Introduction of dynamic turbidity method 鲎 reagent instructions

I. Overview

The sputum test is one of the most sensitive and specific bacterial endotoxin test methods. It is the product of the cell wall of Gram-negative bacteria, commonly referred to as bacterial endotoxin (pyrogen), which reacts with TAL to form an easily distinguishable gel. The simple, economical sputum test has made it widely used for the detection of bacterial endotoxin in injectable pharmaceuticals, biological products, blood products, medical devices and the environment.

The bacterial endotoxin and TAL reaction mixture gradually became cloudy. The higher the bacterial endotoxin concentration, the faster the turbidity changes, and the turbidity change accorded with the second-order reaction kinetic curve. With the aid of the instrument (Note: such as the Tianjin University Radio Factory BET-32C bacterial endotoxin tester), the dynamic turbidimetric test can accurately detect the set turbidity. After the bacterial endotoxin concentration and the reaction time are logarithmically respectively, the two sets of data are statistically analyzed by least squares method to obtain a straight line:

lgT=a+blgC

We call this line a standard curve. Endotoxin levels in unknown samples were determined by comparison to a standard curve.

Second, sputum reagent

The quantitative method 鲎 reagent is completely different from the gel method , reagent, which has high requirements on the purity, solubility, clarity after dissolution and its own color stability in the blood cell extract. The identification value of the quantitative method λ reagent indicates the minimum detection limit of the quantitative guanidine reagent, not the sensitivity. It is calibrated with reference to the US standard endotoxin EC-5, and is marked with EU/ml (eg, the wave card produced by Zhanjiang Marine Biological Products Factory). Quantitative method reagents.).

Third, reconstitution

Before reconstituting, the reagent cartridge is dropped to the bottom of the bottle, and the reagent bottle cap is carefully opened to avoid contact contamination. The small amount of reagent remaining on the reagent bottle cap does not matter. Reconstitute the reagent according to the amount of the label before use. When not in use immediately, seal the bottle mouth with a pyrogen-free sealing film, and gently rotate the reagent bottle until the hydrazine reagent is completely dissolved. Discard the seal if it is broken or dissolved in color or turbidity.

Fourth, storage

The lyophilized quantitative reagent is relatively stable under normal temperature conditions, but should be stored at 20 ° C; avoid long-term placement at temperatures above 25 ° C. The reconstituted hydrazine reagent is preferably stored in a batch to be frozen below -20 ° C during the intermittent use, and may only be used within 7 days, and the hydrazine reagent can only be frozen and thawed once.

Five, warning

Proper application of this test method requires strict compliance with all items in the operating procedures. Sample quantification tests must include a recovery test to verify the eligibility of the recovery rate. All test materials in contact with the sample must be free of endotoxin and the glassware must be effectively detoxified.

Sixth, product interference

The detection method for each sample must be verified to have no significant interference. The interference is usually related to the concentration. LAL can be diluted with water to overcome the interference.

Seven, the most effective dilution

The Chinese Pharmacopoeia 2000 edition stipulates that the endotoxin threshold for intravenously administered drugs is 5 EU/kg.hr, and the intrathecal drug is 0.2 EU/kg.hr. Special limits are also used for some simple drugs. These limits can be used to determine the range of dilution to overcome the interference problem without exceeding the defined endotoxin concentration. The maximum effective dilution factor (MVD) is provided in accordance with the documentation and pharmacopoeia. Formula to calculate:

MVD = endotoxin limit × product titer / minimum detection limit

For example, the endotoxin limit of ceftriaxone sodium (2.0 mg/dose) is 0.20 EU/mg. For example, the minimum detection limit of the quantitative method æ¹› reagent 030329 of Zhanjiang Marine Biological Products Factory is 0.005 EU/ml. First, ceftriaxone sodium was dissolved in 5 ml of test water, and the product titer was 2.0 mg/5 ml = 0.4 mg/ml.

MVD = endotoxin limit × product titer / minimum detection limit

=0.20EU/mg×0.4mg/ml/0.005EU/ml

= 16 times

Eight, daily inspection

The standard curve should include at least three endotoxin concentrations, repeating two tube tests, such as 10, 1.0 and 0.1 EU/ml. For the detection of semi-finished or raw materials, a standard curve of four orders of magnitude from 10 to 0.01 EU/ml may be more suitable. Using the standard curve of three tests, when the standard curve has good consistency, the standard curve can be stored; if the stored standard curve is used, the positive control error must be within ±10%. For recovery testing, the sample positive control tube should contain an endotoxin concentration equal to the midpoint of the standard curve, such as 1.0 EU/ml as described above.

Nine, results

During the whole detection process, the bacterial endotoxin tester automatically monitors the increase of absorbance every 10 seconds. The instrument records the turbidity value corresponding to each time in detail. The time corresponding to the turbidity 0.02 value is called the critical time. The Bacterial Endotoxin Analyzer software automatically calculates the standard curve from the logarithm of the critical time of each standard and its corresponding endotoxin concentration, then displays the characteristics of the standard curve and evaluates whether the standard curve is valid.

BET-32 standard curve analysis

鲎Reagent: 030329 Production: Zhanjiang Marine Biological Products Factory Endotoxin: 2002-12

Regression equation: LogT=2.8849-0.2176LogC

Correlation coefficient: g=-0.9997

In this example, the endotoxin recovery rate of indole propionate for injection was 116%, indicating that the indole propionate for injection had no interference at a concentration of 0.5 mg/ml, and the negative control did not detect the endotoxin value within the prescribed time.