LACTULOSE SOP

1.0  OBJECTIVE:
To lay down a procedure for the active raw material of the Lactulose from the Pharmacopoeial specifications.
2.0  SCOPE:
This SOP shall be applicable in Q.C laboratory.
3.0  RESPONSIBILITY:
3.1  Q.C Analyst.
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, crystalline powder.
5.1.2        Solubility:
5.1.2.1  Material and equipment:
5.1.2.1.1        Glassware (3 test tubes, 1 spatula).
5.1.2.1.2        Methanol.
5.1.2.1.3        Toluene.
5.1.2.1.4        Purified water.
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 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 Toluene in test tube 3 and observe.
5.1.2.4  Observations:
5.1.2.4.1        The sample in test tube 1 containing with purified water is freely soluble.
5.1.2.4.2        The sample in test tube 2 containing with Methanol is sparingly soluble.
5.1.2.4.3        The sample in test tube 3 containing with Toluene is practically insoluble.
5.2  Identification tests:
5.2.1         
5.2.1.1  Material and equipment:
5.2.1.1.1        Glassware (according to requirement).
5.2.1.1.2        Burner.
5.2.1.1.3        10.0ml of purified water.
5.2.1.1.4        3.0ml of cupric tartaric acid.
5.2.1.2  Sample:
5.2.1.2.1        50.0mg.
5.2.1.3  Method:
5.2.1.3.1        Take a test tube add in it 50.0mgof sample.
5.2.1.3.2        Dissolve it in 10.0ml of purified water.
5.2.1.3.3        Add 3.0ml of cupric tartaric acid and heat on burner.
5.2.1.3.4        Observe the changes.
5.2.1.4  Observations:
5.2.1.4.1        The red ppt is formed.
5.2.2         
5.2.2.1  Material and equipment:
5.2.2.1.1        Glassware (according to requirement).
5.2.2.1.2        Water-bath.
5.2.2.1.3        5.0ml of purified water.
5.2.2.1.4        5.0ml of ammonia.
5.2.2.2  Sample:
5.2.2.2.1        0.125g.
5.2.2.3  Method:
5.2.2.3.1        Take a test tube add in it 0.125g of sample.
5.2.2.3.2        Dissolve it in 5.0ml of purified water.
5.2.2.3.3        Add 5.0ml of ammonia and heat on water-bath at 80oC for 10min.
5.2.2.3.4        Observe the changes.
5.2.2.4  Observations:
5.2.2.4.1        A red colour develops.
5.2.3        Specific optical rotation:
5.2.3.1  Material and equipment:
5.2.3.1.1        Polarimeter.
5.2.3.1.2        Analytical weighing balance.
5.2.3.1.3        Glassware (1 beaker of 50.0ml, 1 stirrer, 1 spatula, 1 glass rod).
5.2.3.1.4        0.2ml of concentrated ammonia.
5.2.3.1.5        Purified water.
5.2.3.2  Sample:
5.2.3.2.1        1.25g.
5.2.3.3  Method:
5.2.3.3.1        Take a beaker of 50.0ml and add 1.25g of sample in it.
5.2.3.3.2        Dissolve it in sufficient purified water by using glass rod.
5.2.3.3.3        Add 0.2ml of concentrated ammonia.
5.2.3.3.4        Dilute it to 25.0ml with purified water.
5.2.3.3.5        Firstly clean the Polarimeter with clean dry cloth, according to SOP
5.2.3.3.6        Operate the Polarimeter according to SOP
5.2.3.3.7        Fill the Polarimeter tube with blank solution and determine the observed optical rotation.
5.2.3.3.8        Similarly, fill the Polarimeter tube with sample solution and determine the observed optical rotation.
5.2.3.3.9        Note down the values in annexure-1.
5.2.3.3.10    Calculate the specific optical rotation by using formula:
[α]λ T = α/lc
5.2.3.4  Observations:
5.2.3.4.1        -46.0 to -50.0.
5.3  Assay:
5.3.1        Apparatus:
5.3.1.1  HPLC apparatus.
5.3.1.2  Glassware (according to the requirement).
5.3.1.3  Refractometer (detector).
5.3.1.4  Water-bath.
5.3.1.5  Magnetic stirrer.
5.3.2        Material and reagents:
5.3.2.1  12.5ml of acetonitrile.
5.3.2.2  Purified water.
5.3.2.3  Acetonitrile.
5.3.2.4  Lactulose CRS.
5.3.2.5  Aminopropyl Silica gel for chromatography R (3μm).
5.3.2.6  0.253g of sodium dihydrogen phosphate.
5.3.3        Requirements:
5.3.3.1  Sample:
5.3.3.1.1        1.0g.
5.3.3.2  Test solution:
5.3.3.2.1        Take 50.0ml of beaker and dissolve 1.0g of the substance to be examined in 10.0ml of purified water.
5.3.3.2.2        Add 12.5ml of acetonitrile, with gentle heating on water-bath.
5.3.3.2.3        Dilute it to 25.0ml with the purified water.
5.3.3.3  Reference solutions:
5.3.3.3.1        Reference solution (b):
5.3.3.3.1.1  Take 100ml beaker and dissolve 1.0g of Lactulose CRS in 10.0ml of purified water.
5.3.3.3.1.2  Add 12.5ml of acetonitrile, with gentle heating on water-bath.
5.3.3.3.1.3  Dilute it to 25.0ml with the purified water.
5.3.3.4  Precolumn:
5.3.3.4.1        Size:
5.3.3.4.1.1  Length=0.05m,
5.3.3.4.1.2  θ=4.6mm.
5.3.3.4.2        Stationary phase:
5.3.3.4.2.1  Aminopropyl Silica gel for chromatography R (3μm).
5.3.3.4.3        Temperature:
5.3.3.4.3.1  38±1oC.
5.3.3.5  Column:
5.3.3.5.1        Size:
5.3.3.5.1.1  Length=0.15m,
5.3.3.5.1.2  θ=4.6mm.
5.3.3.5.2        Stationary phase:
5.3.3.5.2.1  Aminopropyl Silica gel for chromatography R (3μm).
5.3.3.5.3        Temperature:
5.3.3.5.3.1  38±1oC.
5.3.3.6  Mobile phase:
5.3.3.6.1        Take a beaker of 1000.0ml and add 0.253g of sodium dihydrogen phosphate in it.
5.3.3.6.2        And dissolve it in 220.0ml of purified water.
5.3.3.6.3        And add 780.0ml of acetonitrile.
5.3.3.7  Flow rate:
5.3.3.7.1        1.0ml/min.
5.3.3.8  Detection:
5.3.3.8.1        Refractometer maintained at a constant temperature.
5.3.3.9  Injection:
5.3.3.9.1        20μL of the test solution and reference solution (b).
5.3.3.10    Run time:
5.3.3.10.1    1.5 times the retention time of Lactulose.
5.3.3.11    Relative retention:
5.3.3.11.1    With reference to Lactulose (retention time=about 18.3min).
5.3.4        Method of analysis:
5.3.4.1  Firstly prepare the test solution, reference solution and mobile phase according to the requirements.
5.3.4.2  The solutions must be free from solid particles.
5.3.4.3  Prepare the apparatus.
5.3.4.4  The mobile phase solvent mixtures must be de-aerated prior to use either by boiling or by applying a partial vacuum to the solvent reservoir.
5.3.4.5  Equilibrate the column with the prescribed mobile phase, flow rate and at temperature specified until a suitable baseline is achieved.
5.3.4.6  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.
5.3.4.7  As the eluate leaves the column it enters a detector, where it is continuously monitored.
5.3.4.8  The electrical signal obtained from detector is amplified and routes to recorder which record the developed spectrum.
5.3.4.9  Calculate the percentage content of Lactulose (C12H22O11) using the declared content of Lactulose CRS.
5.3.5        Limit:
5.3.5.1  95.0% to 102.0% (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 / Lactulose: 2015, pp. 68-70.
8.0  ANNEXURES:
Annexure 1: Specific optical rotation observations and calculations.
Annexure 2: Observations and calculations of HPLC method.
Annexure: 1
Specific optical rotation observations and calculations
Specific optical rotation
Instrument: ___________________                                              Date: _______________
Model: _______________________        Length of Polarimeter tube: ________________
Sample: ________________________________g.
Solvent: ________________________________ml.
Concentration of sample solution: ____________g/ml.
Blank solution:
Sr.#
Blank solution
Temperature
Optical rotation
(α)












                                                                                                 Average: _______________
Optical rotation of blank solution: _______________
Sample solution:
Sr.#
Sample solution
Temperature
Optical rotation
(α)












                                                                                                 Average: _______________
Optical rotation of sample solution: ______________
Optical rotation of substance = Blank solution - Sample solution.



Specific optical rotation of sample solution by using formula:
[α]λ T = α/lc






                                                                      Result: ________________
Remarks: ___________________________________________________________



















Annexure: 2
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
QCA
Quality control active ingredient
F
Format
Q.C
Quality control
Vol
Volume
%
Percentage
B.P
British pharmacopoeia
UV/VIS
Ultraviolet/ visible
μm
Micron
CRS
Chemical reference substance
mg
Milligram
ml
Milliliter
m
Meter
θ
Theta
mm
Millimeter
λ
Lambda
Min
Minutes
ml/min
Milliliter per minutes
μL
Microliter
nm
Nanometer
o
Degree (angle)
l
Length
c
Concentration (g/ml)
g/ml
Gram per milliliter
α
Alpha
λ
Lambda
g/L
Grams per liter


No comments:

Post a Comment