What is 50:50 and hot spike method in BET testing and their importance?

Bacterial Endotoxin Testing is very important test for parenteral preparation. In BET testing generally two methods are followed as mentioned below.

1. 50:50 method
2. Hot spike method


1) 50:50 Method: 

Let's discuss about 50:50 method. In 50:50 method, in negative product control (NPC) sample volume and LRW volume are taken as 50 µL. If we prepare solution of MVD/2 then final results will be given at MVD because after adding same volume of LRW e.g 50 µL and the concentration reduced to half. That's why in 50:50 method we generally perform the BET at MVD/4 or MVD/2 dilution to get the results at MVD/2 or MVD because sample can not be diluted beyond MVD.

2) Hot Spike Method:

In hot spike method,  100 µL sample is required for analysis in negative product control (NPC). Sample is not diluted in hot spike method as there is no addition of LRW in negative product control (NPC). It means if we take MVD/2 sample then testing will be performed at MVD/2. If we take MVD/4 sample then final results will be given at MVD/4. Hot spike method is generally used in the water sample analysis. 

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