MDA-TBA adduct by HPLC

Determination of MDA-TBA adduct by HPLC To avoid spurious absorbance at 532 nm arising from adduct formation of TBA with complexes other than MDA, several methods for the separation of the TBA-MDA complex by reversed-phase HPLC have been developed (Bird et al., 1983 Bird and Draper, 1984 Gilbert et al., 1984 Yu et al., 1986). The methods are very sensitive and specific but because hot acidic conditions are necessary to obtain the adduct there is the possibility of generating MDA artificially. 

Samples are homogenized in 5% TCA, and centrifuged at 1000 xg for 10 min. Adduct is formed with 1% aqueous TBA at 100°C for 30 min, and the pH of the sample is adjusted to pH 1.5 with 4 M HC1 and the sample re-centrifuged. An aliquot of the supernatant fluid is neutralized with 0.15 M NaOH in distilled water/methanol (1 1, v/v) and 10-20 fA then injected onto the column (see below). Alternatively, the adduct may be extracted in n-butanol, the solvent evaporated, and the residue redissolved in mobile phase for injection. The detection limit is about 250 pg of TBA-MDA adduct. 

Mobile phase Flow rate Temperature Detection Sample injection Analysis times Sensitivity 

Note The column should be washed with about 100 ml of methanol after 15-20 determinations. 

Yu et al. (1986) achieved a detection limit of 3.8 pg for the TBA-MDA adduct using fluorometric detection (532 nm excitation 550 nm emission). The adduct was separated on a Zorbax Cl8, 3 //m column (0.63 x 15 cm) or an Altex ODS, 5 /rm analytical column (0.46x25 cm) the columns were eluted with 0.01-0.025 M phosphate buffer (pH 6.5)/methanol (60 40, v/v) at a flow rate of 1.0 ml/min. A presaturation column packed with Corasil silica (35-50 yum) was placed between the pump and the column. Reducing the pH of the mobile phase had little effect on retention time but symmetry and band broadening were worse at more acidic pH probably because the thionyl group of the TBA adduct is increasingly ionized at pH 6.5 and is less attracted to similarly charged residual silanols on the ODS columns. 

---