EUROPEAN PHARMACOPOEIA 6.0Chondroitin sulphate
sodium
retention time less than or equal to 1.5times the retention time of cholesterol.Disregard the peaks due to the internal standard and the solvent.STORAGE
Protected from light.
LABELLING
The label states the source material for the production of cholesterol (for example bovine brain and spinal cord,wool fat or chicken eggs).
IMPURITIES
A.5α-cholest-7-en-3β-ol
(lathosterol),
B.cholesta-5,24-dien-3β-ol (desmosterol),
C.5α-cholesta-7,24-dien-3β-ol.
01/2008:2064吸血鬼日记第二季21
CHONDROITIN SULPHATE SODIUM Chondroitini natrii sulfas
H 2O(C 14H 19NNa 2O 14S)x
DEFINITION Natural copolymer bad mainly on the 2disaccharides:[4)-(β-D -glucopyranosyluronic acid)-(1→3)-[2-(acetylamino)-2-deoxy-β-D -galactopyranosyl 4-sulphate]-(1→]and [4)-(β-D -glucopyranosyluronic acid)-(1→3)-[2-(acetylamino)-2-deoxy-β-D -galactopyranosyl 6-sulphate]-(1→],sodium salt.On complete hydrolysis it liberates D -galactosamine,D -glucuronic acid,acetic acid and sulphuric acid.It is obtained from cartilage of both terrestrial and marine origins.Depending on th
e animal species of origin,it shows different proportions of 4-sulphate and 6-sulphate groups.
Content :95per cent to 105per cent (dried substance).PRODUCTION
The animals from which chondroitin sulphate sodium is derived must fulfil the requirements for the health of animals suitable for human consumption.
CHARACTERS
Appearance :white or almost white,hygroscopic powder.Solubility :freely soluble in water,practically insoluble in acetone and in ethanol (96per cent).
IDENTIFICATION
A.Infrared absorption spectrophotometry (2.2.24).Preparation :discs of potassium bromide R .Comparison :for chondroitin sulphate sodium of
qualifyterrestrial origin u chondroitin sulphate sodium CRS and for chondroitin sulphate sodium of marine origine u chondroitin sulphate sodium (marine)CRS .B.Solution S1(e Tests)gives reaction (b)of sodium (2.3.1).
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C.Examine the electropherograms obtained in the test for related substances.
Results :the principal band in the electropherogram obtained with the test solution is similar in position to the principal band in the electropherogram obtained with reference solution (a).
TESTS
Solution S1.Dissolve 2.500g in 50.0ml of carbon dioxide-free water R .
Solution S2.Dilute 1.0ml of solution S1to 10.0ml with water R .
pH (2.2.3):5.5to 7.5for solution S1.
Specific optical rotation (2.2.7):−20to −30(terrestrial origin)or −12to −19(marine origin)(dried substance),
determined on solution S1.
Intrinsic viscosity :0.01m 3/kg to 0.15m 3/kg.
Test solution (a).Weigh 5.000g (m 0p )of the substance to be
examined and add about 80ml of an 11.7g/l solution of sodium chloride R at room temperature.Dissolve by shaking at room temperature for 30min.Dilute to 100.0ml with an 11.7g/l solution of sodium chloride R .Filter through a 0.45µm filter membrane and discard the first 10ml.The concentration of test solution (a)is only indicative and must be adjusted after an initial measurement of the viscosity of test solution (a).
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Test solution (b).To 15.0ml of test solution (a)add 5.0ml of an 11.7g/l solution of sodium chloride R .
Test solution (c).To 10.0ml of test solution (a)add 10.0ml of an 11.7g/l solution of sodium chloride R .
Test solution (d).To 5.0ml of test solution (a)add 15.0ml of an 11.7g/l solution of sodium chloride R .
Determine the flow-time (2.2.9)for an 11.7g/l solution of sodium chloride R (t 0)and the flow times for the 4test solutions (t 1,t 2,t 3and t 4),at 25.00±0.03°C.U an
appropriate suspended level viscometer (specifications:
viscometer constant =about 0.005mm 2/s 2,kinematic General Notices (1)apply to all monographs and other texts
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Chondroitin sulphate sodium EUROPEAN PHARMACOPOEIA
6.0
viscosity range =1-5mm 2/s,internal diameter of
tube R =0.53mm,volume of bulb C =5.6ml,internal
diameter of tube N =2.8-3.2mm)with a funnel-shaped lower capillary end.U the same viscometer for all measurements;measure all outflow times in triplicate.The test is not valid unless the results do not differ by more than 0.35per cent from the mean and if the flow time t 1is not less than 1.6×t 0and not more than 1.8×t 0.If this is not the ca,adjust the concentration of test solution (a)and repeat the procedure.Calculation of the relative viscosities
wishes是什么意思Since the densities of the chondroitin sulphate solutions and of the solvent are almost equal,the relative viscosities ηr i (being ηr1,ηr2,ηr3and ηr4)can be calculated from the ratio of the flow times for the respective solutions t i (being t 1,t 2,t 3and t 4)to the flow time of the solvent t 0,but taking into account the kinetic energy correction factor for the capillary (B =30800s 3),as shown below:
Calculation of the concentrations
Calculate the concentration c 1(expresd in kg/m 3)of chondroitin sulphate sodium in test solution (a)using the
following expression:
x =percentage content of chondroitin sulphate
sodium as determined in the assay;
h =loss on drying as a percentage.Calculate the concentration c 2(expresd in kg/m 3)of
chondroitin sulphate sodium in test solution (b)using the
following expression:
Calculate the concentration c 3(expresd in kg/m 3
)of chondroitin sulphate sodium in test solution (c)using the following expression:Calculate the concentration c 4(expresd in kg/m 3)of chondroitin sulphate sodium in test solution (d)using the following expression:Calculation of the intrinsic viscosity
The specific viscosity ηs i of the test solution (being ηs1,
ηs2,ηs3and ηs4)is calculated from the relative viscosities
ηr i (being ηr1,ηr2,ηr3and ηr4)according to the following expression:The intrinsic viscosity [η],defined as
is calculated by linear least-squares regression analysis using the following equation:
c i =concentration of the substance to be examine
d expresd in kg/m 3;k H
=
帝国理工学院Huggins’constant.
Related substances .Electrophoresis (2.2.31).
Buffer solution A (0.1M barium acetate pH 5.0).Dissolve 25.54g of barium acetate R in 900ml of water R .Adjust to pH 5.0with glacial acetic acid R and dilute to 1000.0ml with water R .
Buffer solution B (1M barium acetate pH 5.0).Dissolve 255.43g of barium acetate R in 900ml of water R .Adjust to pH 5.0with glacial acetic acid R and dilute to 1000.0ml with water R .
Staining solution .Dissolve 1.0g of toluidine blue R and 2.0g of sodium chloride R in 1000ml of 0.01M hydrochloric acid .Filter.
Test solution .Prepare a 30mg/ml solution of the substance to be examined in water R .
Reference solution (a).Prepare a 30mg/ml solution of chondroitin sulphate sodium CRS in water R .
Reference solution (b).Dilute 2.0ml of reference solution (a)to 100.0ml with water R .
Reference solution (c).Mix equal volumes of reference
test drive
solution (b)and water R .Procedure .Allow the electrophoresis support to cool the
plate to 10°C.Pre-equilibrate the agaro gel for 1min in buffer solution A.Remove excess liquid by careful decanting.Dry the gel for approximately 5min.Place 400ml of buffer solution B into each of the containers of the electrophoresis equipment.Transfer 1µl of each solution to the slots of the agaro gel.Pipette a few millilitres of a 50per cent V/V solution of glycerol R onto the cooled plate of the electrophoresis equipment and place the gel in the middle of the ceramic plate.Place a wick,saturated with buffer solution B,at the positive and negative sides of the agaro gel.Ensure that there is good contact between the electrophoresis buffer and the agaro gel.Perform the electrophoresis under the following conditions:75mA/gel,resulting in a voltage of 100-150V (maximum 300-400V)for
a gel of about 12cm ×10cm.Carry out the electrophoresis for 12min.Place the gel in a mixture consisting of 10volumes of anhydrous ethanol R and 90volumes of
buffer solution A for 2min.Carry out the electrophoresis for 20min.Place the gel in a mixture consisting of 30volumes of anhydrous ethanol R and 70volumes of buffer solution A
for 2min.Carry out the electrophoresis for 20min.Stain the gel in the staining solution for 10min.Desta
in the gel for 15min under running tap water followed by 10-15min with
water R until the band in the electropherogram obtained with reference solution (c)is visible.Allow the gel to dry.System suitability :—the electropherogram obtained with reference solution (c)shows a visible band;—the band in the electropherogram obtained with reference solution (b)is clearly visible and similar in position to the
hannesband in the electropherogram obtained with reference solution (a).
Results :any condary band in the electropherogram obtained with the test solution is not more inten than the band in the electropherogram obtained with reference solution (b)(2per cent).
Protein (2.5.33,Method 2):maximum 3.0per cent (dried
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substance).Test solution .Dilute 1.0ml of solution S1to 50.0ml with 0.1M sodium hydroxide .1526
See the information ction on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.0
Chymotrypsin
Reference solutions .Dissolve about 0.100g of bovine
albumin R ,accurately weighed,in 0.1M sodium hydroxide and dilute to 50.0ml with the same solvent.Carry out all additional dilutions using 0.1M sodium hydroxide .Chlorides (2.4.4):maximum 0.5per cent.
Dilute 1ml of solution S2to 15ml with water R .Do not add diluted nitric acid.Prepare the standard using 5ml of chloride standard solution (5ppm Cl)and 10ml of water R .
Heavy metals (2.4.8):maximum 20ppm.
1.0g complies with test C.Prepare the reference solution using 2ml of lead standard solution (10ppm Pb)R .
Loss on drying (2.2.32):maximum 12.0per cent,determined
on 1.000g by drying in an oven at 105°C for 4h.
Microbial contamination .Total viable aerobic count
(2.6.12)not more than 103micro-organisms per gram.It complies with the tests for Staphylococcus aureus ,
Pudomonas aeruginosa ,Escherichia coli ,Salmonella and Enterobacteria and certain other gram-negative bacteria (2.6.13).
ASSAY
Test solution (a).Weigh 0.100g (m 1)of the substance to be examined,dissolve in water R and dilute to 100.0ml with the same solvent.
Test solution (b).Dilute 5.0ml of test solution (a)to 50.0ml
with water R .
Reference solution (a).Weigh 0.100g (m 0)of chondroitin
sulphate sodium CRS ,previously dried as described in the
test for loss on drying,dissolve in water R and dilute to
100.0ml with the same solvent.
Reference solution (b).Dilute 5.0ml of reference solution (a)to 50.0ml with water R .Titrant solution (a).Weigh 4.000g of cetylpyridinium chloride monohydrate R and dilute to 1000ml with water R .Titrant solution (b).Weigh 1.000g of cetylpyridinium chloride monohydrate R and dilute to 1000ml with water R .
Perform either visual or photometric titration as follows:
Visual titration .Titrate 40.0ml of reference solution (a)and 40.0ml of test solution (a)with titrant solution (a).The solution becomes turbid.At the end point,the liquid appears clear,with an almost-white precipitate in suspension.The
precipitate is more apparent if 0.1ml of a 1per cent solution of methylene blue R is added before star
ting the titration.The precipitated particles are more apparent against the blue background.Photometric titration .Titrate 50.0ml of reference solution (b)and 50.0ml of test solution (b)with titrant solution (b).To determine the end point,u a suitable autotitrator equipped with a phototrode at a suitable wavelength (none is critical)in the visible range.Calculate the percentage content of chondroitin sulphate sodium using the following expression:
v 0=volume of appropriate titrant solution when titrating the appropriate reference solution,in millilitres;
v 1
=volume of appropriate titrant solution when titrating the appropriate test solution,in millilitres;
h =loss on drying of the substance to be examined,as a percentage;
Z
=
percentage content of H 2O(C 14H 19NNa 2O 14S)x in chondroitin sulphate sodium CRS .
STORAGE
In an airtight container,protected from light.
LABELLING The label states the origin of the substance (marine or terrestrial).01/2008:0476
CHYMOTRYPSIN
Chymotrypsinum
[9004-07-3]
DEFINITION
Chymotrypsin is a proteolytic enzyme obtained by the activation of chrymotrypsinogen extracted from the pancreas of beef (Bos taurus L.).It has an activity of not less than 5.0microkatals per milligram.In solution it has maximal enzymic activity at about pH 8;the activity is reversibly inhibited at pH 3,at which pH it is most stable.PRODUCTION
The animals from which chymotrypsin is derived must
fulfil the requirements for the health of animals suitable
for human consumption.Furthermore,the tissues ud shall not include any specified risk material as defined by
any relevant international or,where appropriate,national
legislation.The method of manufacture is validated to demonstrate that the product,if tested,would comply with the following test.Histamine (2.6.10).Not more than 1µg (calculated as
histamine ba)per 5microkatals of chymotrypsin activity.Before carrying out the test,heat the solution of the substance to be examined on a water-bath for 30min.CHARACTERS A white or almost white,crystalline or amorphous powder,sparingly soluble in water.The amorphous form is hygroscopic.IDENTIFICATION
A.Dilute 1ml of solution S (e Tests)to 10ml with water R .In a depression in a white spot plate,mix 0.05ml of this solution with 0.2ml of substrate solution.A purple colour develops.
Substrate solution .To 24.0mg of acetyltyrosine ethyl ester R add 0.2ml of ethanol (96per cent)R ,and swirl until solution is effected.Add 2.0ml of 0.067M phosphate buffer solution pH 7.0R and 1ml o
f methyl red mixed solution R and dilute to 10.0ml with water R .
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General Notices (1)apply to all monographs and other texts
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