Heparin Injection

Action and use

Anticoagulant.

Definition

Heparin Injection is a sterile solution of Heparin Calcium or Heparin Sodium in Water for Injections. The pH of the solution may be adjusted by the addition of a suitable alkali.

Production

The final product is produced from the drug substance where the methods of manufacturing are designed to ensure freedom from contamination by over-sulfated glycosaminoglycans. The production method is validated to demonstrate that the product if tested, would comply with the test for Related substances given below.

The injection complies with the requirements stated under Parenteral Preparations and with the following requirements.

Potency

The estimated potency is not less than 90% and not more than 111% of the stated potency.

Characteristics

A colourless or straw-coloured liquid, free from turbidity and from matter that deposits on standing.

Identification

A. It complies with the requirements described under Assay.

B. Carry out the assay of anti-factor Xa activity of heparin, Appendix XIV J B2. The ratio of anti-factor Xa activity to anti-factor IIa activity, determined as described under Assay, ranges between 0.9 and 1.1.

C. Carry out the method for zone electrophoresis, Appendix III F, using agarose for electrophoresis as the supporting medium. To equilibrate the agarose, and as electrolyte solution, use a mixture of 50 mL of glacial acetic acid and 800 mL of water adjusted to pH 3 by the addition of lithium hydroxide and diluted to 1000 mL with water. Apply separately to the strip 2 µL to 3 µL of each of the following solutions. For solution (1) dilute a volume of the injection with water to give a solution containing 375 IU per mL. For solution (2), dilute heparin sodium EPBRP with an equal volume of water. Pass a current of 1 mA to 2 mA per centimetre of strip width at a potential difference of 300 volts for about 10 minutes. Stain the strips using a 0.1% w/v solution of toluidine blue and remove the excess by washing. The ratio of the distance migrated by the principal band or bands in the gel obtained with solution (1) to the distance migrated by the principal band in the gel obtained with solution (2) is 0.9 to 1.1.

D. When the injection contains Heparin Calcium, it yields reactions A and B characteristic of calcium salts, Appendix VI. When the injection contains Heparin Sodium, it yields reaction A characteristic of sodium salts, Appendix VI.

Tests

Acidity or alkalinity

pH, 5.5 to 8.0, Appendix V L.

Related substances

Carry out the method for liquid chromatography, Appendix III D using the following solutions. Solutions (3) to (6) are stable at room temperature for 24 hours.

(1) For injections containing 25000 IU per mL, dilute 100 µL in 400 µL of water for chromatography. For injections containing 5000 IU or 1000 IU per mL, use 500 µL of each. Mix using a vortex mixer until dissolution is complete.

(2) Mix 500 µL of solution (1) and 250 µL of 1m hydrochloric acid, then add 50 µL of a 25% w/v solution of sodium nitrite. Mix gently and allow to stand at room temperature for 40 minutes before adding 200 µL of 1m sodium hydroxide to stop the reaction.

(3) Dissolve 0.25 g of heparin for physico-chemical analysis EPCRS in water for chromatography and dilute to 2.0 mL with the same solvent. Mix using a vortex mixer until dissolution is complete.

(4) Add 1200 µL of solution (3) to 300 µL of dermatan sulfate and over-sulfated chondroitin sulfate EPCRS. Mix using a vortex mixer to homogenise.

(5) Add 400 µL of solution (3) to 100 µL of water for chromatography and mix using a vortex mixer. Add 250 µL of 1m hydrochloric acid, then add 50 µL of a 25% w/v solution of sodium nitrite. Mix gently and allow to stand at room temperature for 40 minutes before adding 200 µL of 1M sodium hydroxide to stop the reaction.

(6) To 500 µL of solution (4), add 250 µL of 1m hydrochloric acid, then add 50 µL of a 25% w/v solution of sodium nitrite. Mix gently and allow to stand at room temperature for 40 minutes before adding 200 µL of 1m sodium hydroxide to stop the reaction.

chromatographic conditions

(a) Use a stainless steel column (25 cm × 2 mm) packed with anion-exchange resin(9 µm) (Dionex Ionpac AG11-HC is suitable) and a stainless steel pre-column (5 cm × 2 mm) packed with anion-exchange resin (13 µm) (Dionex Ionpac AG11-HC is suitable).

(b) Use gradient elution and the mobile phase described below.

(d) Use a column temperature of 40°.

(e) Use a detection wavelength of 202 nm.

(f) Inject 20 µL of solutions (2), (5) and (6).

Mobile phase

Mobile phase A 0.040% w/v of sodium dihydrogen orthophosphate in water for chromatography adjusted to pH 3.0 with dilute orthophosphoric acid.

Mobile phase B 0.040% w/v of sodium dihydrogen orthophosphateand 14% w/v of sodium perchlorate in water for chromatography, adjusted to pH 3.0 with dilute orthophosphoric acid, filtered and degassed.

Equilibrate the column with the mobile phase in the initial gradient ratio for at least 15 minutes.

When the chromatograms are recorded under the prescribed conditions the retention times relative to heparin (retention time about 26 minutes) are: dermatan sulfate and chondroitin sulfate, about 0.9; over-sulfated chondroitin sulfate, 1.3.

System suitability

The test is not valid unless:

in the chromatogram obtained with solution (5), no peak is present at the retention time of heparin;

in the chromatogram obtained with reference solution (6), the resolution between the peaks due to dermatan sulfate / chondroitin sulfate and over-sulfated chondroitin sulfate is at least 3.0.

Limits

In the chromatogram obtained with solution (2):

the area of the peak due to dermatan sulfate and chondroitin sulfate is not greater than the area of the corresponding peak in the chromatogram obtained with solution (6) (2.0%);

no peaks other than the peak due to dermatan sulfate and chondroitin sulfate are detected.

Bacterial endotoxins

Carry out the test for bacterial endotoxins, Appendix XIV C.

For a preparation supplied in a container with a nominal content of less than 100 mL, dilute the injection if necessary with water BET to give a solution containing 1000 IU of heparin activity per mL (solution A). The endotoxin limit concentration of solution A is 10 IU of endotoxin per mL. Carry out the test using a lysate with a declared sensitivity not less sensitive than 0.0625 IU of endotoxin per mL.

For a preparation supplied in a container with a nominal content of 100 mL or more, the endotoxin limit concentration is 0.25 IU of endotoxin per mL.

For Heparin Injection prepared from Heparin Calcium it may be necessary to add divalent cations in order to achieve the validation criteria.

Assay

Carry out the assay of anti-factor IIa activity of heparin, Appendix XIV J B2. The fiducial limits of error are not less than 80% and not more than 125% of the stated potency.

Storage

Heparin Injection should be kept in a container sealed by fusion of the glass.

Labelling

The strength is stated as the number of IU (Units) in a suitable dose-volume except that for multi-dose containers the strength is stated as the number of IU (Units) per mL.

The label states (1) whether the material is the calcium or sodium salt; (2) when no antimicrobial preservative is present that the preparation contains no antimicrobial preservative and that any portion of the contents not used at once should be discarded.