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内毒素USP36

90〈81〉 Antibiotics—Microbial Assays / Biological Tests

Table A2-1. Test for Outlier MeasurementsUSP 36

In samples from a normal population, gaps equal to or larger than the following values of G 1, G 2, and G 3 occur with a probability P = 0.01, whenoutlier measurements can occur only at one end; or with P = 0.02, when they may occur at either end.

N 89

G 30.6740.643

〈85〉 BACTERIAL ENDOTOXINSTEST

x

with the corresponding texts of the Portions of this general chapter have been harmonized

and/or the not harmonized are marked with symbols (Japanese Pharmacopoeia. Those portions that areEuropean Pharmacopoeia

x

fact. x ) to specify this

x

quantify endotoxins from Gram-negative bacteria usingThe Bacterial Endotoxins Test (BET) is a test to detect or

amoebocyte lysate from the horseshoe crab (phemus or Tachypleus tridentatus). Limulus poly-

nique, which is based on gel formation; the turbidimetricThere are three techniques for this test: the gel-clot tech-

technique, based on the development of turbidity aftercleavage of an endogenous substrate; and the chromogenictechnique, based on the development of color after cleav-age of a synthetic peptide-chromogen complex. Proceed byany of the three techniques for the test. In the event ofdoubt or dispute, the final decision is made based upon thegel-clot limit test unless otherwise indicated in the mono-graph for the product being tested. The test is carried out ina manner that avoids endotoxin contamination.

APPARATUS

als in a hot air oven using a validated process.Depyrogenate all glassware and other heat-stable materi-

x 1

monly used minimum time and temperature is 30 min atx A com-

250and pipet tips for automatic pipetters, use apparatus that is°. If employing plastic apparatus, such as microplates

shown to be free of detectable endotoxin and does not in-terfere in the test. [includes any other receptacle such as a microtiter well.]NOTE —In this chapter, the term “tube”

REAGENTS AND TEST SOLUTIONS

from the lysate of amoebocytes (white blood cells) from theAmoebocyte Lysate—A lyophilized product obtained

horseshoe crab (tridentatus Limulus polyphemus or Tachypleus

tured in accordance with the regulations of the competent). This reagent refers only to a product manufac-

authority. [cans in addition to endotoxins. NOTE —Amoebocyte Lysate reacts to some β

tions that do not react to glucans are available: they areAmoebocyte Lysate prepara--glu-

prepared by removing the G factor reacting to glucans fromAmoebocyte Lysatetem of testing in the presence of glucans.]Amoebocyte Lysate or by inhibiting the G factor reacting sys- and may be used for endotoxin

for Injection or water produced by other procedures thatWater for Bacterial Endotoxins Test (BET)—Use Water

x 1

Heat SterilizationDry-

cles under Sterilization and Sterility Assurance of Compendial Arti-

Unit per mL.〈1211〉. Use Lysate TS having a sensitivity of not less than 0.15 Endotoxin

x 100.617shows no reaction with the lysate employed, at the detec-tion limit of the reagent.or in a buffer recommended by the lysate manufacturer, byLysate TS—Dissolve Amoebocyte Lysate in Water for BET, gentle stirring. Store the reconstituted lysate, refrigerated orfrozen, according to the specifications of the manufacturer. PREPARATION OF SOLUTIONStoxin Stock SolutionStandard Endotoxin Stock Solution—A Standard Endo-ence Standard that has been calibrated to the current WHO is prepared from a USP Endotoxin Refer-International Standard for Endotoxin. Follow the specifica-tions in the package leaflet and on the label for preparationand storage of the toxin is expressed in Endotoxin Units (EU). [Standard Endotoxin Stock SolutionEndotoxin Unit (EU) is equal to one International Unit (IU)NOTE —One USP. Endo-of endotoxin.]dard Endotoxin Stock SolutionStandard Endotoxin Solutions—serial dilutions of for BETStandard Endotoxin Solution vigorously, prepare appropriateAfter mixing the Stan-, using Water activity by adsorption.. Use dilutions as soon as possible to avoid loss ofsolving or diluting drugs using Sample Solutions—Prepare the Sample Solutions by dis-stances or preparations may be more appropriately dis-Water for BET. Some sub-solved, or diluted in other aqueous solutions. If necessary,adjust the pH of the solution to be examined (or dilutionthereof) so that the pH of the mixture of the lysate andSample Solutionlysate manufacturer, usually 6.0–8.0. The pH may be ad- falls within the pH range specified by thejusted by use of an acid, base, or suitable buffer as recom-mended by the lysate manufacturer. Acids and bases maybe prepared from concentrates or solids with in containers free of detectable endotoxin. Buffers must beWater for BETvalidated to be free of detectable endotoxin and interferingfactors. DETERMINATION OF MAXIMUM VALIDDILUTION (MVD)dilution of a specimen at which the endotoxin limit can beThe maximum valid dilution is the maximum allowabledetermined. Determine the MVD from the followingequation:MVD = (endotoxin limit × concentration of (λ) Sample Solution)/drugs, defined on the basis of dose, equals Endotoxin Limit—The endotoxin limit for parenteralK /M x 2x , where K x 2intrathecal (for which ceutical products not administered intrathecally, the endotoxin limit is calcu-K is 0.2 USP-EU/kg of body weight). For radiopharma-lated as 175 EU/intrathecally administered radiopharmaceuticals, the endotoxin limit is ob-V , where V is the maximum recommended dose in mL. Fortained by the formula 14 EU/administered on a per square meter of body surface, the formula is V . For formulations (usually anticancer products)where K = 100 EU/m2 and M is the maximum dose/m2. K /M ,

x

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