TIZANIDINE HYDROCHLORIDE METHOD OF ANALYSIS SOP


1.0  OBJECTIVE:
To lay down a procedure of analytical report for raw active material of the Tizanidine hydrochloride from the Pharmacopoeial specification.
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  Identification tests:
5.1.1        Chlorides test:
5.1.1.1  Material and equipment:
5.1.1.1.1        Centrifuge machine.
5.1.1.1.2        Diluted nitric acid.
5.1.1.1.3        Purified water.
5.1.1.1.4        Silver nitrate.
5.1.1.1.5        Ammonia.
5.1.1.1.6        Excess of 6N ammonium hydroxide.
5.1.1.2  Sample:
5.1.1.2.1        10mg/ml.
5.1.1.3  Method:
5.1.1.3.1        Take a test tube add in it 10mg/ml with the help of pipette.
5.1.1.3.2        Add silver nitrate. Observe the changes.
5.1.1.3.3        A white, curdy ppt is formed that is insoluble in nitric acid but is soluble in a slight excess of 6N ammonium hydroxide.
5.1.1.3.4        Observe the changes.
5.1.1.4  Observations:
5.1.1.4.1        A white, curdy ppt is soluble in a slight excess of 6N ammonium hydroxide.
5.2  pH determination test:
5.2.1        Material and equipment:
5.2.1.1  Glassware (according to the requirement).
5.2.1.2  pH meter.
5.2.2        Sample:
5.2.2.1  10mg/ml.
5.2.3        Method:
5.2.3.1  Firstly clean the pH meter with clean dry cloth, according to SOP
5.2.3.2  Operate the pH meter according to SOP for operation of pH meter i.e. SOP
5.2.3.3  Rejuvenate the electrode before use according to SOP instructions, if there is any need of.
5.2.3.4  Calibrate the electrode of the pH meter according to SOP
5.2.3.5  Perform the test on 10mg/ml solution.
5.2.3.6  Take a beaker of 100.0ml and add 10mg/ml solution in it. Such that it immersed electrodes in it completely.
5.2.3.7  Maintain the temperature of sample at 25oC±2oC.
5.2.3.8  Dip the electrode along with temperature sensor into the sample.
5.2.3.9  When dipping electrode into sample, it must be completely immersed in it.
5.2.3.10 Stir the probe gently in the sample to create a homogeneous sample.
5.2.3.11 Allow the reading to stabilize for a time.
5.2.3.12 Record the observed values of pH & temperature in the respective Annexure-1.
5.2.3.13    Wash the electrodes again after use and store the electrode in storage solution as recommended in SOP of cleaning i.e. SOP. Calibrate the instrument before use according to SOP.
5.2.4        Observation:
5.2.4.1  4.3-5.3.
5.3  Loss on drying:
5.3.1        Material and equipment:
5.3.1.1  Glassware (according to requirement).
5.3.1.2  Analytical weighing balance.
5.3.1.3  Oven.
5.3.2        Sample:
5.3.2.1  0.5g.
5.3.3        Method:
5.3.3.1  Weigh 0.5g of the test sample.
5.3.3.2  Set the oven apparatus. Operate it according to the SOP
5.3.3.3  Place the sample into the tray and dry it.
5.3.3.4  Set the temperature of oven at 105oC for at least 3 hours.
5.3.3.5  And wait till the sample loses its moisture.
5.3.3.6  After 3 hours weigh the sample again by using analytical weighing balance i.e. the final weight.
5.3.3.7  Note down readings on given Annexure-2
5.3.4        Observation:
5.3.4.1  NMT 0.5%.
5.4  Assay:
5.4.1        Buffer:
5.4.1.1  Take beaker of 1000.0ml add 6.8g/L of monobasic potassium phosphate.
5.4.1.2  Adjust pH with5.3N potassium hydroxide to a pH of 7.5±0.05.
5.4.2        Mobile phase:
5.4.2.1  Take a beaker of 100.0ml.
5.4.2.2  Add acetonitrile and buffer (20:80), stir by using magnetic stirrer.
5.4.3        System suitability solution:
5.4.3.1  46μg/ml of USP Tizanidine hydrochloride RS and 0.12μg/ml of USP Tizanidine related compound C RS in mobile phase.
5.4.4        Standard solution:
5.4.4.1  0.046mg/ml of USP Tizanidine hydrochloride RS in mobile phase.
5.4.5        Sample solution:
5.4.5.1  0.046mg/ml of Tizanidine hydrochloride in mobile phase.
5.4.6        Chromatographic system:
5.4.6.1  Mode: Liquid chromatography.
5.4.6.2  Detector: UV 230nm.
5.4.6.3  Column: 4.6mm x 15cm; packing L7.
5.4.6.4  Column temperature: 35o.
5.4.6.5  Flow rate: 1.0ml/min.
5.4.6.6  Injection size: 20μL.
5.4.7        System suitability:
5.4.7.1  Samples: System suitability solution and Standard solution.
[NOTE: The relative retention times for Tizanidine related compound C and Tizanidine are 0.5 and 0.1, respectively.]
5.4.7.2  Suitability requirements:
5.4.7.2.1        Resolution: NLT 6 between Tizanidine and Tizanidine related compound C, System suitability solution.
5.4.7.2.2        Tailing factor: NMT 2.0 for Tizanidine, standard solution.
5.4.7.2.3        Relative standard deviation: NMT 2.0%, standard solution.
5.4.8        Analysis:
5.4.8.1  Samples: Standard solution and sample solution.
5.4.9        Calculate the percentage of Tizanidine  hydrochloride (C9H8ClN5S.HCl) in the portion of Tizanidine  hydrochloride taken:
Result: (rU/rS) x (CS/CU) x 100
rU= peak area of Tizanidine from the sample solution.
rS= peak area of Tizanidine from the standard solution.
CS= concentration of USP of Tizanidine hydrochloride RS in the standard solution (mg/ml).
CU= concentration of the sample solution (mg/ml).
5.4.10    Equilibrate the column and detector with mobile phase at specified flow rate until a constant signal is received.
5.4.11    Separately inject equal volumes about 20μL of the standard solution and sample solution.
5.4.12    Record the chromatogram.
5.4.13    Measure the responses for the major peaks.
5.4.14    Analyze as directed in the monograph.
5.5  Limit:
5.5.1        98.0 to 102.0% on the dried basis.
6.0  REVISION LOG:
Revision No.
Effective Date
Reason
00

New SOP

7.0  REFERENCES:
7.1  USP38NF33 Volume-6 Official Monograph/ Tizanidine hydrochloride: 2015, pp.: 5590-5591.


8.0  ANNEXURES:
Annexure 1: pH measurement.
Annexure 2: Observations of Percentage Loss of drying by using oven.
Annexure 3: Observations and calculations of HPLC method.

Annexure: 1
pH measurement
Sr.#
Temperature
pH

















Result: _________________________________________________________________



Annexure: 2
Observations of percentage loss of drying by using Oven
Percentage loss of drying by using Oven
Weight of Sample = _____________
Time period = _____________
Pressure= _________________
Sr.#
Time (min)
Weight of sample (g)
% Loss of Moisture
Initial weight
Final weight















Average % Loss of Moisture: _____________

% Loss of Moisture:









Remarks: _______________________________________________________________



Annexure: 3
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
Sr.#
Serial number
Q.C
Quality control
Vol
Volume
g
Grams
ml
Milliliters
ppt
Precipitates
mg
Milligrams
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
QCA
Quality control active ingredient
F
Format



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