EUROPEAN PHARMACOPOEIA 11.

0 Water, purified

calculated value for a standardised inoculum. For a freshly Magnesium sulfate, anhydrous 0.024 g
prepared inoculum, growth of the micro-organisms must Sodium pyruvate 0.3 g
be comparable to that obtained with a previously tested
and approved batch of medium. Agar 15.0 g

Table 2249.-1. – Growth promotion of casein soya bean digest Purified water to 1000 mL
agar
Adjust the pH so that after sterilisation it is 7.2 ± 0.2. Sterilise
Preparation of the test
Micro-organism
strain
Growth promotion by heating in an autoclave at 121 °C for 15 min.
Pseudomonas Casein soya bean digestCasein soya bean digest Growth promotion of R2A agar
aeruginosa agar or casein soya bean
agar – Preparation of test strains. Use standardised stable
such as : digest broth ≤ 100 CFU suspensions of test strains or prepare them as stated
ATCC 9027 30-35 °C 30-35 °C in Table 0008.-1. Seed lot culture maintenance
NCIMB 8626 18-24 h ≤ 3 days techniques (seed-lot systems) are used so that the viable
CIP 82.118 micro-organisms used for inoculation are not more than
NBRC 13275 5 passages removed from the original master seed-lot.
Bacillus subtilis Casein soya bean digest Casein soya bean digest Grow each of the bacterial strains separately as described
such as : agar or casein soya bean agar in Table 0008.-1. Use buffered sodium chloride-peptone
digest broth ≤ 100 CFU
ATCC 6633 solution pH 7.0 or phosphate buffer solution pH 7.2 to
30-35 °C 30-35 °C make test suspensions. Use the suspensions within 2 h, or
NCIMB 8054
18-24 h ≤ 3 days within 24 h if stored at 2-8 °C. As an alternative to preparing
CIP 52.62
NBRC 3134 and then diluting a fresh suspension of vegetative cells of
Bacillus subtilis, a stable spore suspension is prepared and
then an appropriate volume of the spore suspension is used
04/2018:0008 for test inoculation. The stable spore suspension may be
maintained at 2-8 °C for a validated period of time.
– Growth promotion. Test each batch of ready-prepared
medium and each batch of medium, prepared either from
dehydrated medium or from the ingredients described.
Inoculate plates of R2A agar separately with a small number
WATER, PURIFIED (not more than 100 CFU) of the micro-organisms indicated
in Table 0008.-1. Incubate under the conditions described
Aqua purificata in the table. Growth obtained must not differ by a factor
greater than 2 from the calculated value for a standardised
H 2O Mr 18.02 inoculum. For a freshly prepared inoculum, growth of the
micro-organisms must be comparable to that obtained with
DEFINITION a previously tested and approved batch of medium.
Water for the preparation of medicines other than those
that are required to be both sterile and apyrogenic, unless Table 0008.-1. – Growth promotion of R2A agar
otherwise justified and authorised. Micro-organism Preparation of the test strain Growth promotion
Pseudomonas Casein soyabean digest agar or R2A agar
Purified water in bulk aeruginosa casein soyabean digest broth ≤ 100 CFU
such as : 30-35 °C 30-35 °C
PRODUCTION ATCC 9027 18-24 h ≤ 3 days
Purified water in bulk is prepared by distillation, by ion NCIMB 8626
exchange, by reverse osmosis or by any other suitable method CIP 82.118
from water that complies with the regulations on water NBRC 13275
intended for human consumption laid down by the competent Bacillus subtilis Casein soyabean digest agar or R2A agar
authority. such as : casein soyabean digest broth ≤ 100 CFU
Purified water in bulk is stored and distributed in conditions ATCC 6633 30-35 °C 30-35 °C
designed to prevent growth of micro-organisms and to avoid NCIMB 8054 18-24 h ≤ 3 days
any other contamination. CIP 52.62
Microbiological monitoring. During production and NBRC 3134
subsequent storage, appropriate measures are taken to Total organic carbon or oxidisable substances. Carry
ensure that the microbial count is adequately controlled and out the test for total organic carbon (2.2.44) with a limit of
monitored. Appropriate alert and action levels are set so 0.5 mg/L or alternatively the following test for oxidisable
as to detect adverse trends. Under normal conditions, an substances : to 100 mL add 10 mL of dilute sulfuric acid R and
appropriate action level is a microbial count of 100 CFU/mL, 0.1 mL of 0.02 M potassium permanganate and boil for 5 min ;
determined by filtration through a membrane with a nominal the solution remains faintly pink.
pore size not greater than 0.45 μm, using R2A agar and
incubating at 30-35 °C for not less than 5 days. The size of the Conductivity. Determine the conductivity off-line or in-line
sample is to be chosen in relation to the expected result. under the following conditions.
R2A agar EQUIPMENT
Yeast extract 0.5 g Conductivity cell :
– electrodes of a suitable material such as stainless steel ;
Proteose peptone 0.5 g
– cell constant : the cell constant is generally certified by the
Casein hydrolysate 0.5 g supplier and is subsequently verified at suitable intervals
Glucose 0.5 g using a certified reference solution with a conductivity less
than 1500 μS·cm− 1 or by comparison with a cell having
Starch 0.5 g a certified cell constant ; the cell constant is confirmed if
Dipotassium hydrogen phosphate 0.3 g the value found is within 2 per cent of the certified value,
otherwise re-calibration must be performed.

General Notices (1) apply to all monographs and other texts 4391
Water, purified EUROPEAN PHARMACOPOEIA 11.0

Conductometer : accuracy of 0.1 μS·cm− 1 or better at the lowest same time in the same manner using a mixture of 4.5 mL of
range. nitrate-free water R and 0.5 mL of nitrate standard solution
System calibration (conductivity cell and conductometer) : (2 ppm NO3) R.
– against one or more suitable certified reference solutions ; Aluminium (2.4.17) : maximum 10 ppb, if intended for use in
– accuracy : within 3 per cent of the measured conductivity the manufacture of dialysis solutions.
plus 0.1 μS·cm− 1. Prescribed solution. To 400 mL of the water to be examined
Conductometer calibration : calibration is carried out for add 10 mL of acetate buffer solution pH 6.0 R and 100 mL of
each range of measurement to be used, after disconnection distilled water R.
of the conductivity cell, using certified precision resistors or Reference solution. Mix 2 mL of aluminium standard
equivalent devices with an uncertainty not greater than 0.1 per solution (2 ppm Al) R, 10 mL of acetate buffer solution
cent of the certified value. pH 6.0 R and 98 mL of distilled water R.
If in-line conductivity cells cannot be dismantled, system Blank solution. Mix 10 mL of acetate buffer solution pH 6.0 R
calibration may be performed against a calibrated and 100 mL of distilled water R.
conductivity-measuring instrument with a conductivity cell Bacterial endotoxins (2.6.14) : less than 0.25 IU/mL, if
placed close to the cell to be calibrated in the water flow. intended for use in the manufacture of dialysis solutions
Temperature measurement : tolerance ± 2 °C. without a further appropriate procedure for removal of
bacterial endotoxins.
PROCEDURE
Measure the conductivity without temperature LABELLING
compensation, recording simultaneously the temperature. The label states, where applicable, that the substance is suitable
Temperature-compensated measurement may be performed for use in the manufacture of dialysis solutions.
after suitable validation.
The water to be examined meets the requirements if the
measured conductivity at the recorded temperature is not Purified water in containers
greater than the value in Table 0008.-2. DEFINITION
Table 0008.-2. – Temperature and conductivity requirements Purified water in bulk that has been filled and stored in
Temperature Conductivity conditions designed to assure the required microbiological
(°C) (μS·cm− 1) quality. It is free from any added substances.
0 2.4
CHARACTERS
10 3.6
Appearance : clear and colourless liquid.
20 4.3
TESTS
25 5.1
It complies with the tests prescribed in the section on Purified
30 5.4 water in bulk and with the following additional tests.
40 6.5 Acidity or alkalinity. To 10 mL, freshly boiled and cooled in
a borosilicate glass flask, add 0.05 mL of methyl red solution R.
50 7.1 The solution is not coloured red.
60 8.1 To 10 mL add 0.1 mL of bromothymol blue solution R1. The
solution is not coloured blue.
70 9.1
Oxidisable substances. To 100 mL add 10 mL of dilute
75 9.7
sulfuric acid R and 0.1 mL of 0.02 M potassium permanganate
80 9.7 and boil for 5 min. The solution remains faintly pink.
90 9.7 Chlorides. To 10 mL add 1 mL of dilute nitric acid R and
0.2 mL of silver nitrate solution R2. The solution shows no
100 10.2 change in appearance for at least 15 min.
Sulfates. To 10 mL add 0.1 mL of dilute hydrochloric acid R
For temperatures not listed in Table 0008.-2, calculate the
and 0.1 mL of barium chloride solution R1. The solution shows
maximal permitted conductivity by interpolation between the
no change in appearance for at least 1 h.
next lower and next higher data points in the table.
Ammonium : maximum 0.2 ppm.
Elemental impurities. If purified water in bulk does not
meet the requirement for conductivity prescribed for Water To 20 mL add 1 mL of alkaline potassium tetraiodomercurate
for injections (0169) in bulk, a risk assessment according to solution R. After 5 min, examine the solution down the vertical
general chapter 5.20. Elemental impurities is carried out. axis of the tube. The solution is not more intensely coloured
The risk assessment should consider the role of water in the than a standard prepared at the same time by adding 1 mL of
manufacturing process, in particular when water is used in a alkaline potassium tetraiodomercurate solution R to a mixture
process but is no longer present in the final product. of 4 mL of ammonium standard solution (1 ppm NH4) R and
16 mL of ammonium-free water R.
CHARACTERS Calcium and magnesium. To 100 mL add 2 mL of ammonium
Appearance : clear and colourless liquid. chloride buffer solution pH 10.0 R, 50 mg of mordant black 11
triturate R and 0.5 mL of 0.01 M sodium edetate. A pure blue
TESTS colour is produced.
Nitrates : maximum 0.2 ppm. Residue on evaporation : maximum 0.001 per cent.
Place 5 mL in a test-tube immersed in iced water, add 0.4 mL Evaporate 100 mL to dryness on a water-bath and dry in an
of a 100 g/L solution of potassium chloride R, 0.1 mL of oven at 100-105 °C. The residue weighs a maximum of 1 mg.
diphenylamine solution R and, dropwise with shaking, 5 mL of
nitrogen-free sulfuric acid R. Transfer the tube to a water-bath Microbial contamination
at 50 °C. After 15 min, any blue colour in the solution is not TAMC : acceptance criterion 102 CFU/mL (2.6.12). Use casein
more intense than that in a reference solution prepared at the soya bean digest agar.

4392 See the information section on general monographs (cover pages)

LABELLING B. Suspend 1 g in 50 mL of water R, boil for 1 min and cool.
The label states, where applicable, that the substance is suitable A thin, cloudy mucilage is formed.
for use in the manufacture of dialysis solutions. C. To 1 mL of the mucilage obtained in identification test B
add 0.05 mL of iodine solution R1. A dark blue colour is
produced, which disappears on heating.
01/2022:0359
TESTS
pH (2.2.3) : 4.5 to 7.0.
Shake 5.0 g with 25.0 mL of carbon dioxide-free water R for
60 s. Allow to stand for 15 min.
WHEAT STARCH (1) ◊Foreign matter. Examined under a microscope using a
50 per cent V/V solution of glycerol R, not more than traces
of matter other than starch granules are present. No starch
Tritici amylum granules of any other origin are present.◊
DEFINITION Total protein : maximum 0.3 per cent (corresponding to
Wheat starch is obtained from the caryopsis of Triticum 0.048 per cent of nitrogen).
aestivum L. (T. vulgare Vill.). Determine the nitrogen content by sulfuric acid digestion as
follows, and calculate the quantity of protein by multiplying
◊PRODUCTION by 6.25.
The gluten content is monitored and stated on the label.◊ Carry out a blank determination by placing 4 g of a powdered
mixture of 100 g of dipotassium sulfate R, 3 g of copper sulfate
♦CHARACTERS pentahydrate R and 3 g of titanium dioxide R, and 3 glass
Appearance : very fine, white or almost white powder that beads in a combustion flask. Wash any adhering particles
creaks when pressed between the fingers. from the neck of the flask with a fine jet of water R. Add
Solubility : practically insoluble in cold water and in ethanol 25 mL of sulfuric acid R, allowing it to run down the sides of
(96 per cent). the flask, and swirl to mix the contents. Close the mouth of
Wheat starch does not contain starch granules of any other the flask loosely, for example by means of a glass bulb with
origin. It may contain a minute quantity, if any, of tissue a short stem, to avoid excessive loss of sulfuric acid. Heat
gradually at first, then increase the temperature until there
fragments of the original plant.♦
is vigorous boiling with condensation of sulfuric acid in the
IDENTIFICATION neck of the flask ; take precautions to prevent the upper part of
A. Microscopic examination (2.8.23) using a 50 per cent V/V the flask from becoming overheated. Continue heating until
solution of glycerol R. It shows large and small granules, a clear solution is obtained and the inside wall of the flask
and, very rarely, intermediate sizes (Figure 0359.-1). The is free from carbonaceous material. Cool, dissolve the solid
large granules, 10-60 μm in diameter, are discoid or, more material by cautiously adding 25 mL of water R to the mixture,
rarely, reniform in surface view. The central hilum and cool again and place in a steam-distillation apparatus. Add
striations are invisible or barely visible and the granules a suitable volume of strong sodium hydroxide solution R to
sometimes show cracks on the edges. In side view, the change the colour of the solution from bluish-green to brown
granules are elliptical and fusiform and the hilum appears or black, and distil immediately by passing steam through the
as a slit along the main axis. The small granules, rounded or mixture. Collect about 40 mL of distillate in 50.0 mL of 0.01 M
polyhedral, are 2-10 μm in diameter. Between orthogonally hydrochloric acid, adding enough water R if necessary to cover
orientated polarising plates or prisms, the granules show a the tip of the condenser. Towards the end of the distillation,
distinct black cross intersecting at the hilum. lower the receiver so that the tip of the condenser is above the
surface of the acid and rinse the end of the condensing tube
with a small quantity of water R. Take precautions to prevent
any water on the outer surface of the condenser from reaching
the contents of the receiver. Titrate the distillate with 0.025 M
sodium hydroxide (n1 mL), using methyl red mixed solution R
as indicator.
Repeat the test adding 3.0 g (m g) of the substance to be
examined to the combustion flask, and using the same volume
of strong sodium hydroxide solution R. Titrate the distillate as
described for the blank determination with 0.025 M sodium
hydroxide (n2 mL). Calculate the percentage content of
nitrogen using the following expression :
0.03503 ( n1 - n2 )
m
Oxidising substances (2.5.30): maximum 20 ppm, calculated
as H2O2.
Sulfur dioxide (2.5.29) : maximum 50 ppm.
Iron (2.4.9) : maximum 10 ppm.
Shake 1.5 g with 15 mL of dilute hydrochloric acid R. Filter.
The filtrate complies with the test.
Loss on drying (2.2.32) : maximum 15.0 per cent, determined
on 1.000 g by drying in an oven at 130 °C for 90 min.
Figure 0359.-1. − Illustration for identification test A of wheat Sulfated ash (2.4.14): maximum 0.6 per cent, determined on
starch 1.0 g.
(1) This monograph has undergone pharmacopoeial harmonisation. See chapter 5.8. Pharmacopoeial harmonisation.

General Notices (1) apply to all monographs and other texts 4393