The analysis of thiamin and its derivatives in whole blood samples under high pH conditions of the mobile phase.
In this study a protocol for the analysis of thiamin and thiamin coenzymes in whole blood was developed. Thiamin and its coenzymes are analyzed by reversed phase liquid chromatography (RPLC), precolumn derivatisation with alkaline potassium ferricyanide and fluorescence detection, all at pH 10. Under these relatively high pH conditions the detectability of the analytes and the robustness of the method were substantially improved. The use of a high pH resistant RPLC column was a crucial step in developing this analysis method. Reproducibility, linearity, recovery, detection limit and column robustness were investigated. The within-batch CV was <2.5%, the between-batch CV <4.5%. The method was linear far above the physiological relevant concentration level. Recovery was almost 100% on an average. The limit of quantification was 1 nmol/l. The robustness of the RPLC column proved to be very high. Up to 1500 injections hardly any substantial changes in retention times and efficiency were observed. In summary: Using a high pH resistant RPLC column resulted in a robust, sensitive and precise method for the analysis of total Vitamin B1 and especially of TDP.
Department of Clinical Chemistry and Hematology, St. Elisabeth Hospital, P.O. Box 90151, 5000 LC Tilburg, The Netherlands.
Bart A J
BA
van Landeghem
Johan
J
Puts
Henk A
HA
Claessens
D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods*; D005453:Fluorescence; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015203:Reproducibility of Results; D013831:Thiamine / Q000097:blood*; D013833:Thiamine Monophosphate / Q000097:blood; D013835:Thiamine Pyrophosphate / Q000097:blood
D013833:Thiamine Monophosphate; D013835:Thiamine Pyrophosphate; D013831:Thiamine