LEVOFLOXACIN HEMIHYDRATE SOP

LEVOFLOXACIN HEMIHYDRATE SOP

1.0  OBJECTIVE:

To lay down a procedure of analytical report for the active raw material of the Levofloxacin hemihydrate 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  Assay:

5.1.1        Apparatus:

5.1.1.1  HPLC apparatus.

5.1.1.2  Glassware (according to the requirement).

5.1.1.3  Spectrophotometer (detector).

5.1.2        Material and reagents:

5.1.2.1  Levofloxacin hemihydrate sample.

5.1.2.2  8.5g/L of ammonium acetate.

5.1.2.3  1.25g/L of cupric sulfate, pentahydrate.

5.1.2.4  1.3g/L of _L-isoleucine.

5.1.2.5  Purified water.

5.1.2.6  Methanol.

5.1.3        Requirements:

5.1.3.1  Buffer:

5.1.3.1.1        8.5g/L of ammonium acetate, 1.25g/L of cupric sulfate, pentahydrate, and 1.3g/L of _L-isoleucine in water.

5.1.3.2  Mobile phase:

5.1.3.2.1          Methanol and Buffer (3:7).

5.1.3.3  Standard solution:

5.1.3.3.1        1mg/ml of USP Levofloxacin in mobile phase.

5.1.3.4  Sample solution:

5.1.3.4.1        1mg/ml of Levofloxacin in mobile phase.

5.1.3.5  Chromatographic system:

5.1.3.5.1        Mode: Liquid chromatography.

5.1.3.5.2        Detector: UV 360nm.

5.1.3.5.3        Column: 4.6mm x 25cm; 5μm packing L1.

5.1.3.5.4        Column temperature: 45^oC.

5.1.3.5.5        Flow rate: 0.8ml/min.

5.1.3.5.6        Injection size: 25μL.

5.1.3.6  System suitability:

5.1.3.6.1        Sample:

5.1.3.6.1.1  Standard solution.

5.1.3.6.2        Suitability requirements:

5.1.3.6.2.1  Tailing factors:

5.1.3.6.2.1.1        0.5-1.5.

5.1.3.6.2.2  Relative standard deviation:

5.1.3.6.2.2.1        NMT 1.0%.

5.1.3.7  Analysis:

5.1.3.7.1        Sample:

5.1.3.7.1.1  Standard solution and sample solution.

5.1.3.7.2        Calculate the percentage of levofloxacin in the portion of Levofloxacin taken.

Result= (r_U/r_S) x (C_S/C_U) x 100.

r_U= peak response of levofloxacin from the sample solution.

r_S= peak response of levofloxacin from the standard solution.

C_S= concentration of USP levofloxacin RS in the standard solution (mg/mL).

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

5.1.4        Procedure:

5.1.4.1  Equilibrate the column and detector with mobile phase at specified flow rate until a constant signal is received.

5.1.4.2  Inject a sample through the injector, or use an auto-sampler.

5.1.4.3  Begin the gradient program.

5.1.4.4  Record the Spectrum.

5.1.4.5  Analyze as directed in the monograph.

5.1.5        Limit:

5.1.5.1  98% to 102% on anhydrous basis.

6.0  REVISION LOG:

Revision No.	Effective Date	Reason
00		New SOP

7.0  REFERENCES:

7.1  USP38NF33 Volume-5 Official Monograph/Levofloxacin: 2015, pp.: 4080-4082.

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 spectrum. 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
QCA	Quality control active ingredient
F	Format
g/L	Gram per liter
mg/ml	Milligram per milliliter
UV	Ultra violet
USP	United states pharmacopoeia
nm	Nanometer
mm	Millimeter
cm	Centimeter
μm	Micron
oC	Degree centigrade
ml/min.	Milliliter per minute
μL	Microliter
NMT	Not more than
%	Percentage
NF	National formulary
BM	Biomark
QCA	Quality control active ingredient
F	Format