Dry-heat destruction kinetics of lipopolysaccharides from Escherichia coli, Serratia marcescens, and Salmonella typhosa at 170 to 250 degrees C are described. The destruction rate seems to follow the second order and can be linearized by the equation, log y = a + b . -10cx. Because c is the slope, 1/c = D3. Both a and b are constant at a given temperature and are linear functions of temperature. The D(3)170, D(3)190, D(3)210, D(3)230, and D(3)250 values for E. coli lipopolysaccharide are 251, 99.4, 33.3, 12.3, and 4.99 min, respectively, with a z value of 46.4 min. The D values for lipopolysaccharides from S. marcescens and S. typhosa are not significantly different from those from E. coli lipopolysaccharide.
Six limulus amebocyte lysate (LAL) preparations obtained from five different suppliers were evaluated for sensitivity, dependability, cost, convenience of use, and correlation with the United States Pharmacopeial (USP) rabbit pyrogen test method. Endotoxins from various gram-negative microorganisms were used for the evaluation. Major differences among the LAL preparations lie in the area of sensitivity. Differences, up to 100-fold, exist in the sensitivity of the various LAL preparations to the same endotoxin. The LAL tests in general were 3 to 300 times more sensitive than was the USP rabbit pyrogen test method. The LAL and the USP rabbit pyrogen test data correlated well when the endotoxin in a relatively pure and undegraded form was examined. However, large discrepancies in correlation were found when partially degraded endotoxins were compared. One LAL preparation responded to both intact and degraded endotoxin, whereas others responded only to intact endotoxin; the latter closely correlated with the febrile response of the rabbit. Therefore, proper selection of an LAL preparation is important for its application in clinical, pharmaceutical, public health, and environmental areas.
A Limulus amoebocyte lysate gel-clotting method for the determination of endotoxin in a small-volume parenteral product has been described. Sample dilution with 0.1 M potassium phosphate monobasic buffer (pH 8.0) effectively eliminated assay interference, whereas dilution with water did not. The threshold pyrogenic dose for Escherichia coli EC-2 and O127:B8 endotoxins was determined to be 1.0 ng of endotoxin per kg of body weight. Not more than 1.0 ng of endotoxin (the threshold pyrogenic dose) per the highest recommended human dose or the USP pyrogen test dose per kg of body weight, whichever dose is more stringent, is a logical limit for the quantity of bacterial endotoxin in small-volume parenteral products. Excellent correlation was attained when this criterion was used to compare the Limulus amoebocyte lysate assay with the USP pyrogen test.
A mathematical model was developed to estimate the number of logarithmic cycles (LDec) of lipopolysaccharide concentration destroyed by a dry-heat sterilization process. The LDec values calculated from the mathematical model agreed well with those obtained from the destruction of lipopolysaccharide by a dry-heat treatment. A discussion of how the mathematical model may be used to evaluate a dry-heat sterilization cycle is presented. This mathematical model and the dryheat destruction curves indicated existence of a maximum LDec value at each temperature. The implications of this finding are discussed.
The chromogenic substrate Limulus amebocyte lysate (LAL) assay method for the detection of endotoxin was automated by a Zymate robotic system. The software developed enables the robot to automatically dilute a stock reference endotoxin standard (20,000 endotoxin units per ml) for the construction of a five-point standard curve, make sample dilutions to the proper testing concentration, and perform chromogenic substrate LAL assays in duplicate. The linearity of the standard curve and the endotoxin concentration in each sample are calculated and results are printed automatically. In 48 min the automated system assays three samples and a reference standard in duplicate along with a water blank. Sensitivity of the assay is a function of incubation time. The assay is linear (r greater than 0.99) in the region of 0 to 1.0 endotoxin units per ml or 0 to 0.2 endotoxin units per ml with incubation times of 10 or 16 min, respectively. The method can be made very sensitive, detecting as low as 0.003 endotoxin units per ml with 30 min of incubation. The precision of the assay method, determined by assaying an endotoxin reference solution eight times, is ca. 6%. The LAL reagent designed for gel-clot assay was modified for the chromogenic substrate assay. We describe the optimum conditions for the performance of the chromogenic substrate LAL assay and stability of the LAL reagent.
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