Electrochemical detection folates

Fig. 6.3.2a-c Chromatogram of 5MTHF using electrochemical detection, a Standard, 100 nM b control cerebrospinal fluid (CSF) c CSF from a patient with cerebral folate deficiency (CFD)... 

Electrochemical detection in LC provides a sensitive assay method for certain vitamins, such as AA, folates, and flavins. AA may be easily detected with femtomolar sensitivity. Sample preparation and matrix interference problems limit the routine applicability of electrochemistry in the analysis of water-soluble vitamins currently to AA. 

Ion-pair HPLC (IP-HPLC) and reversed-phase HPLC (RP-HPLQ have been used to determine folate (Wilson and Horne 1984, 1986), most commonly in conjugation with fluorescence, ultraviolet and UV-visible (UV-vis) spectrometry (Vahteiisto et al. 1996) or electrochemical detection (BCD) systems (Kohashi et al. 1986 Lucock et al. 1989). The pteridine ring structure of the folate can be determined on the basis of its retention time determined by HPLC and spectral characteristics via the diode array detection (DAD) system (Selhub 1989). 

Kohashi, M., Inoue, K., Sotobayashi, H., and Iwai, K., 1986. Microdetermination of folate monoglutamates in serum by liquid-chromatography with electrochemical detection. Journal of Chromatography. 382 303-307. 

Following HPLC separation of different folate forms, a number of detection systems can be used including UV, fluorometric, or electrochemical activity (65). Due to its sensitivity and selectivity, fluorescence detection is most commonly used, particularly for reduced folate forms (86). Quantitation is usually based on external standard methods, although an internal method using a stable isotope label has been recently reported (see below) (87). More cumbersome identification and quantifications are needed in methods in which folates are eluted in clusters (69). It is important that the actual concentrations of folate calibration solutions are determined using known extinction factors (30) because most commercially available folate calibrants are of variable purity (7). 

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