The Limulus Amebocyte Lysate (LAL) test is a technique for detecting endotoxins that uses a reagent formulated from the blood cells of the Limulus horseshoe crab. water for the Bacterial Endotoxin Test (BET) plays an extremely important role in the LAL Test. Although water is often an overlooked substance, the quality of water for the LAL test can have a profound impact on the results; consequently, this post will consider the topic of water.

As a fundamental requirement, water for LAL Tests should be free of LAL-activating substances such as endotoxins and β-glucans. Although this requirement can essentially be achieved through distillation, it is necessary to closely monitor for potential contamination from handling, storage environments, and eluates from containers after the water is distilled.

In many cases, heat treatments such as autoclaving can deactivate small amounts of endotoxins that lead to contamination. Endotoxins are known to be heat-resistant toxins, but they can be deactivated with heat in low-concentration solutions

In contrast to endotoxins, however, aqueous solutions containing β-glucans are extremely stable to heat treatments. Consequently, it is very difficult to eradicate β-glucans through heat treatments. Therefore, it is necessary to pay thorough attention to β-glucan contamination in water for BET.

Another important point that must be considered is how to prevent the water from altering the activity of endotoxin.

For example, trace amounts of iron or aluminum ions mixed in the water may curtail endotoxin activity. As described in Episode 10, our research found that the presence of iron and aluminum ions in the order of micromoles led to a 50% or more decrease in endotoxin activity in aqueous solutions.

Water for injection in some commercially-sold ampoules is actually known to reduce endotoxin activity. We learned that when endotoxins were diluted in water for injection from ampoules, activity fell to 1/100 or less of the initial level (i.e., endotoxicity in a solution prepared with 20 EU/mL fell to 0.2 EU/mL after dilution) (unpublished data).

Indeed, we found that the co-presence of a phosphate buffer suppresses reduction in endotoxin activity points to metal ions as the culprit. This phenomenon discovered by our group is believed to be highly significant because of its great relevance to the issue of reference standard endotoxins for LAL Testing. Nevertheless, in many cases current protocols only emphasize that water for BET must be "endotoxin-free" and neglect any other effects of water on endotoxin activity.

In the Japanese Pharmacopoeia Official Monographs section (Part II)¹⁾, water for injection can be used as water for BET assuming that no gelation is observed when the Bacterial Endotoxin Test is applied using 0.10 mL of said water.

Since the standard endotoxin limit for water for injection is set at ≤0.25 EU/mL, it can be assumed that the criteria for water for BET should be the same. However, the Japanese Pharmacopoeia recommends that "the endotoxin limit should be set at an even lower level when using water for BET with a LAL reagent of high sensitivity," a passage which could be considered as open to interpretation.

The Japanese Industrial Standards²⁾ (JIS) also mentions water for BET, stipulating that it should contain "an endotoxin content of ≤0.006 EU/mL or an endotoxin concentration that is equal to or less than values given in individual specifications." The JIS lists the gel-clot, kinetic turbidimetric, and chromogenic assays, and the organization has adopted a somewhat stricter endotoxin limit for water for BET to facilitate these techniques.

The United States Pharmacopeia³⁾ defines water for BET as "LAL reagent water" that is "sterile water for injection or other water that shows no reaction with the specific LAL reagent with which it is to be used." Moreover, the European Pharmacopoeia⁴⁾ also advises the use of "water LAL" with the same specification.

References

1. Guide to the Japanese Pharmacopoeia 12th Edition (B-529). (1991). Tokyo: Hirokawa-Shoten.
2. General rules for biochemical reagents (JIS K 8008, pp. 23). (1992). Tokyo: Japanese Standards Association.
3. The United States Pharmacopeia 22th, The National Formulary 17th, p.1493-1495, Pharmacopeial Convention Inc., MD (1989).
4. European Pharmacopoeia, V. 2. 1. 9., Maisonneuve S. A., France (1987).