Reconstitution Guide

Reconstitution is the step where a lyophilised peptide is brought back into solution under controlled conditions. It is also where most avoidable variability enters a study, so it is worth doing deliberately and recording carefully.

Equipment

Work at a clean bench with the following to hand:

• Sterile diluent (bacteriostatic water or the diluent specified in your protocol)
• Graduated syringes sized to the volume you are transferring
• Alcohol swabs
• A labelled vial rack
• A batch log sheet for the session

Bring the vial to room temperature slowly rather than warming it directly. Rapid temperature change draws moisture into a cold vial.

Calculating the diluent volume

The only number that matters at this stage is the resulting concentration. For a vial of mass m milligrams taken up in v millilitres of diluent, the concentration is:

concentration (mg/mL) = m / v
concentration (µg/µL) = concentration (mg/mL)

So a 10 mg vial taken up in 2 mL gives 5 mg/mL, which is also 5 µg/µL. If you need a different working concentration, change v — never the number of times you enter the vial.

The Research Solution Calculator on this site performs the same arithmetic and prints the full set of values for your session notes.

Technique

1. Swab the stopper of both the diluent and the peptide vial. Let the alcohol flash off fully.
2. Draw the calculated diluent volume. Expel any air from the syringe before entering the vial.
3. Insert the needle at a slight angle through the centre of the stopper and run the diluent down the inside wall of the vial. Never aim the stream at the powder cake — direct impact is a common cause of foaming and aggregation.
4. Lift the needle clear of the liquid before withdrawing, so you do not pull solution back into the barrel.
5. Swirl the vial gently between finger and thumb until the powder is fully dissolved. Do not shake. If the solution is not clear, keep swirling and allow more time.

Record keeping

Write down, at the time and not afterwards:

• Product name, batch number and fill weight
• Diluent type and volume added
• Resulting concentration
• Date and time of reconstitution
• Operator initials

The batch log sheet supplied in the Reconstitution Kit is laid out for exactly this and gives you a dated record to attach to your notebook.

Storage after reconstitution

Reconstituted solutions are less stable than the lyophilised powder. Follow your protocol's storage instruction; where the protocol does not specify one, keep the solution cold, protected from light, and use it within the shortest window your study allows. Freeze–thaw cycles should be avoided: split into single-use aliquots at the time of reconstitution if a longer working life is needed.