Comparison of three different plasma homocysteine assays with gas chromatography-mass spectrometry

Background: Various methods are available to measure plasma total homocyst(e)ine (tHcy) concentrations, but whether plasma tHcy assays may be used interchangeably is not known. Methods: Results from three different methods [HPLC with fluorescence detection, enzyme immunoassay (EIA), and fluorescence polarization immunoassay (FPIA)] to determine fasting (n = 163) and post-methionine load (n = 80) plasma tHcy concentrations were compared with those obtained by gas chromatographymass spectrometry (GC-MS). Difference plots on nontransformed and log-transformed data were used to assess the agreement between HPLC and GC-MS, EIA and GC-MS, and FPIA and GC- MS. Results: The closest agreement between methods was observed between GC- MS and FPIA for fasting tHcy concentrations, with 95% of the FPIA values between 19% above and 24% below the corresponding GC-MS results. Post- methionine load tHcy concentrations measured by EIA showed the least agreement with GC-MS, with 95% of values measured by EIA ranging between 52% above and 16% below the GC-MS values. With respect to GC-MS, the above- mentioned methods showed a negative bias for fasting tHcy concentrations, but a positive bias for both immunoassays for postmethionine load tHcy concentrations. Conclusions: The agreement among methods is insufficient to allow them to be used interchangeably. The intermethod differences emphasize the need for standardization of plasma tHcy assays.