Comparative studies on vitamin B deficiency inwholeblood of chronically haemodialysed patients: chromatographic, fluorimetricandPCAstudy.

The levels of thiamine diphosphate (ThDP), the most active biologically form of vitamin B, were assessed in whole blood oflong-term haemodialysed patients (n = 50), by applying chromatographic methods based on RP-HPLC technique with isocratic elution and fluorescence detection. The target analyte, thiochrome diphosphate (ThODP), was obtained by pre-column derivatization of vitamin B contained in blood samples, applying deproteination with trichloroacetic acid, following by oxidation with alkaline solution of potassium ferricyanide(III) and stabilization with DTT before assays. A simple and sensitive assay was developed, and the results were referenced to the commercially available test. Steady-state and time-resolved studies on emissive properties of ThODP enabled optimization of the proposed assay. The F-Snedecor test shown no statistically significant differences between both approaches. Assessed parameters of the proposed assay, such as linearity, precision, sensitivity, and recovery, were satisfactory if compared to the reference one. The LOQ value for ThDP in whole blood of studied group of patients was of 0.5 ng/mL and the recovery of88%. The results disclosed high individual variabilities in the interdialytic deficiencies of ThDP among the patients - ranged from afew percent to values close to 100%. A comprehensive clinical data, characterizing patients under study, were processed together, and analysed by employing achemometric discriminative tool, the Principal Components Analysis,to find interdependences among clinical data characterizing patients. The three Principal Components were disclosed, that in sum explained almost 50% of the observed variability of the clinical data set. Among the clinical parameters involved in PCs were dialyzer membrane and type, duration as well as levels of creatinine, haemoglobin, and red blood cells in patients' whole blood.