Limitations of deuterium-labeled internal standards for quantitative electrospray ionization mass spectrometry analysis of fatty acid metabolites.

RATIONALE

The electrospray ionization mass spectrometry (ESI-MS) methodology often shows poor ionization reproducibility in the analysis of biological samples. Therefore, normalization of the measured peak intensities is essential. It is believed that quantitative data with high reproducibility can be obtained by adding a constant amount of an internal standard (IS) material labeled with stable isotopes to each sample, thus allowing the correction of the quantitative value of the target compound by that of the IS. We investigated whether the presence or absence of a labeled IS improves the accuracy of these quantitative values.

METHODS

Triple quadrupole MS coupled with liquid chromatography was used to analyze fatty acid metabolites in biological samples as target compounds. Two independent systems were used to provide a measure of reproducibility in two different laboratories.

RESULTS

Data having poor reproducibility in the raw peak areas were efficiently normalized using the IS, but, crucially, the IS method using stable isotopes was not always necessary. In some cases, the reproducibility was relatively good even without using the IS. In a contaminant matrix, the MS response behavior of the target compound and its stable isotope-labeled material was complicated. Since ion suppression by matrix contaminants was dependent on the concentration of the target compound, the added amounts of the ISs were also important, Furthermore, an equivalent normalization effect was obtained by using a pooled quality control sample as an external standard, thus obviating the need for labeled IS samples, which are often expensive and sometimes not commercially available.

CONCLUSIONS

Our results raise the question as to whether the quantitative method using stable-isotope-labeled ISs is always necessary and beneficial. However, the results obtained in this study cannot be generalized because only fatty acid metabolites were examined using ESI-MS and only a highly substituted deuterium-labeled IS was used.