Sultamicillin Tosilate Dihydrate API BP

KWALITY PHARMACEUTICALS LTD.
MASTER COPY STAMP

TH
6 MILE STONE, VILL. NAG KALAN, MAJITHA ROAD,
AMRITSAR – 143601.(INDIA)
STANDARD TESTING PROCEDURE FOR ACTIVE PHARMA INGREDIENT
NAME OF INGREDIENT SULTAMICILLIN TOSILATE DIHYDRATE API CODE: API1101
STP NO.
KPA/STP/QC/101/4835
PREPARED ON EFFECTIVE DATE NEXT REVIEW DATE REVISION NO.
16-02-2020 18-02-2020 18-02-2022 R0
SUPERSEDES NO: NIL PAGE 1 OF 14
PHARMACOPOEIA REFERENCES : BP SHELF LIFE : 3 YEARS

STORAGE CONDITIONS: PRESERVE IN TIGHT CONTAINERS. STORE AT ROOM TEMPERATURE. PROTECT FROM
LIGHT AND MOISTURE.
Sr.no Test no Tests Specifications Page no.
1 Nil Name of the product Sultamicillin tosilate dihydrate Page no. 1
2 Nil Pharmacopoeia referred BP Page no. 1
3 Nil Pharmacopoeia applied BP 2018 Page no. 1
4 Nil Category Beta-lactamase inhibitor Page no. 1
5 Test 1 Description White or almost white, crystalline powder Page no. 3
6 Test 2 Solubility Practically insoluble in water, sparingly soluble in Page no. 3-4
ethanol (96 percent)
7 Test 3 Identification test By Compare the spectrum with that obtained with Page no. 4
Infrared absorption Sultamicillin tosilate RS or with the reference
spectrophotometry spectrum of Sultamicillin tosilate dihydrate
8 Test 4 Specific optical rotation +178 to +195 (anhydrous substance) Page no. 4-5
9 Test 5 Related substances Page no. 5-8
Impurity B: Not more than 2.0 percent;
Impurity A: Not more than 0.5 percent;
Impurities C, D, E, F, G: For each impurity, not more than 0.5 percent;
Any other impurity: For each impurity, not more than 0.5 percent;
Total: Not more than 4.0 percent;
10 Test 6 Ethyl acetate Maximum 2.0 percent Page no. 8-10
11 Test 7 Water 4.0 percent to 6.0 percent Page no. 10
12 Test 8 Sulfated ash Maximum 0.2 percent Page no. 10-11
13 Test 9 Assay by HPLC Page no. 11-13
14 Test 9 Assay Limit It contains not less than 95.0 percent and not more Page no. 13
than 102.0 percent of Sultamicillin tosilate dihydrate,
(anhydrous substance)
NAME DESIGNATION SIGNATURE DATE

PREPARED BY HARPREET KAUR EXECUTIVE QC 16-02-2020
CHECKED BY KOMALPREET KAUR EXECUTIVE QC 18-02-2020
APPROVED BY T.C. SHARMA SR. QC HEAD 18-02-2020
AUTHORIZED BY TEEKA RAM SHARMA SR.MANAGER QA 18-02-2020
F/KPA/SOP/QA-081-01
KWALITY PHARMACEUTICALS LTD. MASTER COPY STAMP
TH
STP NO.
KPA/STP/QC/101/4835
16-02-2020 18-02-2020 18-02-2022 R0

LIGHT AND MOISTURE.
1 Molecular structure, Molecular formula, Molecular weight, Nomenclature and Category of
Sultamicillin tosilate dihydrate
Molecular Formula : C32H38N4O12S3,2H2O

Molecular weight : 803
Nomenclature: 4-Methylbenzenesulfonate of methylene (2S, 5R, 6R)-6-[[(2R)-aminophenylacetyl]amino]-3,3-
dimethyl-7-oxo-4-thia-1-azabicyclo heptane-2-carboxylate (2S,5R)-3,3-dimethyl-4,4,7-trioxo-4λ6-thia-1-
azabicyclo heptane-2-carboxylate dihydrate.
Category: Beta-lactamase inhibitor.

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LIGHT AND MOISTURE.
Testing parameters
Test 1 Description:
Procedure: By Visual inspection
Limit: White or almost white, crystalline powder.
Test 2 Solubility test:
Practically insoluble in water, sparingly soluble in ethanol (96 percent).
Before to start the solubility test, check these parameters:
Test tubes should be washed completely with distilled water and then dry under oven at 500 C.
Procedure :
1) Practically insoluble in water: Take 100 mg of sample it should dissolve in more than 1000 ml of
distilled water.
2) Sparingly soluble in ethanol (96 percent): Take 100 mg of sample it should dissolve in 3 ml to 10 ml
of ethanol (96 percent).
The following table indicates the meanings of the terms used in statements of approximate solubility’s with
their Descriptive terms:
Approximate volume of solvent in milliliters per gram of solute i.e. Sultamicillin tosilate dihydrate.
very soluble Less than 1ml
freely soluble from 1ml to 10ml
soluble from 10ml to 30 ml
sparingly soluble from 30ml to 100ml

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LIGHT AND MOISTURE.
slightly soluble from 100ml to 1000ml
very slightly soluble from 1000ml to 10,000ml
practically insoluble more than 10,000ml
Test 3 Identification test by IR spectrum:

Procedure: Triturate approximately about 1 mg to 3 mg of Sultamicillin tosilate dihydrate and approximately
300 mg of Potassium Bromide previously dried at 100-105 °C for 2 h. Record the spectrum of sample from
4000 – 650 cm-1.
Observation: Compare the spectrum with that obtained with Sultamicillin tosilate RS or with the reference
spectrum of Sultamicillin tosilate dihydrate.
Test 4 Specific optical rotation
Sample solution: Transfer an accurately weighed 1 gm of Sultamicillin tosilate dihydrate in 50 ml of
volumetric flask; dissolve in 25 ml of dimethylformamide with the help of sonication and dilute with
dimethylformamide to 50 ml, mix it, to obtain concentration 20 mg of Sultamicillin tosilate dihydrate.
Procedure: Determine the zero of the polarimeter and the angle of rotation of polarized light at the
wavelength of the D-line of sodium (l = 589.3 nm) at 20 ± 0.5. Zero is determine when tube is empty.
Calculate the specific optical rotation using the following formula.
For neat liquids:
For substances in solution:

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LIGHT AND MOISTURE.
Where c is the concentration of the solution in grams per liter.

Calculate the content c in grams per liter or the content c¢ in per cent m/m of a dissolved substance using
the following formulae:
a = angle of rotation in degrees read at 20 ± 0.5°C,

l = length in decimeters of the polarimeter tube,
r20 = Density at 20 °C in grams per cubic centimeter. For the purposes of the Pharmacopoeia, density is
replaced by relative density.
Limit: +178 to +195 (anhydrous substance)
Test 5 Related substances: By HPLC
Note: Prepare the solutions immediately before use or keep at 2-8 °C for not more than 6 h.
Chromatographic conditions:
Column: A stainless steel column having (Length 0.10 m × Diameter 4.6 mm) column L1, packed with
stationary phase
Stationary phase: Octadecylsilyl silica gel for chromatography (3.5 µm), L1
Flow rate: 1.0 mL per min.
Detection: Spectrophotometer at 215 nm.
Injection: 5 µL of the blank solution, the test solution and reference solutions (b), (c) and (d).

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LIGHT AND MOISTURE.
Temperature: 25 °C.
Mobile phase A: Transfer an accurately weighed 4.68 g of sodium dihydrogen phosphate in 1000 ml
volumetric flask, dissolve in 500 ml of distilled water with the help of sonication and dilute to 1000 ml with
distilled water. Adjust pH to 3.0 with phosphoric acid.
Mobile phase B: Acetonitrile
Time (min) Mobile phase A (per cent v/v) Mobile phase B (per cent v/v)
0-15 9530 570
15-16 30 70
16-16.5 3095 705
16.5-20 95 5
Solution A: Transfer an accurately measured 200 ml of methanol and 800 ml of acetonitrile in 1000 ml
volumetric flask, and mix (20:80 v/v). Net volume 1000 ml.
Solution B: Transfer an accurately weighed 1.56 g of sodium dihydrogen phosphate in 1000 ml volumetric
flask; dissolve in 900 mL of distilled water with the help of sonication. Add 7.0 mL of phosphoric acid and
dilute to 1000 mL with distilled water.
Blank solution: Transfer an accurately measured 300 ml of Solution B and 700 ml of Solution A in 1000 ml
volumetric flask, and mix (30:70 V/V). Net volume 1000 ml.
Test solution: Transfer an accurately weighed 70.0 mg of Sultamicillin tosilate dihydrate in 50 ml volumetric
flask, dissolve in 35 ml of solution A and sonicate for about 1 min. Add 13 mL of solution B, mix and sonicate
for about 1 min and dilute to 50 ml with solution B and mix, to obtain a concentration about 1.4 mg per ml of
Sultamicillin tosilate dihydrate.

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LIGHT AND MOISTURE.
Reference solution (a): Transfer an accurately weighed 70.0 mg of Sultamicillin in 50 ml volumetric flask,
dissolve in 35 ml of solution A and sonicate for about 1 min. Add 13 mL of solution B, mix and sonicate for
about 1 min and dilute to 50 ml with solution B and mix, to obtain a concentration about 1.4 mg per ml of
Sultamicillin.
Preparation of sodium hydroxide solution (0.4 gm per litre): Transfer an accurately weighed 40 mg
of sodium hydroxide in 100 ml volumetric flask; dissolve in 50 mL of distilled water with the help of
sonication and dilute to 100 mL with distilled water.
Preparation of hydrochloric acid solution (0.36 gm per litre): Transfer an accurately weighed 0.015 ml of
hydrochloric acid in 50 ml volumetric flask; dissolve in 20 mL of distilled water with the help of sonication and
Reference solution (b): Suspend 15 mg of Sultamicillin tosilate dihydrate in 100 ml volumetric flask; dissolve
in 20 mL of sodium hydroxide solution (0.4 gm per L) and sonicate in an ultrasonic bath for about 5 min. Add
20 mL of hydrochloric acid solution (0.36 gm per L) and dilute to 100.0 mL with water.
Reference solution (c): Transfer an accurately weighed 0.200 g Sultamicillin tosilate dihydrate in 100 ml
volumetric flask; dissolve in 70.0 mL of solution A and sonicate for about 1 min. Add 25.0 mL of solution B,
mix and sonicate for about 1 min. Dilute to 100.0 mL with solution B and mix. Dilute 1.0 mL of this solution to
100.0 mL with the blank solution.
Reference solution (d): Transfer an accurately weighed 32.3 mg of ampicillin trihydrate CRS (impurity B) and
7.0 mg of sulbactum CRS (impurity A) in 1000 ml beaker; dissolve in 500 ml of distilled water and dilute to
1000 mL with distilled water.
Relative retention: With reference to Sultamicillin (retention time = about 9.3 min): impurity A = about 0.41;

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LIGHT AND MOISTURE.
ampicillin penicilloic acid = about 0.47; tosilate = about 0.50; impurity B = about 0.55; impurity C = about
0.94; impurity D = about 1.09; impurity F = about 1.23; impurity E = about 1.26; impurity G = about 1.42.
System suitability: reference solution (b):
Resolution: minimum 2.5 between the peaks due to ampicillin penicilloic acid and tosilate and minimum 2.5
between the peaks due to tosilate and impurity B.
Limits:
Impurity B: Not more than the area of the corresponding peak in the chromatogram obtained with reference
solution (d) (2.0 percent);
Impurity A: Not more than the area of the corresponding peak in the chromatogram obtained with reference
solution (d) (0.5 percent);
Impurities C, D, E, F, G: For each impurity, not more than 0.5 times the area of the peak due to Sultamicillin in
the chromatogram obtained with reference solution (c) (0.5 percent);
Any other impurity: For each impurity, not more than 0.5 times the area of the peak due to Sultamicillin in
the chromatogram obtained with reference solution (c) (0.5 percent);
Total: Not more than 4 times the area of the peak due to Sultamicillin in the chromatogram obtained with
reference solution (c) (4.0 percent);
Disregard limit: 0.1 times the area of the peak due to Sultamicillin in the chromatogram obtained with
reference solution (c) (0.1 percent).
Test 6 Ethyl acetate: By Head space Gas Chromatography:
Column:
Material: fused silica;

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LIGHT AND MOISTURE.
size: l = 50 m, Ø = 0.32 mm;
Stationary phase: poly (dimethyl) siloxane (Film thickness: 1.8 µm or 3 µm).
Carrier gas: Helium for chromatography.
Linear velocity: 35 cm/s.
Split ratio: 1:5
Static head-space conditions that may be used.
Equilibration temperature: 105⁰ C
Equilibration time: 45 min
Transfer-line temperature: 110⁰ C
Pressurization time: 30 s
Temperature:
Time (min) Temperature (0 C)
Column 0-6 70
6-16 70→220
16-18 220
Injection port 140
Detector 250
Detection: Flame ionization.
Injection: 1 ml.
Solvent mixture: Transfer an accurately measured 1 ml of distilled water and 99 ml of dimethylformamide in
100 ml volumetric flask, mix it. Net volume 100 ml.

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LIGHT AND MOISTURE.
Test solution: Transfer an accurately weighed 200 mg of Sultamicillin tosilate dihydrate in 10 ml volumetric
flask and dissolve in 7.0 mL of solvent mixture, mix it.
Reference solution: Transfer an accurately weighed 200 mg of ethyl acetate in 250 ml volumetric flask;
dissolve in 240 mL of solvent mixture and dilute to 250.0 mL with the solvent mixture. Further transfer 5.0 mL
of this solution in 10 ml volumetric flask and dilute to 7.0 mL with solvent mixture.
Note: Immediately close the vials with a tight rubber membrane stopper coated with polytetrafluoroethylene
and secure with an aluminium crimped cap. Shake to obtain a homogeneous solution
Relative retention: With reference to dimethylformamide (retention time = about 14 min): ethyl
acetate = about 0.7.
Limit: Maximum 2.0 percent
Test 7 Water:
Procedure: Water content is determined by using 200 mg of Sultamicillin tosilate dihydrate previously dried
with the help of Karl fisher apparatus.
Limit: 4.0 percent to 6.0 percent.
Test 8 Sulfated ash:
Procedure: Ignite a porcelain crucible at 600 ± 500C for 30minutes, cool the crucible in desiccator and weigh
it. Weigh about 1000 mg of the sample in the crucible. Moisten the sample with 1.0 ml of sulfuric acid, then
heat gently at a temperature as low as practicable until the sample is thoroughly charred and ignite in muffle
furnace at 600 ± 500C, until the residue is completely incinerated. Ensure that flames are not produced at any
time during the procedure. Cool the crucible in a desiccators, weigh the crucible with residue and calculate
the percentage of residue using the following formula:

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LIGHT AND MOISTURE.
Calculation :
Calculate the % of residue by the following formula:
–
%=
If the amount of residue exceed from limit, repeat the moistening with sulfuric acid, heating and igniting as
before, using 30 minutes ignition period, until two consecutive weighing of the residue do not differ by more
than 0.5mg
Limit: Maximum 0.2 percent.
Test 9 Assay:
Chromatographic conditions:
Column: A stainless steel column having (Length 0.10 m × Diameter 4.6 mm) column L1, packed with
stationary phase
Stationary phase: Octadecylsilyl silica gel for chromatography (3.5 µm), L1
Flow rate: 1.0 mL per min.
Detection: Spectrophotometer at 215 nm.
Injection: 5 µL
Temperature: 25 °C.
Mobile phase A: Transfer an accurately weighed 4.68 g of sodium dihydrogen phosphate in 1000 ml
volumetric flask, dissolve in 500 ml of distilled water with the help of sonication and dilute to 1000 ml with
distilled water. Adjust pH to 3.0 with phosphoric acid.
Mobile phase B: Acetonitrile

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LIGHT AND MOISTURE.
Time (min) Mobile phase A (per cent v/v) Mobile phase B (per cent v/v)
0-15 9530 570
15-16 30 70
16-16.5 3095 705
16.5-20 95 5
Solution A: Transfer an accurately measured 200 ml of methanol and 800 ml of acetonitrile in 1000 ml
volumetric flask, and mix (20:80 v/v). Net volume 1000 ml.
Solution B: Transfer an accurately weighed 1.56 g of sodium dihydrogen phosphate in 1000 ml volumetric
flask; dissolve in 900 mL of distilled water with the help of sonication. Add 7.0 mL of phosphoric acid and
Blank solution: Transfer an accurately measured 300 ml of Solution B and 700 ml of Solution A in 1000 ml
volumetric flask, and mix (30:70 V/V). Net volume 1000 ml.
Test solution: Transfer an accurately weighed 70 mg of Sultamicillin tosilate dihydrate in 50 ml volumetric
flask, dissolve in 35 ml of solution A and sonicate for about 1 min. Add 13 mL of solution B, mix and sonicate
for about 1 min and dilute to 50 ml with solution B and mix, to obtain a concentration about 1.4 mg per ml of
Sultamicillin tosilate dihydrate.
Reference solution: Transfer an accurately weighed 70.0 mg of Sultamicillin in 50 ml volumetric flask,
dissolve in 35 ml of solution A and sonicate for about 1 min. Add 13 mL of solution B, mix and sonicate for
about 1 min and dilute to 50 ml with solution B and mix, to obtain a concentration about 1.4 mg per ml of
Sultamicillin.
Relative retention: With reference to Sultamicillin (retention time = about 9.3 min)

F/KPA/SOP/QA-081-01
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LIGHT AND MOISTURE.
Calculate the percentage content of Sultamicillin tosilate (C 32H38N4O12S3) from the declared content of
Sultamicillin tosilate CRS
Limit: It contains not less than 95.0 percent and not more than 102.0 percent of Sultamicillin tosilate
dihydrate, (anhydrous substance).

F/KPA/SOP/QA-081-01
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LIGHT AND MOISTURE.
REVISION/ REVIEW HISTORY
Sr. No. Date Details & reason for revision / review Revision no.
1. 18-02-2020 New STP R0

F/KPA/SOP/QA-081-01

Sultamicillin Tosilate Dihydrate API BP

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KWALITY PHARMACEUTICALS LTD.

MASTER COPY STAMP

PHARMACOPOEIA REFERENCES : BP SHELF LIFE : 3 YEARS

NAME DESIGNATION SIGNATURE DATE

PHARMACOPOEIA REFERENCES : BP SHELF LIFE : 3 YEARS

Molecular Formula : C32H38N4O12S3,2H2O

NAME DESIGNATION SIGNATURE DATE

PHARMACOPOEIA REFERENCES : BP SHELF LIFE : 3 YEARS

NAME DESIGNATION SIGNATURE DATE

PHARMACOPOEIA REFERENCES : BP SHELF LIFE : 3 YEARS

Test 3 Identification test by IR spectrum:

For substances in solution:

NAME DESIGNATION SIGNATURE DATE

PHARMACOPOEIA REFERENCES : BP SHELF LIFE : 3 YEARS

Where c is the concentration of the solution in grams per liter.

a = angle of rotation in degrees read at 20 ± 0.5°C,

NAME DESIGNATION SIGNATURE DATE

PHARMACOPOEIA REFERENCES : BP SHELF LIFE : 3 YEARS

NAME DESIGNATION SIGNATURE DATE

PHARMACOPOEIA REFERENCES : BP SHELF LIFE : 3 YEARS

NAME DESIGNATION SIGNATURE DATE

PHARMACOPOEIA REFERENCES : BP SHELF LIFE : 3 YEARS

NAME DESIGNATION SIGNATURE DATE

PHARMACOPOEIA REFERENCES : BP SHELF LIFE : 3 YEARS

NAME DESIGNATION SIGNATURE DATE

PHARMACOPOEIA REFERENCES : BP SHELF LIFE : 3 YEARS

NAME DESIGNATION SIGNATURE DATE

PHARMACOPOEIA REFERENCES : BP SHELF LIFE : 3 YEARS

NAME DESIGNATION SIGNATURE DATE

PHARMACOPOEIA REFERENCES : BP SHELF LIFE : 3 YEARS

NAME DESIGNATION SIGNATURE DATE

PHARMACOPOEIA REFERENCES : BP SHELF LIFE : 3 YEARS

NAME DESIGNATION SIGNATURE DATE

PHARMACOPOEIA REFERENCES : BP SHELF LIFE : 3 YEARS

REVISION/ REVIEW HISTORY

NAME DESIGNATION SIGNATURE DATE

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