Mercapturic acids of styrene in man: Comparability of the results obtained by LC/MS/MS and by HPLC-fluorimeter, and stability of samples under different storage conditions

Two analytical methods (HPLC-fluorimeter [HPLC-FLD] and tandem mass spectrometry LC/MS/MS) are available to assay phenyl-hydroxyethylmercapturic acids (PHEMAs), the mercapturic acids of styrene in humans. In the past, each method was used to check different populations of subjects, but until now no attempt has been made to compare the two methods. This study was designed to verify whether the two methods actually give comparable results. The influence of different conditions of sample storage in altering the concentration of PHEMAs was also investigated. Urine samples were collected at the beginning and at the end of the workshift from 10 workers exposed to different levels of styrene. Each sample was analysed both by LC/MS/MS after storage under different conditions (respectively, at -20 and +4°C, and after repeated freezing-thawing cycles), and by HPLC-FLD (in the same conditions of storage). Strong correlations were found between the two methods both for total PHEMAs and for each of the isomers measured, including the minor (S,R)-M1. Also an alternative approach, the Bland-Altman test, confirmed the agreement between the two methods. The different storage conditions tested did not decrease the concentration of PHEMAs but, surprisingly, a clear trend to increase was shown, particularly for (R,R)-M1, (S,R)-M2 and (R,R)-M2 in samples stored at +4°C for 1 week. In conclusion, the study demonstrates that the methods give comparable results. Indirectly, this confirms also the main characteristics of PHEMAs, showed in the previous experiments: low biotransformation rates of styrene into PHEMAs; large inter-individual variability; and the presence of a clear preference in the excretion of the isomers deriving from (S)-styrene oxide. PHEMAs appear stable under different storage conditions, but further studies are needed to explain the increase of levels that occurs when samples are not kept frozen. To avoid pre-analytical errors, samples collected for biomonitoring or research purposes should be frozen as soon as possible, and thawed only one time just before the analysis.