CHROMOGENIC METHOD IN ENDOTOXIN TESTING FOR INTRAVENA INJECTION PREPARATION
Sohadi Warya, Iyan Sopyan, Insan Sunan K., Dzikry Ilhami Faculty of Pharmacy Padjadjaran University
Abstract
A chromogenic endotoxin test using Limulus Amoebocyte Lysate has been done. The objective was study the application of the chromogenic method of endotoxin determination of ascorbic acid injection dosage form. In the chromogenic method the colour was formed as a result as the reaction between endotoxin and LAL reagent; the colour intensity and the speed of colour formation rate is proportional to the concentration of endotoxin. In this test, Escherichia coli endotoxin standard with concentration of 50, 5, 0.5, 0.05, and 0.005 EU/mL were applied. From the test, it was found that endotoxin content 1.686 and 0.324 EU/mL for Product A and Product B respectively.
Key words : Chromogenic, Endotoxin, Ascorbic Acid injection, Limulus Amoebocyte Lysate
INTRODUCTION
Injection is a sterile dosage in form of solution, emulsion, suspension or powder that must be dissolved or suspended before use, which is injected into the tissue by ripping into the skin or through the skin or mucous membranes (Depkes RI, 1979). As sterile preparation, injection must meet the following requirements: germ-free, free of solvents that are physiologically not neutral, isotonic, isohydri, free from floating materials and for large volume injection should be free of pyrogens (Voigt, 1995).
Pyrogen is a substance capable of causing fever and ever contaminate pharmaceutical preparations (Suwandi, 1988). Gram negative bacteria sre the most potent procedure of endotoxin (Jones, 2001). Usually, human are sensitive to endotoxin that often cause pyrogenic respond. The exist of endotoxin in blood stream can cause fever, inflamation, and often shock (Joiner, et al., 2002).
To guarantee that there is no pyrogen in an injection preparation, endotoxin testing should be done. Endotoxin test is an importan part of quality assurance or quality control of large volume injection dosage. The first endotoxin test conduted was rabbit test according to the USP. In this time, in vitro test had been developed which have higher sensitivity than the rabbit test, the test using Limulus Amoebocyte Lysate (LAL). LAL is a liquid extract of crab’s (Mottar, et al., 2006). There are several types of endotoxin test using LAL, such as gel-clot method, chromogenic method and turbidimetry method (The Official Compendia of Standards, 2002).
Method that to be used in this research is the chromogenic method. This method measures the reaction time required for the formation of colour intensity after the release of colours from the peptide complex chromogenic appropriate by endotoxin lysate using a spectrophotometer set at wavelength 405 nm (Her Majesty's Stationery Office, 1980). The stronger the colour, the greater the measured absorbance value. Endotoxin concentration of endotoin in units of ng/mL or EU/ml (Akers, 1994).
MATERIALS AND METHODS
Instrument for this research are cuvet, sterile syringe, thermometer, test tube, refrigator, laminar airflow (LAF), Uv-vis Spectrophotometer (Specord, Analytical Zena), Peristaltic pump, watrbath (Memert), and all general labolatory glasses equipment.
Material : A class vitamin C injection, B class Vit C injection, Pyrocrome LAL test, (chromogenic test), LAL reconstruction Buffer, endotoxin control standard, (e . Coli), LAL reagent water, 70% alcohol spiritus,
Method :
Endotoxin preparation and LAL Kit :
Endotoxin preparation. Preparation endotoxin standard solution in concentration of 50 Eu/ml ; 5 Eu/ml ; 0,5 Eu/ml ; 0,05 Eu/ml, and 0,05 eu/ml to make endotoxin standard curve,
LAL preparation. Pyrochrome LAL 60- test kit reagent used kin this research pyrocrome is soluted in 3,2 ml LAL reconstruction buffer after being soluted, LAL is used to test endotoxin in vitamin C injection
Chromgenic Method Procedure
Wavelengteh determination.
Prepareation the endotoxin standard solution as test solution, pathogen free aqua pro injection with LAL reagent blanko, blanko is sterilized and store in same temperature, as test solution than the test solution and blanko solution, are put in each cuvet to detarmaining the wavelength that make maximum absorbation, the absorbation is being measued at 405 nm wavelength.
How to make standard curve/standard curve process.
Put 50 eu/ml endotoxin standard solution that has been prepared as many as 0,3 ml into cuvet. After that add LAL reagent as many s 0,3 ml in to the cuvet and than put cuvet to Uv-vis spectrophotometer which has been connected to waterbath with 370C temperature,
measure the initial time when the maximum peak absorbance wave is formed. Do the same treatment for 5, 0.5, 0.05, and 0,005 eu/ml standard solution. thus obtained caliberation curve in each initial time when the peak formed
Level evdotoxin in vit C injection. Determiantion a sample of 0,3 ml vit c injection is inserted in to cuvet, then add 0,3 ml LAL reagent put the cuvet into Uv-vis spectrophotometer which has beeb connected to waterbath in 370C, measured the initial time
when the curve peak is formed. Do the same treatment to every vit C injection Product twice each product
Data Analysis. To find out the endotoxin level in vit c injection, data that was obtained is calculated using linier regression equation in calaiberation curve
RESULT AND DISCUSSION Onset Time Endotoxin Standard Solution
Concentration (EU/mL) Onset Time (second)
0.05 3181
0.05 2065
0.5 1312
5 837
y = - 0.1 9 2x + 3.0 6 1 R2= 10 0 0
0.0 0 0 0.5 0 0 1.0 0 0 1.5 0 0 2.0 0 0 2.5 0 0 3.0 0 0 3.5 0 0 4.0 0 0
-3 -2 -1 0 1 2
L o g o f C o n c e n tra tio n
L o g o f O n s e t T im e
Standard Curve of Endotoxin
Endotoxin standard curve generated from dilution of the concentration of endotoxin in 50 EU/mL, 5 EU/mL, 0,5 EU/mL, 0,05 EU/mL and 0,005 EU/mL which reacted with pyrochrome Measurement of onset time (time of initial formation of colour) endotoxin standard solution carried out at wavelength of 405 nm.
Provided linier regression equation y = -0,192 x + 3,061. Where y = Log T (time of initial formation of colour) and x = Log C (endotoxin cncentration) with r = 1,000.
Onset Time Vitamin C Injection Preparation at 405 nm Wavelength
Vit. C Injection Preparation
Onset Time (second)
I II Mean
Product A 1150 932 1041
Product B 1502 1355 1428,5
The result of the endotoxin in the injection dosage of vitamin C with pyrochrome produce yellow colour.
(product A) (product B)
From the graph and the picture above can be seen that the onset times dosage injections of vitamin C of product A is faster than product B. And intensity of the colour formed on the injection dosage of vitamin C of product A is more powerful than product B. This shows that product A have more endotoxin than product B. Because the less time the formation of colour and stronger the colour produced showed greater levels of endotoxin contained in a sample.
The level of endotoxin contained in the dosage of vitamin C injection of product A as much as 1.686 EU/mL and product B as much as 0.324 EU/mL.
0 20 0 40 0 60 0 80 0 100 0 120 0 140 0 160 0 Product A Product B
Vitamin C Injection Preparation
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