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Printed by: Anandkumar Joshi Official Date: Official as of 01-May-2018 Document Type: USP @2023 USPC
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2 s, and at least a 10-s delay. For each sample, an initial

Dalteparin Sodium short spectrum is collected (1 scan), and the water
resonance is then suppressed by selective irradiation
during the relaxation delay. Final spectra are recorded
over 32 scans. For all samples, the TSP methyl signal
should be set to 0.00 ppm. Record the 1H NMR spectrum
of the Standard solution. Collect the 1H NMR spectrum
with a spectral window of at least 10 to −2 ppm and
without spinning. The Standard solution shall be run at
least daily when the Sample solution is being run. All
spectra are phased, and linear baseline correction is
applied to all spectra before peak identification.
Suitability requirements
Chemical shift: The TSP methyl signal should be set to
0.00 ppm for all samples.
Chemical shifts for system suitability: The ppm values
CAS RN®: 9041-08-1. for the methyl group of N-acetyl, the H-2 of N-sulfo
DEFINITION glucosamine, the H-2 of glucuronic acid plus 3-O-sulfo
Dalteparin Sodium is the sodium salt of a low molecular weight glucosamine, the H-1 of iduronic acid, and the H-1 of
heparin obtained by nitrous acid depolymerization of 3-O sulfo glucosamine of dalteparin in the Standard
heparin from porcine intestine or intestinal mucosa. Heparin solution are present at 2.05, 3.28, 3.39, 5.01, and 5.51,
source material used in the manufacture of Dalteparin respectively. Two additional signals, corresponding to
the H-1 of the 2-O-sulfo iduronic acid linked to the

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Sodium complies with the compendial requirements stated
in the Heparin Sodium monograph. Dalteparin Sodium is terminal 2, 5-anhydromannitol and the H-1 of 2-O-sulfo
produced by a validated manufacturing and purification iduronic acid are located at 5.18–5.22 ppm. The ppm
procedure under conditions shown to minimize the presence values of these signals do not differ by more than
of species containing the N–NO group. The majority of the ±0.03 ppm, Standard solution.
[NOTE—Depending on specific sample makeup and
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 chains. The weight-average molecular weight (M w)
ci instrument parameters, including the field strength of
the NMR instrument, the two signals associated with
the H-1 of 2-O-sulfo iduronic acid at 5.18–5.22 ppm
ranges between 5600 Da and 6400 Da, with a characteristic may appear well separated or as a main signal with a
value of about 6000 Da. The percentage of chains lower than shoulder.]
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molecular weight 3000 Da is NMT 13.0%, and the Analysis
percentage of chains higher than molecular weight 8000 Da Sample: Sample solution
ranges between 15.0% and 25.0%. The degree of sulfation Record the 1H NMR spectra of the Sample solution.
is NLT 1.8/disaccharide unit. The potency is NLT 110 and Acceptance criteria: The ppm values for the methyl group
NMT 210 Anti-Factor Xa International Units (IU)/mg of of N-acetyl, the H-2 of N-sulfo glucosamine, the H-2 of
glucuronic acid plus 3-O-sulfo glucosamine, the H-1 of
O

activity, calculated on the dried basis. The anti-factor IIa iduronic acid and the H-1 of the 2-O-sulfo iduronic acid
activity is NLT 35 IU/mg and NMT 100 IU/mg, calculated on linked to the terminal anhydromannitol, the H-1 of
the dried basis. The ratio of anti-factor Xa activity to 2-O-sulfo iduronic acid and the H-1 of 3-O sulfo
anti-factor IIa activity is between 1.9 and 3.2. glucosamine of dalteparin in the Sample solution are present
IDENTIFICATION at 2.05, 3.28, 3.39, 5.01, 5.18–5.22, and 5.51, respectively.
• A. 1H NMR SPECTRUM The ppm values of these signals do not differ by more than
Standard solution: Dissolve 15 mg of USP Dalteparin ±0.03 ppm.
Sodium RS in 0.7 mL of deuterium oxide with deuterated • B. MOLECULAR WEIGHT DISTRIBUTION AND
trimethylsilylpropionic (TSP) acid sodium salt. The sample WEIGHT-AVERAGE MOLECULAR WEIGHT
is freeze-dried to remove exchangeable protons. Redissolve (See Low Molecular Weight Heparin Molecular Weight
the sample and repeat the freeze-drying step twice more Determinations á209ñ.)
before transferring the sample into an NMR tube. Acceptance criteria: The weight-average molecular
Sample solution: Dissolve 15 mg of Dalteparin Sodium in weight (M w) ranges between 5600 Da and 6400 Da, with a
0.7 mL of deuterium oxide (99.9%) with deuterated TSP. characteristic value of about 6000 Da. The percentage of
The sample is freeze-dried to remove exchangeable chains lower than the molecular weight 3000 Da (M 3000)
protons. Redissolve the sample and repeat the is NMT 13.0%, and the percentage of chains higher than
freeze-drying step twice more before transferring the the molecular weight 8000 Da (M 8000) ranges between
sample into an NMR tube. 15.0% and 25.0%.
Instrumental conditions • C. ANTI-FACTOR XA TO ANTI-FACTOR IIA RATIO
(See Nuclear Magnetic Resonance Spectroscopy á761ñ.) (See Anti-Factor Xa and Anti-Factor IIa Assays for
Mode: NMR, pulsed (Fourier transform) Unfractionated and Low Molecular Weight Heparins á208ñ,
Frequency: NLT 500 MHz for 1H Anti-Factor Xa and Anti-Factor IIa Assays for Low Molecular
Temperature: 30° Weight Heparins.)
System suitability Acceptance criteria: The ratio of the numerical value of the
Samples: Standard solution and Sample solution anti-factor Xa activity, in Anti-Factor Xa IU/mg, to the
Transfer the Standard solution and the Sample solution to numerical value of the anti-factor IIa activity, in
NMR tubes of 5 mm in diameter. Using a pulsed (Fourier Anti-Factor IIa IU/mg, as determined by the Anti-Factor Xa
transform) NMR spectrometer operating at NLT Activity and Anti-Factor IIa Activity assays, is NLT 1.9 and
500 MHz for 1H, acquire a free induction decay (FID) with NMT 3.2, respectively.
NLT 32 scans using a 90° pulse, an acquisition time of NLT

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Printed by: Anandkumar Joshi Official Date: Official as of 01-May-2018 Document Type: USP @2023 USPC
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• D. IDENTIFICATION TESTS—GENERAL: Meets the Column regeneration: 1 M sodium chloride (NaCl) at

requirements for Sodium Content 0.5 mL/min for about 1 h. After regeneration, wash the
column with water and re-equilibrate with Mobile phase.
ASSAY Flow rate: 0.5 mL/min
• ANTI-FACTOR XA ACTIVITY Injection volume: 25 µL
(See Anti-Factor Xa and Anti-Factor IIa Assays for Run time: 10 min
Unfractionated and Low Molecular Weight Heparins á208ñ, System suitability
Anti-Factor Xa and Anti-Factor IIa Assays for Low Molecular Samples: Calibration standard solutions and Sample
Weight Heparins, Anti-Factor Xa Activity for Low Molecular solution
Weight Heparin.) Suitability requirements
Analysis: Proceed as directed in the chapter. Column efficiency: NLT 4000 theoretical plates for the
Acceptance criteria: The potency is NLT 110 and NMT nitrite peak for all Calibration solutions and Sample
210 Anti-Factor Xa IU/mg on the dried basis. solution runs
OTHER COMPONENTS Tailing factor: Between 0.8 and 1.2 for all Calibration
• NITROGEN DETERMINATION, Method II á461ñ: 1.5%–2.5% on solutions and Sample solution runs
the dried basis Relative standard deviation: Inject Calibration standard
• SODIUM CONTENT solutions with 25 ng/mL concentration at least six times.
Cesium chloride solution: 1.27 mg/mL of cesium chloride Calculate the relative standard deviation % (%RSD) of
in 0.1 M hydrochloric acid the nitrite peak areas of the last six injections. The %RSD
Standard solution A: 0.0025% of sodium chloride in is NMT 2%.
Cesium chloride solution Analysis
Standard solution B: 0.0050% of sodium chloride in Samples: Calibration standard solutions and Sample

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Cesium chloride solution solution
Standard solution C: 0.0075% of sodium chloride in Plot the areas of the nitrite peaks from the chromatograms
Cesium chloride solution of the Calibration standard solutions against respective
Sample solution: Transfer 50.0 mg of Dalteparin Sodium concentrations of nitrite. Draw a best-fit regression line
to a 100-mL volumetric flask, and dissolve in and dilute with through the points. The correlation coefficient is NLT
0.995. Calculate the concentration of nitrite from the
Cesium chloride solution to volume.
Analysis
Samples: Cesium chloride solution, Standard solution A,
Standard solution B, Standard solution C, and Sample
ci areas of the nitrite peak in the chromatogram of the
Sample solution.
Acceptance criteria: NMT 5 ppm
solution • BORON
Concomitantly determine the absorbances of the Cesium [NOTE—Use only plastic labware, avoid glass.]
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chloride solution (blank), the Sample solution, and the Blank: 1% (v/v) solution of nitric acid in water
Standard solutions at 330.3 nm, using a sodium Calibration solution: Prepare a 11.4-µg/mL solution of USP
hollow-cathode lamp and an air–acetylene flame. Using Boric Acid RS in the Blank.
the absorbances of Standard solutions A, B, and C, Standard solution A: Dissolve 0.2500 g of USP Low
determine the sodium content in the Sample solution after Molecular Weight Heparin for Boron Analysis RS in about
an appropriate blank correction. 2 mL of water, add 100 µL of nitric acid, and dilute with the
O

Acceptance criteria: 10.5%–13.5% on the dried basis Blank to 10.00 mL.

Standard solution B: Dissolve 0.2500 g of USP Low
IMPURITIES Molecular Weight Heparin for Boron Analysis RS in about
• LIMIT OF NITRITES 2 mL of Blank, add 10 µL of a 5.7-mg/mL solution of USP
Mobile phase: Dissolve 13.6 g of sodium acetate trihydrate Boric Acid RS, and dilute with the Blank to 10.00 mL. This
in 900 mL of water in a 1000-mL volumetric flask. Adjust solution contains 1 µg/mL of boron.
with orthophosphoric acid to a pH of 4.3, and dilute with Sample solution: Dissolve 0.2500 g of Dalteparin Sodium
water to 1000 mL. Filter through a 0.45-µm membrane. in about 2 mL of water, add 100 µL of nitric acid, and dilute
Nitrite stock standard solution: Dissolve 0.075 g of sodium with the Blank to 10.00 mL.
nitrite in a 1000-mL volumetric flask with carbon Analysis
dioxide-free water (0.05 g/L of nitrite). Samples: Blank, Calibration solution, Standard solution A,
Nitrite standard solution: Dilute 1 mL of Nitrite stock Standard solution B, and Sample solution
standard solution in a 100-mL volumetric flask with carbon Boron is determined by measurement of the emission from
dioxide-free water (500 ng/mL of nitrite). inductively coupled plasma (ICP) at 249.733 nm or a
Calibration standard solutions: Dilute Nitrite standard suitable wavelength. Use an appropriate apparatus with
solution in carbon dioxide-free water to prepare four settings that have been optimized as directed by the
solutions with the final nitrite concentrations of 2.5, 5, 15, manufacturer.
and 25 ng/mL. Calculate the content of boron in Dalteparin Sodium using
Sample solution: Weigh 80.0 mg of Dalteparin Sodium the following correction factor:
into a 20-mL volumetric flask, and dissolve in carbon
dioxide-free water. F = (r SB – r SA) × 2/(r C – r B)
Chromatographic system
(See Chromatography á621ñ, System Suitability.) r SB = response of boron from Standard solution B
Mode: LC r SA = response of boron from Standard solution A
Detector: Electrochemical detector containing a working rC = response of boron from the Calibration solution
electrode (glassy carbon type) with the potential of rB = response of boron from the Blank
+1.00 V against a silver–silver chloride reference electrode
Column: 3-mm × 15-cm; 5-µm packing L92 Acceptance criteria: NMT 1 ppm
Column temperature: 30 ± 5°

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Printed by: Anandkumar Joshi Official Date: Official as of 01-May-2018 Document Type: USP @2023 USPC
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SPECIFIC TESTS hydroxide added in 100-µL portions. [NOTE—Prepare the

• ANTI-FACTOR IIA ACTIVITY sodium hydroxide solution in carbon dioxide-free water.]
(See Anti-Factor Xa and Anti-Factor IIa Assays for Record the buret reading and the conductivity meter
Unfractionated and Low Molecular Weight Heparins, á208ñ, reading after each addition of the sodium hydroxide
Anti-Factor Xa and Anti-Factor IIa Assays for Low Molecular solution.
Weight Heparins, Anti-Factor IIa Activity for Low Molecular Plot the conductivity measurements on the y-axis against
Weight Heparin.) the volumes of sodium hydroxide added on the x-axis.
Acceptance criteria: NLT 35 and NMT 100 Anti-Factor IIa The graph will have three linear sections—an initial
IU/mg on the dried basis downward slope, a middle slight rise, and a final rise. For
• MOLAR RATIO OF SULFATE TO CARBOXYLATE each of these sections, draw the best-fit straight lines using
Mobile phase: Carbon dioxide-free water linear regression analysis. At the points where the first and
Sample solution: 50 mg of Dalteparin Sodium in 10 mL of second straight lines intersect and where the second and
carbon dioxide-free water third lines intersect, draw perpendiculars to the x-axis to
Chromatographic system determine the volumes of sodium hydroxide taken up by
(See Chromatography á621ñ, System Suitability.) the sample at those points. The point where the first and
Mode: LC second lines intersect corresponds to the volume of
Detector: Ion sodium hydroxide taken up by the sulfate groups (V S).
Column: Two columns: one 1.5-cm × 2.5-cm column, The point where the second and third lines intersect
packed with an anion-exchange resin L64 packing, and corresponds to the volume of sodium hydroxide
one 1.5-cm × 7.5-cm column, packed with a consumed by the sulfate and the carboxylate groups
cation-exchange resin L65 packing.1 The outlet of the together (V T).
anion-exchange column is connected to the inlet of the Calculate the molar ratio of sulfate to carboxylate:

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cation-exchange column.
Flow rate: 1 mL/min Result = V S/(V T − V S)
Analysis
Sample: Sample solution Acceptance criteria: The molar ratio of sulfate to
[NOTE—Regenerate the anion-exchange column and the carboxylate is NLT 1.8.
cation-exchange column with 1 N sodium hydroxide • PH á791ñ: 5.5–8.0 for a 1.0% solution in water
and 1 N hydrochloric acid, respectively, between two
injections.]
With the valve in the inject position, inject the Sample
ci • LOSS ON DRYING á731ñ
Sample: 1 g
Analysis: Dry the Sample under vacuum at 70° for 6 h.
solution into the anion-exchange column, and collect the Acceptance criteria: NMT 10%
eluate from the cation-exchange column in a beaker at • BACTERIAL ENDOTOXINS TEST á85ñ: It contains NMT
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the outlet until the ion detector reading returns to the 0.01 USP Endotoxin Unit/IU of anti-factor Xa activity.
baseline value. Quantitatively transfer the eluate to a
titration vessel containing a magnetic stirring bar, and ADDITIONAL REQUIREMENTS
dilute with carbon dioxide-free water to about 60 mL. • PACKAGING AND STORAGE: Preserve in tight, light-resistant
Position the titration vessel on a magnetic stirrer, and containers, and store below 40°, preferably at room
immerse the electrodes. Note the initial conductivity temperature.
O

reading, and titrate with approximately 0.1 N sodium • LABELING: Label to state the number of Anti-factor Xa
International Units of activity per mg.
1 The • USP REFERENCE STANDARDS á11ñ
procedure is based on analyses performed with two columns: one
1.5-cm × 2.5-cm packed with anion-exchange resin Dowex 1X8 (200– USP Boric Acid RS
400 mesh) and the other 1.5-cm × 7.5-cm packed with cation-exchange USP Dalteparin Sodium RS
resin Dowex 50WX2 (100–200 mesh). USP Low Molecular Weight Heparin for Boron Analysis RS

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