AMLODIPINE BESILATE SOP

AMLODIPINE BESILATE SOP

1.0 OBJECTIVE:

To lay down a procedure of analytical report for the active raw material of the Amlodipine besilate from the Pharmacopoeial specifications.

2.0 SCOPE:

This SOP shall be applicable in Q.C laboratory.

3.0 RESPONSIBILITY:

3.1 Q.C Analysts.

4.0 ACCOUNTABILITY:

4.1 Q.C Manager.

5.0 PROCEDURE:

5.1 Characters:

5.1.1 Appearance:

5.1.1.1 White or almost white powder.

5.1.2 Solubility:

5.1.2.1 Material and equipment:

5.1.2.1.1 Glassware (test tubes, spatula).

5.1.2.1.2 Purified water.

5.1.2.1.3 Methanol.

5.1.2.1.4 Anhydrous ethanol.

5.1.2.1.5 2-propanol.

5.1.2.2 Sample:

5.1.2.2.1 Small quantity.

5.1.2.3 Method:

5.1.2.3.1 Take 4 test tubes and add small quantity of sample for testing solubility according to B.P specifications.

5.1.2.3.2 Add purified water in test tube 1 and observe.

5.1.2.3.3 Add methanol in test tube 2 and observe.

5.1.2.3.4 Add anhydrous ethanol in test tube 3 and observe.

5.1.2.3.5 Add 2-propanol in test tube 4 and observe.

5.1.2.4 Observations:

5.1.2.4.1 The sample in test tube 1 containing with water is slightly soluble.

5.1.2.4.2 The sample in test tube 2 containing with methanol is freely soluble.

5.1.2.4.3 The sample in test tube 3 containing with anhydrous ethanol is sparingly soluble.

5.1.2.4.4 The sample in test tube 4 containing with 2-propanol is slightly soluble.

5.2 Assay:

5.2.1 Apparatus:

5.2.1.1 Liquid chromatography apparatus.

5.2.1.2 Glassware (according to the requirement).

5.2.1.3 UV/VIS Spectrophotometer (detector).

5.2.2 Material and reagents:

5.2.2.1 Octadecylsilyl silica gel for chromatography (5μm).

5.2.2.2 2.3g/L solution of ammonium acetate.

5.2.2.3 Methanol.

5.2.3 Requirements:

5.2.3.1 Sample:

5.2.3.1.1 50.0mg (sample to be examined).

5.2.3.1.2 50.0mg (amlodipine besilate CRS) for reference solution.

5.2.3.2 Test solution:

5.2.3.2.1 Test solution (a):

5.2.3.2.1.1 Take 100ml of beaker and dissolve 50.0mg of the substance to be examined in the mobile phase.

5.2.3.2.1.2 And dilute to 50.0ml with the mobile phase.

5.2.3.2.2 Test solution (b):

5.2.3.2.2.1 Take a beaker of 100ml and dilute 5.0ml of test solution (a) to 100.0ml with mobile phase.

5.2.3.3 Reference solutions:

5.2.3.3.1 Reference solution (e):

5.2.3.3.1.1 Take 100ml beaker and dissolve 50.0mg of amlodipine besilate CRS in the mobile phase.

5.2.3.3.1.2 Dilute 5.0ml of the solution to 100.0ml with the mobile phase.

5.2.3.4 Column:

5.2.3.4.1 Size:

5.2.3.4.1.1 Length=0.25m,

5.2.3.4.1.2 θ=4.0mm.

5.2.3.4.2 Stationary phase:

5.2.3.4.2.1 Octadecylsilyl silica gel for chromatography (5μm).

5.2.3.4.3 Temperature:

5.2.3.4.3.1 30^oC.

5.2.3.5 Mobile phase:

5.2.3.5.1 2.3g/L solution of ammonium acetate, methanol (30:70 v/v).

5.2.3.6 Flow rate:

5.2.3.6.1 1.5ml/min.

5.2.3.7 Detection:

5.2.3.7.1 Spectrophotometer at 237nm.

5.2.3.8 Injection:

5.2.3.8.1 20μL of the test solution (b) and reference solution (e).

5.2.3.9 Run time:

5.2.3.9.1 Twice the retention time of amlodipine.

5.2.3.10 Relative retention:

5.2.3.10.1 With reference to amlodipine (retention time=about 20min).

5.2.4 Method of analysis:

5.2.4.1 Carry out the test protected from light.

5.2.4.2 Firstly prepare the test solution, reference solution and mobile phase according to the requirements.

5.2.4.3 The solutions must be free from solid particles.

5.2.4.4 Prepare the apparatus.

5.2.4.5 The mobile phase solvent mixtures must be deaerated prior to use either by boiling or by applying a partial vacuum to the solvent reservoir.

5.2.4.6 Equilibrate the column with the prescribed mobile phase, flow rate and at temperature specified until a suitable baseline is achieved.

5.2.4.7 Test solution of the mixture to be separated is now introduced into the mobile phase with the help of an injector just before entering the separating column.

5.2.4.8 As the eluate leaves the column it enters a detector, where it is continuously monitored at the specified λ.

5.2.4.9 The electrical signal obtained from detector is amplified and routes to recorder which record the developed chromatogram.

5.2.4.10 Calculate the percentage content of Amlodipine besilate (C₂₆H₃₁ClN₂O₈S) from the declared content of amlodipine besilate CRS.

5.2.5 Limit:

5.2.5.1 97% to 102% (anhydrous substance).

6.0 REVISION LOG:

Revision No.	Effective Date	Reason
00		New SOP

7.0 REFERENCES:

7.1 The British Pharmacopoeia. Vol I., Official Monograph /Amlodipine besilate: 2015, pp. 53-54.

8.0 ANNEXURES:

Annexure 1: Observations and calculations of HPLC method.

Annexure: 1

Observations and calculations of HPLC method

Analysis on HPLC

Instrument: ___________________ Date: _________________

Model: ___________________

Column size:	Length=
Column size:	θ=
Stationary phase:
Temperature:
Mobile phase:
Flow rate:
Injection size:
Detector:
Wavelength:	λ=

Sample solution: _______________________

Reference standard solution: ______________

Impurities: ____________________________

(calculate each component calculation separately)

OBSERVATIONS:

Attach chromatogram.

CALCULATIONS:

1. Retention time: n= no. of peak

Retention time of unretained peak (t_m)= _____________

No. of peaks	Retention time of peak of interest (t_r)_n	Height of peak of interest (h)_n	Width of peak of interest (w)_n	Area of peak of interest A=1/2(h x w)

2. Retention volume:

Flow rate= _______________ml/min.

No. of peaks	Retention time of peak of interest (t_r)_n	Retention volume = retention time x flow rate

3. Retention factor:

Retention time of unretained peak (t_m)= _____________

No. of peaks	Retention time of peak of interest (t_r)_n	Retention factor of a component k= (t_r-t_m)/t_m

4. Separation factor (α):

No. of peaks	Retention factor of a component (k_n)	Relative retention of two adjacent peaks α = k₂/k₁

5. Resolution:

Retention time of unretained peak (t_m)= _____________

No. of peaks	Retention time of peak of interest (t_r)_n	Width of peak of interest (w)_n	Resolution R_s = 2 (t_r2-t_r1) (w₁-w₂)

6. Efficiency:

No. of peaks or components	Retention time of peak of interest (t_r)_n	Width of peak of interest (w)_n	Efficiency (No. of theoretical plates) N= 16 (t_r/w)²

7. Height equivalent to a theoretical plate (HETP):

Length of column = ________________________

No. of peaks or components	No. of theoretical plates (N)	Height equivalent to a theoretical plate HETP = L/N

8. Symmetry factor (tailing factor):

No. of peaks or components	Distance from the peak max. to leading edge of the peak (f)	Width w		Symmetry factor
No. of peaks or components		At 5%	At 10%	A_s = w_5% 2f	A_s = w_10% 2f

9. Response factor & Relative response factor:

Conc. (mg/ml)= ___________________

No. of peak	Peak area	Response factor = (peak area/conc.)	Relative response factor = (response factor of impurity/response factor of API)

10. Relative standard deviation (%RSD):

Use formula of relative standard deviation where it is required i.e.,

11. Percentage of content:

Percentage content = (r_U/r_S) x (C_S/C_U) x 100.

r_U= peak response of substance from the sample solution.

r_S= peak response of substance from the standard solution.

C_S= concentration of substance in the standard solution (mg/mL).

C_U= concentration of substance in the sample solution (mg/mL).

RESULTS:

________________________________________________________________________________________________________________________________________________

9.0 ABBREVIATIONS:

Abbreviation	Expanded Form
SOP	Standard operating procedure
&	And
No.	Number
Ltd.	Limited
Q.C	Quality control
B.P	British pharmacopoeia
μm	Micrometer
g/L	Gram per liter
mg	Milligram
CRS	Chemical reference solution
ml	Milliliter
m	Meter
θ	Theta
mm	Millimeter
^oC	Degree centigrade
v/v	Volume by volume
ml/min	Milliliter per minute
nm	Nanometer
μL	Microliter
Min	Minute
λ	Lambda
%	Percentage
UV/VIS	Ultraviolet/ visible
Vol	Volume
QCA	Quality control active ingredient
F	Format

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