10.6 m hcl

Furosine (FUR) was measured in NFDM as described by Resmini et al. (1990) with minor modifications. Between 110 and 140 mg of NFDM was accurately weighed into a screw-cap Pyrex tube with a PTFE-lined septa along with 8 mL of 10.6 M HCl (Sigma Aldrich). Nitrogen gas was bubbled through the samples for 2 min and then capped and heated to 110°C for 23 h. After cooling to room temperature, the hydrolysate was filtered and 0.5 mL of filtrate was loaded onto a solid-phase extraction column (500 mg Discovery DSC-18, Supelco) previously conditioned with 5 mL of methanol followed by 10 mL of water. The FUR was eluted with 3 mL of 3 M HCl. Detection was performed with a Waters HPLC system (Milford, MA). A FUR dedicated column was used (Altima C8, 250 × 4.6 mm, 5μm, Alltech-Grace, Columbia, MD) with isocratic conditions. The mobile phase consisted of 0.4% acetic acid (Sigma Aldrich) and the column temperature was 35°C. Ultraviolet detection was performed at 280 nm. A 5-point external standard curve ranging from 50 to 400 ng was constructed for quantification.

The method of Resmini et al. (1990) was followed to analyze furosine with minor modifications. An amount of NFDM/MPC70 corresponding to 40 to 50 mg of protein was placed in a screw-cap Pyrex tube with a PTFE-lined septa along with 8 mL of 10.6 M HCl (Sigma Aldrich). Nitrogen was bubbled through the samples for 2 min and then capped and heated to 110°C for 23 h. After cooling, the hydrolysate was filtered and 0.5 mL was loaded onto a 500-mg reversed-phase solidphase extraction column (Discovery DSC-18, Supelco) previously conditioned with 5 mL of methanol and 10 mL of water. Furosine was eluted with 3 mL of 3 M HCl. Furosine was separated and detected with a Waters HPLC system (Milford, MA). A furosine-dedicated column was used (Altima C8, 250 × 4.6 mm, 5 μm, Alltech-Grace, Columbia, MD) with isocratic conditions. The mobile phase consisted of 0.4% acetic acid (Sigma Aldrich) and the column temperature was 35°C. The UV detection was performed at 280 nm. A 5-point external standard curve ranging from 50 to 400 ng was constructed for quantification of both NFDM and MPC70.