Bioequivalence and Bioavailability Forum

Hi Norah,

❝ I'm preparing a BE protocol regarding, enteric delaying release aspirin […] Could you please tell me what are you recommendations to avoid the degradation of active ingredient in metabolite.

In my CRO we had two methods (anticoagulant K₃EDTA or Li-heparin). Example for 10 mL vacutainers.

1. Stabilizer H₃PO₄
   Prepare 5% orthophosporic acid (59 mL 85% o-H₃PO₄ p.a. diluted to 1000 mL).
   Caution: Even diluted phosphoric acid is strongly acidic; wear gloves and eye protection.
   
2. Stabilizer potassium fluoride
   Prepare cold saturated KF solution (100 g KF p.a. in 100 ml water).
   Caution: KF is poisonous. Observe the usual rules.
   

Penetrate the stopper of 10 mL vacutainers (e.g., by a 200–500 µL HPLC syringe with a thin needle) and inject 100 µl of the respective stabilizer. Use these for all samples.

Handling & storage

Put blood samples immediately after draw in an ice-bath. Allow 10 minutes for equilibration and centrifuge at ~2000×g at +4 ℃ for 20 minutes.

1. Mix plasma with 5% H₃PO₄ 1:1 (exactly!). Store at ≤20 ℃.
   
2. Store at ≤70 ℃.

If you have no problems with sensitivity I would suggest #1. However, the final dilution step is labour-intensive. #2 gives a ~2times lower LOQ but the deep-freezer will suck up a lot of electricity…