Reconstituting a lyophilized vial

Watch the handling

Supplies

• Lyophilized vial
  Sealed vial with the freeze-dried cake. Check the label, lot and the certificate of analysis before opening.
• Sterile diluent
  The diluent named on the product documentation. Bacteriostatic water contains benzyl alcohol; sterile water does not.
• Graduated syringes
  Sized to the volume being measured. U-100 insulin syringes are graduated in units where 100 units = 1 mL.
• Alcohol prep pads
  For wiping vial stoppers before each puncture.
• Nitrile gloves
  Clean gloves for aseptic handling.
• Sharps container
  Puncture-resistant container for used needles.
• Labels and a fine marker
  Record contents, concentration and the date of reconstitution on the vial.
• Refrigerator thermometer
  Confirms cold storage stays within the range the documentation gives.

The steps

1. 
   Step 1

   Prepare a clean workspace

   Wash hands, put on clean gloves and lay out the supplies on a clean surface. Let a refrigerated vial reach room temperature so condensation does not form on the stopper.

2. 
   Step 2

   Wipe both stoppers

   Pop off the small plastic flip caps (the aluminum crimp ring stays on) and wipe each exposed rubber stopper with a fresh alcohol pad. Let them air-dry. Flip caps cannot be put back once removed.

3. 
   Step 3

   Draw the diluent

   Insert a new sterile needle through the center of the diluent vial's rubber stopper, turn the vial upside down and draw the volume you chose; the needle stays in the stopper the whole time. Use the calculator to see what concentration that volume gives.

4. 
   Step 4

   Add it slowly down the wall

   Insert the needle straight down through the center of the powder vial's rubber stopper. Tilt the vial and angle the needle tip toward the inside glass wall so the diluent runs gently down the wall rather than onto the cake. A vacuum in the vial may pull liquid in; control the plunger, then withdraw the needle straight up.

5. 
   Step 5

   Swirl, do not shake

   Gently roll or swirl the vial until the cake dissolves. Shaking can foam the solution and may damage some peptides. Inspect: the solution should be clear, without particles.

6. 
   Step 6

   Label and refrigerate

   Write the concentration and date on the label, store as the documentation directs (usually refrigerated, protected from light) and discard used sharps in a sharps container.

The arithmetic

Concentration = mass in the vial ÷ volume of diluent. Everything else follows from that.

Mass in the vial (as labeled)

2 mg5 mg10 mg20 mg30 mg50 mg

Other: mg

Diluent added

1 mL2 mL3 mL5 mL

Other: mL

Concentration

5 mg/mL

5,000 mcg/mL

In 10 units (0.1 mL)

500 mcg

In 1 unit (0.01 mL)

50 mcg

Find the volume for an amount you enter

mcg

Computed from your inputs. Not a dose. Verify before use.

What each syringe mark contains at this concentration

Units (U-100)	5	10	20	25	50	100
mL	0.05	0.1	0.2	0.25	0.5	1
mcg	250	500	1,000	1,250	2,500	5,000

Arithmetic on the numbers selected. A unit is a volume mark (100 units = 1 mL), not an amount of compound. General reconstitution steps · Full calculator

Micrograms in 10 units, common combinations

Reading a U-100 syringe

• 100 units = 1 mL; 50 units = 0.5 mL; 10 units = 0.1 mL; 1 unit = 0.01 mL.
• 0.3 mL syringes are usually marked every half or one unit; 1 mL syringes every two units.
• A unit is a volume, not an amount of compound. The amount in a given volume depends on the concentration you made.

Storage after reconstitution

• Lyophilized (unmixed) vials: typically stored cold and dry, away from light, as the documentation directs. Many are stable longer frozen.
• Reconstituted vials: typically refrigerated (about 2–8 °C), protected from light and not frozen.
• Bacteriostatic water contains benzyl alcohol, which inhibits bacterial growth in multi-use vials; sterile water has no preservative.
• Transport: an insulated case with a cold pack keeps vials cool; avoid direct contact between ice packs and glass.
• Keep a fridge thermometer next to the vials; household refrigerators can swing several degrees.

Mistakes and troubleshooting

Added the wrong volume of diluent

The concentration changes, not the amount of compound in the vial. Recalculate with the volume actually added and relabel the vial.

Shook the vial and it foamed

Let it settle in the refrigerator. Foam is a sign of agitation; some peptides aggregate when shaken. Note it on the label.

The solution is cloudy or has particles

Do not use it. Cloudiness can indicate incomplete dissolution, contamination or degradation. Record the lot and contact the supplier with the certificate of analysis.

The powder cake looks melted or shrunken before reconstitution

This can indicate the vial was exposed to heat or moisture. Record the lot and condition on arrival.

Liquid sprayed back when the needle was removed

Pressure built up in the vial. Withdraw a small volume of air while adding diluent to keep pressure neutral.

Confused units with mL

On a U-100 syringe, 100 units = 1 mL and 10 units = 0.1 mL. A unit is a volume mark, not an amount of compound.

Froze a reconstituted vial

Freeze–thaw cycles can degrade peptides in solution. Follow the documentation; most reconstituted vials are kept refrigerated, not frozen.

Lost track of when it was mixed

Always date the label. Stability after reconstitution is limited and differs between compounds and diluents.

Frequently asked questions