HPLC-FDHigh-Performance Liquid Chromatography with Fluorescence Detection
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In survey 2, only the mean value of the pool with lowest tHcy concentration (pool A) was significantly lower (P = 0.027) by the amino acid analyzer method compared with the mean value obtained by HPLC-FD with TCEP/SBDF as the reduction/ derivatization agents.
At 33 [micro]mol/L tHcy, values obtained by the FPIA method were significantly lower (P = 0.029) compared with values obtained by HPLC-FD with TBP/SBDF as the reduction/ derivatization agents.
To confirm the observation that tHcy values obtained by the FPIA method were lower at increased tHcy concentrations compared with values obtained by HPLC-FD (TBP/SBDF), we reanalyzed each of the 10 pools by both methods over 10 days.
In our laboratory, intralaboratory precision was also somewhat lower for the FPIA method (CV, 3.8%) compared with the HPLC-FD (TBP/SBDF) method (CV, 6.7%).
However, when the results of the three surveys were combined to obtain a larger sample number, it is of interest to note that for tHcy concentrations >30 [micro]mol/L, values obtained by the FPIA method were significantly lower than values obtained by HPLC-FD using TBP/SBDF as the reduction/ derivatization agents.
Because clinical studies have associated ranges of tHcy concentrations with the risk for CAD, our observation that results from the FPIA method were significantly different from those obtained by the HPLC-FD (TBP/SBDF) method warrants further investigation into the accuracy of tHcy measurements.
An aliquot of each specimen was analyzed for tHcy in the CDC NHANES laboratory by an HPLC-FD assay (7) with plasma QC pools at three concentrations (6.9, 13.4, and 29.2 [micro]mol/L).
The within-method variation was lowest for the FPIA assay (laboratories 4B, 8B, 13, and 14) and highest for the HPLC-FD assay using NaB[H.sub.4]/MBrB (laboratories 10 and 11).
Using the standard errors of the mean differences, we computed the 95% confidence intervals; these showed an apparent positive bias for HPLC-ED, HPLC-FD using NaB[H.sub.4]/MBrB (however, only for laboratory 10), and EIA, and an apparent negative bias for HPLC-FD using trialkylphosphine/ABD-F.
Ninety-five percent of tHcy determinations by HPLC-FD using trialkylphosphine/ABD-F were 2.05 to 0.33 [micro]mol/L lower than concentrations determined by GC/MS.
When we grouped laboratories by method type and performed a two-way ANOVA with laboratory and method as variables to compare methods individually, we found no significant differences among the methods except that HPLC-FD trialkylphosphine/ABD-F (laboratory 6) gave significantly lower results than HPLC-FD NaBH4/ MBrB (laboratories 10 and 11) and HPLC-ED (laboratories 1-3).
In the present study, in which each laboratory and method used its own calibrators, the results of the two laboratories performing HPLC-FD with NaBH4/MBrB demonstrated that laboratory-to-laboratory variations within one method can exceed among-method variations.