Calculation of Related Substances by HPLC

1.0 OBJECTIVE 

To lay down the procedure for the Calculation of Related Substances by HPLC

2.0 SCOPE

This SOP is applicable to Calculation of Related Substances by HPLC in company.

3.0 BACKGROUND 

NIL

4.0 RESPONSIBILITY

4.1 QC-Chemist

4.2 Head-QC

5.0 PROCEDURE

5.1 Rosuvstatin calcium related substances method. The impurities and their acceptance criteria is as given in the following table. 

1. Anti Isomer-NMT 0.20 %

2. Lactone Impurity- NMT 0.15%

3. Major unknown individual impurity- NMT 0.10%

4. Total Impurities- NMT 1.0%

5.2 Chromatographic Conditions:

HPLC with UV detector at wavelength of 248 nm, Column is Phenomenex , C18, 250x 4.6mm, 5 microns, Column temperature 25 degrees, injection volumen 20 microleters, runtime 70 min. Diluent 50:50 ACN and water, Mobile phase Water: ACN: Methanol: TrimethylAmine at 450: 250:350:1 v/v and adjust the pH to 6.5 with Glacial acetic acid.

Gradient Program:

Time    Flow Gradient (ml/min)

0.01    1.0

20       1.0

25       1.8

65       1.8

67       1.0

70       1.0

5.3 Sample Preparation:

25 mg of Ruvastatin calcium in 50 ml of volumetric flask. Dissolve the substance in dilutent with 20-30 ml and then makeup the volume with mobile phase.

5.4 System suitability stock solution:

25 mg of Anti isomer of RC, dissolve with diiluent and makeup with mobile phase as mentioned in the sample paparation.

5.5 System suitability Solution:

25 mg of working standard in 50 ml flask and dissove with diluent and makeuo to 50 ml as mentioned in the sample preparation.

5.6 Procedure:

Inject the sample into the chromatogram as mentioned in the chromatographic conditions ad run the chromatogram for 10 minutes.

Calculate the impurities with the following formula

% of known impurities = Ri / Rs * 100 * RF

% of unknown impurities= ri/Rs * 100 * RF

Total impurities = sum of all known and unknown impurities

RF is response factor for impurities

Ri = Area of each impurity peak in the chromatogram of the sample solution

Rs= Sum of all areas of Rosuvastatin calcium and all other impurity peaks in the sample solution

Let us start the calculation with typical chromatogram

5.7 Before going for calculation we need to have the value of Response factor for each impurity Anti isomer is 1.32 and for lactone impurity is 1.00 

From the above chromatogram 

The area of anti isomer = 29208

The area of Lactone Impurity = 8178

The sum of all areas of Rosuvastatin calcium and all other impurity peaks in the sample solution = 22107163

then calculate anti isomer

29208/22107163 x 100 x 1.32 = 0.174

Lactone impurity

8178/22107163 x 100 x 1.00 = 0.03

Largest unknown impurity

4541/ 22107163 x 100 = 0.02

6.0 ANNEXURES

Nil