Need support for learning, How to prepare stock solution for 10, 100 and 1000 micro-gram/kg API drug for intravenous administration, sub-acute study in both rats and mice. Would appreciate if one can provide complete answer.
If the question is about concentration, it can be easily done by using repetitive dilutions. For example, mixing 1 mL of 1000 mg/mL solution and 9 mL of solvent will provide 10 mL of 100 mg/mL solution, and so on.
Dear Avinash,
First question to answer: How many animals are you using? How much volume of each solution (10, 100 and 1000µg/kg) are you injecting and how many times into the same animal?
Second Q: what is the average weight of the lab animals (mice and/or rats) you are using?
Third Q: Do you have any constraint regarding the amount of API (for instance only a limited amount is available or it is very expensive)?
You need to know the number of animals you will be using as well as the injected volume. Example: Let’s assume your mice have an average weight of 20 g and the rats, of 200g and that the injected volume (IV) is 0.15 mL for mice and 0.40 mL for rats).
Example with mice:
The concentrations of 1000, 100 and 10 µg/Kg transform to:
1000 µg/1000g * 20g / 0.15mL = 133.3 µg/mL
100 µg/1000g * 20g / 0.15mL = 13.33 µg/mL
10 µg/1000g * 20g / 0.15mL = 1.333 µg/mL
Let’s suppose you will be using 15 mice in each group and each animal will be injected three times (once daily). Therefore you will need 6.75 mL of each solution (15 mice * 3 inj/mouse * 0.15 mL/inj).
Let's say, v1, v2 and v3 are the volumes you need for each final solution (133.3, 13.33 and 1.333µg/mL respectively) .
Taking into consideration the above example, v1=v2=v3 = 6.75 mL
Multiply the volume by its concentration and you get the exact mass of API (expressed in mg) required to prepare each one of them:
mA0 (mg) = (133.3*v1 + 13.33*v2 + 1.333*v3) * 1000 = 0.999 mg
Now, round up mA0 to a value as small as possible, but higher than mAPI (I would say, at least about 10% more), lets call it mA1 and find out a volume "Vss" such as the stock solution "SS" has a concentration not lower than 133.3 µg/mL (in your case):
mA1/Vss => 133.3 µg/mL.
If mA1 is larger than 10mg and Vss > 10 mL prepare your SS such as its concentration matches 133.3µg/mL. Otherwise and if the drug is expensive or the amount available limited, you need to dilute SS to get the 133.3 µg/mL sol.
We would need (without taking into account any losses) about 1 mg of the drug.
Depending on your laboratory conditions you could prepare, for instance, 15 mL of a 133.3 µg/mL by weighing 2.00 mg of drug and dissolving in 15mL which would match your most concentrated solution.
In this example, we will weight 10.0 mg of drug and dissolve it in 5.0 mL to get a 2000µg/mL stock solution.
Now, depending on the glassware and equipment available you should choose the actual volumes to prepare the 133.3, 13.33 and 1.333µg/mL solutions that need to be not less than v1, v2 and v3.
Prepare the more concentrated solution (133.3µg/ml) by diluting SS (if needed) to get a final volume larger than v3 + v2/10. (6.75+0.675=7.43mL)
For instance, using a micropipette, take 667µL of the stock solution (2000µg/mL) and dilute it to 10.0mL. You will get: 0.667mL * 2.00mg/mL / 10.0mL= 133.4µg/mL
Now, round up v2 to a suitable (larger) volume to match your glassware, pipettes or micropipettes available (v2a). For instance v2a=10 mL.
Take "v2a/10" mL (10mL/10=1mL) from the 133.4µg/ml solution into a suitable flask and complete the volume with the solvent. You get now 1mL * 133.4/10 mL=13.34 µg/mL.
Now, round up v3 to a suitable (larger) volume to match your glassware, pipettes or micropipettes available (v3a). For instance v3a=10 mL.
Take "v3a/10" mL (10mL/10=1mL) from the 13.34µg/ml solution into a suitable flask and complete the volume with the solvent. You get now 1mL * 13.34/10 mL=1.334 µg/mL.
You should take into account that the volumes prepared for each solution are enough to account for the volume actually needed to be injected plus the volume needed to prepared the next less concentrated solution.
For rats, just start from the beginning replacing the average weight and injected volumes by the appropriated ones and continue the calculations.
Hope its clear and helpful, Luis
Thank you for your detailed and clear explanation. it was very helpful and understandable.
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