CAP CODE Pyrochrome® Chromogenic Endotoxin Testing


or the Detection and Quantification of Gram Negative BacterialEndotoxins (Lipopolysaccharides)

The Limulusamebocyte lysate (LAL) test, when used according to U.S. Food and DrugAdministration (FDA) guidelines (1), may be substituted for the U.S. Pharmacopeia (USP)Pyrogen Test (rabbit fever test) for the end-product testing of “human injectable drugs(including biological products), animal injectable drugs, and medical devices.” The LAL test isrecommended for the quantitation of endotoxin in raw materials used in production,including water, and for in-process monitoring of endotoxin levels. The USP BacterialEndotoxins Test (2) is the official LAL test referenced in specific USP monographs.

Summary of Test

Limulusamebocyte lysate is an aqueous extract of blood cells (amebocytes) from thehorseshoe crab Limulus polyphemus. In the presence of endotoxin, factors in LAL are activatedin a proteolytic cascade that results in the cleavage of a colorless artificial peptide substratepresent in Pyrochrome LAL. Proteolytic cleavage of the substrate liberates p-nitroaniline(pNA), which is yellow and absorbs at 405 nm. The test is performed by adding a volume ofPyrochrome to a volume of specimen and incubating the reaction mixture at 37°C. The greaterthe endotoxin concentration in the specimen, the faster pNA will be produced. Pyrochromecan be used to quantify endotoxin concentration in two ways (3).In the kinetic method, the time taken to reach a particular absorbance at 405 nm (the onsettime) is determined. Higher endotoxin concentrations give shorter onset times. The assayrequires specialized instrumentation to incubate multiple samples at a controlled temperature(usually 37°C) and to take optical density readings at regular intervals. Standard curves may beconstructed by plotting the log onset time against the log concentration of standard endotoxinand are used to calculate endotoxin concentrations in specimens.Alternatively, in the endpoint chromogenic method, the amount of pNA released can bemeasured following a fixed incubation period. A standard curve, consisting of measuredoptical density plotted against known standard endotoxin concentration, is used to determineconcentrations in specimens. For samples that absorb at 405-410 nm, a diazo-couplingmodification may be used. In this method, pNA (formed during the endpoint chromogenicmethod) is reacted with nitrite in HCl and then with N-(1-Naphthyl)-ethylenediamine (NEDA)to form a diazotized magenta derivative that absorbs at a range between 540-550 nm.The Pyrochrome LAL methods are rapid, specific, easy to perform, and highly sensitive. Thedetection limit depends on the method and instrumentation used and may be as sensitive as0.005 Endotoxin Units (EU) per mL.

History and Biologic Principle

Howell described the clotting of Limulusblood in 1885 (4). In the 1950’s, at the MarineBiological Laboratory, Woods Hole, MA, Bang discovered that gram negative bacteria causeLimulusblood to clot (5). Levin and Bang later determined that the reaction is enzymatic andthat the enzymes are located in granules in the amebocytes (6, 7). They showed that clotting isinitiated by a unique structural component of the bacterial cell wall called endotoxin orlipopolysaccharide (8). Current understanding is that the reaction consists of a cascade ofenzyme activation steps terminating in the cleavage of the peptide, coagulogen. The insolublecleavage product of coagulogen (coagulin) coalesces by ionic interaction. If sufficient coagulinforms, turbidity appears followed by the formation of a gel-clot. This interaction forms thebasis of an assay for endotoxin termed the Limulusamebocyte lysate (LAL) test. In 1977,Japanese investigators discovered that endotoxin-activated LAL would also cleave smallchromogenic peptides which contained an amino acid cleavage site similar to coagulogen andthe chromophore, paranitroanilide (9). Cleavage results in the release of pNA, which is yellowand absorbs light at 405 nm. In this chromogenic adaptation of the LAL assay, coagulogenconcentration is reduced by dilution to minimize interference when chromogenic substrate isadded to the LAL. Thus, when endotoxin is added to chromogenic LAL, a color is formed inpreference to turbidity or a gel-clot. In all versions of the LAL assay (gel-clot, turbidimetric, andchromogenic), the greater the amount of endotoxin present, the faster the endpoint (gel-clot,turbidity, or color) develops. More information about the LAL assay types, reaction, andapplications is available in the literature (10, 11, 12).

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