Analytical methods to measure plasma total homocyst(e)ine (tHcy) concentrat
ions are reviewed. High-pressure liquid chromatography (HPLC) with fluorome
tric detection is the most widely used method to determine plasma tHcy conc
entrations. Both monobromobimane and ammonium 7-fluorobenzo-2-oxa-1,3-diazo
le-4-sulphonate (SBD-F) are popular thiol-specific fluorogenic agents suita
ble for tHcy analysis. Monobromobimane has the advantage that it is highly
reactive towards thiols, but its hydrolysis products are also fluorogenic,
thus necessitating complex chromatography to obtain satisfactory separation
between the compounds of interest and interferents. SBD-F does not show fl
uorescence, thus allowing isocratic separation of SBD-F derivatized thiols,
SBD-thiol adducts are light sensitive and require protection against light
to ensure reliable results.
HPLC with electrochemical detection is also often used and has the advantag
e that no derivatization of thiols is required prior to detection. A recent
ly reported liquid chromatography electrospray tandem mass spectrometric as
say has the potential to become the reference method for plasma tHcy analys
is.
Other methods to measure plasma tHcy concentrations include gas-liquid chro
matography, capillary electrophoresis, and immunoassays. Fluorescence polar
ization immunoassay compares well with gas chromatography-mass spectrometry
and may become the method of choice in routine diagnostic clinical chemist
ry laboratories.
Instability of tHcy in whole blood as well as postprandial and orthostatic
variation are preanalytical factors that should be accounted for in plasma
tHcy analysis. Between-method and between-laboratory variations in serum tH
cy analysis are not yet satisfactory; certified reference material and stan
dardization of the plasma tHcy assay will be essential to reduce between-la
boratory bias.