My calibration graph must start from at least 50 ppb. Also when I am analysing it in food samples, I need to detect at least 20 microgram pf B12 per Kg of food. Thats certainly very difficult. Does any one use an AOAC HPLC method ?
If you have to analyse vitamins from food samples at 50 ppb level I think it is difficult to analyse with HPLC-UV detector. The reason you may not get the sensitivity and linearity at this level using HPLC.
You may use HPLC-MS(TQD/SQD) configuration which can detect at lower ppb level vitamins with very high accuracy and precision from complex food samples
I have attached a application note of all water soluble vitamin analysis from food sample. This u may use as a reference for HPLC-UV method development.