AZITHROMYCIN SOP

AZITHROMYCIN SOP

1.0  OBJECTIVE:

To lay down a procedure of analytical report for the active raw material of the Azithromycin dihydrate 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        Anhydrous ethanol.

5.1.2.1.4        Methylene chloride.

5.1.2.2  Sample:

5.1.2.2.1        Small quantity.

5.1.2.3  Method:

5.1.2.3.1        Take 3 test tubes and add small quantity of sample for testing solubility according to B.P specifications.

5.1.2.3.2        Add purified water, anhydrous ethanol and methylene chloride in each test tube separately in a small volume and observe the solubility of the sample.

5.1.2.4  Observations:

5.1.2.4.1        The sample in test tube 1 containing with water is practically insoluble.

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

5.1.2.4.3        The sample in test tube 3 containing with methylene chloride is freely soluble.

5.2  Assay:

5.2.1        Apparatus:

5.2.1.1  HPLC apparatus.

5.2.1.2  Glassware (according to the requirement).

5.2.1.3  Spectrophotometer (detector).

5.2.2        Material and reagents:

5.2.2.1  Azithromycin dihydrate sample.

5.2.2.2  Octadecylsilyl vinyl polymer for chromatography (5μm).

5.2.2.3  Acetonitrile.

5.2.2.4  6.7g/L solution of dipotassium hydrogen phosphate.

5.2.2.5  Phosphoric acid.

5.2.2.6  560g/L solution of potassium hydroxide.

5.2.3        Requirements:

5.2.3.1  Solution A:

5.2.3.1.1        Take 1000ml of beaker and mix 60 volumes of acetonitrile and 40 volumes of a 6.7g/L solution of dipotassium hydrogen phosphate adjusted to pH 8.0 with phosphoric acid.

5.2.3.2  Sample:

5.2.3.2.1        53.0mg (sample to be examined).

5.2.3.2.2        53.0mg (Azithromycin CRS) for reference solution.

5.2.3.3  Test solution:

5.2.3.3.1        Test solution:

5.2.3.3.1.1  Take 100ml of beaker and dissolve 53.0mg of the substance to be examined in 2ml of acetonitrile.

5.2.3.3.1.2  And dilute to 100.0ml with the solution A.

5.2.3.4  Reference solutions:

5.2.3.4.1        Reference solution:

5.2.3.4.1.1  Take 100ml beaker and dissolve 53.0mg of Azithromycin CRS in 2ml of acetonitrile.

5.2.3.4.1.2  And dilute to 100.0 ml with solution A.

5.2.3.5  Column:

5.2.3.5.1        Size:

5.2.3.5.1.1  Length=0.25m,

5.2.3.5.1.2  θ=4.6mm.

5.2.3.5.2        Stationary phase:

5.2.3.5.2.1  Octadecylsilyl vinyl polymer for chromatography (5μm).

5.2.3.5.3        Temperature:

5.2.3.5.3.1  40^oC.

5.2.3.6  Mobile phase:

5.2.3.6.1        Mix 60 volumes of acetonitrile and 40 volumes of a 6.7g/L solution of dipotassium hydrogen phosphate adjusted to pH 11.0 with 560g/L solution of potassium hydroxide.

5.2.3.7  Flow rate:

5.2.3.7.1        1.0ml/min.

5.2.3.8  Detection:

5.2.3.8.1        Spectrophotometer at 210nm.

5.2.3.9  Injection:

5.2.3.9.1        10μL.

5.2.3.10 Run time:

5.2.3.10.1    1.5 times the retention time of Azithromycin.

5.2.3.11 Retention time Azithromycin:

5.2.3.11.1    About 10min.

5.2.4        Method of analysis:

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

5.2.4.2  The solutions must be free from solid particles.

5.2.4.3  Prepare the apparatus.

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

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

5.2.4.6  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.

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

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

5.2.4.9  Calculate the percentage content of Azithromycin (C₃₈H₇₂N₂O₁₂) from the declared content of Azithromycin CRS.

5.2.5        Observations:

5.2.5.1  96% 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 /Azithromycin: 2015, pp. 225-227.

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= 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 (tm)= _____________ No. of peaks Retention time of peak of interest (tr)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 (tr)n Retention volume = retention time x flow rate 3.      Retention factor: Retention time of unretained peak (tm)= _____________ No. of peaks Retention time of peak of interest (tr)n Retention factor of a component k= (tr-tm)/tm 4.      Separation factor (α): No. of peaks Retention factor of a component (kn) Relative retention of two adjacent peaks α = k2/k1 5.      Resolution: Retention time of unretained peak (tm)= _____________ No. of peaks Retention time of peak of interest (tr)n Width of peak of interest (w)n Resolution Rs = 2 (tr2-tr1)         (w1-w2) 6.      Efficiency: No. of peaks or components Retention time of peak of interest (tr)n Width of peak of interest (w)n Efficiency (No. of theoretical plates) N= 16 (tr/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 At 5% At 10% As = w5%        2f As = w10%        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 = (rU/rS) x (CS/CU) x 100. rU= peak response of substance from the sample solution. rS= peak response of substance from the standard solution. CS= concentration of substance in the standard solution (mg/mL). CU= 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	Micron/ micrometer
g/L	Gram per liter
ml	Milliliter
mg	Milligram
CRS	Chemical reference solution
m	Meter
θ	Theta
mm	Millimeter
oC	Degree Celsius
ml/min	Milliliter per minute
nm	Nanometer
μL	Microliter
Min	Minute
λ	Lamda
%	Percentage
Vol	Volume
QCA	Quality control active ingredient
F	Format