurement results and, therefore, needs to be assessed carefully when developing analytical methods for the measurement of fatty acid profiles.Disclaimer: The findings and conclusions in this report are those of the author(s) and do not necessarily represent the official position of the Centers for Disease Control and Prevention/the Agency for Toxic Substances and Disease Registry. Use of trade names is for identification only and does not imply endorsement by the Centers for Disease Control and Prevention, the Public Health Service, and the US Department of Health and Human Services.

Keywords

• Internal standards
• Isotope dilution
• Gas chromatography
• Mass spectrometry
• Fatty acids