A: Add 2 mL of water to the total content of a single-dose container, or to 0.4 mL from a multiple-dose container, and 1 mL of 2% w/v protamine sulfate solution in a glass test tube, and mix. A creamy white precipitate is formed.

B: Ultraviolet Absorption 197U—

C: It meets the requirements of the test for Sodium 191.

Mobile phase— Prepare a filtered and degassed mixture of water, acetonitrile, and methanol (80:15:5 v/v).

Standard solution— Transfer about 75 mg, accurately weighed, of USP Benzyl Alcohol RS to a 50-mL volumetric flask, and dilute with Mobile phase to volume.

Test solutions— Transfer 5.0 mL of the Injection to a 50-mL volumetric flask. Dilute with Mobile phase 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 packing L7. 1 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. Calculate the percentage of benzyl alcohol in the portion of enoxaparin sodium solution taken by the formula: in which AT and AS are areas of the benzyl alcohol peaks in the chromatograms of the Test solution and the Standard solution, respectively; and M is the mass of the benzyl alcohol dissolved to prepare the Standard solution. The percentage (w/v) of benzyl alcohol in the Injection is not less than 1.35% and not more than 1.65%.

Mobile phase— Prepare a 3.0 mM sodium carbonate solution. Make adjustments if necessary.

Standard sulfate stock solution— Prepare a solution of sodium sulfate in Mobile phase in a suitable sulfate-free container such that the concentration of sulfate is accurately known at about 1 g per L. Transfer about 5 g, accurately weighed, of the solution to a similar container, and add Mobile phase to obtain about 25 g of solution.

Standard solutions— Prepare standard solutions at concentrations of 0.1 µg per g, 0.5 µg per g, 1 µg per g, 2 µg per g, 4 µg per g, and 5 µg per g by appropriate dilution of the Standard sulfate stock solution in Mobile phase.

System suitability solution— Prepare a solution containing 3 µg per mL of sulfate anion and 5 µg per mL of oxalate anion.

Test solutions— Transfer about 200 mg of a 100 mg per mL Injection, accurately weighed, to a suitable previously tared sulfate-free vial. Add Mobile phase to obtain a total mass, MS, of about 20 g.

Chromatographic system (see Chromatography 621)— The ion chromatograph is equipped with a conductivity detector and a 4-mm × 5-cm anion-exchange guard column, a 4-mm × 25-cm anion-exchange analytical column, both containing L61 packing (see Chromatographic Reagents under Reagents, Indicators, and Solutions), and a micromembrane anion autosuppressor2 or a suitable chemical suppression system. The flow rate is about 2.0 mL per minute.

Procedure— Chromatograph about 25 µL of the System suitability solution. The resolution between the sulfate and oxalate peaks is greater than 1. Separately inject 25 µL of the Standard solutions and the Test solution into the chromatograph, and plot the standard curve of sulfate peak height as a function of sulfate concentration (in µg per g) in the Standard solutions. From the sulfate peak height in the chromatogram determine the concentration of sulfate, T, in µg per g, in the Test solution using the standard curve. Calculate the percentage of free sulfate content (w/w) in the Injection taken by the formula: in which m is the mass, in mg, of Injection aliquoted to prepare the Test solution. The percentage of free sulfate is not more than 0.12%.