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:
Average: _______________
Optical
rotation of blank solution: _______________
Sample solution:
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:
___________________
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)=
_____________
2.
Retention volume:
Flow rate= _______________ml/min.
3.
Retention factor:
Retention time of unretained peak (tm)=
_____________
4.
Separation factor (α):
5.
Resolution:
Retention time of unretained peak (tm)=
_____________
6.
Efficiency:
7.
Height equivalent to a theoretical plate (HETP):
Length of column = ________________________
8.
Symmetry factor (tailing factor):
9.
Response factor & Relative response factor:
Conc. (mg/ml)= ___________________
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:
________________________________________________________________________________________________________________________________________________
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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
|