Plasma samples (500 [micro]L) were mixed with 1500 [micro]L of ice-cold 100 g/L TCA containing 15 nmol/L isoriboflavin, which served as an internal standard.
Isoriboflavin served as the reference calibrator for riboflavin, FMN, and FAD, and riboflavin was used as the reference calibrator for isoriboflavin.
To study the stability of the internal standard in TCA used for plasma protein precipitation, we studied aliquots of 100 g/L TCA containing 150 nmol/L isoriboflavin incubated for 1, 2, 4, 7, and 14 days.
We investigated the stability of the vitamers and the internal standard in TCA-treated plasma before neutralization by adding 500 nmol/L riboflavin, FMN, FAD, or isoriboflavin to the samples.