FP-089 REV-06 - Final

Avra Laboratories Pvt. Ltd.
,
STANDARD SPECIFICATION AND TEST PROCEDURE
Title: Irinotecan Hydrochloride USP (400016)
Department Quality Control Issue Date 17/06/2018
STP No. FP-089 Revision No. 06 Effective Date 18/06/2018
Issued to department Copy No. Review Date 17/06/2021
SPECIFICATIONS
S. No. TEST SPECIFICATION
1.0 Description Pale yellow to yellow crystalline powder

2.0 Solubility Slightly soluble in water, methanol and ethanol.
Identification:
The Infrared absorption spectrum should be concordant
 By IR
with that of reference spectrum of Irinotecan
Hydrochloride (400016).
The retention time of the major peak in the chromatogram
 By HPLC
of the test solution corresponding to the Irinotecan
3.0
Hydrochloride (400016) peak in the identification
solution, as obtained in the test for limit of Irinotecan
Hydrochloride (400016) enantiomer by HPLC (% w/w)
test.
 By Chloride Test
2 mg/mL solution meets the requirements of the test.
4.0 Water content by KF (% w/w) 7.00 - 9.00 %
5.0 Residue on ignition (% w/w) NMT 0.10 %
6.0 Heavy metals NMT 10 ppm
7.0 Related substances by HPLC (%w/w)
Irinotecan related compound - B
7.1 NMT 0.15 %
(400014)
Irinotecan related compound - C
7.2 NMT 0.10 %
(1001302)
7.3 Any unspecified impurity NMT 0.10 %
7.4 Total impurities NMT 0.50 %
NLT 98.0 % and NMT 102.0 % (Calculated on anhydrous
8.0 Assay by HPLC (% w/w)
basis)
9.0 Limit of Irinotecan Hydrochloride (400016) enantiomer by HPLC (%w/w)
Irinotecan related compound - D
9.1 NMT 0.15 %
(400065)
10.0 Microbial Enumeration tests:
Prepared By Reviewed By Reviewed By Approved By

Name B. Venkata Krishna T. Brahma Reddy B. Mallesh A.V. Ramesh
Designation Sr. Executive Manager Executive Dy. General Manager
Department Quality Control Quality Control Quality Assurance Quality Assurance
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Format No.: F-02-02/QC-011 Effective date: 17/10/2015 Page 1 of 44

Avra Laboratories Pvt. Ltd.,
S. No. TEST SPECIFICATION
Total aerobic microbial count NMT 1000 cfu/g

Total combined yeasts and moulds NMT 100 cfu/g
Microorganism:
Bile tolerant gram negative
Should be absent
bacteria
Escherichia coli Should be absent
Pseudomonas aeruginosa Should be absent
Salmonella species Should be absent
Staphylococcus aureus Should be absent
Clostridia Should be absent
Candida albicans Should be absent
11.0 Residual solvents:
Method-I (by GC-HS):
Methanol NMT 3000 ppm
Ethanol NMT 5000 ppm
Acetone NMT 5000 ppm
Dichloromethane (DCM) NMT 600 ppm
Ethyl acetate NMT 5000 ppm
Tetrahydrofuran (THF) NMT 720 ppm
Benzene* NMT 2 ppm
Toluene NMT 890 ppm
N,N - Dimethylformamide (DMF) NMT 880 ppm
Method-II (by HPLC):
Formic acid NMT 5000 ppm
Trifluoroacetic acid (TFA) NMT 1000 ppm
Acetic acid NMT 5000 ppm
Additional test parameter
X-ray diffraction pattern of the sample preparation should
**12.0 X-ray diffraction (polymorphism) show significant characteristic 2θ values of form-B at
7.6°, 8.30°, 9.55°, 11.0°, 12.40° (±0.2°).
Note: 1. (*) Class 1 solvent might be present in another solvent. (e.g. toluene or acetone containing Benzene)
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2. (**) indicates in-house specification

Impurity Profile:
Irinotecan related compound - B (400014):
(S)-4,11-diethyl-4,9-dihydroxy-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14
(4H,12H)-dione
Molecular formula : C22H20N2O5
Molecular weight : 392.4
Irinotecan related compound - C (1001302):
(S)-9-[(1,4'-bipiperidine)-1'-carbonyloxy]-4-methyl-11-ethyl-3,4,12,14-tetrahydro-4-hydroxy-
3,14-dioxo-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline hydrochloride
Molecular formula : C32H43ClN4O9
Irinotecan related compound - D (400065):
(R)-9-[(1,4'-Bipiperidine)-1'-carbonyloxy]-4,11-diethyl-3,4,12,14-tetrahydro-4-hydroxy-3,14-
dioxo-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin hydrochloride, trihydrate
Molecular formula : C33H45ClN4O9
TEST PROCEDURE
1.0 Description:
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1.1 Apparatus:
1.1.1 Watch glass
1.1.2 Spatula
1.1.3 Analytical balance
1.2 Reagents: Nil
1.3 Procedure:
1.3.1 Take 1 g or sufficient quantity of sample in a previously cleaned and
dried watch glass / petri dish and spread to a uniform layer by using
clean, dry spatula.
Observe the following under diffused daylight
 The nature of the substance (solid, powder, etc)
 The color of substance
 Any extraneous matter present
 Any lumps or non-homogeneity.
2.0 Solubility:
2.1 Apparatus
2.1.1 Test tube
2.1.3 Measuring cylinder
2.2 Reagents
2.2.1 Ethanol (AR grade)
2.2.2 Methanol (AR grade)
2.2.3 Distilled water
2.3 Procedure:

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2.3.1 Slightly soluble in Ethanol: Take about 100 mg of sample, transfer to

test tube, add 100 mL of ethanol and shake well to dissolve and
observe the solubility. The sample solution should be slightly soluble
and clear without any suspended / foreign particles.
2.3.2 Slightly soluble in Methanol: Take about 100 mg of sample, transfer
to test tube, add 100 mL of methanol and shake well to dissolve and
observe the solubility. The sample solution should be slightly soluble
and clear without any suspended / foreign particles.
2.3.3 Slightly soluble in water: Take about 100 mg of sample, transfer to
test tube, add 100 mL of water and shake well to dissolve and observe
the solubility. The sample solution should be slightly soluble and clear
without any suspended / foreign particles.
3.0 Identification :
 Identification by IR :
3.1 Apparatus:
3.1.1 Analytical Balance
3.1.2 FT- IR spectrophotometer
3.1.3 Mortar and pestle
3.1.4 KBr press and die
3.2 Reagents:
3.2.1 Potassium bromide (Spectroscopy grade)
3.3 Procedure:
3.3.1 Transfer 1 to 2 mg of Irinotecan Hydrochloride (400016) standard
sample to mortar and pestle.
3.3.2 Mix it with 200 to 300 mg of potassium bromide previously dried at
105ºC.
3.3.3 Grind the mixture carefully and spread it uniformly in a suitable disc.
3.3.4 Compress under vacuum at a pressure of about 800 Mpa (8 t. Cm-2), so
that uniform disc is formed.

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3.3.5 Remove the disc carefully from the die and record the IR spectrum
between 4000 cm-1 and 650 cm-1 (2.5 m to 15.4 m) using IR
spectrophotometer.
3.3.6 Repeat the procedure from step 3.3.1 to 3.3.5 to record the IR
spectrum of the sample instead of Irinotecan Hydrochloride (400016)
standard sample.
3.3.7 The IR spectrum of sample should exhibits maxima only at the same
wavelengths as that of Irinotecan Hydrochloride (400016) standard.
 Identification by HPLC
3.4 Apparatus:
3.4.1 HPLC equipped with UV-VIS (or) PDA detector
3.4.3 Vacuum pump
3.4.4 Filtration flask
3.4.6 Sonicator
3.4.7 Volumetric flask
3.4.8 Graduated pipette
3.5 Reagents:
3.5.1 Ethanol (HPLC grade)
3.5.2 n-Hexane (HPLC grade)
3.5.3 Diethylamine (AR grade)
3.6 Procedure:

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3.6.1 The retention time of the major peak in the chromatogram of the test
solution corresponding to the Irinotecan Hydrochloride (400016)
[(S)-Enantiomer] peak as obtained in the test for Limit of Irinotecan
Hydrochloride (400016) enantiomer.
 Identification by Chloride test:
3.7 Apparatus:
3.7.1 Analytical Balance
3.7.2 Test tubes
3.8 Reagents:
3.8.1 2 M Nitric Acid
3.8.2 0.1 M Silver nitrate
3.8.3 6 N Ammonium hydroxide
3.9 Procedure:
3.9.1 Dissolve 20 mg of Irinotecan Hydrochloride (400016) test sample in
10 mL of water (2mg/mL) in a test tube.
3.9.2 Add 1 mL of 2 M Nitric acid and 0.2 mL of 0.1 M silver nitrate.
3.9.3 A white curdy precipitate is formed.
3.9.4 Formation of curdy white precipitate shows the presence of chlorides.
3.9.5 The precipitate is not dissolved in nitric acid and soluble in 6 N
ammonium hydroxide.
4.0 Moisture content by KF (% w/w):

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4.1 Apparatus :
4.1.1 Digital KF
4.2 Reagents :
4.2.1 Karl Fischer reagent
4.2.2 Methanol (AR Grade)
4.3 Procedure :
4.3.1 Weigh and transfer about 200 mg of sample (W) into the titration
vessel, which contains approximately 50 mL of methanol previously
neutralized with factor determined Karl Fischer reagent.
4.3.2 Titrate the sample with Karl Fischer reagent till the end point reached.
Note the volume of Karl Fischer reagent consumed for sample (V).
4.3.3 Use di-sodium tartrate for Karl Fischer reagent standardization factor
(F) for calculation.
4.3.4 Then calculate the % of moisture content in the sample by using the
following formula,
Calculation:
Moisture content (%) = [(V x F)/W] x 100
Where,
V = Volume of Karl Fischer reagent consumed for sample
F = Karl Fischer reagent standardization factor
W = Weight of sample taken in milligrams.
Note: Repeat twice the moisture content analysis and report the average result.
Average moisture content (%) = Trial-1 + Trial-2
2
5.0 Residue on ignition:

5.1 Apparatus:
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5.1.1 Silica / platinum crucible

5.1.2 Desiccator containing Silica gel
5.1.4 Muffle furnace
5.1.6 Hot Plate
5.2 Reagents:
5.2.1 Sulfuric acid (AR Grade)
5.3 Procedure:
5.3.1 Heat a silica or platinum crucible to redness in a muffle furnace at
600±50o C for 30 minutes.
5.3.2 Allow the crucible to cool to room temperature in a desiccator.
5.3.3 Take the weight of the empty crucible and note (W1).
5.3.4 Transfer approximately 1 g of the substance to the crucible being
examined and weigh the crucible and contents accurately and note
(W2).
5.3.5 Moisten the substance with 1 mL of sulfuric acid, heat gently at low
temperature on hot plate until the substance is thoroughly charred.
5.3.6 Cool, moisten the residue with a few drops of sulfuric acid, heat gently
until white fumes are no longer evolved.
5.3.7 Ignite at 600  50C, until all black particles have disappeared.
5.3.8 Conduct the ignition in a place protected from air currents.
5.3.9 Allow the crucible to cool and weigh (W3).
Calculation:

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The amount of residue present in substance is expressed by:

= (W3 - W1) x 100
(W2 - W1)
5.3.10 If the residue exceeds the specification limit, add a few drops of
sulfuric acid and heat. Ignite as before allow cooling and noting the
weight (W4).
5.3.11 Repeat the operation for another 30 min (W 5) until two successive
weighing do not differ by more than 0.5 mg.
Calculation:
The amount of residue present in substance is expressed by:
(W5 - W1) x 100
(W2 - W1)
Where
W1 = Weight of the empty crucible
W2 = Weight of the crucible with sample
W3 = Weight of the crucible with residue (Initial)
W4 = Weight of the crucible with residue (Second)
W5 = Weight of the crucible with residue (Third)
Note: Incase after second ignition difference is more than 0.5mg,
repeat the procedure until successive weighing do not differ by more
than 0.5 mg, and consider that last two weights as W4 and W5
continuously.
6.0 Heavy Metals :

6.1 Apparatus :
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6.1.2 Measuring cylinders
6.1.3 Nessler cylinders
6.1.6 Silica or platinum crucible
6.1.7 Muffle furnace
6.2 Reagents:
6.2.1 Lead nitrate (AR grade)
6.2.2 Conc .Nitric acid (AR grade)
6.2.3 Glacial Acetic acid (AR grade)
6.2.4 Ammonium hydroxide (Concentrated ammonia solution) (AR grade)
6.2.5 Conc. Hydrochloric acid (AR grade)
6.2.6 Conc. Sulfuric acid (AR grade)
6.2.7 Sodium hydroxide (AR grade)
6.2.8 Ammonium acetate (AR grade)
6.2.9 Thioacetamide (AR grade)
6.2.10 Glycerin (AR grade)
6.2.11 Water
6.3 Procedure:
6.3.1 Lead nitrate stock solution :
Dissolve 159.8 mg of lead nitrate in 100 mL of purified water to which
has been added 1 mL of concentrated nitric acid, then dilute with water

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to 1000 mL. Prepare and store this solution in glass containers free
from soluble lead salts.
6.3.2 Preparation standard Lead solution:
On the day of use dilute 10 mL of lead nitrate stock solution with
purified water to 100 mL. Each mL of standard lead solution contains
the equivalent of 10 μg of lead. A comparison solution prepared on the
basis of 100 μL of standard lead solution per g of substance being
tested contains the equivalent of 1 part of lead per million parts of
substance being tested.
6.3.3 Preparation of 6 N Hydrochloric acid:
To a 100 mL volumetric flask containing 10 mL of purified water,
slowly add 51.6 mL of concentrated hydrochloric acid. Cool and then
dilute to volume with purified water.
6.3.4 Preparation of Acetic acid 1 N:
slowly add 5.8 mL of glacial acetic acid. Cool it and then dilute to
volume with purified water and mix.
6.3.5 Preparation of 6 N Ammonium hydroxide:
slowly add 41 mL of ammonium hydroxide (concentrated ammonia
solution) and then dilute to volume with purified water.
6.3.6 Preparation pH 3.5 Acetate buffer:
Weigh accurately and transfer about 25 g of ammonium acetate in a
100 mL beaker, dissolve it in 25 mL of purified water, and then add to
it 38 mL of 6 N hydrochloric acid. Adjust if necessary with 6 N
ammonium hydroxide or 6 N hydrochloric acid to a pH of 3.5. Transfer
the solution in a 100 mL volumetric flask along with the washings,
dilute to volume with purified water and mix.
6.3.7 Preparation of Thioacetamide TS:
Weigh accurately and transfer about 4 g of thioacetamide in 100 mL
volumetric flask dissolve and dilute to volume with purified water.

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6.3.8 Preparation of Glycerin base TS:

To 200 g of glycerin add purified water to bring the total weight to 235
g. Add 140 mL of 1 N sodium hydroxide and 50 mL of purified water.
6.3.9 Preparation of Thioacetamide-Glycerin base TS:
Mix 0.2 mL of thioacetamide TS and 1 mL of glycerin base TS, and
heat in a boiling water bath for 20 seconds. Use the mixture
immediately.
6.3.10 Preparation of standard solution:
Into a 50 mL color-comparison tube pipet 2.0 mL of standard lead
solution (20 g of Pb) and dilute with purified water to 25 mL. Using a
pH meter or short-range pH indicator paper as external indicator, adjust
with 1 N acetic acid or 6 N ammonium hydroxide to a pH between 3.0
and 4.0, dilute with water to 40 mL and mix.
6.3.11 Preparation of test solution :
Weigh accurately 1 g of sample and transfer in a silica or platinum
crucible, add sufficient amount of concentrated sulfuric acid to wet the
substance, and carefully ignite at a low temperature until thoroughly
charred. (The crucible may be loosely covered with suitable lid during
the charring). Add to the carbonized mass 2 mL of concentrated nitric
acid and 5 drops of sulfuric acid, and heat cautiously until white fumes
no longer are evolved. Ignite preferably in muffle furnace at 500°C to
600°C, until the carbon is completely burned off. Cool add 4 mL of 6
N hydrochloric acid cover digest on a steam bath for 15 minutes,
uncover and slowly evaporate on a steam bath to dryness. Moisten the
residue with 1 drop of hydrochloric acid, add 10 mL of hot water and
digest for 2 minutes. Add 6 N ammonium hydroxide dropwise until the
solution is just alkaline to litmus paper dilute with water to 25 mL and
adjust with 1 N acetic acid to a pH between 3.0 and 4.0 using short-
range pH indicator paper as an external indicator. Filter if necessary,
rinse the crucible and the filter with 10 mL of water, combine the
filtrate and rinsing in a 50 mL color-comparison tube, dilute with water
to 40 mL, and mix.
6.3.12 Procedure:

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To each of the tubes containing the standard preparation and the Test
preparation add 2 mL of pH 3.5 acetate buffer then add 1.2 mL of
thioacetamide-glycerin base TS dilute with water to 50 mL mix allow
to stand for 2 minutes, and view downward over a white surface: The
color of the solution from the test preparation is not darker than that of
solution from the standard preparation.
7.0 Related Substances by HPLC ( %w/w) (Organic impurities procedure-1) :
7.1 Apparatus:
7.1.1 HPLC equipped with UV-VIS detector
7.1.2 Column (250mm X4.6 mm X 5 µm) with packing L1
7.1.4 pH meter
7.1.5 Vacuum pump
7.1.7 Sonicator
7.2 Reagents:
7.2.1 Mono basic sodium phosphate monohydrate (HPLC grade)
7.2.2 1-Octane sulfonic acid sodium salt mono hydrate (HPLC grade)
7.2.3 Hydrochloric acid (Min 35 %) (Excel AR grade)
7.2.4 Acetonitrile (HPLC grade)
7.2.5 Methanol (HPLC grade)
7.2.6 Water (HPLC/WPS/Nanopure)
7.3 Procedure:
7.3.1 Preparation of solution A:
Weigh accurately and transfer about 2.80 g of mono basic sodium
phosphate monohydrate and 1.80 g of 1-Octane sulfonic acid sodium

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salt mono hydrate into a 1 L volumetric flask. Dissolve in 750 mL of

water and sonicate. Dilute up to the mark with water and mix well.
7.3.2 Preparation of mobile phase:
Mix solution A, Acetonitrile and Methanol in the ratio (solution A:
Acetonitrile: Methanol (59:17:24, v/v). Filter through 0.45 μm
filtration unit, degas it by sonication.
7.3.3 Diluent:
Use mobile phase adjusted with diluted hydrochloric acid to a pH 3.65
± 0.15.
7.3.4 Elution: Isocratic elution
7.3.5 Preparation of standard stock solution:
Weigh accurately and transfer about 10 mg of Irinotecan
Hydrochloride (400016) standard sample into a 100 mL volumetric
flask dissolve in diluent. Dilute up to the mark with diluent and mix
well to get a solution with a concentration of about 0.1 mg/mL.
Take about 1.0 mL of (0.1mg/mL) standard stock solution and dilute to
50 mL with diluent (0.002mg/mL).
7.3.7 Preparation of sensitivity solution:
Take about 2.5 mL of 0.002 mg/mL standard solution and dilute to 10
mL with diluent (0.0005 mg/mL).
7.3.8 System suitability stock solution:
Weigh accurately and transfer about each 5.00 mg of Irinotecan related
compound-B (400014) & Irinotecan related compound-C (1001302)
into a 50 mL volumetric flask. Add about 25 mL of methanol dissolve
and sonicate. Dilute up to the mark with methanol and mix well to get
a solution with a concentration of about 0.1 mg/mL.
Take 1.0 mL of 0.1 mg/mL above solution and dilute to 10 mL with
methanol (0.01mg/mL).
7.3.9 Preparation of system suitability solution:

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Take about 1.0 mL of 0.01 mg/mL above system suitability stock

solution and dilute to 10 mL with diluent (0.001mg/mL).
7.3.10 Preparation of test solution (Duplicate preparation):
Weigh accurately and transfer about 25.00 mg of Irinotecan
Hydrochloride (400016) test sample into a 25 mL volumetric
flask. Add 15 mL of diluent and sonicate for 1 min and make up to
the mark with diluent and sonicate till get clear solution. Make
duplicate preparations.
7.3.11 HPLC chromatographic conditions:
Column : Waters, symmetry-C18 or equivalent
Column dimensions : 250 mm x 4.6 mm, 5m
Flow rate : 1.50 mL/min
Wavelength : UV at 255 nm
Column oven Temperature : 40°C
Injection volume : 15 L
Run time : 40.00 min
7.3.12 Injection sequence:

Solution Number of injections
Blank 01+
Sensitivity solution 01
System suitability solution 01

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Standard solution 06
Sample solution preparation-1 01
Sample solution preparation-2 01
7.3.13 Acceptance criteria for system suitability:
Acceptance
S. No. Parameter
criteria
Resolution between Irinotecan related
01 compound - B & C in system suitability NLT 1.1
solution.
Relative standard deviation (area’s) in standard
02 NMT 2.0 %
solution.
03 Signal to noise ratio in sensitivity solution NLT 10
Impurity calculation:
ATest WImp 1 25
Impurity (%) = --------- x -------- x ------ x ------- x P
AStd 100 50 WTest
Where,
A Test = Peak area response of impurity peak in sample solution.
AStd = Average peak area response of standard peak in
Standard solution from 6 replicate injections
W Test = Weight of test sample (mg)
WImp = Weight of standard
P = Potency of standard
7.3.14 Acceptance criteria:

Note: Disregard any impurity peaks less than 0.05 %
Name RT(about) RRT Acceptance criteria NMT (%)
Irinotecan related compound - B (400014) 7.4 min 0.55 0.15

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Irinotecan related compound - C (1001302) 8.2 min 0.60 0.10

Irinotecan Hydrochloride (400016) 14.0 min 1.00 -----
Any unspecified impurity ------ ------ 0.10
Total impurities ------ ------ 0.5
7.3.15 Related substances LOD & LOQ Levels :
Name LOD LOQ
Irinotecan related compound - B (400014) 0.02 % 0.05%
Irinotecan related compound - C (1001302) 0.02 % 0.05%
Irinotecan Hydrochloride (400016) 0.02 % 0.05%
7.3.16 Reference chromatogram : System suitability solution
8.0 Assay by HPLC (%w/w) :

8.1 Apparatus:
8.1.1 HPLC equipped with UV-VIS detector.
8.1.2 Column 4.6 mm X 250 cm X 5 µm with packing L1
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8.1.4 pH meter
8.1.5 Vacuum pump
8.1.7 Sonicator
8.2 Reagents:
8.2.1 Mono basic sodium phosphate monohydrate (HPLC grade)
8.2.2 1-Octanesulfonic acid sodium salt monohydrate (HPLC grade)
8.2.3 Hydrochloric acid (Min 35%) (Excel AR grade)
8.2.4 Acetonitrile (HPLC grade)
8.2.6 Water (HPLC/WPS/Nanopure)
8.3 Procedure:
8.3.1 Preparation of solution A:
Weigh and Transfer about 2.80 g of mono basic sodium phosphate
monohydrate and 1.80 g of 1-Octane sulfonic acid sodium salt mono
hydrate into a 1 L volumetric flask. Add 750 mL of water dissolve and
sonicate. Dilute up to the mark with water and mix well.
8.3.2 Preparation of mobile phase:
Mix solution A, Acetonitrile and Methanol in the ratio (solution A:
Acetonitrile: Methanol (59:17:24,v/v). Filter through 0.45 μm filtration
unit, degas it by sonication.
8.3.3 Diluent: Use mobile phase adjusted with diluted hydrochloric
acid to a pH 3.65 ± 0.15.
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flask. Add 15 mL of diluent and sonicate for 1 min and make up to the
mark with diluent and sonicate till get clear solution.
8.3.6 Preparation test solution(Prepare in duplicate):
Hydrochloride (400016) test sample into a 25 mL volumetric flask.
Add 15 mL of diluent and sonicate for 1 min and make up to the mark
with diluent and sonicate till get clear solution. Make duplicate
preparations.
Column : Waters, Symmetry-C18 or equivalent
Column dimensions : 250mm x 4.6 mm, 5m, packing L1
Column oven Temperature : 40°C
Retention Time:
S.No. Name RT (about)
1.0 Irinotecan Hydrochloride(400016) 14.0 min

Blank 01+
Standard solution-1 05
Standard solution-2 01
Sample solution-1 01
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Sample solution-2 01
Standard solution-1 (after every 12 samples
01
injections and after the final sample in the sequence)
8.3.9 Acceptance criteria for System Suitability:
Parameter Acceptance criteria
Tailing factor for main principal peak for first
NMT 1.5
standard injection.
Relative standard deviation (area’s) in standard
NMT 2.0 %
solution.
Theoretical plates for main principal peak for first
NLT 5000
standard injection.
The recovery difference between two standards. NMT 1.00 %
8.3.10 Calculation:
Calculate the % of assay Irinotecan Hydrochloride (400016) present in the
sample
Asa Wsd 100
% Assay = ------- x --------- x ---------------- x Standard potency
(Anhydrous basis) Asd Wsa (100-MC)
Where,
Asa = Average area of Irinotecan Hydrochloride (400016) peak in sample solution
Asd = Average area of Irinotecan Hydrochloride (400016) peak in standard solution
Wsd = Weight of standard solution, in mg
Wsa = Weight of sample solution, in mg
8.3.11 Calculate the % difference between two standard solutions
using following formula
% difference between two standard solution =100- [A2 x W1 x 100]
As x W2
Where,

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A2: Average Area of Irinotecan Hydrochloride (400016) peak in standard solution-2

As: Average Area of Irinotecan Hydrochloride (400016) peak in standard solution-1
W1: Weight of Irinotecan Hydrochloride (400016) in standard solution-1 (mg)
W2: Weight of Irinotecan Hydrochloride (400016) in standard solution-2 (mg)
8.3.12 Reference chromatogram : Standard solution
9.0 Limit of Irinotecan Hydrochloride (400016) Enantiomer (%w/w):

9.1 Apparatus:
9.1.3 Column 4.6 mm X 250 mm X10µm packing L40
9.1.4 Vacuum pump
9.1.6 Sonicator
9.2 Reagents:
9.2.1 Ethanol (HPLC grade)
9.2.2 n-Hexane (HPLC grade)
9.2.3 Diethylamine (AR grade)
9.3 Procedure:
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9.3.1 Preparation of mobile Phase:

Mix Ethanol and n-Hexane in the ratio (Ethanol:n-Hexane)
(500:500, v/v)) and add 2 mL of diethyl amine and mix well. Filter
through 0.45 μm filtration unit, degas it by sonication.
9.3.2 Diluent: Ethanol : diethylamine (250:1, v/v)
9.3.4 Preparation of system suitability solution:
Hydrochloride (400016) standard and 5.00 mg of Irinotecan related
compound - D (400065) into a 50 mL volumetric flask. Add about 25
mL of diluent dissolve and sonicate. Dilute up to the mark with diluent
and mix well to get a solution with a concentration of 0.1 mg/mL each.
9.3.5 Preparation of identification solution:
flask. Add about 5 mL of diluent dissolve and sonicate. Dilute up to the
mark with diluent and mix well to get a solution with a concentration
of 1 mg/mL.
Weigh accurately and transfer about 5.00 mg of Irinotecan related
compound - D (400065) into a 50 mL volumetric flask. Add about 25
mL of diluent dissolve and sonicate. Dilute up to the mark with diluent
and mix well to get a solution with a concentration of 0.1 mg/mL.
 Take 1.0 mL of 0.1mg/mL of standard stock solution and dilute to
10 mL with diluent (0.01mg/mL).
 Take 1.0 mL of 0.01mg/mL of standard stock solution and dilute
to 10 mL with diluent (0.001mg/mL).
Take 1.5 mL of 0.01mg/mL of standard stock solution and dilute to 10
mL with diluent (0.0015mg/mL).

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9.3.8 Preparation of sensitivity Solution :

Take 5.0 mL of 0.001mg/mL of standard solution and dilute to 10 mL
with diluent (0.0005mg/mL).
9.3.9 Preparation of test solution:
Hydrochloride (400016) test sample into a 10 mL volumetric flask and
add about 5 mL of diluent to dissolve and sonicate till get clear
solution. Dilute up to the mark with diluent and mix well, to get a
solution with a concentration of 1 mg/mL.
Column : Diacel, Chiralcel-OD or equivalent
Column dimensions : 250 mm x 4.6 mm, 10 m packing L40
Cooler temperature : 15°C
Blank 01+
Sensitivity solution 01
System suitability solution 01
standard solution 06
Identification Solution 01
Sample solution 01
9.3.12 Acceptance criteria for System Suitability:
S. No. Parameter Acceptance

criteria
Resolution between Irinotecan related Compound -
01 D (400065) and Irinotecan Hydrochloride (400016) NLT 2.5
in system suitability solution.
02 Relative standard deviation (area’s) in standard NMT 5.0
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solution.
Irinotecan related compound - D (400065) peak in
03 Should be visible
Sensitivity solution.
Name RRT RT(about)
Irinotecan related compound - D (400065) 0.70 12.5 min
Irinotecan Hydrochloride (400016) 1.00 9.1 min
9.3.14 Enantiomer calculation :

Ru Cs
----- x ----- x 100
Rs Cu
Ru: Peak area of Irinotecan related compound - D from the test solution
Rs: Peak area of Irinotecan related compound - D from the Standard solution
Cs: Conc. of Irinotecan related compound - D in the standard solution (mg/mL)
Cu: Conc. of Irinotecan Hydrochloride (400016) in the test solution (mg/mL)
9.3.15 Irinotecan Hydrochloride (400016) Enantiomer LOD & LOQ

Levels :
Name LOD LOQ

R-Enantiomer 0.003 % 0.01%
9.3.16 Reference chromatogram : Resolution solution

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10.0 Microbial enumeration test :

10.1 Microbial enumeration test by pour plate method:
10.1.1 Total aerobic microbial count & total combined molds and
yeast:
10.1.2 Sample preparation:
Weight 10 g of the sample and aseptically pour it in 100 mL Soya bean
casein digest broth if necessary adjust pH to 6 - 8 and mix properly.
After sample taken for total aerobic count, remaining sample was kept
for enrichment at 30-35oC for 18-24 hrs.
10.1.3 Total aerobic microbial count: Plate out 1 mL of sample directly in
duplicate petri plates. Bulk seed the petri plates with molten Soya bean
casein digest agar and incubates at 30o – 35oC for 5 days. Count the
colonies developed in the petri plates to calculate colony forming
unit/g.
10.1.4 Total combined molds and yeast: Plate out 1 mL of sample directly in
duplicate petri plates. Bulk seed the petri plates with molten
Sabouraud’s dextrose agar and incubate at 20o – 25oC for 7 days.
Count the colonies developed in the petri plates to calculate cfu (colony
forming unit)/g.

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10.1.5 After incubation count the no of colonies under the colony counter.
10.1.6 If the fungal colonies appear in the Soya bean casein digest agar plate,
count them as bacterial colonies, and if the bacterial colonies are found
in Sabouraud’s dextrose agar, count them as fungal colonies.
Name Acceptance criteria
Total aerobic microbial count 1000 cfu/g
Total combined molds and 100 cfu/g
yeast
10.2 Pathogens :
10.2.1 Test for Bile tolerant gram negative bacteria: Prepare sample by
taking 1g of sample into 10 mL Soya bean casein digest broth (SCDB)
and it was kept at 20-25oC for 2 hrs to resuscitate the bacteria.
After that take 10 mL of sample, inoculate into 100 mL of Entero
bacteria enrichment broth mossel and incubate at 30-35oC for 24-48
hrs.
After suitable incubation, take the Enterobacteria enrichment broth
mossel from the incubator, cyclomix the tube and take loop full of
suspension, and streak on sterile pre-incubated Violet red bile glucose
agar, and incubate at 30-35oC for 18-24 hrs.
If colonies forming then we conclude it as Bile tolerant gram negative
bacteria is present.
If no colonies are observed on the Violet red bile glucose agar, we
resulted as absence of Bile tolerant gram negative bacteria.
10.2.2 Test for Escherichia coli: Take 1 mL of suspension from enriched
Soya bean casein digest broth and transfer to 100 mL of sterile pre-
incubated MacConkey’s broth and incubated at 43oC for 24 - 48 hours.
After suitable incubation take the broth from the incubator and take
loop full of suspension, and streak on sterile pre-incubate agar, and
incubate at 30-35oC for 3days.

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If colonies forming like brick red may have surrounding zone of

precipitated bile, then we go for confirmation test.
If no characteristic colonies are observed in the MacConkey’s agar, we
resulted as absence of Escherchia coli.
10.2.3 Test for Pseudomonas aeruginosa: Take loop full of suspension from
the incubated enriched Soyabean casein digest broth (SCDB), and
streak on sterile pre-incubated Cetrimide agar, incubated at 30-35oC for
18 – 72 hours.
If colonies appear greenish and exhibit a greenish fluorescence under
ultra violet light, then we go for confirmation test.
If no characteristic colonies are observed in Cetrimide agar, we
resulted as absence of Pseudomonas aeruginosa.
10.2.4 Test for Salmonella species: Take 0.1 mL of suspension from the
incubated enriched Soyabean casein digest broth (SCDB), and transfer
to 10 mL sterile Rappaport vasilidis broth and incubated at 30-35 oC for
18-24 hours.
After incubation, take loop full of suspension and streak on sterile pre-
incubated Xylose lysine deoxy cholate agar medium, and incubated at
30-35oC for 18- 48hours.
If colonies appear with red with or without black center, then we go for
confirmation test.
If no characteristic colonies observed in Xylose-lysine deoxy cholate
agar medium, we resulted as absence of Salmonella species.
10.2.5 Test for Staphylococcus aureus: Take loop full of suspension from
the incubated enriched Soyabean casein digest broth (SCDB), and
streak on sterile pre-incubated Mannitol salt Agar, incubated at 30-
35oC for 18-72 hours.
If colonies appear with yellow colonies with yellow zones, then we go
for confirmation test.
If no characteristic colonies observed in Mannitol salt agar medium, we
resulted as absence of Staphylococcus aureus.

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10.2.6 Test for Clostridia:

Sample Preparation: Take 2 g of sample and dissolve in 20 mL of
Reinforced medium for clostridia and mix properly. Divide the sample
into two portions to obtain 10 mL in one tube and 10 mL in another
tube. Heat one portion at 80oC for 10 min, and cool rapidly. Do not
heat another portion (10 mL).
Incubate the both tubes under anaerobic conditions at 30-35oC for 48-
72 hours. After incubation, sub culture from each container on
Columbia agar, and incubate under anaerobic conditions at 30-35oC for
48 hrs.
If colonies appear in anaerobic condition, then go for confirmation
tests.
If no colonies observed in columbia agar medium, we resulted as
absence of clostridia.
10.2.7 Test for Candida albicans: Take 1 g of sample and dissolve in 100 mL
of Sabouraud’s dextrose broth. Incubate the tube at 30-35 oC for 3-5
days.
After incubation, subculture on Sabouraud’s dextrose agar (SDA), and
incubate at 30-35oC for 24-48 hrs.
If white color colonies appear may indicate the presence of Candida
albicans. Then we go for confirmation tests.
If no characteristic colonies observed in Sabouraud’s dextrose agar
(SDA) medium, we resulted as absence of Candida albicans.
All the above tests are done and documented as online in analysis raw
data sheet.
10.2.8 Confirmation Test:
Escherichia coli: Presence of Escherichia coli shall be confirmed by
gram staining (gram-positive rods) and by streaking a loopful of the
suspected colonies from MacConkey’s agar to Levine eosin methylene
blue agar. Incubate the plates at 30-35°C for 24-48 hours. If after
incubation, plates shows colonies of following characteristics presence
of Escherichia coli is confirmed.

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Levine eosin methylene blue agar colonies with characteristic of

metallic sheen under reflected light and blue-black appearance under
transmitted light Escherichia coli is confirmed.
Pseudomonas aeruginosa: From the selective media plates pick the
suspected colonies and perform gram staining (gram-negative rods).
Oxidase test: It shall be performed to confirm identification as follow,
with the aid of an inoculating loop, transfer suspected colonies from
the surface of Cetrimide agar to strip or discs of filter paper
impregnated with N, N-dimethyl-p-phenylenediamine hydrochloride. If
a pink colour is produced within 5 to 10 seconds, the presence
of Pseudomonas aeruginosa is confirmed.
Salmonella species: From the selective media plates pick the
suspected colonies and go for confirmatory tests with the following
biochemical/media and by gram staining (gram-negative rods).
Individually transfer the suspected colonies from Xylose lysine deoxy
cholate agar to the slant of Triple sugar-iron agar with inoculating loop
and then stabbing with inoculating straight wire well in the butt.
Incubate at 30-35° C for 24-48 hours. After incubation, examine the
tube of triple sugar iron agar medium for the presence of microbial
growth and for the following physical characteristics.
(a) Slant surface: Alkaline reaction (red color).
(b) Butt: Acid reaction (yellow color) and/or gas bubble (with or
without concomitant blackening).
If the butt, slant of triple sugar iron agar shows growth and physical
characteristics confirming to the above descriptions the presence of
Salmonella species is indicated.
Staphylococcus aureus: From the selective media plates pick the
suspected colonies and go for confirmatory tests. If colonies are found
confirming to the above descriptions identification shall be performed
by a coagulase test as follow. With the aid of an inoculating loop,
individually transfer suspected colonies from the agar surface of the
Mannitol salt Agar to separate tubes containing 0.5 mL of mammalian
plasma (preferably rabbit or horse). Incubate in a water-bath / incubator
at 37oC for 3 to 24 hours, in parallel with positive control using known

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strain of Staphylococcus aureus and negative control using plasma

alone. Examine after 3 hours and at suitable intervals thereafter for the
presence of coagulation. If coagulation in any degree is observed, the
presence of Staphylococcus aureus is indicated. And perform the gram
staining for the presence of gram positive cocci.
Clostridia: Gram staining (gram positive rods)
Catalase test: From the selective media plate pick the suspected
colonies by sterile inoculation loop and dip in H 2O2 solution which is
taken in test tube. If the inoculum is not producing any bubbles in H 2O2
solution it is confirmation of presence of Clostridia.
Candida albicans: From the selective media plates pick the suspected
colonies and go for confirmatory test by streaking on pre incubated
Sabouraud’s chloramphenicol agar/Sabouraud’s dextrose agar plate,
and incubate at 30-35oC for 24-48 hrs.
The colonies are white to cream colored, smooth and yeast-like in
appearance.
Simple staining: Pick the suspected colony from the media plate, and
make smear on a microscopic glass slide. Then observe under light
microscope.
The cells with budding, presence of Candida albicans are confirmed.
11.0 Residual Solvents :

Method-I (by GC-HS) :
11.1 Apparatus:
11.1.1 Gas chromatograph equipped with flame ionization detector with
Head Space
11.2 Reagents:
11.2.1 Methanol (HPLC Grade)
11.2.2 Acetone (HPLC Grade)
11.2.3 Ethanol (HPLC Grade)
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11.2.4 Dichloromethane (DCM) (HPLC Grade)

11.2.5 Ethyl acetate (HPLC Grade)
11.2.6 Tetrahydrofuran (THF) (HPLC Grade)
11.2.7 Benzene (HPLC Grade)
11.2.8 Toluene (HPLC Grade)
11.2.9 N,N - Dimethylformamide (DMF) (HPLC grade)
11.2.10 Dimethyl sulfoxide (GC Grade)
11.3 Procedure:
11.3.1 GC Chromatographic Conditions
Column : Dikma,DM-624 capillary column
or equivalent
Column dimension : 30 m x0.32 mm x 1.8 m
Column initial temp : 32C
Column temp program : 32C/20min@20C- 220/5min
Carrier gas : Nitrogen
Detector : FID
Column Flow : 0.80 mL/min
Det. Temp : 250C
Inj. Temp : 180C
Split ratio : 1:10
Run time : 34.4 min
HS 20 parameter:
Oven Temperature : 90°C
Sample line Temperature : 100°C
Transfer Line Temperature : 110°C
Shaking level :5
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Multi injection count :1

Pressurise gas Pressure : 7.3 psi
Equilibrating time : 20.00min
Pressurising time : 1.00 min
Pressure Equili.Time : 0.10 min
Load time : 0.50 min
Load Equilb time : 0.50 min
Injection time : 1.00 min
Needle flush time : 3.00 min
G.C. Cycle Time : 60.00 min
11.3.2 Retention time:
Solvent name RT(about)

Methanol 6.1 min
Ethanol 9.1 min
Acetone 10.5 min
Dichloromethane (DCM) 12.6 min
Ethyl acetate 21.7 min
Tetrahydrofuran (THF) 22.2 min
Benzene 23.9 min
Toluene 28.7 min
N,N-Dimethylformamide(DMF) 29.7 min
(DDD(DMF(DMF)
11.3.3 Diluent: Dimethylsulfoxide

11.3.4 Blank:
Transfer 2mL of the Dimethylsulfoxide into the headspace vial of about
20 mL capacity and seal the vial with crimp cap and septa immediately.

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Stock solution - 1:
Weigh and transfer about 300.00 mg of Methanol, 500.00 mg of
Acetone, 500.00 mg of Ethanol 60.00 mg of Dichloromethane (DCM),
500.00 mg of Ethyl acetate, 72.00 mg of Tetrahydrofuran (THF) and
89.00 mg of Toluene, 88.00 mg of N,N- Dimethylformamide (DMF)
into a 100 mL volumetric flask containing 10 mL of diluent and make
up to volume with same diluent.
11.3.6 Preparation of Benzene stock solution (Stock solution - 2) :
Weigh accurately and transfer about 20.00 mg of Benzene into a 100
mL volumetric flask containing 10 mL of diluent and make up to
volume with same diluent.
From the above solution 1 mL was transferred into a 100 mL
volumetric flask and make up to the volume with diluent.
11.3.7 Preparation of standard solution :

From the stock solutions-1&2 each 5 mL was transferred into a 50
mL volumetric flask and make up to the volume with diluent and
shake well.
Pippete out 2 mL of standard solution in six different 20 mL head
space vials and closed immediately with Teflon septum and crimp the
aluminum cap with a crimper.
11.3.8 Preparation of sample solution (prepare in duplicate):
Weigh and transfer about 200.00 mg of Irinotecan Hydrochloride
(400016) test sample into a headspace vial, add 2 mL of diluent and
seal the vial immediately. Make duplicate preparations.
11.3.9 System suitability criteria:
Resolution between peaks of Ethyl acetate and Tetrahydrofuran (THF)
peak Should be NLT 1.5 in standard solution

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The RSD for peak area of each solvent should be NMT 15.0 % in
standard solution.
11.3.10Procedure:
Place the sealed vials of sample preparations and run the headspace
analyzer using the above GC parameters. Record the chromatograms
and the peak responses. Identify the solvent peak in the sample solution
based on the retention times obtained in standard chromatogram and
note down the area response. Make blank correction if necessary.
11.3.11 Injection Sequence :
Diluent(blank) +1
Standard preparation 6
Diluent(blank) 1
Sample preparation-1 1
11.3.12 Calculation:
AT WS 5 2 p
Solvent (ppm) = ------- x --------- x ------ x -------- x ------- x 106
AS 100 50 WT 100
Where,
AT = Peak area response of respective solvent peak in sample preparation.
AS = Average peak area response of respective solvent peak in standard
preparation.
WT = Weight of sample in mg
WS = Weight of standard (respected solvent) in mg

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P = Purity of respective standard solvent.

Note: Calculate the ppm with individual injection and report the
average of two injections
Benzene:
AT WS 1 5 2 p
Benzene (ppm) = ------- x --------- x ------ x --- x -------- x ------- x 106
AS 100 100 50 WT 100
Where,
AT = Peak area response of respective solvent peak in sample preparation.
AS = Average peak area response of respective solvent peak in standard
preparation.
WT = Weight of sample in mg.
WS = Weight of standard (respected solvent) in mg.
P = Purity of respective standard solvent.
Note: Calculate the ppm with individual injection and report the average of
two injections.
11.3.13 Residual solvents by GC-HS LOD & LOQ Levels:
LOD LOQ
Solvent Name
(ppm) (ppm)
Methanol 9 30
Ethanol 16 52
Acetone 16 52
Dichloromethane (DCM) 6 20
Ethyl acetate 15 50
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Tetrahydrofuran (THF) 2 8
Benzene 0.2 0.7
Toluene 2.7 9
N,N- Dimethylformamide (DMF) 13 44
Method - II (by HPLC):

11.4 Apparatus:
11.4.3 Vacuum pump
11.4.7 Sonicator
11.5 Reagents:
11.5.2 Water (Nanopure/WPS/HPLC grade)
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11.5.3 Hydrochloric acid (Min 35 %) (AR grade)

11.5.4 Acetic acid (HPLC grade)
11.5.5 Ortho phosphoric acid (Min 85%) (HPLC grade)
11.5.6 Trifluoroacetic acid (HPLC grade)
11.5.7 Formic acid (AR grade)
11.6 Procedure :
11.6.1 Preparation of mobile phase A :
Take 1000 mL of water, filter through 0.45 μm filtration unit and add
1.0 mL of orthophosphoric acid and degas it by sonication.
11.6.2 Preparation of mobile phase B:
Take sufficient quantity of Methanol, filter through 0.45 μm membrane
filtration unit and degas it by sonication.
11.6.3 Diluent:
8.9 mL of Conc. HCL in 1000 mL Water.
11.6.4 Gradient elution:
Time/B%: 0.01/0, 10/0, 11/90, 15/90, 16/0, 30/0, 30.01/Stop.
Weigh and transfer about 250.00 mg of Formic acid, 50.00 mg of
Trifluoroacetic acid and 250.00 mg of Acetic acid into a 100 mL
volumetric flask containing 10 mL of diluent and make up to volume
with same diluent.
From the stock solution 5 mL was transfer into a 50 mL volumetric
flask and make up to the volume with diluent.
11.6.7 Preparation of sample solution (prepare in duplicate):
Weigh accurately and transfer about 250.00 mg of 400016 test sample
into a 5 mL volumetric flask. Dissolve and make up to volume with the

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diluent and filtered with 0.22 micron syringe filter. Make duplicate
preparations.
Column : Phenomenex Synergy 4µ Hydro-RP
80 A or equivalent
Column dimensions: 250 mm x 4.6 mm, 4 m
11.6.9 Injection Sequence :

Diluent(blank) +1
Standard preparation 6
Diluent(blank) 1
 The resolution between Formic acid and Trifluoroacetic

acid (TFA) should be NLT 1.2.
 The relative standard deviation (RSD) of peak area of
Solvents should be NMT 5.0 %.
11.6.10 Calculations :
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After blank correction, disregard all peaks except formic acid,

trifluoroacetic acid and acetic acid. In the chromatogram obtained from
test preparation, calculate the quantity, in ppm, of formic acid,
trifluoroacetic acid and acetic acid in the portion of sample by the
following formula.
AT WS 5 5 p
Solvent (ppm) = ------- x -------- x ----- x ------ x ------ x 106
AS 100 50 WT 100
Where,
AT = Area of respective residual solvent peak in the
chromatogram obtained from test preparation.
AS= Average area of respective residual solvent peak in the
chromatograms obtained from standard preparation
WT= Weight of test sample taken in mg for Test preparation
WS= Weight of respective solvent standard taken in mg for
standard preparation
P= Percentage potency or purity of respective solvent standard
(As-is basis)
Note: Calculate the ppm with individual injection and report
the average ppm of two injections.
11.6.11Residual solvents by HPLC LOD & LOQ Levels:
Solvent LOD (ppm) LOQ (ppm)

Formic acid 7.6 25.0
Trifluoroacetic acid (TFA) 7.6 25.0
Acetic acid 7.6 25.0
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11.6.12Retention times:
Solvent name RT (about)

Formic acid 3.5 min
Trifluoroacetic acid (TFA) 4.1 min
Acetic acid 5.8 min
11.6.13 Reference chromatogram : Standard solution
12.0 X-ray diffraction (Polymorphism):

12.1 Apparatus:
12.1.1 Bruker AXS, DB Advance
12.2 Reagents: Nil
12.3 Procedure:
12.3.1 Place the sample on a sample holder, pack and smoothen its
surface with a polishing glass microscope slide and record the X-
ray diffractogram. X-ray diffraction pattern of the sample
preparation should show significant characteristic 28 values of
Form-B at 7.6°,8.30°,9.55°, 11.00° and 12.40°, (±0.2°).
12.3.2 Instrumental parameters:
Detector : Lynx Eye
X-Ray source : Copper
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Voltage : 40 kV
Current : 30 mA
2 Theta start : 2°
2 Theta End : 50°
Divergence slit : 0.3°
Scan speed (Step time) : 0.3 Sec/Step
Increment (Step size) : 0.02°
Scan type-locked coupled : Continuous
Rotation : On
Note: Instrumental parameters can be changed as per the make
of instrument.
Note: The above X-ray diffraction test carried at outside
laboratory (third party analysis).
Revision history:
Supersede Current
S. No. STP No. Reason for change
Revision No. Revision No.
1 -- FP-089 00 First issue
Revision no. changed from 00 to 01 due to:
2 00 FP-089 01 1. Residual solvents by GC, HPLC
Specifications and test procedure added.
3 01 FP-089 02 Revision no. changed from 01 to 02 due to:
1. Compound name is changed as per the
monograph in US Pharmacopeia.
2. LOD, LOQ values are incorporated for related
substance, enantiomer and residual solvents.
3. X-ray diffraction test parameter and method is
incorporated in STP.
4. Solubility test specification is revised for better
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Supersede Current
S. No. STP No. Reason for change
Revision No. Revision No.
clarity.
5. Refer change control No. CC/2/QCP/15/024
for more details.
1. TFA residual solvent specification changed
4 02 FP-089 03 from 5000 ppm to 1000 ppm.
2. Refer change control No. CC/2/QCP/15/027
for more details.
1. Residual solvents by GC HT3 parameters changed
5 03 FP-089 04 to HS 20 parameters.
2. For more details refer change control
No. CC/2/QCP/17/010.
Revision No. changed from 04 to 05 due to :
1. In Residual solvents GCHS test Hexanes are
6 04 FP-089 05 excluded.
2. For more details refer change control No.
CC/2/QCP/17/029.
Revision No. changed from 05 to 06 due to :

1. Chemical names given uniformly.
7 05 FP-089 06 2. Reference chromatograms changed.
3. For more details refer change control No.
CC/2/QCP/18/030.

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FP-089 REV-06 - Final

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Avra Laboratories Pvt. Ltd.

S. No. TEST SPECIFICATION

1.0 Description Pale yellow to yellow crystalline powder

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S. No. TEST SPECIFICATION

Total aerobic microbial count NMT 1000 cfu/g

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2. (**) indicates in-house specification

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2.3.1 Slightly soluble in Ethanol: Take about 100 mg of sample, transfer to

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4.0 Moisture content by KF (% w/w):

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5.0 Residue on ignition:

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5.1.1 Silica / platinum crucible

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The amount of residue present in substance is expressed by:

6.0 Heavy Metals :

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6.1.1 Analytical balance

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6.3.8 Preparation of Glycerin base TS:

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salt mono hydrate into a 1 L volumetric flask. Dissolve in 750 mL of

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Take about 1.0 mL of 0.01 mg/mL above system suitability stock

7.3.12 Injection sequence:

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7.3.14 Acceptance criteria:

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Irinotecan related compound - C (1001302) 8.2 min 0.60 0.10

7.3.16 Reference chromatogram : System suitability solution

8.0 Assay by HPLC (%w/w) :

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8.3.8 Injection sequence:

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A2: Average Area of Irinotecan Hydrochloride (400016) peak in standard solution-2

9.0 Limit of Irinotecan Hydrochloride (400016) Enantiomer (%w/w):

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9.3.1 Preparation of mobile Phase:

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