MOXIFLOXACIN HYDROCHLORIDE SOP

1.0  OBJECTIVE:
To lay down a procedure of analytical report for the active raw material of the Moxifloxacin HCl 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  Light yellow or yellow powder.
5.1.1.2  Crystalline.
5.1.1.3  Slightly hygroscopic.
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        Ethanol (96%).
5.1.2.1.4        Acetone.
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, ethanol (96%) and acetone 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 sparingly soluble.
5.1.2.4.2        The sample in test tube 2 containing with ethanol (96%) is slightly soluble.
5.1.2.4.3        The sample in test tube 3 containing with acetone is practically soluble.
5.2  Identification tests:
5.2.1        Specific optical rotation:
5.2.1.1  Material and equipment:
5.2.1.1.1        Glassware (according to requirement).
5.2.1.1.2        Polarimeter.
5.2.1.1.3        Acetonitrile.
5.2.1.1.4        Purified water.
5.2.1.2  Sample:
5.2.1.2.1        0.2g.
5.2.1.3  Method:
5.2.1.3.1        Take a beaker and dissolve 0.2g in 20ml of a mixture of equal volumes of acetonitrile and water.
5.2.1.3.2        Determine the zero of the Polarimeter and the angle of rotation of polarized light at the wavelength of the D-line of sodium (λ=589nm) and at 20±0.5oC.
5.2.1.3.3        Determine the zero of the apparatus with the tube closed, for solids filled with prescribed solvent.
5.2.1.3.4        Calculate the specific optical rotation by using formula according to B.P monograph.
5.2.1.4  Observations:
5.2.1.4.1        -125 to -138 (anhydrous substance).
5.2.2         
5.2.2.1  Material and equipment:
5.2.2.1.1        Glassware (according to requirement).
5.2.2.1.2        Dilute nitric acid.
5.2.2.1.3        Purified water.
5.2.2.1.4        Silver nitrate.
5.2.2.1.5        Ammonia.
5.2.2.2  Sample:
5.2.2.2.1        50mg.
5.2.2.3  Method:
5.2.2.3.1        Dissolve 50mg in 5ml of water, add 1ml of dilute nitric acid, mix, allow it to stand for 5 min and filter. The filtrate gives chlorides reaction according to B.P specifications.
5.2.2.3.2        Use 2ml of the above solution.
5.2.2.3.3        Acidify with dilute nitric acid.
5.2.2.3.4        And add 0.4ml of silver nitrate.
5.2.2.3.5        Shake and allow it to stand.
5.2.2.3.6        A curdled, white ppt is formed.
5.2.2.3.7        Centrifuge it, by using centrifuge machine i.e. SOP
5.2.2.3.8        Wash the ppt with 3 quantities, each of 1ml, of water.
5.2.2.3.9        Carry out this operation rapidly is subdued light, degrading the fact that the supernatant solution may not become perfectly clear.
5.2.2.3.10    Suspend the precipitate in 2ml of water and add 1.5ml of ammonia.
5.2.2.4  Observations:
5.2.2.4.1        The precipitate dissolves easily with the possible exception of a few large particles which dissolves slowly.
5.3  Other tests:
5.3.1        pH:
5.3.1.1  Material and equipment:
5.3.1.1.1        Glassware (according to the requirement).
5.3.1.1.2        pH meter.
5.3.1.1.3        Carbon dioxide-free water.
5.3.1.2  Sample:
5.3.1.2.1        0.10g
5.3.1.3  Method:
5.3.1.3.1        Dissolve 0.10g in 50ml of carbon dioxide-free water.
5.3.1.3.2        Use pH meter for determining pH of the sample as per SOP of operation of pH meter i.e. SOP
5.3.1.3.3        Calibrate the instrument before use according to SOP No
5.3.1.3.4        Measurements are made at the same temperature 25-35oC.
5.3.1.3.5        Immerse the electrodes in the solutions to be examined and take reading.
5.3.1.3.6        And follow the B.P conditions accordingly.
5.3.1.4  Observation:
5.3.1.4.1        3.9 to 4.6.
5.4  Assay:
5.4.1        Apparatus:
5.4.1.1  HPLC apparatus.
5.4.1.2  Glassware (according to the requirement).
5.4.2        Material and reagents:
5.4.2.1  Moxifloxacin hydrochloride sample.
5.4.2.2  End-capped phenylsilyl silica gel for chromatography (5μm).
5.4.2.3  Tetrabutylammonium hydrogen sulfate.
5.4.2.4  Potassium dihydrogen phosphate.
5.4.2.5  Phosphoric acid.
5.4.2.6  Anhydrous sodium sulfite.
5.4.2.7  Methanol.
5.4.2.8  0.5g/L of tetrabutylammonium hydrogen sulfate.
5.4.2.9  1.0g/L of potassium dihydrogen phosphate.
5.4.2.10    3.4g/L of phosphoric acid.
5.4.2.11    Purified water.
5.4.3        Requirements:
5.4.3.1  Solution A:
5.4.3.1.1        Take 1000ml of beaker and dissolve 0.5g of tetrabutylammonium hydrogen sulfate and 0.1g of potassium dihydrogen phosphate in about 500ml of water.
5.4.3.1.2        Add 2ml of phosphoric acid and 0.50g of anhydrous sodium sulfite.
5.4.3.1.3        And then dilute to 1000ml with water.
5.4.3.2  Sample:
5.4.3.2.1        50.0mg (sample to be examined).
5.4.3.2.2        50.0mg (Moxifloxacin hydrochloride CRS) for reference solution.
5.4.3.3  Test solution:
5.4.3.3.1        Test solution (a):
5.4.3.3.1.1  Take 100ml of beaker and dissolve 50.0mg of the substance to be examined in solution A.
5.4.3.3.1.2  And dilute to 50.0ml with the same solution.
5.4.3.3.2        Test solution (b):
5.4.3.3.2.1  Take a beaker and dilute 2.0ml of test solution (a) to 20.0ml with solution A.
5.4.3.4  Reference solutions:
5.4.3.4.1        Reference solution (a):
5.4.3.4.1.1  Take 100ml beaker and dissolve 50.0mg of Moxifloxacin hydrochloride CRS in solution A and dilute to 50.0ml with the same solution.
5.4.3.4.1.2  Dilute 2.0ml of this solution to 20.0ml with solution A.
5.4.3.5  Column:
5.4.3.5.1        Size:
5.4.3.5.1.1  Length=0.25m,
5.4.3.5.1.2  θ=4.6mm.
5.4.3.5.2        Stationary phase:
5.4.3.5.2.1  End-capped phenylsilyl silica gel for chromatography (5μm).
5.4.3.5.3        Temperature:
5.4.3.5.3.1  45oC.
5.4.3.6  Mobile phase:
5.4.3.6.1        Mix 28 volumes of methanol and 72 volumes of solution containing 0.5g/L of tetrabutylammonium hydrogen sulfate, 1.0g/L of potassium dihydrogen phosphate and 3.4g/L of phosphoric acid.
5.4.3.7  Flow rate:
5.4.3.7.1        1.3ml/min.
5.4.3.8  Detection:
5.4.3.8.1        Spectrophotometer at 293nm.
5.4.3.9  Injection:
5.4.3.9.1        10μL of the test solution (b) and reference solution (a).
5.4.3.10    Run time:
5.4.3.10.1    2.5 times the retention time of Moxifloxacin.
5.4.4        Method of analysis:
5.4.4.1  Firstly prepare the test solution, reference solution and mobile phase according to the requirements.
5.4.4.2  The solutions must be free from solid particles.
5.4.4.3  Prepare the apparatus.
5.4.4.4  The mobile phase solvent mixtures must be deaerated prior to use either by boiling or by applying a partial vacuum to the solvent reservoir.
5.4.4.5  Equilibrate the column with the prescribed mobile phase, flow rate and at temperature specified until a suitable baseline is achieved.
5.4.4.6  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.
5.4.4.7  As the eluate leaves the column it enters a detector, where it is continuously monitored at the specified λ.
5.4.4.8  The electrical signal obtained from detector is amplified and routes to recorder which record the developed chromatogram.
5.4.4.9  Calculate the percentage content of Moxifloxacin HCl (C21H25ClFN3O4).
5.4.5        Limit:
5.4.5.1  98% 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 II., Official Monograph /Moxifloxacin hydrochloride: 2015, pp. 318-319.
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.,
PIC
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
HCl
Hydrochloric acid
Q.C
Quality control
%
Percentage
B.P
British pharmacopoeia
g
Grams
ml
Milliliter
λ
Lamda
oC
Degree Celsius
mg
Milligram
Min
Minute
ppt
Precipitate
μm
Micron/ micrometer
g/L
Gram per liter
CRS
Chemical reference solution
m
Meter
θ
Theta
mm
Millimeter
ml/min
Milliliter per minute
nm
Nanometer
μL
Microliter
Vol
Volume
QCA
Quality control active ingredient
F
Format
mg/ml
Milligram per milliliter
UV
Ultra violet


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