Enoxaparin Sodium
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[9041-08-1].
» Enoxaparin Sodium is the sodium salt of a depolymerized heparin. It is obtained by alkaline depolymerization of heparin benzyl ester. The starting material, heparin, is obtained exclusively from porcine intestinal mucosa. Enoxaparin Sodium consists of a complex set of oligosaccharides that have not yet been completely characterized. The majority of the components have a 4-enopyranose uronate structure at the non-reducing end of their chain. About 20 percent of the materials contain a 1,6-anhydro derivative on the reducing end of the chain, the range being between 15 and 25 percent. The weight-average molecular weight of Enoxaparin Sodium is 4,500 Da, the range being between 3,800 and 5,000 Da; about 16 percent have a molecular weight of less than 2,000 Da, the range being between 12.0 and 20.0 percent; about 74 percent have a molecular weight between 2,000 and 8,000 Da, the range being between 68.0 and 82.0 percent. Not more than 18.0 percent have a molecular weight higher than 8,000 Da. When prepared as a solution, the solution is analyzed for clarity and degree of color using a validated method. The degree of sulfation is not less than 1.8 per disaccharide unit. It has a potency of not less than 90 and not more than 125 Anti-Factor Xa International Units (IU) per mg, and not less than 20.0 and not more than 35.0 Anti-Factor IIa IU per mg, calculated on the dried basis. The ratio of anti-factor Xa activity to anti-factor IIa activity is between 3.3 to 5.3.
Packaging and storage— Preserve in tight containers, and store below 40, preferably at room temperature.
Identification—
A: Ultraviolet Absorption 197U—
Solution: 500 µg per mL.
Medium: 0.01 N hydrochloric acid. The spectra exhibit maxima at 231 ± 2 nm.
B: 13C NMR spectrum (see Nuclear Magnetic Resonance 761)—
Standard solution— Dissolve 200 mg of USP Enoxaparin Sodium RS in a mixture of 0.2 mL of deuterium oxide and 0.8 mL of water. Add 0.05 mL of deuterated methanol to serve as an internal reference.
Test solution— Dissolve 200 mg of Enoxaparin Sodium in a mixture of 0.2 mL of deuterium oxide and 0.8 mL of water. Add 0.05 mL of deuterated methanol.
Procedure— Transfer the Standard solution and the Test solution to NMR tubes of 5-mm diameter. Using a pulsed (Fourier transform) NMR spectrometer operating at not less than 75 MHz for 13C, record the 13C NMR spectra of the Standard solution and the Test solution at 40. The spectra are similar.
C: The ratio of the numerical value of the anti-factor Xa activity, in Anti-Factor Xa IU per mg, to the numerical value of the anti-factor IIa activity, in Anti-Factor IIa IU per mg, as determined by the Assay (anti-factor Xa activity) and the Anti-factor IIa activity, respectively, is not less than 3.3 and not more than 5.3.
D: Molecular weight distribution and weight-average molecular weight—
Mobile phase— Prepare a 0.5 M lithium nitrate solution. Pass through a membrane filter having a porosity of 0.45 µm or less, and degas with helium.
Standard solution— Dissolve about 10 mg of USP Enoxaparin Sodium RS, accurately weighed, in 1 mL of Mobile phase.
Test solution— Dissolve about 10 mg of Enoxaparin Sodium, accurately weighed, in 1 mL of Mobile phase.
Chromatographic system (see Chromatography 621)— The high performance size exclusion chromatograph is equipped with a differential refractive index detector, a 6- × 40-mm guard column and two 7.8- × 300-mm analytical columns in series, both analytical and guard columns prepacked with packing L59, and used at room temperature. The flow rate is about 0.6 mL per minute maintained constant to±1.0%.
Procedure— Reconstitute one vial each of USP Enoxaparin Sodium Molecular Weight Calibrant A RS and USP Enoxaparin Sodium Molecular Weight Calibrant B RS in 1 mL of Mobile phase. Separately inject 20 µL of USP Enoxaparin Sodium Molecular Weight Calibrant A RS and USP Enoxaparin Sodium Molecular Weight Calibrant B RS, record the chromatograms, and measure the retention times. Inject in duplicate 20 µL of each of the Standard solution and the Test solution, and record the chromatograms for a length of time to ensure complete elution, including salt and solvent peaks. Calculate the total area under each of the Standard solution and Test solution chromatograms, excluding salt and solvent peaks at the end.
Calibration curve— Plot the retention times on the x-axis against the peak molecular weights on the y-axis for the peaks in the chromatograms of USP Enoxaparin Sodium Molecular Weight Calibrant A RS and USP Enoxaparin Sodium Molecular Weight Calibrant B RS, and fit the data to a third-order polynomial using a suitable gel permeation chromatography (GPC) software.
Calculations— Compute the data, using the same GPC software and determine the weight-average molecular weight, MW, for each of the duplicate chromatograms of the Standard solution and the Test solution, and take the average for each solution. Correct the mean value of MW to the nearest 50. The Chromatographic system is suitable if MW of USP Enoxaparin Sodium RS is within 150 Da of the labeled MW value. The MW for the Test solution is between 3,800 and 5,000 Da. Using the same software, determine for each of the duplicate Test solution chromatograms the percentage of Enoxaparin Sodium chains with molecular weights lower than 2000 Da, M2000, the percentage of Enoxaparin Sodium chains with molecular weights in the range 2000 to 8000 Da, M2000–8000, and the percentage of Enoxaparin Sodium chains with molecular weights greater than 8000 Da, M8000. Average the duplicate values and express to the nearest 0.5%. M2000 is between 12.0% and 20.0%, M2000–8000 is between 68.0% and 82.0%, and M8000 is not more than 18.0%.
E: It responds to the test for Sodium 191.
Specific absorbance (see Spectrophotometry and Light-Scattering 851)—
Test solution— Dissolve 50.0 mg of Enoxaparin Sodium in 100 mL of 0.01 N hydrochloric acid.
Procedure— Obtain the UV spectra of the Standard solution and the Test solution between 200 nm and 300 nm against 0.01 N hydrochloric acid blank. Calculate the specific absorbance at the wavelength of maximum absorbance at 231 ± 2 nm, with reference to the dried substance, using the following formula:
A × 100 × 1000 / [ M × l × (100 – E)]
in which A is the absorbance at the wavelength of maximum absorbance; M is the weight, in mg, of Enoxaparin Sodium in the Test solution; l is the pathlength (typically l = 1 cm); and E is the loss on drying, in percent. The specific absorbance is between 14.0 and 20.0, calculated on the dried basis.
Bacterial endotoxins 85— It contains not more than 0.01 USP Endotoxin Unit per IU of anti-factor Xa activity.
pH 791: between 6.2 and 7.7 of a 10.0% solution in water.
Loss on drying 731— Dry 1 g in a vacuum at 70 for 6 hours: it loses not more than 10.0% of its weight.
Nitrogen content, Method II 461: between 1.8% and 2.5%, calculated on the dried basis.
Heavy metals, Method I 231— Prepare a 5% solution in water: the limit is not more than 0.0030%.
Sodium content (see Spectrophotometry and Light-Scattering 851)—
Cesium chloride solution— Prepare a solution of cesium chloride in 0.1 N hydrochloric acid containing 1.27 mg per mL.
Standard solutions— Dissolve an accurately weighed quantity of sodium chloride in Cesium chloride solution to obtain a solution having a known concentration of about 0.2% sodium. Dilute accurately measured volumes of this solution with Cesium chloride solution having known concentrations of 0.0025%, 0.0050%, and 0.0075% of sodium.
Test solution— Transfer an accurately weighed quantity of about 50.0 mg of Enoxaparin Sodium to a 100-mL volumetric flask, and dissolve in and dilute with Cesium chloride solution to volume.
Procedure— Concomitantly determine the absorbances of the Cesium chloride solution (blank), Test solution, and Standard solutions at 330.3 nm using a sodium hollow-cathode lamp and an air–acetylene flame. Using the absorbances of Standard solutions, determine the sodium content in the Test solution after appropriate blank correction. The sodium content, calculated on the dried basis, is between 11.3% and 13.5%.
Molar ratio of sulfate to carboxylate (see Chromatography 621)—
Mobile phase: carbon dioxide-free water.
Test solution— Dissolve an accurately weighed quantity of about 50 mg of Enoxaparin Sodium in 10 mL of carbon dioxide-free water.
Chromatographic system— The liquid chromatographic system consists of two peristaltic pumps, a six-port injection valve, an ion detector, and two columns—one 1.5- × 2.5-cm column packed with an anion-exchange resin L64 packing and one 1.5- × 7.5-cm column packed with a cation-exchange resin L65 packing. The outlet of the anion-exchange column is connected to the inlet of the cation-exchange column. The flow rate is about 1 mL per minute.
Procedure— [note—Regenerate the anion-exchange column and the cation-exchange column with 1 N sodium hydroxide and 1 N hydrochloric acid, respectively, between two injections.] Inject the Test solution into the anion-exchange column, and collect the eluate from the cation-exchange column in a beaker at the outlet until the ion detector reading returns to the baseline value. Quantitatively transfer the eluate to a titration vessel containing a magnetic stirring bar, and dilute with carbon dioxide-free water to about 60 mL. Position the titration vessel on a magnetic stirrer and immerse the electrodes. Note the initial conductivity reading and titrate with approximately 0.1 N sodium hydroxide added in 100-µL portions. [note—Prepare the sodium hydroxide solution in carbon dioxide-free water.] Record the burette reading and the conductivity meter reading after each addition of the sodium hydroxide solution.
Calculations— Plot the conductivity measurements on the y-axis against the volumes of sodium hydroxide added on the x-axis. The graph will have three linear sections—an initial downwards slope, a middle slight rise, and a final rise. For each of these sections draw the best-fit straight lines using linear regression analysis. At the points where the first and second straight lines intersect and where the second and third lines intersect, draw perpendiculars to the x-axis to determine the volumes of sodium hydroxide taken up by the sample at those points. The point where the first and second lines intersect corresponds to the volume of sodium hydroxide taken up by the sulfate groups (VS). The point where the second and third lines intersect corresponds to the volume of sodium hydroxide consumed by the sulfate and the carboxylate groups together (VT). Calculate the molar ratio of sulfate to carboxylate by the formula:
VS / (VT – VS)
The molar ratio of sulfate to carboxylate is not less than 1.8.
Benzyl alcohol content—
Mobile phase— Prepare a filtered and degassed mixture of water, acetonitrile, and methanol (80:15:5 v/v).
Standard solution— Dissolve 100 mg of USP Benzyl Alcohol RS in 200 mL of water. Transfer 5 mL of this solution to a 25-mL volumetric flask, and dilute with water to volume.
Test solution— Weigh 0.5 g of Enoxaparin Sodium into a 10-mL volumetric flask, and dissolve in 5.0 mL of 1 N sodium hydroxide. Allow to stand at room temperature for about 1 hour. Add 1.0 mL of glacial acetic acid, dilute with water to volume, and mix.
Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 256-nm detector and a 4.6-mm × 15-cm stainless steel column that contains L7 packing. The flow rate is about 1.0 mL per minute maintained constant to ±10%.
Procedure— Separately inject equal volumes (about 20 µL) of the Standard solution and the Test solution, record the chromatograms, and measure the peak responses.
Calculation— Calculate the percentage of benzyl alcohol in Enoxaparin Sodium taken by the formula,
(AT × CS)/(AS × CT)
in which AT is the benzyl alcohol peak area in the Test solution; CS is the concentration, in mg per mL, of benzyl alcohol; AS is the area of the benzyl alcohol peak in the Standard solution; and CT is the concentration, in mg per mL, of Enoxaparin Sodium. The percentage of benzyl alcohol is not more than 0.1%.
Anti-factor IIa activity—
Acetic acid solution, pH 7.4 Polyethylene glycol 6000 buffer, pH 7.4 Buffer, pH 8.4 Buffer, and Human antithrombin III solution— Proceed as directed under Assay (anti-factor Xa activity), except that the concentration of the Human antithrombin III solution is 0.5 Antithrombin III Unit per mL.
Thrombin human solution— Reconstitute thrombin human (see Reagent Specifications in the section Reagents, Indicators and Solutions) in water, and dilute in pH 7.4 Polyethylene glycol 6000 buffer to obtain a solution having a concentration of 5 Thrombin Units per mL.
Chromogenic substrate solution— Prepare a solution of a suitable chromogenic substrate for an amidolytic test (see Reagent Specifications in the section Reagents, Indicators, and Solutions) for thrombin in water to obtain a concentration of about 3 mM. Immediately before use, dilute with pH 8.4 Buffer to 0.5 mM.
Standard solutions— Dilute USP Enoxaparin Sodium Solution for Bioassays RS with pH 7.4 Buffer to obtain four dilutions having concentrations in the range between 0.015 and 0.075 IU of anti-factor IIa activity per mL.
Test solutions— Proceed as directed under Standard solutions to obtain concentrations of Enoxaparin Sodium similar to those obtained for the Standard solutions.
Procedure— Proceed as directed under Assay (anti-factor Xa activity), except to use Thrombin human solution instead of Factor Xa solution and to use the Human antithrombin III solution as described above.
Calculations— For each series, calculate the regression of the absorbance against log concentrations of the Test solutions and of the Standard solutions, and calculate the potency of the enoxaparin sodium in IU of anti-factor IIa activity per mg using statistical methods for parallel-line assays. The four independent dilution estimates are then combined to obtain the final weighted mean. Then calculate the confidence limits. Express the anti-factor IIa activity of Enoxaparin Sodium per mg, calculated on the dried basis. It has a potency of not less than 20.0 and not more than 35.0 anti-Factor IIa IU per mg.
Assay (anti-factor Xa activity)—
Acetic acid solution— Transfer 42 mL of glacial acetic acid to a 100-mL volumetric flask, dilute with water to volume, and mix.
pH 7.4 Polyethylene glycol 6000 buffer— Dissolve 6.08 g of tris(hydroxymethyl)aminomethane and 8.77 g of sodium chloride in 500 mL of water. Add 1.0 g of polyethylene glycol 6000, adjust with hydrochloric acid to a pH of 7.4, and dilute with water to 1000 mL.
pH 7.4 Buffer— Dissolve 6.08 g of tris(hydroxymethyl)aminomethane and 8.77 g of sodium chloride in 500 mL of water. Adjust with hydrochloric acid to a pH of 7.4, and dilute with water to 1000 mL.
pH 8.4 Buffer— Dissolve 3.03 g of tris(hydroxymethyl)aminomethane, 5.12 g of sodium chloride and 1.40 g of edetate sodium in 250 mL of water. Adjust with hydrochloric acid to a pH of 8.4, and dilute with water to 500 mL.
Human antithrombin III solution— Reconstitute a vial of antithrombin III (see Reagent Specifications in the section Reagents, Indicators, and Solutions) in water to obtain a solution containing 5 Antithrombin III Units per mL. Dilute this solution with pH 7.4 Polyethylene glycol 6000 buffer to obtain a solution having a concentration of 1.0 Antithrombin III Unit per mL.
Factor Xa solution— Reconstitute an accurately weighed quantity of bovine factor Xa (see Reagent Specifications in the section Reagents, Indicators, and Solutions) in pH 7.4 Polyethylene glycol 6000 buffer to obtain a solution that gives an increase in absorbance value at 405 nm of not more than 0.20 absorbance units per minute when assayed as described below but using as an appropriate volume (V, in µL) of pH 7.4 Buffer instead of V µL of the enoxaparin solution.
Chromogenic substrate solution— Prepare a solution of a suitable chromogenic substrate for amidolytic test (see Reagent Specifications in the section Reagents, Indicators, and Solutions) for factor Xa in water to obtain a concentration of about 3 mM. Dilute with pH 8.4 Buffer to obtain a solution having a concentration of 0.5 mM.
Standard preparations— Dilute USP Enoxaparin Sodium Solution for Bioassays RS with pH 7.4 Buffer to obtain four dilutions in the concentration range between 0.025 and 0.2 USP Anti-Factor Xa IU per mL.
Assay preparations— Proceed as directed for Standard preparations to obtain concentrations of Enoxaparin Sodium similar to those obtained for the Standard preparations.
Procedure— Label 18 suitable tubes: B1 and B2 for blanks; T1, T2, T3, and T4 each in duplicate for the dilutions of the Assay preparations; and S1, S2, S3, and S4 each in duplicate for the dilutions of the Standard preparations. [note—Treat the tubes in the order B1, S1, S2, S3, S4, T1, T2, T3, T4, T1, T2, T3, T4, S1, S2, S3, S4, B2.] To each tube add the same volume, V, (20 to 50 µL) of Human antithrombin III solution and an equal volume, V, of either the blank, pH 7.4 Buffer, or an appropriate dilution of the Assay preparations or the Standard preparations. Mix, but do not allow bubbles to form. Incubate at 37 for 1.0 minute. Add to each tube volume 2V (40 to100 µL) of Factor Xa solution, and incubate for 1.0 minute. Add 5V (100 to 250 µL) volume of Chromogenic substrate solution. Stop the reaction after 4.0 minutes with 5V (100 to 250 µL) volume of Acetic acid solution. Measure the absorbance of each solution at 405 nm using a suitable spectrophotometer (see Spectrophotometry and Light-Scattering 851) against blank B1. The reading of blank B2 relative to the blank B1 is not more than ±0.05 absorbance units.
Calculations— For each series, calculate the regression of the absorbance against log concentrations of the Assay preparations and of the Standard preparations, and calculate the potency of the enoxaparin sodium in IU of anti-factor Xa activity per mL using statistical methods for parallel-line assays. The four independent log relative potency estimates are then combined to obtain the final geometric mean. Its confidence limits are calculated. Express the anti-factor Xa activity of Enoxaparin Sodium per mg, calculated on the dried basis. The potency is not less than 90 and not more than 125 Anti-Factor Xa IU per mg.
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Topic/Question Contact Expert Committee
Monograph Anita Y. Szajek, Ph.D.
Senior Scientist
1-301-816-8325		 (BBBBP05) Biologics and Biotechnology - Blood and Blood Products
Reference Standards Lili Wang, Technical Services Scientist
1-301-816-8129
RSTech@usp.org
85 Radhakrishna S Tirumalai, Ph.D.
Senior Scientist
1-301-816-8339		 (MSA05) Microbiology and Sterility Assurance
USP32–NF27 Page 2253
Pharmacopeial Forum: Volume No. 33(1) Page 52
Chromatographic Column—
Chromatographic columns text is not derived from, and not part of, USP 32 or NF 27.