Chromogenic Method In Endotoxin Testing For Intravena Injection Preparation.
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 gelclot 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).
Preparation that will be tested the level of endotoxin in this research is the small
volumes injection containing Vitamin C.
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 Uvvis spectrophotometer which has been connected to waterbath with 37 0C 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 37 0C, 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
50
546
4.0 0 0
L o g o f O n se t T im e
3.5 0 0
3.0 0 0
2.5 0 0
2.0 0 0
1.5 0 0
y = - 0.1 9 2x + 3.0 6 1
R 2 = 10 0 0
1.0 0 0
0.5 0 0
0.0 0 0
-3
-2
-1
0
1
2
L o g o f C o n c e n tra tio n
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
Onset Time (second)
Vit. C Injection
Preparation
I
II
Mean
Product A
1150
932
1041
Product B
1502
1355
1428,5
160
0
140
0
120
0
100
0
O
n
s
et
Ti
m
e
80
0
60
0
40
0
20
0
0
Product
A
Vitamin C Injection
Preparation
Product
B
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.
REFERENCES
Akers, M.J. 1994. Parenteral Quality Control. Marcel Dekker, Inc. New York
Departemen Kesehatan Republik Indonesia. 1978. Formularium Nasional. Edisi II.
Departemen Kesehatan Republik Indonesia. Jakarta. Hal : 324
Departemen Kesehatan Republik Indonesia. 1979. Farmakope Indonesia. Edisi ketiga.
Jakarta : Departemen Kesehatan Republik Indonesia. Hal : 13-14
Her Majesty's Stationery Office. 1980. British Pharmacopeia. Volume II. Her Majesty's
Stationery Office. London. Page : A225
Joiner, Timothy J., Paul F. Kraus, Thomas C. Kupiec, Ph.D. 2002. Comparison of Endotoxin
Testing Methods for Pharmaceutical Products. International Journal of Pharmaceutical
Compounding Vo.6/No.6. Page : 408-409
Jones, Marty, BS Pharm, PharmD. 2001. Bacterial Endotoxins and Pyrogens. International
Journal of Pharmaceutical Compounding Vol.5/No.4. Page : 258-263
Mottar, J, De Block J, Merchiers M, Vantomme K, Moermans R. 2006. Routine Limulus
Amoebocyte Lysate (LAL) test. http://www.ncbi.nlm.nih.gov /entrez/query.fcgi?
db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list
uids=8320369&query_hl=2&itool=pubmed_docsum. [30 November 2006]
Suwandi, U. 1988. Uji Pirogenitas dengan Kelinci dan Limulus Amebocyte Lysate.
Http://www.kalbefarma.com/files/cdk/files/52_08_UjiPirogenitasdenganKelincidanLi
mulus.pdf/52_08_UjiPirogenitasdenganKelincidanLimulus.html. [30 November 2006]
The Official Compendia of Standards. 2002. The United States Pharmacopeia 25 : The
National Formulary 23. Volume 23. Twinbrook Parkway : United States Pharmacopeial
Convention, Inc. Page : 1889-1892
Voigt, R. 1995. Buku Pelajaran Teknologi Farmasi. Edisi kelima. Gadjah Mada University
Press. Yogyakarta. Hal : 462 463
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 gelclot 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).
Preparation that will be tested the level of endotoxin in this research is the small
volumes injection containing Vitamin C.
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 Uvvis spectrophotometer which has been connected to waterbath with 37 0C 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 37 0C, 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
50
546
4.0 0 0
L o g o f O n se t T im e
3.5 0 0
3.0 0 0
2.5 0 0
2.0 0 0
1.5 0 0
y = - 0.1 9 2x + 3.0 6 1
R 2 = 10 0 0
1.0 0 0
0.5 0 0
0.0 0 0
-3
-2
-1
0
1
2
L o g o f C o n c e n tra tio n
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
Onset Time (second)
Vit. C Injection
Preparation
I
II
Mean
Product A
1150
932
1041
Product B
1502
1355
1428,5
160
0
140
0
120
0
100
0
O
n
s
et
Ti
m
e
80
0
60
0
40
0
20
0
0
Product
A
Vitamin C Injection
Preparation
Product
B
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.
REFERENCES
Akers, M.J. 1994. Parenteral Quality Control. Marcel Dekker, Inc. New York
Departemen Kesehatan Republik Indonesia. 1978. Formularium Nasional. Edisi II.
Departemen Kesehatan Republik Indonesia. Jakarta. Hal : 324
Departemen Kesehatan Republik Indonesia. 1979. Farmakope Indonesia. Edisi ketiga.
Jakarta : Departemen Kesehatan Republik Indonesia. Hal : 13-14
Her Majesty's Stationery Office. 1980. British Pharmacopeia. Volume II. Her Majesty's
Stationery Office. London. Page : A225
Joiner, Timothy J., Paul F. Kraus, Thomas C. Kupiec, Ph.D. 2002. Comparison of Endotoxin
Testing Methods for Pharmaceutical Products. International Journal of Pharmaceutical
Compounding Vo.6/No.6. Page : 408-409
Jones, Marty, BS Pharm, PharmD. 2001. Bacterial Endotoxins and Pyrogens. International
Journal of Pharmaceutical Compounding Vol.5/No.4. Page : 258-263
Mottar, J, De Block J, Merchiers M, Vantomme K, Moermans R. 2006. Routine Limulus
Amoebocyte Lysate (LAL) test. http://www.ncbi.nlm.nih.gov /entrez/query.fcgi?
db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list
uids=8320369&query_hl=2&itool=pubmed_docsum. [30 November 2006]
Suwandi, U. 1988. Uji Pirogenitas dengan Kelinci dan Limulus Amebocyte Lysate.
Http://www.kalbefarma.com/files/cdk/files/52_08_UjiPirogenitasdenganKelincidanLi
mulus.pdf/52_08_UjiPirogenitasdenganKelincidanLimulus.html. [30 November 2006]
The Official Compendia of Standards. 2002. The United States Pharmacopeia 25 : The
National Formulary 23. Volume 23. Twinbrook Parkway : United States Pharmacopeial
Convention, Inc. Page : 1889-1892
Voigt, R. 1995. Buku Pelajaran Teknologi Farmasi. Edisi kelima. Gadjah Mada University
Press. Yogyakarta. Hal : 462 463