Dalteparin sodium EUROPEAN PHARMACOPOEIA
Reference solution (a).Dilute 2.5mL of dimethylamine
solution R (impurity D)to 100.0mL with water R (solution A).Firmly attach the septum and cap to a 20mL vial.Using a 10μL syringe,inject 10μL of solution A into the vial.
Reference solution (b).Firmly attach the septum and cap to a 20mL vial.Using a 10μL syringe,inject 10μL of solution A and 10μL of a 10g/L solution of triethylamine R into the vial.Column :
–material :fused silica;
–size :l =30.0m,Ø=0.53mm;
–stationary phase :base-deactivated polyethyleneglycol R (ﬁlm thickness 1.0μm).
Carrier gas :helium for chromatography R .Flow rate :13mL/min.Split ratio :1:1.
Static head-space conditions that may be used :–equilibration temperature :60°C;–equilibration time :10min;
–transfer-line temperature :90°C;–pressurisation time :30s.Temperature :
35→165Injection port 180Detector
Detection :ﬂame ionisation.Injection :1mL.
System suitability :reference solution (b):
–resolution :minimum 2.5between the peaks due to impurity D and triethylamine.Limit :
–impurity D :not more than the area of the corresponding peak in the chromatogram obtained with reference solution (a)(0.05per cent).
Water (2.5.12):maximum 0.5per cent,determined on 1.00g.Sulfated ash (2.4.14):maximum 0.1per cent,determined on 1.0g.
Dissolve 0.150g in 30mL of anhydrous acetic acid R .Titrate with 0.1M perchloric acid ,determining the end-point potentiometrically (2.2.20).
1mL of 0.1M perchloric acid is equivalent to 18.22mg of C 6H 10N 6O.
At a temperature of 2°C to 8°C,protected from light.IMPURITIES
Specified impurities :A,B,D .
Other detectable impurities (the following substances would,if present at a sufﬁcient level,be detected by one or other of the tests in the monograph.They are limited by the general acceptance criterion for other/unspeciﬁed impurities and/or by the general monograph Substances for pharmaceutical use (2034).It is therefore not necessary to identify these impurities for demonstration of compliance.See also 5.10.Control of impurities in substances for pharmaceutical use ):C
. A.3,7-dihydro-4H -imidazo[4,5-d ]-1,2,3-triazin-4-one
DALTEPARIN SODIUM Dalteparinum
Dalteparin sodium is the sodium salt of a low-molecular-mass heparin that is obtained by nitrous acid depolymerisation of heparin from porcine intestinal mucosa.The majority of the components have a 2-O -sulfo-α-L -idopyranosuronic acid structure at the non-reducing end and a
6-O -sulfo-2,5-anhydro-D -mannitol structure at the reducing end of their chain.
Dalteparin sodium complies with the monograph
Low-molecular-mass heparins (0828)with the modifications and additional requirements below.
The mass-average relative molecular mass ranges between 5600and 6400,with a characteristic value of about 6000.The degree of sulfatation is 2.0to 2.5per disaccharide unit.The potency is not less than 110IU and not more than 210IU of anti-factor Xa activity per milligram,calculated with reference to the dried substance.The anti-factor IIa activity is not less than 35IU/mg and not more than 100IU/mg,calculated with reference to the dried substance.The ratio of anti-factor Xa activity to anti-factor IIa activity is between 1.9and 3.2.
Dalteparin sodium is produced by a validated manufacturing and puriﬁcation procedure under conditions designed to minimise the presence of N-NO groups.
The manufacturing procedure must have been shown to
reduce any contamination by N-NO groups to approved limits using an appropriate,validated quantiﬁcation method.1988
See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 8.0Dalteparin
Carry out identiﬁcation test A as described in the monograph Low-molecular-mass heparins (0828)using dalteparin sodium CRS .
Carry out identiﬁcation test C as described in the monograph
Low-molecular-mass heparins (0828).The following
The mass-average relative molecular mass ranges between 5600
and 6400.The mass percentage of chains lower than 3000is
not more than 13.0per cent.The mass percentage of chains
higher than 8000ranges between 15.0per cent and 25.0per
Appearance of solution .Dissolve 1g in 10mL of water R .The solution is clear (2.2.1)and not more intensely coloured than intensity 5of the range of reference solutions of the most appropriate colour (2.2.2,Method II ).Nitrite .Not more than 5ppm.Examine by liquid chromatography (2.2.29).Rinse all volumetric flasks at least three times with water R before the preparation of the solutions.Test solution.Dissolve 80.0mg of the substance to be examined in water R and dilute to 10.0mL with the same solvent.Allow to stand for at least 30min.
Reference solution (a).Dissolve 60.0mg of sodium nitrite R in water R and dilute to 1000.0mL with the same solvent.For the preparation of reference solution (b),use a pipette previously rinsed with reference solution (a).
Reference solution (b).Dilute 1.00mL of reference solution (a)to 50.0mL with water R .Before preparing reference solutions (c),(d)and (e),rinse all pipettes with reference solution (b).
Reference solution (c).Dilute 1.00mL of reference solution (b)to 100.0mL with water R (corresponding to 1ppm of nitrite in the test sample).Reference solution (d).Dilute 3.00mL of reference solution (b)to 100.0mL with water R (corresponding to 3ppm of nitrite
in the test sample).
Reference solution (e).Dilute 5.00mL of reference solution (b)to 100.0mL with water R (corresponding to 5ppm of nitrite
in the test sample).
The chromatographic procedure may be carried out using:–a column 0.125m long and 4.3mm in internal diameter
packed with a strong anion-exchange resin;–as mobile phase at a ﬂow rate of 1.0mL/min a solution consisting of 13.61g of sodium acetate R dissolved in water R ,adjusted to pH 4.3with phosphoric acid R and diluted to 1000mL with water R ;–as detector an appropriate electrochemical device with the following characteristics and settings:a suitable working electrode,a detector potential of +1.00V versus Ag/AgCl reference electrode and a detector sensitivity of 0.1μA full scale.Inject 100μL of reference solution (d).When the
chromatograms are recorded in the prescribed conditions,the retention time for nitrite is 3.3to 4.0min.The test is not valid unless:
–the number of theoretical plates calculated for the nitrite peak is at least 7000per metre per column (dalteparin sodium will block the binding sites of the stationary phase,which will cause shorter retention times and lower separation efﬁciency for the analyte;the initial
performance of the column may be partially restored using a 58g/L solution of sodium chloride R at a ﬂow rate of 1.0mL/min for 1h;after regeneration the column is rinsed with 200mL to 400mL of water R );–the symmetry factor for the nitrite peak is less than 3;
–the relative standard deviation of the peak area for nitrite
obtained from 6injections is less than 3.0per cent.
Inject 100μL each of reference solutions (c)and (e).The test is not valid unless:
–the correlation factor for a linear relationship between concentration and response for reference solutions (c),(d)and (e)is at least 0.995;–the signal-to-noise ratio for reference solution (c)is not less than 5(if the noise level is too high,electrode recalibration is recommended);–a blank injection of water R does not give rise to spurious peaks.Inject 100μL of the test solution.Calculate the content of nitrite from the peak areas in the chromatogram obtained with reference solutions (c),(d)and (e).
Boron .Not more than 1ppm,determined by inductively coupled plasma atomic emission spectroscopy.
Boron is determined by measurement of the emission from an inductively coupled plasma (ICP)at a wavelength speciﬁc to boron.The emission line at 249.733nm is used.Use an
appropriate apparatus,whose settings have been optimised as directed by the manufacturer.Test solution.Dissolve 0.2500g of the substance to be
examined in about 2mL of water for chromatography R ,add 100μL of nitric acid R and dilute to 10.00mL with the same
solvent.Reference solution (a).Prepare a 1per cent V/V solution of nitric acid R in water for chromatography R (blank).Reference solution (b).Prepare a 11.4μg/mL solution of boric
acid R in a 1per cent V/V solution of nitric acid R in water for chromatography R (STD cal ).Reference solution (c).Dissolve 0.2500g of a reference
dalteparin sodium with no detectable boron in about 2mL of
water for chromatography R ,add 100μL of nitric acid R and dilute to 10.00mL with the same solvent (STD 0).Reference solution (d).Dissolve 0.2500g of a reference dalteparin sodium with no boron detected in about 2mL of a 1per cent V/V solution of nitric acid R in water for
chromatography R ,add 10μL of a 5.7mg/mL solution of boric acid R and dilute to 10.00mL with the same solvent (STD 1).This solution contains 1μg/mL of boron.
Calculate the content of boron in the substance to be examined,using the following correction factor:
Loss on drying (2.2.32).Not more than 5.0per cent,determined on 1.000g by drying in an oven at 60°C over diphosphorus pentoxide R at a pressure not exceeding 670Pa for 3h.
General Notices (1)apply to all monographs and other texts