Drug Making Protocol – Jonathan Chen, 5/26/2009
Methacholine Chloride
1. Take 5 mL of sterile USP grade water and inject into 100mg Provocholine (brand-name for USP
certified methacholine chloride). Shake well. Label as Vial A; 20mg/mL
2. Take 0.1 mL of Vial A + 4.9 mL of sterile water and mix into sterile container. Shake well. Label as
Vial B; 0.4 mg/mL
3. Take 0.1 mL of Vial B + 4.9 mL of sterile water and mix into sterile container. Shake well. Label
as Vial C; 8ug/mL
4. Take 0.1 mL of Vial C + 3.9 mL of Lactated Ringer’s (if you want ultra sterile, use a 0.22 um filter).
Label as Vial D; 0.2ug/mL (or 1 micromolar). Shake well.
Cocktail (Sato’s concentration)
1. Take 0.02 mL (0.0198176 if exact) of 0.2 mg/mL isuprel (isoproterenol) + 0.02 mL (0.0183 mL if
exact) of 25 mg/mL aminophylline + 0.03 mL (0.023772 if exact) of 0.4 mg/mL atropine + 0.13
mL of Lactated Ringer’s to make 0.2 mL of solution.
2. Inject this whole solution intra dermally.
Cocktail (Jeff Wine concentration, which is double Sato’s amount but only 0.1 mL is injected under the
skin).
1. Take 0.4 mL of isuprel + 0.37 mL of aminophylline + 0.48 mL of atropine + 0.75 mL of Lactated
Ringer’s to make 2 mL of “Jeff wine cocktail” solution.
2. Shake well.
3. Take out 0.1 mL of solution to inject
How to make Cocktail Dilutions
1. Follow above JW cocktail directions to make 1:1 cocktail.
2. Dilute as necessary; it may take many steps. For example, to make 1/500 cocktail dilution, may
need to do a 1:50 dilution, followed by a 1:10 dilution.
3. For the LAST dilution, make sure you add more atropine so that the relative amounts of atropine
are the same as for 1:1 cocktail. This is to ensure that the cholinergic receptors are blocked.
4. Hint for atropine: For a Jeff Wine cocktail, an injection of 0.1 mL requires 0.023772 mL (to keep
the same amount of drugs as Sato’s) of atropine in it. Therefore, if your resultant dilution will be
2.0 mL, you need to have a final atropine volume of 20 x 0.023772= 0.47544 mL or 0.48 mL of
atropine
Storage Instructions: Everything should go in the 4 degree Celsius refrigerator.
1: Cocktail: may turn yellow over 1-2 months. 2: Aminophylline: may re-crystallize over 1-2 months
3: Methacholine: nothing special noted so far