EUROPEAN PHARMACOPOEIA 10.

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

Casein hydrolysate 0.5 g

Glucose 0.5 g
04/2017:0169 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
administration when water is used as vehicle (water for – Preparation of test strains. Use standardised stable
injections in bulk) and for dissolving or diluting substances suspensions of test strains or prepare them as stated
or preparations for parenteral administration (sterilised water in Table 0169.-1. Seed lot culture maintenance
for injections). techniques (seed-lot systems) are used so that the viable
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 4203
Water for injections EUROPEAN PHARMACOPOEIA 10.0

Table 0169.-1. – Growth promotion of R2A agar Table 0169.-2. – Stage 1

Temperature and conductivity requirements (for
Micro-organism Preparation of the test Growth promotion non-temperature-compensated conductivity measurements)
strain
Pseudomonas Casein soyabean digest R2A agar Temperature Conductivity
aeruginosa agar or casein soyabean ≤ 100 CFU (°C) (µS·cm− 1)
such as : digest broth
30-35 °C 0 0.6
ATCC 9027 30-35 °C
≤ 3 days
NCIMB 8626 18-24 h 5 0.8
CIP 82.118 10 0.9
NBRC 13275
15 1.0
Bacillus subtilis Casein soyabean digest R2A agar
such as : agar or casein soyabean ≤ 100 CFU 20 1.1
digest broth
ATCC 6633 30-35 °C
30-35 °C 25 1.3
NCIMB 8054 ≤ 3 days
18-24 h
CIP 52.62 30 1.4
NBRC 3134
35 1.5
Total organic carbon (2.2.44) : maximum 0.5 mg/L. 40 1.7
Conductivity. Determine the conductivity off-line or in-line 45 1.8
under the following conditions.
50 1.9
EQUIPMENT
Conductivity cell : 55 2.1
60 2.2
– electrodes of a suitable material such as stainless steel ;
65 2.4
– cell constant : the cell constant is generally certified by the
70 2.5
supplier and is subsequently verified at suitable intervals
using a certified reference solution with a conductivity less 75 2.7
than 1500 µS·cm− 1 or by comparison with a cell having a
80 2.7
certified cell constant. The cell constant is confirmed if
the value found is within 2 per cent of the certified value, 85 2.7
otherwise re-calibration must be performed.
90 2.7
Conductometer : accuracy of 0.1 µS·cm− 1 or better at the lowest 95 2.9
range.
100 3.1
System calibration (conductivity cell and conductometer) :
– against one or more suitable certified reference solutions ; Stage 2
– accuracy : within 3 per cent of the measured conductivity 4. Transfer a sufficient amount of the water to be examined
plus 0.1 µS·cm− 1. (100 mL or more) to a suitable container, and stir the test
sample. Adjust the temperature, if necessary, and while
Conductometer calibration : calibration is carried out for maintaining it at 25 ± 1 °C, begin vigorously agitating the
each range of measurement to be used, after disconnection test sample while periodically observing the conductivity.
of the conductivity cell, using certified precision resistors or When the change in conductivity (due to uptake of
equivalent devices with an uncertainty not greater than 0.1 per atmospheric carbon dioxide) is less than 0.1 µS.cm− 1 per
cent of the certified value. 5 min, note the conductivity.
If in-line conductivity cells cannot be dismantled, system
calibration may be performed against a calibrated 5. If the conductivity is not greater than 2.1 µS.cm− 1, the
conductivity-measuring instrument with a conductivity cell water to be examined meets the requirements of the test for
placed close to the cell to be calibrated in the water flow. conductivity. If the conductivity is greater than 2.1 µS.cm− 1,
proceed with stage 3.
Temperature measurement : tolerance ± 2 °C.
Stage 3
PROCEDURE
Stage 1 6. Perform this test within approximately 5 min of the
1. Measure the conductivity without temperature conductivity determination in step 5 under stage 2, while
compensation, recording simultaneously the temperature. maintaining the sample temperature at 25 ± 1 °C. Add a
Temperature-compensated measurement may be recently prepared saturated solution of potassium chloride R
performed after suitable validation. to the test sample (0.3 mL per 100 mL of the test sample),
and determine the pH (2.2.3) to the nearest 0.1 pH unit.
2. Using Table 0169.-2, find the closest temperature value
that is not greater than the measured temperature. The 7. Using Table 0169.-3, determine the conductivity limit
corresponding conductivity value is the limit at that at the measured pH value in step 6. If the measured
temperature. conductivity in step 4 under stage 2 is not greater than
the conductivity requirements for the pH determined, the
3. If the measured conductivity is not greater than the value water to be examined meets the requirements of the test for
in Table 0169.-2, the water to be examined meets the conductivity. If either the measured conductivity is greater
requirements of the test for conductivity. If the conductivity than this value or the pH is outside the range of 5.0-7.0,
is higher than the value in Table 0169.-2, proceed with the water to be examined does not meet the requirements
stage 2. of the test for conductivity.

4204 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 10.0 Water for injections

Table 0169.-3. – Stage 3 Sterilised water for injections

pH and conductivity requirements (for atmosphere-
and temperature-equilibrated samples) DEFINITION
pH Conductivity Water for injections in bulk that has been distributed into
(µS·cm− 1) suitable containers, closed and sterilised by heat in conditions
5.0 4.7 which ensure that the product still complies with the test for
bacterial endotoxins. Sterilised water for injections is free
5.1 4.1 from any added substances.
5.2 3.6 Examined in suitable conditions of visibility, it is clear and
5.3 3.3 colourless.
5.4 3.0
Each container contains a sufficient quantity of water for
injections to permit the nominal volume to be withdrawn.
5.5 2.8

5.6 2.6
TESTS
Acidity or alkalinity. To 20 mL add 0.05 mL of phenol
5.7 2.5
red solution R. If the solution is yellow, it becomes red
5.8 2.4 on the addition of 0.1 mL of 0.01 M sodium hydroxide ;
if red, it becomes yellow on the addition of 0.15 mL of
5.9 2.4
0.01 M hydrochloric acid.
6.0 2.4
Conductivity : maximum 25 µS·cm− 1 for containers with a
6.1 2.4 nominal volume of 10 mL or less ; maximum 5 µS·cm− 1 for
containers with a nominal volume greater than 10 mL.
6.2 2.5
Use equipment and the calibration procedure as defined
6.3 2.4 under Water for injections in bulk, maintaining the sample
6.4 2.3 temperature at 25 ± 1 °C.
6.5 2.2 Oxidisable substances. For containers with a nominal volume
less than 50 mL : heat 100 mL to boiling with 10 mL of dilute
6.6 2.1 sulfuric acid R, add 0.4 mL of 0.02 M potassium permanganate
6.7 2.6 and boil for 5 min ; the solution remains faintly pink.
For containers with a nominal volume equal to or greater than
6.8 3.1
50 mL : heat 100 mL to boiling with 10 mL of dilute sulfuric
6.9 3.8 acid R, add 0.2 mL of 0.02 M potassium permanganate and
boil for 5 min ; the solution remains faintly pink.
7.0 4.6
Chlorides (2.4.4) : maximum 0.5 ppm for containers with a
nominal volume of 100 mL or less.
15 mL complies with the limit test for chlorides. Prepare
CHARACTERS the standard using a mixture of 1.5 mL of chloride standard
solution (5 ppm Cl) R and 13.5 mL of water R. Examine the
Appearance : clear and colourless liquid. solutions down the vertical axes of the tubes.
For containers with a nominal volume greater than 100 mL,
use the following test : to 10 mL add 1 mL of dilute nitric
TESTS acid R and 0.2 mL of silver nitrate solution R2. The solution
shows no change in appearance for at least 15 min.
Nitrates : maximum 0.2 ppm.
Nitrates : maximum 0.2 ppm.
Place 5 mL in a test-tube immersed in iced water, add 0.4 mL Place 5 mL in a test-tube immersed in iced water, add 0.4 mL
of a 100 g/L solution of potassium chloride R, 0.1 mL of of a 100 g/L solution of potassium chloride R, 0.1 mL of
diphenylamine solution R and, dropwise with shaking, 5 mL of diphenylamine solution R and, dropwise with shaking, 5 mL of
nitrogen-free sulfuric acid R. Transfer the tube to a water-bath nitrogen-free sulfuric acid R. Transfer the tube to a water-bath
at 50 °C. After 15 min, any blue colour in the solution is not at 50 °C. After 15 min, any blue colour in the solution is not
more intense than that in a reference solution prepared at the more intense than that in a reference solution prepared at the
same time in the same manner using a mixture of 4.5 mL of same time in the same manner using a mixture of 4.5 mL of
nitrate-free water R and 0.5 mL of nitrate standard solution nitrate-free water R and 0.5 mL of nitrate standard solution
(2 ppm NO3) R. (2 ppm NO3) R.
Aluminium (2.4.17) : maximum 10 ppb, if intended for use in Sulfates. To 10 mL add 0.1 mL of dilute hydrochloric acid R
the manufacture of dialysis solutions. and 0.1 mL of barium chloride solution R1. The solution shows
no change in appearance for at least 1 h.
Prescribed solution. To 400 mL of the water to be examined Aluminium (2.4.17) : maximum 10 ppb, if intended for use in
add 10 mL of acetate buffer solution pH 6.0 R and 100 mL of the manufacture of dialysis solutions.
distilled water R.
Prescribed solution. To 400 mL of the water to be examined
Reference solution. Mix 2 mL of aluminium standard add 10 mL of acetate buffer solution pH 6.0 R and 100 mL of
solution (2 ppm Al) R, 10 mL of acetate buffer solution pH 6.0 R distilled water R.
and 98 mL of distilled water R. Reference solution. Mix 2 mL of aluminium standard
solution (2 ppm Al) R, 10 mL of acetate buffer solution pH 6.0 R
Blank solution. Mix 10 mL of acetate buffer solution pH 6.0 R and 98 mL of distilled water R.
and 100 mL of distilled water R.
Blank solution. Mix 10 mL of acetate buffer solution pH 6.0 R
Bacterial endotoxins (2.6.14) : less than 0.25 IU/mL. and 100 mL of distilled water R.

General Notices (1) apply to all monographs and other texts 4205
Water for preparation of extracts EUROPEAN PHARMACOPOEIA 10.0

Ammonium : for containers with a nominal volume less than Reference solution. Dissolve 0.163 g of potassium nitrate R
50 mL : maximum 0.6 ppm ; for containers with a nominal and 0.149 g of potassium bromide R in water R and dilute to
volume equal to or greater than 50 mL : maximum 0.2 ppm. 100.0 mL with the same solvent. Dilute 5.0 mL of the solution
Containers with a nominal volume less than 50 mL : to 20 mL to 100.0 mL with water R.
add 1 mL of alkaline potassium tetraiodomercurate solution R ; Column :
after 5 min, examine the solution down the vertical axis of – size : l = 0.25 m, Ø = 4 mm ;
the tube ; the solution is not more intensely coloured than a – stationary phase : anion-exchange resin R3.
standard prepared at the same time by adding 1 mL of alkaline
Mobile phase : dissolve 0.265 g of anhydrous sodium
potassium tetraiodomercurate solution R to a mixture of 4 mL
carbonate R and 0.210 g of sodium hydrogen carbonate R in
of ammonium standard solution (3 ppm NH4) R and 16 mL
water R and dilute to 1000.0 mL with the same solvent.
of ammonium-free water R.
Flow rate : 1.2 mL/min.
Containers with a nominal volume equal to or greater
than 50 mL : to 20 mL add 1 mL of alkaline potassium Detection : conductivity detector, using a self-regenerating
tetraiodomercurate solution R ; after 5 min, examine the anion suppressor.
solution down the vertical axis of the tube ; the solution is not Injection : 25 µL.
more intensely coloured than a standard prepared at the same Run time : twice the retention time of nitrate.
time by adding 1 mL of alkaline potassium tetraiodomercurate Relative retention with reference to nitrate (retention
solution R to a mixture of 4 mL of ammonium standard time = about 7 min) : bromide = about 0.9.
solution (1 ppm NH4) R and 16 mL of ammonium-free water R. System suitability : reference solution :
Calcium and magnesium. To 100 mL add 2 mL of ammonium – resolution : minimum 2.0 between the peaks due to bromide
chloride buffer solution pH 10.0 R, 50 mg of mordant black 11 and nitrate.
triturate R and 0.5 mL of 0.01 M sodium edetate. A pure blue Limit :
colour is produced.
– nitrate : maximum 50 ppm.
Residue on evaporation : maximum 4 mg (0.004 per cent) for
containers with a nominal volume of 10 mL or less ; maximum Microbiological monitoring. Appropriate measures are taken
3 mg (0.003 per cent) for containers with a nominal volume to ensure that the microbial count is adequately controlled
greater than 10 mL. and monitored. Appropriate alert and action levels are set so
as to detect adverse trends.
Evaporate 100 mL to dryness on a water-bath and dry in an
Under normal conditions, an appropriate action level is a
oven at 100-105 °C.
microbial count of 100 CFU/mL, determined by filtration
Particulate contamination : sub-visible particles (2.9.19). It through a membrane with a nominal pore size not greater than
complies with test A or test B, as appropriate. 0.45 µm, using casein soya bean digest agar and incubating at
Sterility (2.6.1). It complies with the test for sterility. 30-35 °C for not less than 5 days.
Bacterial endotoxins (2.6.14) : less than 0.25 IU/mL. The size of the sample is to be chosen in relation to the
expected result.
Casein soya bean digest agar
Pancreatic digest of casein 15.0 g
04/2012:2249
Papaic digest of soya bean 5.0 g

Sodium chloride 5.0 g

Agar 15.0 g

WATER FOR PREPARATION OF Purified water to 1000 mL

EXTRACTS Adjust the pH so that after sterilisation it is 7.3 ± 0.2. Sterilise

in an autoclave using a validated cycle.
Aqua ad extracta praeparanda Growth promotion of casein soya bean digest agar
– Preparation of test strains. Use standardised stable
DEFINITION suspensions of test strains or prepare them as stated
Water intended for the preparation of Herbal drug extracts in Table 2249.-1. Seed lot culture maintenance
(0765) complies with the sections Purified water in bulk or techniques (seed-lot systems) are used so that the viable
Purified water in containers in the monograph Purified water micro-organisms used for inoculation are not more than
(0008), or is water intended for human consumption of a 5 passages removed from the original master seed-lot.
quality equivalent to that defined in Directive 98/83/EC which Grow each of the bacterial strains separately as described
is monitored according to the Production section described in Table 2249.-1. Use buffered sodium chloride-peptone
below. solution pH 7.0 or phosphate buffer solution pH 7.2 to
make test suspensions. Use the suspensions within 2 h, or
PRODUCTION within 24 h if stored at 2-8 °C. As an alternative to preparing
When water intended for human consumption is used as water and then diluting a fresh suspension of vegetative cells of
for preparation of extracts it is a clear, colourless liquid. It is Bacillus subtilis, a stable spore suspension is prepared and
stored (where necessary) and distributed under conditions then an appropriate volume of the spore suspension is used
designed to prevent growth of micro-organisms and to avoid for test inoculation. The stable spore suspension may be
other contamination. maintained at 2-8 °C for a validated period of time.
For monitoring purposes, the following tests are carried out at – Growth promotion. Test each batch of ready-prepared
regular intervals to demonstrate consistency in the quality of medium and each batch of medium, prepared either from
the water used for the preparation of extracts. dehydrated medium or from the ingredients described.
Conductivity (2.2.38) : maximum 2500 µS·cm− 1, measured at Inoculate plates of casein soya bean digest agar separately
20 °C. with a small number (not more than 100 CFU) of the
micro-organisms indicated in Table 2249.-1. Incubate
Nitrate. Liquid chromatography (2.2.29). under the conditions described in this table. Growth
Test solution. The substance to be examined. obtained must not differ by a factor greater than 2 from the

4206 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 10.0 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 solution pH 7.0 or phosphate buffer solution pH 7.2 to
ATCC 6633
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.
WATER, PURIFIED Inoculate plates of R2A agar separately with a small number
(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 4207
Water, purified EUROPEAN PHARMACOPOEIA 10.0

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 pH 6.0 R
cent of the certified value. 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.

4208 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 10.0 Wheat starch

LABELLING TESTS
The label states, where applicable, that the substance is suitable pH (2.2.3) : 4.5 to 7.0.
for use in the manufacture of dialysis solutions.
Shake 5.0 g with 25.0 mL of carbon dioxide-free water R for
60 s. Allow to stand for 15 min.
01/2020:0359
◊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
granules of any other origin are present.◊
WHEAT STARCH (1) Total protein : maximum 0.3 per cent (corresponding to
0.048 per cent of nitrogen).
Tritici amylum Determine the nitrogen content by sulfuric acid digestion as
follows, and calculate the quantity of protein by multiplying
DEFINITION
by 6.25.
Wheat starch is obtained from the caryopsis of Triticum
aestivum L. (T. vulgare Vill.). 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 beads
Appearance : very fine, white or almost white powder that in a combustion flask. Wash any adhering particles from the
creaks when pressed between the fingers. neck into the flask with 25 mL of sulfuric acid R, allowing it to
Solubility : practically insoluble in cold water and in ethanol run down the sides of the flask, and swirl to mix the contents.
(96 per cent). Close the mouth of the flask loosely, for example by means of a
glass bulb with a short stem, to avoid excessive loss of sulfuric
Wheat starch does not contain starch granules of any other acid. Heat gradually at first, then increase the temperature
origin. It may contain a minute quantity, if any, of tissue until there is vigorous boiling with condensation of sulfuric
fragments of the original plant.♦ acid in the neck of the flask ; take precautions to prevent the
IDENTIFICATION upper part of the flask from becoming overheated. Continue
A. Microscopic examination (2.8.23) using a 50 per cent V/V heating until a clear solution is obtained. Cool, dissolve the
solution of glycerol R. It shows large and small granules, solid material by cautiously adding 25 mL of water R to the
and, very rarely, intermediate sizes (Figure 0359.-1). The mixture, cool again and place in a steam-distillation apparatus.
large granules, 10-60 µm in diameter, are discoid or, more Add a suitable volume of strong sodium hydroxide solution R
rarely, reniform in surface view. The central hilum and to change the colour of the solution from bluish-green to
striations are invisible or barely visible and the granules brown or black, and distil immediately by passing steam
sometimes show cracks on the edges. In side view, the through the mixture. Collect about 40 mL of distillate in
granules are elliptical and fusiform and the hilum appears 50.0 mL of 0.01 M hydrochloric acid, adding enough water R
as a slit along the main axis. The small granules, rounded or if necessary to cover the tip of the condenser. Towards the
polyhedral, are 2-10 µm in diameter. Between orthogonally end of the distillation, lower the receiver so that the tip of the
orientated polarising plates or prisms, the granules show a condenser is above the surface of the acid. Take precautions to
distinct black cross intersecting at the hilum. 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.
Sulfated ash (2.4.14): maximum 0.6 per cent, determined on
1.0 g.
Figure 0359.-1. − Illustration for identification test A of wheat
starch Microbial contamination
B. Suspend 1 g in 50 mL of water R, boil for 1 min and cool. TAMC : acceptance criterion 103 CFU/g (2.6.12).
A thin, cloudy mucilage is formed. TYMC : acceptance criterion 102 CFU/g (2.6.12).
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 Absence of Escherichia coli (2.6.13).
produced, which disappears on heating. ◊Absence of Salmonella (2.6.13).◊
(1) This monograph has undergone pharmacopoeial harmonisation. See chapter 5.8. Pharmacopoeial harmonisation.

General Notices (1) apply to all monographs and other texts 4209