Warfarin sodium clathrate EUROPEAN PHARMACOPOEIA 11.

Column :
– size : l = 0.25 m, Ø = 4.0 mm ;
– stationary phase : cyanosilyl silica gel for chromatography R
(5 μm) ;
– temperature : 30 °C.
C. (3E)-4-phenylbut-3-en-2-one (benzalacetone). Mobile phase : glacial acetic acid R, acetonitrile R, water R
(1:25:75 V/V/V).
Flow rate : 1.5 mL/min.
04/2014:0699 Detection : spectrophotometer at 260 nm.
Injection : 20 μL.
Run time : twice the retention time of warfarin.
Identification of impurities: use the chromatogram obtained
with reference solution (a) to identify the peaks due to
WARFARIN SODIUM CLATHRATE impurities B and C.
Relative retention with reference to warfarin
Warfarinum natricum clathratum (retention time = about 9 min) : impurity B = about
0.4 ; impurity C = about 0.6.
System suitability : reference solution (a) :
– resolution : minimum 2.0 between the peaks due to
impurities B and C.
Limits :
– correction factors : for the calculation of content,
multiply the peak areas of the following impurities by
DEFINITION the corresponding correction factor : impurity B = 0.5 ;
impurity C = 0.4 ;
Mixture, in the form of a clathrate, of warfarin sodium (sodium
2-oxo-3-[(1RS)-3-oxo-1-phenylbutyl]-2H-1-benzopyran- – impurities B, C : for each impurity, not more than 1.5 times
4-olate) and propan-2-ol in molecular proportions 2:1 the area of the principal peak in the chromatogram
(equivalent to about 92 per cent of warfarin sodium). obtained with reference solution (b) (0.15 per cent) ;
Content : – unspecified impurities : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
– warfarin sodium : 98.0 per cent to 102.0 per cent (anhydrous with reference solution (b) (0.10 per cent) ;
and propan-2-ol-free substance) ;
– total : not more than 3 times the area of the principal peak
– propan-2-ol : 8.0 per cent to 8.5 per cent. in the chromatogram obtained with reference solution (b)
CHARACTERS (0.3 per cent);
Appearance : white or almost white, hygroscopic, crystalline – disregard limit : 0.5 times the area of the principal peak in
powder. the chromatogram obtained with reference solution (b)
(0.05 per cent).
Solubility : very soluble in water, freely soluble in ethanol
(96 per cent), soluble in acetone, very slightly soluble in Phenolic ketones. Dissolve 1.25 g in a 20 g/L solution of
methylene chloride. sodium hydroxide R and dilute to 10.0 mL with the same
solvent. The absorbance (2.2.25) is maximum 0.20 measured
IDENTIFICATION at 385 nm within 15 min of preparing the solution.
A. Infrared absorption spectrophotometry (2.2.24). Propan-2-ol (2.4.24, System A) : 8.0 per cent to 8.5 per cent.
Comparison : warfarin sodium clathrate CRS. Water (2.5.12) : maximum 0.3 per cent, determined on 2.500 g.
B. Propan-2-ol (see Tests).
ASSAY
C. It gives reaction (b) of sodium (2.3.1).
Dissolve 0.100 g in 0.01 M sodium hydroxide and dilute
TESTS to 100.0 mL with the same solvent. Dilute 10.0 mL of the
Appearance of solution. The solution is clear (2.2.1) and solution to 100.0 mL with 0.01 M sodium hydroxide. Dilute
colourless (2.2.2, Method II). 10.0 mL of this solution to 100.0 mL with 0.01 M sodium
hydroxide. Measure the absorbance (2.2.25) at the absorption
Dissolve 1.0 g in water R and dilute to 20 mL with the same maximum at 308 nm.
solvent.
Calculate the percentage content of warfarin sodium
pH (2.2.3) : 7.6 to 8.6. (C19H15NaO4) taking the specific absorbance to be 431.
Dissolve 1.0 g in carbon dioxide-free water R and dilute to
100 mL with the same solvent. STORAGE
Related substances. Liquid chromatography (2.2.29). In an airtight container, protected from light.
Solvent mixture : methanol R, water R (25:75 V/V). IMPURITIES
Test solution. Dissolve 40.0 mg of the substance to be Specified impurities : B, C.
examined in the solvent mixture and dilute to 50.0 mL with
Other detectable impurities (the following substances would,
the solvent mixture.
if present at a sufficient level, be detected by one or other of
Reference solution (a). Dissolve 2 mg of 4-hydroxycoumarin R the tests in the monograph. They are limited by the general
(impurity B) and 2 mg of benzalacetone R (impurity C) in acceptance criterion for other/unspecified impurities and/or
25 mL of methanol R and dilute to 100 mL with water R. by the general monograph Substances for pharmaceutical
Reference solution (b). Dilute 1.0 mL of the test solution to use (2034). It is therefore not necessary to identify these
100.0 mL with the solvent mixture. Dilute 1.0 mL of this impurities for demonstration of compliance. See also 5.10.
solution to 10.0 mL with the solvent mixture. Control of impurities in substances for pharmaceutical use) : A.

4386 See the information section on general monographs (cover pages)

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EUROPEAN PHARMACOPOEIA 11.0 Water for injections

in-process monitoring of the electrical conductivity, and

regular monitoring of total organic carbon and microbial
contamination are applied.

The first portion of water obtained when the system begins to

function is discarded.
A. (5RS)-3-(2-hydroxyphenyl)-5-phenylcyclohex-2-enone,
Water for injections in bulk is stored and distributed in
conditions designed to prevent growth of micro-organisms
and to avoid any other contamination.
Microbiological monitoring. During production and
subsequent storage, appropriate measures are taken to
ensure that the microbial count is adequately controlled and
monitored. Appropriate alert and action levels are set so
B. 4-hydroxy-2H-1-benzopyran-2-one (4-hydroxycoumarin), as to detect adverse trends. Under normal conditions, an
appropriate action level is a microbial count of 10 CFU per
100 mL when determined by filtration through a membrane
with a nominal pore size not greater than 0.45 μm, using R2A
agar, using at least 200 mL of water for injections in bulk and
incubating at 30-35 °C for not less than 5 days. For aseptic
processing, stricter alert levels may need to be applied.

R2A agar
C. (3E)-4-phenylbut-3-en-2-one (benzalacetone). Yeast extract 0.5 g

Proteose peptone 0.5 g

Casein hydrolysate 0.5 g

Glucose 0.5 g
04/2017:0169
corrected 11.0 Starch 0.5 g

Dipotassium hydrogen phosphate 0.3 g

Magnesium sulfate, anhydrous 0.024 g

Sodium pyruvate 0.3 g

WATER FOR INJECTIONS Agar 15.0 g

Purified water to 1000 mL

Aqua ad iniectabile
Adjust the pH so that after sterilisation it is 7.2 ± 0.2. Sterilise
H 2O Mr 18.02 by heating in an autoclave at 121 °C for 15 min.

DEFINITION Growth promotion of R2A agar

Water for the preparation of medicines for parenteral – Preparation of test strains. Use standardised stable
administration when water is used as vehicle (water for suspensions of test strains or prepare them as stated
injections in bulk) and for dissolving or diluting substances in Table 0169.-1. Seed lot culture maintenance
or preparations for parenteral administration (sterilised water techniques (seed-lot systems) are used so that the viable
for injections). micro-organisms used for inoculation are not more than
5 passages removed from the original master seed-lot.
Water for injections in bulk Grow each of the bacterial strains separately as described
in Table 0169.-1. Use buffered sodium chloride-peptone
PRODUCTION solution pH 7.0 or phosphate buffer solution pH 7.2 to
make test suspensions. Use the suspensions within 2 h, or
Water for injections in bulk is obtained from water that within 24 h if stored at 2-8 °C. As an alternative to preparing
complies with the regulations on water intended for human and then diluting a fresh suspension of vegetative cells of
consumption laid down by the competent authority or from Bacillus subtilis, a stable spore suspension is prepared and
purified water. It is produced either : then an appropriate volume of the spore suspension is used
– by distillation in an apparatus of which the parts in contact for test inoculation. The stable spore suspension may be
with the water are of neutral glass, quartz or a suitable maintained at 2-8 °C for a validated period of time.
metal and which is fitted with an effective device to prevent
the entrainment of droplets ; or – Growth promotion. Test each batch of ready-prepared
medium and each batch of medium, prepared either from
– by a purification process that is equivalent to distillation. dehydrated medium or from the ingredients described.
Reverse osmosis, which may be single-pass or double-pass, Inoculate plates of R2A agar separately with a small number
coupled with other appropriate techniques such as (not more than 100 CFU) of the micro-organisms indicated
electro-deionisation, ultrafiltration or nanofiltration, is in Table 0169.-1. Incubate under the conditions described
suitable. Notice is given to the supervisory authority of the in the table. Growth obtained must not differ by a factor
manufacturer before implementation. greater than 2 from the calculated value for a standardised
For all methods of production, correct operation monitoring inoculum. For a freshly prepared inoculum, growth of the
and maintenance of the system are essential. In order to ensure micro-organisms must be comparable to that obtained with
the appropriate quality of the water, validated procedures, a previously tested and approved batch of medium.

General Notices (1) apply to all monographs and other texts 4387
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