3.2. S.4.3.4 Validation of The GC Method For Determination of The Residual Solvents (Methanol, Ethanol) of Ofloxacin by GC

Zhejiang East-Asia Pharmaceutical Co., Ltd.
Coastal Industrial City, Pubagang town, Sanmen county, Zhejiang, China. P.C.: 317100.
3.2. S.4.3.4 Validation of the GC method for determination of the residual

solvents (Methanol, ethanol) of Ofloxacin by GC
1. Reagent and substances
Name Supplier Grade Batch No. Expiry date
Absolute methanol Shanghai Lingfeng AR 20151208 May 31, 2017

Zhejiang
Absolute ethanol AR 20150720 Apr.14, 2017
Linganqingshan
n-propanol Xilong Chemical AR 150104 Sep.16, 2017
1% Sodium
Zhejiang East-Asia Solution Y161123 Feb.22, 2017
hydroxide solution
Ofloxacin Zhejiang East-Asia Sample DC-002-1611001-1 -
2. Instruments
Instrument Type Code number Calibrated expiry date
GC Agilent 7890B QC-155 Jun.11, 2017
GC Agilent 7890B QC-489 May 30, 2017
Electronic balance XPE105 QC-139 Apr.12, 2017
Code Model
Chromatographic column GC069 DB-624 30m×0.53mm×3.0µm
Chromatographic column GC057 DB-624 30m×0.53mm×3.0µm
3. Operation conditions
3.1 Chromatograph 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: 95ºC
Ofloxacin DMF Open part (USP)

Date: Apr.24, 2017
Transfer line temperature: 105ºC

Equilibrium time: 28min
Injection size: 1mL
4 Validation data analysis

4.1 System suitability
4.1.1 Method
Internal standard solution: weigh accurately 56.49mg of n-propanol into a 100mL volumetric
flask, dilute to volume with 1% Sodium hydroxide solution, mix well, then transfer 8.0mL of this
solution and dilute to 1000mL with 1% Sodium hydroxide solution, mix well.
Reference solution: Weigh accurately 99.83mg of absolute methanol and 99.44mg of absolute
ethanol into a 100mL volumetric flask, dilute to volume with internal standard solution, mix well;
transfer accurately 1.0mL of this solution into a 100mL volumetric flask, dilute to volume with
internal standard solution, mix well.
Inject the above solution into chromatograph and record chromatograms.
4.1.2 Acceptance criteria

Resolution between methanol peak and ethanol peak should be not less than 1.5; RSD of peak area
ratio for each solvent to internal standard obtained from reference solution should be not more than
5%.
4.1.3 Results
Resolution between methanol peak and ethanol peak is 11.2; RSD of peak area ratio for methanol to
internal standard is 1.0%; RSD of peak area ratio for ethanol to internal standard is 0.74%.
4.1.4 Evaluation
Meet the requirements.
4.2 Specificity
4.2.1 Method
Preparation of solution:
Methanol location solution: take an appropriate amount of absolute methanol into a 20mL
headspace vial, add 2.0mL of internal standard solution, mix well and seal.
Ethanol location solution: take an appropriate amount of absolute ethanol into a 20mL headspace
Date: Apr.24, 2017
vial, add 2.0mL of internal standard solution, mix well and seal.
n-propanol location solution: take an appropriate amount of n-propanol into a 20mL headspace
Sample solution: Weigh accurately 40.85mg of sample (Batch No.: DC-002-1611001-1) into a
20mL headspace vial, add 2.0mL of internal standard solution, seal.
Mixture solution (1): take appropriate amount of absolute methanol, absolute ethanol and
n-propanol into a 20mL headspace vial with 2.0mL of internal standard solution, seal.
Mixture solution (2): weigh accurately 40.00mg sample (Batch No.: DC-002-1611001-1) into a
20mL headspace vial, add 2.0mL of internal standard solution, take appropriate amount of absolute
methanol, absolute ethanol and n-propanol into it, seal.
Inject the above solutions into the chromatograph, and record the chromatograms.

Blank solvent should no interference on the determination of sample; resolution between methanol
peak and ethanol peak should be not less than 1.5.
4.2.3 Results
Table-1 Location of solvents
Sequence No. RT(min) RRT(min) Resolution
Methanol 2.706 0.51 -
Ethanol 3.660 0.68 11.2
n-propanol 5.356 1.00 20.6
4.2.4 Evaluation
Blank solvent has no interference on the determination of sample; resolution between each solvent
peak meet requirement.
4.3 Linearity, accuracy, recovery precision and range

4.3.1 Method
Preparation for solutions:

Date: Apr.24, 2017
Reference solution (200%): weigh accurately 99.76mg of absolute methanol and 100.34mg of
absolute ethanol into a 100mL volumetric flask, dilute to volume with internal standard solution,
mix well; transfer accurately 1.0mL of this solution into a 100mL volumetric flask, dilute to volume
with internal standard solution, mix well.
LOQ solution: weigh accurately 100.25mg of absolute methanol and 50.24mg of absolute ethanol
into a 10mL volumetric flask, dilute to volume with internal standard solution, mix well; transfer
accurately 1.0mL of this solution into a 100mL volumetric flask, dilute to volume with internal
standard solution, mix well, then transfer accurately 1.0mL of this solution into a 100mL
volumetric flask, dilute to volume with internal standard solution, mix well.
Reference solution (20%): Transfer accurately 2.0mL reference solution (200%) into a 20mL
Sample solution: weigh 39.43mg, 39.44mg sample (Batch No.: DC-002-1611001-1) into two
20mL of headspace vial, respectively, add accurately 2.0mL of internal standard solution, mix well
and seal.
LOQ test solution: weigh 40.61mg, 39.75mg, 39.80mg of sample (Batch No.: DC-002-1611001-1)
into three 20mL of headspace vial, respectively, transfer accurately 2.0mL of LOQ solution into
them, mix well and seal.
20% test solution: weigh 40.21mg, 40.42mg, 39.42mg of sample (Batch No.: DC-002-1611001-1)
into three 20mL of headspace vial, respectively, transfer accurately 2.0mL of reference solution
(20%) into them, mix well and seal.
Date: Apr.24, 2017
(150%), into them, mix well and seal.
Inject the above solutions into chromatograph and record the chromatograms.

Linearity correlation coefficient should be ≥ 0.990; the y-axis intercept/100% response value should
be < 10%; RSD of response factor should be ≤10%; Recovery for each concentration should be
90% to 108%. RSD of recovery precision should be ≤ 10%.
4.3.3 Result
Table-2 Linearity, accuracy, recovery precision and range of Methanol
Measured value
Recovery rate= × 100% ;
Addition value
Measured peak area / int ernal s tan dard peak area
Measured value= × Reference solution concentration×
Re ference solution peak area / int ernal s tan dard peak area
Dilution volume
Methanol peak area in reference solution/ internal standard peak area: 0.54, Concentration of methanol in reference solution:
10.050µg/mL. Peak area of sample/ internal standard peak area: 0.014.
Measured peak
Theoretical addition Addition Average Recovery RSD of
area/ internal Measured Recovery Response
concentration value peak precision response
standard peak value (µg) rate (%) factor
(µg/mL) (µg) area (%) factor (%)
area
0.054 2.01 100
1.002 2.004 0.049 2.22 1.82 91 4.9 2.22
0.053 1.97 98
0.102 3.80 95
1.995 3.990 0.099 4.35 3.68 92 1.6 2.18
0.101 3.76 94 7.1
0.265 9.86 99
4.988 9.976 0.288 12.08 10.72 107 4.0 2.42
0.281 10.46 105
0.555 20.66 104

9.976 19.952 25.01 1.6 2.51
0.576 21.44 107

Date: Apr.24, 2017
0.571 21.25 107
0.864 32.16 107
14.964 29.928 0.877 38.14 32.64 108 2.5 2.55
0.829 30.86 103
Linear equation y=2.5842x-0.657 Correlation coefficient r=0.9999

linearity range:
Average of recovery rate: 101% y-axis intercept/100% response value×100 =-2.6%
1.002µg/mL-14.964µg/mL
Table-3 Linearity, accuracy, recovery precision and range of Ethanol

Measured value
Recovery rate= × 100% ;
Addition value
Measured peak area / int ernal s tan dard peak area
Measured value= × Reference solution concentration×
Re ference solution peak area / int ernal s tan dard peak area
Dilution volume
Ethanol peak area in reference solution/ internal standard peak area: 1.15, Concentration of ethanol in reference solution:
9.991µg/mL. Peak area of sample/ internal standard peak area: 0.062.
Measured peak
Theoretical addition Addition Average Recovery RSD of
area/ internal Measured Recovery Response
concentration value peak precision response
standard peak value (µg) rate (%) factor
(µg/mL) (µg) area (%) factor (%)
area
0.059 1.03 103
0.502 1.004 0.054 2.44 0.94 94 5.2 4.86
0.059 1.03 103 3.7
0.225 3.91 97
2.007 4.014 9.77 1.2 4.87
0.225 3.91 97

Date: Apr.24, 2017
0.228 3.96 99
0.548 9.52 95
5.017 10.034 0.568 24.64 9.87 98 3.1 4.91
0.582 10.11 101
1.177 20.45 102
10.034 20.068 1.185 51.83 20.59 103 0.98 5.17
1.163 20.21 101
1.802 31.31 104
15.051 30.102 1.797 79.67 31.22 104 1.1 5.26
1.767 30.70 102
Linear equation y=5.3179x-1.0146 Correlation coefficient r=0.9997

linearity range:
Average of recovery rate: 100% y-axis intercept/100% response value×100 =-2.0%
0.502µg/mL-15.051µg/mL
4.3.4 Evaluation
Linear equation of methanol: y=2.5842x-0.657; correlation coefficient is 0.9999; RSD of recovery
precision is < 10%; y-axis intercept/100% response value is -2.6%; RSD of response factor is <
10%; average recovery rate is 101%.
Linear equation of ethanol: y=5.3179x-1.0146; correlation coefficient is 0.9997; RSD of recovery
precision is < 10%; y-axis intercept/100% response value is -2.0%; RSD of response factor is <
10%; average recovery rate is 100%.
Meet the requirement.

Date: Apr.24, 2017
4.4 LOQ
4.4.1Method
In the system suitability item, S/N of methanol is 146.7, S/N of ethanol is 283.7, and prepare the
LOQ solution according to the results.
LOQ solution: weigh accurately 100.25mg of absolute methanol and 50.24mg of ethanol into a
10mL volumetric flask, dilute to volume with internal standard solution, mix well, transfer
accurately 1.0mL of this solution into a 100mL volumetric flask, dilute to volume with internal
standard solution, mix well, then transfer accurately 1.0mL of this solution into a 100mL
Inject the above solutions into chromatograph, record the chromatograms.

Signal to noise (S/N) of each solvent should be ≥10; RSD of peak area should be ≤ 10%.
4.4.3 Result
Table-4 S/N of LOQ
Relative
Name S/N Average
concentration (%)
Methanol 18.8 18.9 18.2 17.4 16.1 22.1 18.6 0.0010025
Ethanol 16.5 17.0 16.1 14.5 14.4 19.8 16.4 0.0005024
Table-5 Peak area of LOQ
Name Peak area RSD (%)
Methanol 3.98 4.37 4.29 4.20 4.15 4.31 3.3
Ethanol 4.33 4.75 4.41 4.10 4.31 4.50 4.9
4.4.4 Evaluation
LOQ of methanol is 0.001%, and LOQ of ethanol is 0.0005%.
4.5 LOD
4.5.1 Method
LOD solution: transfer accurately 5.0mL of LOQ solution into a 25mL volumetric flask, dilute to
volume with internal standard solution, mix well. Prepare in parallel.

Date: Apr.24, 2017
Inject above solutions into chromatograph and record the chromatogram.

S/N of each solvent should be 3±1.
4.5.3 Result
Table-6 S/N of LOD
Name S/N Average Relative concentration (%)
Methanol 3.8 3.1 2.4 3.1 0.0002005
Ethanol 2.7 2.1 2.1 2.3 0.00010048
4.5.4 Evaluation
LOD of methanol is 0.0002% and LOD of ethanol is 0.0001%.
4.6 Instrument precision and method precision

4.6.1 Method
Solution preparation for instrument precision:
Sample solution: weigh accurately 1000.78mg of sample (Batch No.: DC-002-1611001-1) into a
50mL volumetric flask, dissolve and dilute to volume with reference solution, mix well.
Solution preparation for method precision:
Sample solution (1): weigh accurately 40.62mg of sample (Batch No.: DC-002-1611001-1) into a
20mL headspace vial, add accurately 2.0mL of reference solution to dissolve, seal.

Date: Apr.24, 2017
4.6.2 Result
RSD of test result should be ≤10%.
4.6.3 Result
See table 7, 8.
Table 7-Test result of instrument precision
Injections 1 2 3 4 5 6 RSD (%)
Methanol (%) 0.053 0.053 0.053 0.053 0.054 0.053 0.77
Ethanol (%) 0.054 0.054 0.054 0.053 0.053 0.054 0.96
Table 8-Test result of method precision

Sample Sample Sample Sample Sample Sample
RSD
solution solution solution solution solution solution Average
(%)
(1) (2) (3) (4) (5) (6)
Methanol (%) 0.050 0.051 0.052 0.053 0.052 0.051 0.052 2.0
Ethanol (%) 0.052 0.052 0.054 0.053 0.054 0.053 0.053 1.7
4.6.4 Evaluation
Meet the requirement.
4.7 Intermediate precision

4.7.1 Method
Two analysts test the same sample using the different instruments at different time.
Analyst 1: Youjuan Chen (Refer to method precision item)
Analyst 2: Xiaofen Luo
Date: Apr.24, 2017
Methanol location solution: take an appropriate amount of absolute methanol into a 20mL
headspace vial, add 2.0mL of internal standard solution, mix well and seal.
Ethanol location solution: take an appropriate amount of absolute ethanol into a 20mL headspace
n-propanol location solution: take an appropriate amount of n-propanol into a 20mL headspace

RSD of test result obtained from each analyst should be≤ 10%. AD of the average value of test
result between two analysts should be≤ ±0.2S.
4.7.3 Result
Result of system suitability: Resolution between methanol peak and ethanol peak is 11.2, RSD of
peak area ratio for methanol to internal standard is 2.9%; RSD of peak area ratio for ethanol to
internal standard is 1.4%.
Table 9-Test result of intermediate precision
Date: Apr.24, 2017
Sample Sample Sample Sample Sample Sample

AD between
solution solution solution solution solution solution Average RSD
two analysts
(1) (2) (3) (4) (5) (6)
Methanol (%) 0.052 0.053 0.052 0.057 0.052 0.055 0.054 3.9 0.002
Ethanol (%) 0.054 0.055 0.054 0.058 0.054 0.056 0.055 2.9 0.002
4.7.4 Evaluation
Meet the requirements.
4.8Robustness
4.8.1Method
Changing the following chromatographic condition, and analyze the sample, respectively.
Test condition 1: The flow rate change to 4.4mL/min and 4.8mL/min, other parameters are same.
Test condition 2: The heating rate change to 18ºC/min and 22ºC/min, other parameters are same.
Test condition 3: The injection port temperature change to 168ºC/min and 172ºC/min, other
parameters are same.
Test condition 4: The detector temperature change to 248ºC/min and 252ºC/min, other parameters
are same.
Test condition 5: The oven temperature change to 80ºC and 90ºC, other parameters are same.
Test solution 6: The equilibrium time change to 26min and 30min, other parameters are same.
Test condition 7: Use different code of chromatographic column, other parameters are same.
Preparation of solution:
4.8.2Acceptance criteria
When test condition changed, the system suitability test result should meet the requirements, AD of
Date: Apr.24, 2017
each solvent between condition changed and normal condition should be ≤±0.2S.
4.8.3Result
Table 10-Test result of Robustness
Sample batch no. Methanol (%) Ethanol (%)
Normal condition DC-002-1611001-1 0.055 0.056

System suitability: Resolution between methanol peak and ethanol peak is 11.2, RSD of peak area ratio
for methanol to internal standard is 1.4%; RSD of peak area ratio for replicate ethanol to internal standard
is 1.5%.
Flow rate: 4.4mL/min DC-002-1611001-1 0.054 0.056
for methanol to internal standard is 0.92%; RSD of peak area ratio for ethanol to internal standard is 1.4%.
AD compared with normal condition (%) -0.001 0
Flow rate: 4.8mL/min DC-002-1611001-1 0.054 0.056

Heating rate: 18ºC/min DC-002-1611001-1 0.054 0.055

for methanol to internal standard is 0.78%; RSD of peak area ratio for ethanol to internal standard is
0.38%.
AD compared with normal condition (%) -0.001 -0.001
Heating rate: 22ºC/min DC-002-1611001-1 0.055 0.056

0.71%.
AD compared with normal condition (%) 0 0
Injection port temperature: 168ºC DC-002-1611001-1 0.053 0.055

Injection port temperature: 172ºC DC-002-1611001-1 0.054 0.055

Detector temperature: 248ºC DC-002-1611001-1 0.054 0.055

Detector temperature: 252ºC DC-002-1611001-1 0.055 0.056

Date: Apr.24, 2017
for methanol to internal standard is 0; RSD of peak area ratio for ethanol to internal standard is 0.
AD compared with normal condition (%) 0 0
Oven temperature: 80ºC DC-002-1611001-1 0.053 0.055

Oven temperature: 90ºC DC-002-1611001-1 0.054 0.056

0.71%.
Equilibrium time: 26min DC-002-1611001-1 0.052 0.055

Equilibrium time: 30min DC-002-1611001-1 0.055 0.057

AD compared with normal condition (%) 0 0.001
Chromatographic column code: GC097 DC-002-1611001-1 0.053 0.054

4.8.4 Evaluation
When test condition changed, the system suitability test results meet the requirements, all of AD of
test result of methanol and ethanol between condition changed and normal conditions are less than
±0.2S. Meet the requirements.
4.9 Solution stability

4.9.1 Method

Date: Apr.24, 2017

Put them respectively for 0h, 6h, 12h, 18h, 24h and 48h at room temperature, analysis, respectively,
and record the chromatograms.
.
4.9.2Acceptance criteria
Within a certain time interval, AD of test result compared with 0h should be not more than ±0.2S.
4.9.3Result
Table 11- Stability test result of reference solution
Peak area ratio of methanol to Peak area ratio of ethanol to
Holding time
internal standard internal standard
0h 0.57 1.16
6h 0.59 1.19
12h 0.59 1.21
18h 0.58 1.20
24h 0.59 1.20
48h 0.55 1.17
RSD (%) 2.8 1.6
Table 12- Stability test result of sample solution
Holding time Methanol (%) Ethanol (%)
0h 0.053 0.054
6h 0.052 0.054
12h 0.051 0.051
18h 0.053 0.053
24h 0.053 0.052
48h 0.052 0.053
AD compared with 0h (%) -0.001 -0.001
4.9.4Evaluation
Putting the sample solution for 48h at room temperature, AD of test result for each solvent
compared with 0h is less than ±0.2S, certify that the sample solution is stable within 48h.
Date: Apr.24, 2017
4.10 Solution stability

4.10.1 Method
Sample solution: weigh accurately 40mg of sample into a 20mL headspace vial, add accurately
2.0mL of internal standard solution, mix well and seal.
Inject above solutions into chromatograph and record the chromatograms.
4.10.2 Results
See the following table-13
Table-13 Result of sample test
Sample weight(mg) Methanol (%) Ethanol (%)
40.93 Not detected 0.0019

DC-002-1611001-1 0.0020

DC-002-1611002-1 0.0020

DC-002-1611003-1 0.0014
System suitability: Resolution between methanol peak and ethanol peak is 11.1, RSD of peak area
ratio for methanol to internal standard is 2.1%; RSD of peak area ratio for ethanol to internal
standard is 1.1%.
4.10.3 Evaluation
According to the test results of three consecutive batches of sample, the residual ethanol are less
than the limit and the residual methanol are all not detected.
4.11 Validation results summary

Table-14
Validation items Acceptable criteria Validation results

Date: Apr.24, 2017
System suitability Meet the requirements Meet the requirement

Blank solvent should have no
interference on the determine of
Specificity Meet the requirement
sample, resolution between each
solvent should be not less than 1.5.
LOQ of methanol is 0.001% (S/N=18.6);
S/N: ≥10
LOQ LOQ of ethanol is 0.0005% (S/N=16.4)
RSD of peak area: ≤20%
RSD of peak area are both ≤20%.
LOD of methanol is 0.0002% (S/N=3.1);
LOD S/N=3±1
LOD of methanol is 0.0001% (S/N=2.3)
Correlation coefficient of methanol is
Linear correlation coefficient:
0.9999; y-axis intercept/100% response
≥0.990,
value is -2.6%; average recovery rate is
Y-intercept/100% response value:
Linearity, accuracy, 101%;
<10%,
recovery precision Correlation coefficient of ethanol is 0.9997;
RSD of response factor: ≤10%,
and range y-axis intercept/100% response value is
Recovery of each concentration:
-2.0%; average recovery rate is 100%.
90%-108%,
RSD of response factor are both < 10%;
RSD of recovery precision: ≤10%.
RSD of recovery precision are both < 10%.
Instrument precision RSD: ≤10% Meet the requirement
Method precision RSD: ≤10% Meet the requirement

RSD of test result obtained from each
Intermediate analyst should be≤ 10%. AD of the
Meet the requirement
precision average value of test result between
two analysts should be≤ ±0.2S.
The results of system suitability test
could meet the requirements when
the analysis parameter changed.
Robustness Meet the requirement
The AD between changed condition
and normal condition should be ≤ ±
0.2S.
Within a certain time interval, AD of AD of test result for each solvent is less
Solution stability test result compared with 0h should than ±0.2S after the sample solution
be ≤ ±0.2S standing for 48h at room temperature.
4.12 Evaluation and advice

Base on the validation results for the validated items performed for system suitability, specificity,
LOQ, LOD, linearity range, accuracy, precision (Instrument precision, method precision and
intermediate precision), robustness and solution stability, the analytical method is certified that it is
suitable for determination of residual solvents (Methanol and ethanol) in Ofloxacin.
5 Validation conclusion
It is verified that the analytical method for determination of residual solvents (Methanol and ethanol)
in Ofloxacin is shown in below:
a. Chromatograph conditions
Column: fused quartz capillary column DB-624 30m×0.53mm×3.0µm

Date: Apr.24, 2017
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
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; transfer accurately 1.0mL of this solution and add internal
standard solution to dilute to 100mL, mix well. Transfer accurately 2.0mL of reference solution into
five 20mL headspace vials, respectively, seal.
Sample solution: Weigh accurately 40mg of sample into a 20mL headspace vial, transfer 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
Date: Apr.24, 2017
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,
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.

Date: Apr.24, 2017

3.2. S.4.3.4 Validation of The GC Method For Determination of The Residual Solvents (Methanol, Ethanol) of Ofloxacin by GC

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Zhejiang East-Asia Pharmaceutical Co., Ltd.

3.2. S.4.3.4 Validation of the GC method for determination of the residual

Name Supplier Grade Batch No. Expiry date

Absolute methanol Shanghai Lingfeng AR 20151208 May 31, 2017

Ofloxacin Zhejiang East-Asia Sample DC-002-1611002-1 -

Ofloxacin Zhejiang East-Asia Sample DC-002-1611003-1 -

Instrument Type Code number Calibrated expiry date

GC Agilent 7890B QC-155 Jun.11, 2017

GC Agilent 7890B QC-489 May 30, 2017

Electronic balance XPE105 QC-139 Apr.12, 2017

Chromatographic column GC069 DB-624 30m×0.53mm×3.0µm

Chromatographic column GC057 DB-624 30m×0.53mm×3.0µm

Ofloxacin DMF Open part (USP)

Transfer line temperature: 105ºC

4 Validation data analysis

4.1.2 Acceptance criteria

4.2.2 Acceptance criteria

Sequence No. RT(min) RRT(min) Resolution

Methanol 2.706 0.51 -

Ethanol 3.660 0.68 11.2

n-propanol 5.356 1.00 20.6

4.3 Linearity, accuracy, recovery precision and range

Ofloxacin DMF Open part (USP)

4.3.2 Acceptance criteria

1.002 2.004 0.049 2.22 1.82 91 4.9 2.22

1.995 3.990 0.099 4.35 3.68 92 1.6 2.18

0.101 3.76 94 7.1

4.988 9.976 0.288 12.08 10.72 107 4.0 2.42

0.281 10.46 105

0.555 20.66 104

Ofloxacin DMF Open part (USP)

0.571 21.25 107

0.864 32.16 107

14.964 29.928 0.877 38.14 32.64 108 2.5 2.55

0.829 30.86 103

Linear equation y=2.5842x-0.657 Correlation coefficient r=0.9999

Table-3 Linearity, accuracy, recovery precision and range of Ethanol

0.502 1.004 0.054 2.44 0.94 94 5.2 4.86

0.059 1.03 103 3.7

Ofloxacin DMF Open part (USP)

5.017 10.034 0.568 24.64 9.87 98 3.1 4.91

0.582 10.11 101

1.177 20.45 102

10.034 20.068 1.185 51.83 20.59 103 0.98 5.17

1.163 20.21 101

1.802 31.31 104

15.051 30.102 1.797 79.67 31.22 104 1.1 5.26

1.767 30.70 102

Linear equation y=5.3179x-1.0146 Correlation coefficient r=0.9997

Ofloxacin DMF Open part (USP)

4.4.2 Acceptance criteria

Ethanol 16.5 17.0 16.1 14.5 14.4 19.8 16.4 0.0005024

Table-5 Peak area of LOQ

Name Peak area RSD (%)

Methanol 3.98 4.37 4.29 4.20 4.15 4.31 3.3

Ethanol 4.33 4.75 4.41 4.10 4.31 4.50 4.9

Ofloxacin DMF Open part (USP)