Zhejiang East-Asia Pharmaceutical Co., Ltd.

Coastal Industrial City, Pubagang town, Sanmen county, Zhejiang, China. P.C.: 317100.

3.2. S.4.2 Analytical Procedure

【Definition】
Ofloxacin contains NLT 98.5% and NMT 101.5% of C18H20FN3O4, calculated on the dried basis.

【Description】
Appearance: Ofloxacin occurs as pale yellowish-white to light yellowish-white crystals or
crystalline powder.
Procedure:
Take an appropriate amount of sample and spread on a white background, observe the appearance
of the sample visually under fluorescent.

Solubility: It is sparingly soluble in chloroform, slightly soluble in alcohol, in methanol and in

water.
Procedure:
Grind the sample into fine powder, take appropriate amount of sample to a certain volume of
solvent at 25ºC±2ºC, shake strongly for 30s every 5min, observe the solubility of solution in 30min.

【Optical rotation】-1o ~ +1o (25°C)

Weigh accurately 1.0g sample in a 100mL volumetric flask, add chloroform to dissolve and dilute to
volume, mix well. Take the solution and conduct test according to Optical Rotation Determination.
Calculation formula:
100α
Optical rotation[α ]tD =
lc × ( 1 - Loss on drying )
In which,
D--- D line of sodium spectrum;
t---The temperature of measurement;
l---The thickness of the layer of sample solution;
α---The observed optical rotation;
c---The weight of test sample in 100mL solution, g.

Ofloxacin DMF Open part (USP)

Date: Apr.24, 2017
 Zhejiang East-Asia Pharmaceutical Co., Ltd.
Coastal Industrial City, Pubagang town, Sanmen county, Zhejiang, China. P.C.: 317100.

【Identification】
A. Infrared absorption
Procedure:
Triturate 1-1.5 mg of the substance to be examined with 200-300 mg of finely powdered and dried
potassium bromide R (ratio which compared to the examined substance: 200:1) in an agate mortar,
carefully grind the mixture and spread it uniformly and transfer it into the compression mold
immediately to crush into thin disc, take it out and place it on the infrared instrument. Scan the
prepared sample in the infrared region of 4000-400cm-1 according to the instrument parameters.
Then take 1-1.5mg ofloxacin reference substance and operate with the same method. (Generally the
reference spectrum was made once a year and saved in the computer for comparison). Compare the
infrared spectrum of the sample with that of the reference substance.

B. UV test
Weigh 13.4mg of sample into a 100mL volumetric flask, add 0.1mol/L hydrochloric acid solution
to dissolve and dilute to volume, transfer 5mL of this solution and add 0.1mol/L hydrochloric acid
to dilute to 100mL, used as test solution. Take test solution and scan the sample at the wavelength
coverage of 220nm-350nm according to Ultraviolet spectrophotometry

【Related substances】
a. Chromatographic system
Chromatographic column: Inertsil ODS-3V 4.6×150mm 5μ (Or other suitable column)
Mobile phase: Acetonitrile-buffer (Dissolve 3.1g of ammonium acetate and 5.4g of sodium
perchlorate in 1000mL water, adjust with phosphoric acid to a pH of 2.2) (240:1300)
Column temperature: 45°C
Flow rate: about 0.5mL/min
Detector: UV 294nm
Injection volume: 10μL
b. Procedure
Diluent: Acetonitrile and water (1: 6)
System suitability test solution: weigh 10.0mg of ofloxacin impurity A RS (Desmethyl ofloxacin)
and 10.0mg of ofloxacin RS into a 100mL volumetric flask, dissolve and dilute to volume with
diluent, mix well. Transfer 10mL of this solution and add diluent to make exactly 50mL, then
transfer 1.0mL of this solution into a 50mL volumetric flask, dilute to volume with diluent, mix

Ofloxacin DMF Open part (USP)

Date: Apr.24, 2017
 Zhejiang East-Asia Pharmaceutical Co., Ltd.
Coastal Industrial City, Pubagang town, Sanmen county, Zhejiang, China. P.C.: 317100.

well.
Standard solution: weigh 20mg of ofloxacin RS into a 100mL volumetric flask, dissolve and dilute
to volume with diluent, mix well, used as stock solution. Transfer 1.0mL of stock solution and add
diluent to make exactly 50mL, then transfer 1.0mL of this solution into a 10mL volumetric flask,
dilute to volume with diluent, mix well.
Test solution: weigh 20mg of sample into a 100mL volumetric flask, dissolve and dilute to volume
with diluent, mix well.
System suitability test: inject system suitability test solution into chromatograph and record
chromatograms. Resolution between ofloxacin impurity A peak and ofloxacin peak should be not
less than 2.0.
Repeatability test: inject standard solution into chromatograph, replication injection for 6 times,
and record chromatograms. RSD of peak area for ofloxacin should be not more than 3.0%.
Sample test: inject test solution and standard solution into chromatograph, record chromatograms
until 2.5times of the retention time of ofloxacin peak.
Sequence of injection: blank solvent (1-3needles) →system suitability test (1needle) →
repeatability test (6needles) →standard solution (1needle) →test solution (1needle)
Calculation formula:
AS × C R
Individual impurity = × 100%
AR × C S
( Total peak area of test solution - main peak area in test solution ) × CR
Total impurities( Sum of individual impurity ) = × 100%
AR × CS

In which,
CR---Concentration of standard solution, mg/mL;
CS---Concentration of test solution, mg/mL;
AR---Peak area of ofloxacin in standard solution;
AS---Peak area of individual impurity in test solution.
Acceptance criteria:
Individual impurities: NMT0.3%, total impurities: NMT0.5%.

【Loss on drying】
Take 1.0g of sample, accurately weigh, put in a platymorphia weighing bottle which has reached to
constant weight at 105ºC, dry in the drying oven at 105ºC for 3 hours, cover the weighing
bottle when take it out, then put into the desiccator, cool to room temperature, after weigh
accurately, put it in the drying oven again and dry to constant weight at 105ºC.

Ofloxacin DMF Open part (USP)

Date: Apr.24, 2017
 Zhejiang East-Asia Pharmaceutical Co., Ltd.
Coastal Industrial City, Pubagang town, Sanmen county, Zhejiang, China. P.C.: 317100.

Calculate formula:
W1 - W2
Loss on drying   100%
W1 - W
In which:
W1---Weight of sample and weighing bottle, g;
W2---Weight of (sample and weighing bottle) constant weight after drying, g;
W---Weight of weighing bottle, g.

【Residue on ignition】
(1) Empty crucible with constant weight
Take a clean crucible and place it in the muffle furnace, cover the lid obliquely on the crucible.
Ignite the crucible at 550~650ºC for about 30min, stop heating, take out the crucible when the
muffle furnace cool to 300ºC, and put it in the desiccator, cover the lid, cool to room temperature
(generally about 60min). Weigh the crucible accurately (accurate to 0.1mg). Repeat the operation
with the same condition to constant weight, standby application.
(2) Weigh testing sample
Take 1.0g of sample and put it into the crucible which has been ignited to constant weight, weigh
accurately.
(3) Charring
Put the crucible within sample on electric stove or gaslight and ignite gently (avoiding sudden
expansion and burning of the testing sample) until the sample is thoroughly charred as black and
fumes no longer evolved, cool to room temperature(The above procedure should be performed at
fume cupboard ).
(4) Incinerated
Unless otherwise directed, moisten the charring sample with 1.0mL of sulfuric acid, heat on the
electric stove or gaslight until sulfuric acid vapor and white fumes are no longer evolved. Put the
crucible into the muffle furnace, cover the lid obliquely on the crucible, ignite at 550-650ºC for
about 60min until the residue is completely incinerated.
(5) Constant weight
Repeat the procedure of stop heating, place in muffle furnace until to constant weight.
Calculation formula:
W2  W
Residue on ignition   100%
W1  W

Ofloxacin DMF Open part (USP)

Date: Apr.24, 2017
 Zhejiang East-Asia Pharmaceutical Co., Ltd.
Coastal Industrial City, Pubagang town, Sanmen county, Zhejiang, China. P.C.: 317100.

In which,
W---The weight of crucible, g.
W1---The weight of sample and crucible before ignition, g,
W2---The weight of sample and crucible after ignition, g,

【Heavy metals】
Test solution: take 1.0g of sample and put into the platinum crucible, weigh accurately,add
sufficient sulfuric acid to wet the substance, and carefully ignite at a low temperature until
thoroughly charred. Add to the carbonized mass 2mL of nitric acid and 5 drops of sulfuric acid, and
heat cautiously until white fumes no longer are evolved. Ignite, preferably in a muffle furnace, at
500°C~600ºC, until the carbon is completely burned off. Cool, add 4mL of 6mol/L hydrochloric
acid, cover, heat on a water bath for 15minutes, uncover, and slowly evaporate on a water bath to
dryness. Moisten the residue with 1 drop of hydrochloric acid, add 10mL of hot water, and digest
for 2minutes. Add 6mol/L ammonium hydroxide dropwise until the solution is just alkaline to
litmus paper, dilute with water to 25mL, and adjust with 1mol/L acetic acid to a pH of 3.0 ~4.0,
using short-range pH indicator paper as an external indicator. Filter if necessary, rinse the crucible
and the filter with 10mL of water, combine the filtrate and washing liquor in a 50mL
color-comparison tube, dilute with water to 40mL, and mix well.
Standard solution: pipet 1.0mL of Standard Lead Solution (10µg of Pb) into a 50mL
color-comparison tube, and dilute with water to 25mL. Using a short-range pH indicator paper as
external indicator, adjust with 1mol/L acetic acid or 6mol/L ammonium hydroxide to a pH of 3.0
~4.0, dilute with water to 40mL, and mix well.
Test method: To each tube add 2mL of pH3.5 acetate buffer, then add 1.2mL of thioacetamide TS,
dilute with water to 50mL, mix, allow to stand for 2minutes. Observe from up to down on a white
background: the color of the Test solution is not darker than that of the Standard solution.

【Arsenic】
Standard solution: take accurately 3.0mL of standard arsenic solution into a generator bottle, add
2mL sulfuric acid, mix well, add 30% hydrogen peroxide with a same amount of that to prepare test
solution. Heat the mixture solution until strong fume produced, cool, add 10mL water carefully,
heat again and until strong fume produced, add 10mL water again, repeat the operation to remove
trace hydrogen peroxide, cool, add water to 35mL.
Test solution: weigh 3.0g of sample into a generator bottle, add 5mL of sulfuric acid and a few

Ofloxacin DMF Open part (USP)

Date: Apr.24, 2017
 Zhejiang East-Asia Pharmaceutical Co., Ltd.
Coastal Industrial City, Pubagang town, Sanmen county, Zhejiang, China. P.C.: 317100.

crystal ball, heat to starting to carbonize (The temperature should be not more than 120ºC),
dropwise add 30% hydrogen peroxide carefully, make the reaction quiet down, drip, shake and heat
at the same time, until the color of solution change to colorless or slightly grass color, cool, add
10mL water carefully, shake well, and evaporate to produce strong fume, repeat this operation, until
hydrogen peroxide is decomposed completely, cool, add 10mL water carefully, shake well, then
dilute to 35mL with water.
Test method: add 20mL of 3.5mol/L sulfuric acid solution, 2mL of potassium iodide TS, 0.5mL of
acid stannous chloride TS and 1mL isopropanol, mix well, allow to stand for 30min at room
temperature, put cotton with lead acetate into pipe, have a length of 2mm, connect gas-guide with
absorber tube, transfer 3mL of silver diethyldithiocarbamate TS to absorber tube. Add 3.0g of zinc
for arsenic analysis to generator bottle, and immediately connect with gas-guide tube, allow to stand
for 45min at room temperature, and rotate the arsenic generator bottle every 10min gently.
Disconnect the absorber tube from gas-guide tube and generator bottle, put the obtained absorber
tube of test solution and that of standard solution on the white background. Observe the color of test
solution produced and that of standard solution produced from up to down, and the color of test
solution produced should be not more intense than that of standard solution produced.

【Residual solvents】
Test method (Gas Chromatography):
a. GC conditions:
Column: fused quartz capillary column DB-624 30m×0.53mm×3.0µm
Carrier gas: nitrogen (N2), flow rate: 4.6mL/min (Constant current mode)
Split ratio: 5:1
Column temperature: 35ºC (3min) →90ºC (20ºC/min) →200ºC (40ºC/min) →2min.
Injection port temperature: 170ºC
Detector temperature: 250ºC
Headspace injection condition
Oven temperature: 85ºC
Quantitative line temperature (or injector temperature): 95ºC
Transfer line temperature: 105ºC
Equilibrium time: 28min
Injection size: 1mL
If it is CTC, the headspace injection condition is:
Oven temperature: 85ºC
Ofloxacin DMF Open part (USP)
Date: Apr.24, 2017
 Zhejiang East-Asia Pharmaceutical Co., Ltd.
Coastal Industrial City, Pubagang town, Sanmen county, Zhejiang, China. P.C.: 317100.

Injector temperature: 95ºC

CTC purging pressure: 0.5bar
Equilibrium time: 28min
Injection size: 1mL

b. Procedure
Internal standard solution: weigh accurately 56mg of n-propanol into a 100mL volumetric flask
with about 20mL 1% sodium hydroxide solution, dilute to volume with 1% sodium hydroxide
solution, mix well, then pipet 2.0mL of this solution and dilute to 250mL with 1% sodium
hydroxide solution, mix well.
Blank solution: Pipet accurately 2.0mL of internal standard solution into a 20mL headspace vial,
seal.
Methanol location solution: take an appropriate amount of methanol into a 20mL headspace vial,
and pipet accurately 2.0mL of internal standard solution into it, seal.
Ethanol location solution: take an appropriate amount of ethanol into a 20mL headspace vial, and
pipet accurately 2.0mL of internal standard solution into it, seal.
Reference solution: Weigh accurately 100mg of absolute methanol and 100mg of absolute ethanol
into a 100mL volumetric flask with about 20mL of internal standard solution, dilute to volume with
internal standard solution, mix well; pipet accurately 1.0mL of this solution and add internal
standard solution to dilute to 100mL, mix well. Pipet accurately 2.0mL of reference solution into
five 20mL headspace vials, respectively, seal.
Sample solution: Weigh accurately40mg of sample into a 20mL headspace vial, pipet accurately
2.0mL of internal standard solution to dissolve, seal.
System suitability test: According to the above GC and headspace injection condition, inject blank
solution, location solutions and reference solution (5 replication injections), respectively, record the
chromatograms (RRT of methanol is 0.5, RRT of ethanol is 0.6, RRT of n-propanol is 1.0).
Resolution between methanol peak and ethanol should be not less than 2.0; RSD of peak area ratio
of methanol or ethanol to the internal standard should be not more than 5%.
Determination of sample: inject the sample solution according to the above condition, record the
chromatograms, read the peak area.
Calculation formula:
R U ×C S
Re sidual solvents = × 100%
RS × CU
In which,
Ofloxacin DMF Open part (USP)
Date: Apr.24, 2017
 Zhejiang East-Asia Pharmaceutical Co., Ltd.
Coastal Industrial City, Pubagang town, Sanmen county, Zhejiang, China. P.C.: 317100.

RU---Peak area ratio of methanol or ethanol to the internal standard, corrected for blank,
from the sample solution;
RS--- Peak area ratio of methanol or ethanol to the internal standard, corrected for blank,
from the reference solution;
CS---Concentration of corresponding solvent in reference solution;
CU---Concentration of sample in the sample solution.
Acceptance criteria:
Methanol: NMT0.005%, ethanol: NMT0.05%.

【Assay】
Take about 0.2g of sample, weigh accurately, dissolve with 50mL glacial acetic acid, titrate with
perchloric acid VS (0.1mol/L) according to potentiometric titration determination, calibrate the
titration result by blank test. Each 1mL of 0.1 mol/L perchloric acid VS is equivalent to 36.14mg of
C18H20FN3O4.
Calculation formula:
( V - V0 ) × C × 36.14
X = %
m( 1 - Loss on drying )
In which,
V0---The consumed volume of perchloric acid VS which titrate blank, mL;
V---The consumed volume of perchloric acid VS which titrate sample, mL;
C---The concentration of perchloric acid VS, mol/L;
m---Weight of sample, g.
36.14---Each 1mL perchloric acid VS (0.1mol/L) is equivalent to 36.14mg of
C18H20FN3O4.
Acceptance criteria: 98.5%-101.5%, calculate on the dried basis.

Ofloxacin DMF Open part (USP)

Date: Apr.24, 2017