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内毒素USP36

90〈81〉 Antibiotics—Microbial Assays / Biological Tests USP 36

Table A2-1. Test for Outlier Measurements

In samples from a normal population, gaps equal to or larger than the following values of G1, G2, and G3 occur with a probability P = 0.01, when outlier measurements can occur only at one end; or with P = 0.02, when they may occur at either end.

N34567

G10.9870.8890.7810.6980.637

N8910

G20.6810.6340.597

N111213

G30.6740.6430.617

shows no reaction with the lysate employed, at the detec-〈85〉 BACTERIAL ENDOTOXINS tion limit of the reagent.

Lysate TS—Dissolve Amoebocyte Lysate in Water for BET, TEST or in a buffer recommended by the lysate manufacturer, by

gentle stirring. Store the reconstituted lysate, refrigerated or

frozen, according to the specifications of the manufacturer. x Portions of this general chapter have been harmonized

with the corresponding texts of the European Pharmacopoeia PREPARATION OF SOLUTIONS

and/or the Japanese Pharmacopoeia. Those portions that are

not harmonized are marked with symbols (x x) to specify this

Standard Endotoxin Stock Solution—A Standard Endo-fact.x

toxin Stock Solution is prepared from a USP Endotoxin Refer-The Bacterial Endotoxins Test (BET) is a test to detect or

ence Standard that has been calibrated to the current WHO quantify endotoxins from Gram-negative bacteria using

International Standard for Endotoxin. Follow the specifica-amoebocyte lysate from the horseshoe crab (Limulus poly-

tions in the package leaflet and on the label for preparation phemus or Tachypleus tridentatus).

and storage of the Standard Endotoxin Stock Solution. Endo-There are three techniques for this test: the gel-clot tech-

toxin is expressed in Endotoxin Units (EU). [NOTE—One USP nique, which is based on gel formation; the turbidimetric

Endotoxin Unit (EU) is equal to one International Unit (IU) technique, based on the development of turbidity after

of endotoxin.]

cleavage of an endogenous substrate; and the chromogenic

technique, based on the development of color after cleav-Standard Endotoxin Solutions—After mixing the Stan-age of a synthetic peptide-chromogen complex. Proceed by dard Endotoxin Stock Solution vigorously, prepare appropriate any of the three techniques for the test. In the event of serial dilutions of Standard Endotoxin Solution, using Water doubt or dispute, the final decision is made based upon the for BET. Use dilutions as soon as possible to avoid loss of

gel-clot limit test unless otherwise indicated in the mono-activity by adsorption.

graph for the product being tested. The test is carried out in Sample Solutions—Prepare the Sample Solutions by dis-a manner that avoids endotoxin contamination.solving or diluting drugs using Water for BET. Some sub-

stances or preparations may be more appropriately dis-

solved, or diluted in other aqueous solutions. If necessary, APPARATUS adjust the pH of the solution to be examined (or dilution

thereof) so that the pH of the mixture of the lysate and Depyrogenate all glassware and other heat-stable materi-Sample Solution falls within the pH range specified by the als in a hot air oven using a validated process.x1x A com-lysate manufacturer, usually 6.0–8.0. The pH may be ad-monly used minimum time and temperature is 30 min at justed by use of an acid, base, or suitable buffer as recom-250°. If employing plastic apparatus, such as microplates mended by the lysate manufacturer. Acids and bases may and pipet tips for automatic pipetters, use apparatus that is be prepared from concentrates or solids with Water for BET shown to be free of detectable endotoxin and does not in-in containers free of detectable endotoxin. Buffers must be terfere in the test. [NOTE—In this chapter, the term “tube”validated to be free of detectable endotoxin and interfering includes any other receptacle such as a microtiter well.]factors.

REAGENTS AND TEST SOLUTIONS DETERMINATION OF MAXIMUM VALID

DILUTION (MVD) Amoebocyte Lysate—A lyophilized product obtained

from the lysate of amoebocytes (white blood cells) from the The maximum valid dilution is the maximum allowable horseshoe crab (Limulus polyphemus or Tachypleus dilution of a specimen at which the endotoxin limit can be tridentatus). This reagent refers only to a product manufac-determined. Determine the MVD from the following

tured in accordance with the regulations of the competent equation:

authority. [NOTE—Amoebocyte Lysate reacts to some β-glu-

cans in addition to endotoxins. Amoebocyte Lysate prepara-MVD = (endotoxin limit × concentration of Sample Solution)/ tions that do not react to glucans are available: they are(λ)

prepared by removing the G factor reacting to glucans from

Amoebocyte Lysate or by inhibiting the G factor reacting sys-Endotoxin Limit—The endotoxin limit for parenteral tem of Amoebocyte Lysate and may be used for endotoxin drugs, defined on the basis of dose, equals K/M

x2

testing in the presence of glucans.]

x, where K Water for Bacterial Endotoxins Test (BET)—Use Water

x2

intrathecal (for which K is 0.2 USP-EU/kg of body weight). For radiopharma-for Injection or water produced by other procedures that ceutical products not administered intrathecally, the endotoxin limit is calcu-

lated as 175 EU/V, where V is the maximum recommended dose in mL. For x1Dry-intrathecally administered radiopharmaceuticals, the endotoxin limit is ob-Heat Sterilization under Sterilization and Sterility Assurance of Compendial Arti-tained by the formula 14 EU/V. For formulations (usually anticancer products) cles 〈1211〉. Use Lysate TS having a sensitivity of not less than 0.15 Endotoxin administered on a per square meter of body surface, the formula is K/M,

Unit per mL.x where K = 100 EU/m

2 and M is the maximum dose/m2.x

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