Reconstitution

A lyophilised peptide arrives as a freeze-dried solid, often as a barely visible film at the bottom of the vial rather than as an obvious powder. Reconstitution is the step that turns it into a solution you can measure. It is simple, and there are only two places it usually goes wrong: the diluent, and the speed at which it is added.

Choosing a diluent

Bacteriostatic water is the default. The 0.9 percent benzyl alcohol it carries is a preservative, which means one vial can be entered repeatedly without the solution becoming a growth medium. That is what makes it suitable for a vial you will draw from more than once.

Sterile water carries no preservative. It is the right choice when the benzyl alcohol would interfere with the compound or the assay, and the wrong choice for anything you intend to enter more than once.

The procedure

1. Let the vial reach room temperature. Opening cold glass draws condensation into the vial, and moisture is what degrades lyophilised powder.
2. Wipe the stopper with a 70 percent isopropyl pad and let it air dry. Seventy percent is more effective than higher concentrations, which evaporate before they act.
3. Add the diluent down the inside wall. Angle the needle so the stream runs down the glass rather than landing directly on the powder. A jet directed onto the solid is the most common cause of denaturation at this step.
4. Leave it alone. Do not shake. Most peptides dissolve within a minute or two at room temperature. Swirl gently only if material remains after several minutes.
5. Label and refrigerate. Record the compound, the fill weight, the diluent volume, the resulting concentration and the date.

Working out the concentration

Concentration is fill weight divided by diluent volume. A 10 mg vial with 2 ml of bacteriostatic water gives 5 mg/ml. To find the volume to draw for a given amount, divide that amount by the concentration. On a U-100 insulin syringe, multiply the volume in millilitres by 100 to get the unit marking, because 100 units is exactly 1 ml.

Vial	Diluent	Concentration	250 mcg draws to
5 mg	1 ml	5.00 mg/ml	5 units
5 mg	2 ml	2.50 mg/ml	10 units
10 mg	2 ml	5.00 mg/ml	5 units
10 mg	5 ml	2.00 mg/ml	12.5 units

The reconstitution calculator does this for any combination and marks the result on a drawn syringe, including a warning when the draw falls below the range where the graduations can be read accurately.

After reconstitution

A reconstituted vial is refrigerated from that point on. It is no longer stable at ambient temperature the way the lyophilised powder was, and it should not be frozen and thawed repeatedly. If a stock will be drawn from over a long period, aliquoting it into smaller sterile vials limits how many times any one vial is punctured. See storage and handling for what changes once the compound is in solution.