What We Need to Know in a Recovery Test
This article was written by Dr. Masakazu Tsuchiya, FUJIFILM Wako Pure Chemical Corporation, for Vol. 64, No. 2(April 1996) of Wako Junyaku Jiho.
The content of this article is from the time of publication. It is not the latest information due to new knowledge and changes in regulatory rules after original publication.
We often get these questions that need to be addressed when performing an endotoxin recovery test: 1) whether is it necessary to classify and distinguish the effects of a sample, and 2) what type of endotoxin standard should be used. When answering these questions, one must consider the state of endotoxin to be measured. The biological activity of endotoxin cannot be simplified into an absolute amount, as the size of micelles is not constant. This property makes quantification more complex.
Because the activity of endotoxin can change when endotoxin is added to a sample, one must determine whether the activity should be measured before or after the addition. One might choose to use the LAL test to measure the amount of endotoxin detected in the sample since this amount reflects the biological activity of endotoxin in the sample at the time of measurement.
Alternatively, one might determine the potential activity before endotoxin is altered in the sample since the activity may be changed by the sample. In this case, one would estimate the potential activity of endotoxin before being altered by the sample by dividing the measured amount by the recovery rate.
The former does not take into account the possible change in the activity of endotoxin in a sample if the sample is transferred to another environment. For example, if the test drug is a pharmaceutical, its administration into the body may increase the activity of endotoxin as it comes in contact with body fluid.
We examined the reduction of endotoxin activity by metal ions and demonstrated that the change in the outcome of the LAL test was equivalent to that of the rabbit pyrogen test. This suggests that the measurement of endotoxin activity after the change may be valid. Note, however, that the activity of endotoxin may change further.
The latter assumes that there is a constant change in the activity of endotoxin in a sample regardless of the type of endotoxin during recovery testing. However, it is difficult to predict the properties of endotoxin already present in the sample. Therefore, the estimated activity of endotoxin added to the sample may not truly reflect the activity prior to its addition.
While both methods may be valid, we cannot conclude which method is more suitable at this point. I believe that in the context of endotoxin recovery testing, there has not been a lot of focus on the potential change in the activity of endotoxin. Because of the significance of endotoxin measurements, we need to consider what the measured endotoxin level actually indicates.
Let us go back to the focus of this article, which is to discuss what we need to know in a recovery test. A recovery test is one of the tests performed to confirm the effectiveness of the LAL test. Change in the activity of endotoxin is a critical component to consider in the measurement of endotoxin activity by the LAL test.
However, since the potential for change in endotoxin activity is not fully understood, it is not appropriate to use it as a factor to determine the effectiveness of LAL tests. Instead, what we need to do at this point is to examine the effects of a sample on the reaction system under conditions that don't significantly alter endotoxin activity.