1 -alkylthio-2-alkylisoindole

Figure 20.2 Derivatization of pantothenic acid to fluorescent compound 1-alkylthio-2-alkylisoindole.

The OPA reagent for HPLC is prepared according to the method of Benson and Hare (21). The fluorescence reaction is performed in a 55 C water bath. OPA reacts with the guanidino group of TTX presumably to form a fluorescent product, l-alkylthio-2-alkylisoindole (22, 23). TTX is monitored at 453 nm with 332-nm excitation. Peak areas are calculated by a data processing system of the analyzer. 

Add 2 pL of working reagent A for every 10 pL of standard or microdialysate sample to form electroactive I-alkylthio-2-alkylisoindoles. Allow the derivatization to proceed for 2 mm at 4 C (see Notes 8 and 9)... 

Figure 12. Formation of a l-alkylthio-2-alkylisoindole by reaction of OPA, a thiol and a primary amine.

Fluorescent compounds can be synthesized from pantothenic acid and the first HPLC-fluorimetric method was reported in 1995 (Blanco et al. 1995). In principle, pantothenic acid can be hydrolysed to (3-alanine and pantoic acid by hot alkaline hydrolysis and the product (3-alanine is reacted with u-phthal-dialdehyde and 3-mercaptopropionic acid (3-MPA) to form a fluorescent compound, l-alkylthio-2-alkylisoindole. This fluorescence is monitored at an excitation wavelength of 345 nm and emission wavelength of 455 nm (Figure 20.2). This method can determine the total pantothenic acid contents in foodstuffs and urinary pantothenic acid levels (Pakin et al. 2004 Takahashi et al. 2009). 

Analytical conditions are shown in Table 20.4. A urine sample was directly injected into this system. Urinary pantothenic acid is separated in the reversed phase column and hydrolysed to fl-alanine and pantoic acid. The product P-alanine reacts with o-phthaldialdehyde (OPA) and 3-mercaptopropionic acid (3-MPA), and is derivatized to l-alkylthio-2-alkylisoindole which is detected by fluorescence (Takahashi et al. 2009). 

Determination of urinary pantothenic acid by HPLC-fluorimetric method. A 20 pL of urine sample was injected into HPLC system shown in Figure 20.3. Urinary pantothenic acid was detected as a sharp peak of fluorescent compound l-alkylthio-2-alkylisoindole by fluorescence and its retention time was at 14 min (Takahashi et al. 2009). 

FIGURE 2.2 Reaction of o-phlhalaldehyde in the presence of 2-mercaptoetfaanol to form a highly fluorescent l-(alkylthio)-2-alkylisoindole. 

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