Calcitonin (Salmon) Injection

Hormone.

Calcitonin (Salmon) Injection is a sterile solution of Calcitonin (Salmon) in Water for Injections.

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

90.0 to 115.0% of the stated amount of the peptide.

A colourless solution.

In the Assay, the retention time of the principal peak in the chromatogram obtained with solution (1) is similar to that of the principal peak in the chromatogram obtained with solution (2).

pH, 3.9 to 4.5, Appendix V L.

Carry out the method for liquid chromatography, Appendix III D, using the following solutions.

(1) Dilute the injection, if necessary, with a 0.1m solution of sodium dihydrogen orthophosphate adjusted to pH 4.0 with orthophosphoric acid to give a final concentration of 10 µg of calcitonin (salmon) per mL.

(2) 10 µg of calcitonin (salmon) BPCRS per mL in a 0.1m solution of sodium dihydrogen orthophosphate adjusted to pH 4.0 with orthophosphoric acid.

(3) Heat the injection at 75° for 15 hours and, if necessary, dilute as described for solution (1).

(a) Use a stainless steel column (25 cm × 4.6 mm) packed with octadecylsilyl silica gel for chromatography (5 µm) (Vydac C18, 300 Å wide pore column for proteins and peptides is suitable).

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

(c) Use a flow rate of 1 mL per minute.

(d) Use a column temperature of 40°.

(e) Use a detection wavelength of 220 nm.

(f) Inject 200 µL of each solution.

Mobile phase A  100 volumes of a 0.363% w/v solution of tetramethylammonium hydroxide pentahydrate and 150 volumes of acetonitrile adjusted to pH 2.5 with orthophosphoric acid.

Mobile phase B  50 volumes of acetonitrile and 450 volumes of a 0.402% w/v solution of tetramethyl-ammonium hydroxide pentahydrate adjusted to pH 2.5 with orthophosphoric acid.

Use the following gradient.

In the chromatogram obtained with solution (3) the peak due to calcitonin C is the largest peak to elute after the injection buffer salts and before the principal peak with a relative retention to that of calcitonin (salmon) of between 0.5 and 0.6.

The test is not valid unless the resolution factor between the peaks due to calcitonin C and calcitonin (salmon) in the chromatogram obtained with solution (3) is at least 3.0.

In the chromatogram obtained with solution (1) the area of any peak corresponding to calcitonin C is not greater than 7% by normalisation.

Carry out the method for liquid chromatography, Appendix III D, using the following solutions.

(1) Dilute the injection, if necessary, with mobile phase A to give a final concentration of 10 µg of calcitonin (salmon) per mL.

(2) Dissolve the contents of a vial of N-acetyl-cys¹calcitonin EPCRS in 0.4 mL of mobile phase A, dilute to 40 mL with mobile phase A and add 0.1 mL of solution (1).

(a) Use a stainless steel column (25 cm × 4.6 mm) packed with octadecylsilyl silica gel for chromatography (5 µm).

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

(c) Use a flow rate of 1 mL per minute.

(d) Use a column temperature of 65°.

(e) Use a detection wavelength of 220 nm.

(f) Inject 200 µL of each solution.

Mobile phase A  Dissolve 3.26 g of tetramethylammonium hydroxide pentahydrate in 900 mL of water, adjust the pH to 2.5 with orthophosphoric acid and mix with 100 mL of acetonitrile for chromatography.

Mobile phase B  Dissolve 1.45 g of tetramethylammonium hydroxide pentahydrate in 400 mL of water, adjust the pH to 2.5 with orthophosphoric acid and mix with 600 mL of acetonitrile for chromatography.

Use the following gradient.

When the chromatogram for solution (2) is recorded in the prescribed conditions, the relative retention time of N-acetyl-cys¹ calcitonin is about 1.15 relative to the principal peak.

The test is not valid unless the resolution factor between the peaks corresponding to calcitonin and N-acetyl-cys¹ calcitonin is at least 5.0 and the symmetry factor for the N-acetyl-cys¹ calcitonin peak is not greater than 2.5. If necessary, adjust the initial ratio of A:B in the mobile phase.

In the chromatogram obtained with solution (1):

the area of any secondary peak is not greater than 3% of the total area of all the peaks;

the sum of the areas of any such peaks is not greater than 5% of the total area of all the peaks by normalisation;

disregard any peak with an area less than 0.1 % of that of the principal peak.

Carry out the method described under the test for Calcitonin C.

Calculate the content of calcitonin (salmon) from the areas of the peak due to calcitonin (salmon) and that of any peak due to calcitonin C, using the declared content of C₁₄₅H₂₄₀N₄₄O₄₈S₂ in calcitonin (salmon) BPCRS.

Calcitonin (Salmon) Injection should be protected from light and stored at a temperature of 2° to 8°.

The label states the strength as the number of IU (Units) per mL. The label also states the equivalent number of micrograms of the peptide per mL.