HPLC-FDHigh-Performance Liquid Chromatography with Fluorescence Detection
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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.
8%) compared with the HPLC-FD (TBP/SBDF) method (CV, 6.
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.
Method Reduction/ Mean [+ or -] SD of respective Derivatization n pools, [micro]mol/L (CV, %) agents Survey 1 A B Within method HPLC-FD NaB[H.
Ninety-five percent of tHcy determinations by HPLC-FD using trialkylphosphine/ABD-F were 2.
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.
When compared with GC/MS, HPLC-FD using TCEP/ SBD-F and FPIA showed virtually no apparent bias and narrow limits of agreement.
Three methods performed best regarding analytical imprecision: GC/MS, FPIA, and HPLC-FD using TCEP/SBD-F.
The following three methods performed best regarding apparent analytical bias (vs GC/MS): FPIA and HPLC-FD using TCEP/SBD-F or TBP/SBD-F.