Dequalinium chloride

EUROPEAN PHARMACOPOEIA 8.0

B. (1R,3s,5S)-3-(10,11-dihydro-5H-dibenzo[a,d][7]annulen5-yloxy)-8-methyl-8-azabicyclo[3.2.1]octane
(pseudodeptropine),

C. 10,11-dihydro-5H-dibenzo[a,d][7]annulen-5-ol
(dibenzocycloheptadienol),

D. (1R,3r,5S)-3-(10,11-dihydro-5H-dibenzo[a,d][7]annulen5-yloxy)-8-azabicyclo[3.2.1]octane (demethyldeptropine).
01/2008:1413
corrected 6.0

DEQUALINIUM CHLORIDE
Dequalinii chloridum

C30H40Cl2N4
[522-51-0]

Mr 527.6

DEFINITION
1,1-(Decane-1,10-diyl)bis(4-amino-2-methylquinolinium)
dichloride (dried substance).
Content : 95.0 per cent to 101.0 per cent.
CHARACTERS
Appearance : white or yellowish-white powder, hygroscopic.
Solubility : slightly soluble in water and in ethanol (96 per
cent).
IDENTIFICATION
First identification : B, E.
Second identification : A, C, D, E.
A. Ultraviolet and visible absorption spectrophotometry
(2.2.25).
Test solution. Dissolve about 10 mg in water R and dilute to
100 mL with the same solvent. Dilute 10 mL of the solution
to 100 mL with water R.
Spectral range : 230-350 nm.
Absorption maxima : at 240 nm and 326 nm.
Shoulder : at 336 nm.
General Notices (1) apply to all monographs and other texts

Absorbance ratios:
A240/A326 = 1.56 to 1.80 ;
A326/A336 = 1.12 to 1.30.
B. Infrared absorption spectrophotometry (2.2.24).
Spectral range : 600-2000 cm 1.
Comparison : dequalinium chloride CRS.
C. To 5 mL of solution S (see Tests) add 5 mL of potassium
ferricyanide solution R. A yellow precipitate is formed.
D. To 10 mL of solution S add 1 mL of dilute nitric acid R. A
white precipitate is formed. Filter and reserve the ltrate
for identication test E.
E. The ltrate from identication test D gives reaction (a) of
chlorides (2.3.1).
TESTS
Solution S. Dissolve 0.2 g in 90 mL of carbon dioxide-free
water R, heating if necessary, and dilute to 100 mL with the
same solvent.
Appearance of solution. Solution S is clear (2.2.1) and
colourless (2.2.2, Method II).
Acidity or alkalinity. To 5 mL of solution S add 0.1 mL
of bromothymol blue solution R1. Not more than 0.2 mL
of 0.01 M hydrochloric acid or 0.01 M sodium hydroxide is
required to change the colour of the indicator.
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 10.0 mg of the substance to be
examined in the mobile phase and dilute to 10.0 mL with the
mobile phase.
Reference solution (a). Dissolve 10.0 mg of dequalinium
chloride for performance test CRS in the mobile phase and
dilute to 10.0 mL with the mobile phase.
Reference solution (b). Dissolve 10.0 mg of dequalinium
chloride CRS in the mobile phase and dilute to 10.0 mL with
the mobile phase. Dilute 1.0 mL of the solution to 50.0 mL
with the mobile phase.
Column :
size : l = 0.25 m, = 4.6 mm ;
stationary phase : end-capped octadecylsilyl silica gel for
chromatography R.
Mobile phase : dissolve 2 g of sodium hexanesulfonate R in
300 mL of water R ; adjust to pH 4.0 with acetic acid R and add
700 mL of methanol R.
Flow rate : 1.5 mL/min.
Detection : spectrophotometer at 240 nm.
Injection : 10 L.
Run time : 5 times the retention time of dequalinium chloride.
System suitability : reference solution (a) :
peak-to-valley ratio : minimum 2.0, where Hp = height above
the baseline of the peak due to impurity B and Hv = height
above the baseline of the lowest point of the curve
separating this peak from the peak due to dequalinium
chloride. If necessary, adjust the concentration of methanol
in the mobile phase.
Limits :
impurity A : not more than 0.5 times the area of the
principal peak in the chromatogram obtained with
reference solution (b) (1 per cent) ;
total of impurities other than A : not more than 5 times the
area of the principal peak in the chromatogram obtained
with reference solution (b) (10 per cent) ;
disregard limit : 0.025 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
(0.05 per cent).
Readily carbonisable substances. Dissolve 20 mg in 2 mL of
sulfuric acid R. After 5 min the solution is not more intensely
coloured than reference solution BY4 (2.2.2, Method I).
1999

3-O-Desacyl-4-monophosphoryl lipid A

Loss on drying (2.2.32): maximum 7.0 per cent, determined

on 1.000 g by drying at 105 C at a pressure not exceeding
0.7 kPa.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g.
ASSAY
In order to avoid overheating in the reaction medium, mix
thoroughly throughout and stop the titration immediately after
the end-point has been reached.
Dissolve 0.200 g in 5 mL of anhydrous formic acid R and add
50 mL of acetic anhydride R. Titrate with 0.1 M perchloric
acid, determining the end-point potentiometrically (2.2.20).
1 mL of 0.1 M perchloric acid is equivalent to 26.38 mg of
C30H40Cl2N4.

EUROPEAN PHARMACOPOEIA 8.0

activities of the parent LPS. It consists of a mixture of

congeners, all containing a backbone of 16-linked
disaccharide of 2-deoxy-2-aminoglucose phosphorylated at
the 4-position, but differing in the fatty acid substitutions at
the 2, 2 and 3 positions. The immunostimulatory activities
of 3-O-desacyl-4-monophosphoryl lipid A combined
with the vaccine include up-regulation of co-stimulatory
molecules on antigen-presenting cells and secretion of
pro-inammatory cytokines, resulting in an enhanced
immune response of the Th1-type against the antigens.
3-O-desacyl-4-monophosphoryl lipid A is a lyophilised
powder or a sterile liquid.
Requirements given in the sections up to and including the
section Triethylamine salt of 3-O-desacyl-4-monophosphoryl
lipid A also apply to formulations that do not proceed to the
3-O-desacyl-4-monophosphoryl lipid A liquid bulk.

STORAGE
In an airtight container.

PRODUCTION
GENERAL PROVISIONS
The production method shall have been shown to yield
IMPURITIES
consistently a 3-O-desacyl-4-monophosphoryl lipid A
Specified impurities : A.
comparable in structure and function with a preparation of
Other detectable impurities (the following substances would,
3-O-desacyl-4-monophosphoryl lipid A used as adjuvant in
if present at a sufcient level, be detected by one or other of
the particular vaccine of proven clinical efcacy and safety
the tests in the monograph. They are limited by the general
in man.
acceptance criterion for other/unspecied impurities and/or
During development studies, and wherever revalidation is
by the general monograph Substances for pharmaceutical use
necessary, a test for residual endotoxin activity is carried out
(2034). It is therefore not necessary to identify these impurities
by injecting intravenously 12-day-old embryonated hens
for demonstration of compliance. See also 5.10. Control of
eggs with 0.1 mL of dilutions of the test sample (8 eggs
impurities in substances for pharmaceutical use) : B, C.
per dilution) of 3-O-desacyl-4-monophosphoryl lipid A.
Eggs are candled and read for mortality at 18-24 hours
post-inoculation and the chick embryo 50 per cent lethal dose
(CELD50) is calculated. The residual endotoxin activity of the
3-O-desacyl-4-monophosphoryl lipid A is acceptable if the
CELD50 is more than 100 g.
An endotoxin standard of Salmonella typhimurium is prepared
and selected dilutions are injected into each group of 8 eggs.
A. 2-methylquinolin-4-amine,
For a test to be valid, the CELD50 of the endotoxin standard
must not be more than 0.05 g.
Reference preparation : a batch of 3-O-desacyl-4monophosphoryl lipid A shown to be comparable
in structure and function with a preparation of
3-O-desacyl-4-monophosphoryl lipid A used as adjuvant in
the particular vaccine of proven clinical efcacy and safety in
B. 4-amino-1-[10-[(2-methylquinolin-4-yl)amino]decyl]-2man or a batch representative thereof.
methylquinolinium chloride,
BACTERIAL SEED LOTS
The bacterial strain used for master seed lots shall be identied
by historical records that include information on its origin and
the tests used to characterise the strain, in particular genotypic
and phenotypic information. Only a working seed lot that
complies with the following requirements may be used.
Identication. The working seed lot is identied by suitable
methods such as Gram staining and fatty acid proling (5.1.6).
Microbial Purity. Each seed lot complies with the
C. 1-[10-(4-amino-2-methylquinolinio)decyl]-4-[[10requirements for absence of contaminating organisms.
(4-amino-2-methylquinolinio)decyl]amino]-2Purity of bacterial cultures is veried by methods of suitable
methylquinolinium trichloride.
sensitivity and specicity.
07/2011:2537 PROPAGATION AND HARVEST
The bacteria is grown using a suitable liquid medium. At the
of cultivation, the culture is tested for purity and yield.
3-O-DESACYL-4-MONOPHOSPHORYL end
The culture medium is separated from the bacterial mass by a
LIPID A
suitable method, for example ltration. Only a harvest that is
consistent with respect to the proles for growth rate, pH, and
O2-consumption may be used for the extraction of LPS.
Adeps A 3-O-desacyl-4TRIETHYLAMINE SALT OF 3-O-DESACYL-4monophosphorylatus
MONOPHOSPHORYL LIPID A
DEFINITION
LPS is extracted from the bacterial cells by successive
3-O-Desacyl-4-monophosphoryl lipid A is a detoxied
alcohol and chloroform-methanol extractions and is then
derivative of the lipopolysaccharide (LPS) of Salmonella
converted to 3-O-desacyl-4-monophosphoryl lipid A by
minnesota, strain R595, which retains the immunostimulatory hydrolysis, then puried and salied by triethanolamine

2000

See the information section on general monographs (cover pages)