Background: Current sampling for total homocysteine (tHcy) is
problematic, requiring plasma separation within 15 min. The aim of this study was to
develop a liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method for the
measurement of tHcy in plasma and dried blood spots and to determine whether the
dried blood spot concentration could be used to predict plasma concentrations of
tHcy.
Methods: LC-MS/MS methodology was optimized to measure tHcy in plasma
and dried blood spots. Fifty blood samples collected from heart transplant patients
were used to form dried blood spots and for plasma analysis. Plasma tHcy was also
measured using the Abbott IMx1 method and values were compared to the tHcy
concentrations determined in plasma and dried blood spots using LC-MS/MS
methodology.
Results: The plasma tHcy LC-MS/MS results compared well with the IMx
values: LC-MS/MS=1·18(IMx)-0·44 (r
2=0·915). The within-batch precision (n =10) of the
plasma LC-MS/MS method was < 2·0% at 14·6 and 37·7 µmol/L, respectively; the
between-batch precision (n=10) was 5·0 and 8·0%, respectively, at
these concentrations. The method was found to be sensitive down to 1 µmol/L and
linear up to at least 100 µmol/L. Dried blood spot LC-MS/MS results were considerably
lower than the plasma IMx values (P < 0·0001): dried blood spot
LC-MS/MS=0·33IMx+1·77 (r
2=0·682). The within-batch precision (n=20) of the dried
blood spot LC-MS/MS method was 7·3% and 4·7% at concentrations of 4·0 and 7·9 µmol/L,
respectively; the between-batch precision was 12·6% and 7·9% at concentrations of 5·1
and 8·0 µmol/L, respectively. To assess whether dried blood spots are suitable as a
screening test to predict plasma tHcy concentrations, arbitary cut-off levels were
compared. If it is assumed that a plasma tHcy concentration of >15 µmol/L is
raised, a dried blood spot result of >6·8 µmol/L has a sensitivity and specificity
in detecting a raised plasma tHcy of 83·3% and 96·2%, respectively, and a positive
and negative predictive value of 95% and 86%, respectively, with an efficiency of
90%. Use of a dried blood spot cut-off concentration of 6·2 µmol/L for predicting
high plasma tHcy concentrations (above 15 µmol/L) has a sensitivity and specificity
of 95·8% and 73·1%, respectively, positive and negative predictive values of 76% and
95%, respectively, and an efficiency of 84%.
Conclusions: We have developed a precise and accurate LC-MS/MS method
for measuring plasma tHcy concentrations, which uses a small volume of plasma and is
suitable for routine use. A satisfactory LC-MS/MS method for the measurement of tHcy
in dried blood spots was also developed; this method might be useful in routine
screening for raised plasma concentrations of tHcy.
