Analytical Method Validation Report of Saxagliptin Tablet

Analytical Method Validation Report
Saglip Tablet
Document No. CCL-AMVR-170-A
2015
ANALYTICAL METHOD VALIDATION REPORT
Saglip Tablets
DOCUMENT NO.: CCL-AMVR-170-A
Page 1 of 25

Saglip Tablet
Table of Contents
1
INTRODUCTION...................................................................................................... 3
ANALYTICAL PROCEDURE........................................................................................3
2.1
ASSAY
PROCEDURE.................................................................................................................... 3
VALIDATION OF METHOD........................................................................................3
3.1
SPECIFICITY.............................................................................................................................. 4
3.2
LINEARITY................................................................................................................................ 5
3.3
RANGE.................................................................................................................................... 7
3.4
ACCURACY............................................................................................................................... 8
3.5
PRECISION............................................................................................................................. 10
3.5.1 Repeatability................................................................................................................... 11
3.5.2 Intermediate precision.................................................................................................... 14
3.6
LIMIT OF DETECTION (LOD) AND LIMIT OF QUANTITATION (LOQ).....................................................17
3.7
ROBUSTNESS.......................................................................................................................... 17
3.8
SYSTEM SUITABILITY................................................................................................................. 23
3.9
CONCLUSION.......................................................................................................................... 23
3.10 DEVIATION............................................................................................................................. 23
Page 2 of 25

Saglip Tablet
Introduction
Analytical Procedure
The analytical method for Saglip Tablet was validated and found to be valid for assay and
stability studies due to its accuracy and specificity. Forced degradation studies of finished
pharmaceutical product (Saglip tablet) were performed, the resolution between degraded
products and principle peak was found to be >2. Peak purity and relative retention times
confirmed the specificity of method. Method was investigated for its range, linearity,
accuracy, precision, robustness, and LOQ, LOD and solution stability. All the parameters
were performed on Agilent 1260 series HPLC equipped with Auto sampler, column oven,
quaternary low pressure gradient pump and PDA detector for peak purity purposes
2.1
Assay procedure
Buffer: Dissolve 2.72 grams of Potassium Dihydrogen Phosphate in 1000 mL of distilled

water and adjust the pH 4.6 with dilute Sodium Hydroxide solution.
Diluent: 0.1N HCl
Mobile phase: Buffer: Acetonitrile (80:20)
Standard solution: Weigh on analytical balance, working standard of Saxagliptin HCl
equivalent to 25mg of Saxagliptin and transfer it into a 50ml volumetric flask, make up the
volume with diluent and sonicate for 10 minutes. Take 10ml from this solution into a 100ml
volumetric flask and make up the volume with diluent.
Sample solution: Weigh on analytical balance, ground powder of tablets equivalent to
5mg of Saxagliptin and transfer it into a 100ml volumetric flask, make up the volume with
diluent and sonicate for 10 minutes (50ppm).
Mode: LC
Detector: UV 213 nm
Column: C18
Column temperature: Ambient
Flow rate: 1.5 mL/min
Injection size: 10 L
System suitability requirements
Tailing factor: NMT 2.0
Relative standard deviation: NMT 2.0%
Calculate the percentage of the labeled amount of Saxagliptin in the portion of Tablets
taken:
Result = (rU/rS) x (CS/CU) x 100/1.116
rU = peak response from the Sample solution
rS = peak response from the Standard solution
CS = concentration of Saxagliptin HCl RS in the Standard solution (mg/mL)
CU = nominal concentration of Saxagliptin in the Sample solution (mg/mL)
Acceptance criteria: 90.0%110.0%
Page 3 of 25

Saglip Tablet
Validation of Method
Following Validation characteristics were considered for analytical method validation:
1)
2)
3)
4)
5)
3.1
Specificity (Identification)
Linearity
Range
Accuracy (Recovery)
Precision
i. Repeatability
ii. Intermediate Precision
6) LOD and LOQ
7) Robustness
8) Solution stability
9) System suitability
Specificity
Samples of drug product were exposed to heat, light, acid, base, and oxidizing agent to
produce 10%30% degradation of the active. The degraded samples were then analyzed
using the method to determine if there were interferences with the active or related
compound peaks.
The following degradation conditions were selected:
Obtain or prepare solutions of ~0.1 N HCL, 1 N HCL, 2 N HCL ~0.15 N NaOH, 1 N NaOH, 2
N NaOH ~1% H2O2, 5% H2O2, 10% H2O2 and purified water.
Stress
Condition
Unstress
ed
sample
weight
taken
(mg)
Chrom. No.
1
231.0
1
2
3
4
0.1 N HCl
226.0
5
5
6
7
1 N HCl
229.9
4
8
9
2 N HCl
229.8
2
Page 4 of 25
10
11
Table 1
FORCED DEGRADATION FOR SAXAGLIPTIN
Theoretic
al
Mea
No. of degradation
Content
Peak
n
product
(g
Area
Area
(Area; RT)
mL1)
606.
7
608.
607.2
0
2.49
5
606.
5
666.
7
667.
668.4
0
2.43
4
671.
1
669.
5
668.
669.1
0
2.48
3
669.
4
675.
675.8
0
2.48
8
673.
Amount
recovere
d
(g
mL1)
Recover
y (%)
2.76
99.52%
0.48%
3.04
111.95
%
-11.95%
3.05
110.17
%
-10.17%
3.08
111.33
%
Degradat
ion
-11.33%

Saglip Tablet
12
13
0.1N
NaOH
228.7
1
1N
NaOH
233.5
5
2N
NaOH
227.2
1%
H2O2
229.9
9
14
15
16
17
18
19
20
21
22
23
24
25
5%
H2O2
231.2
8
26
27
28
10 %
H2O2
232.3
7
29
30
34
Standard
35
36
37
38
25.12
39
Table 1
FORCED DEGRADATION FOR SAXAGLIPTIN
8
677.
7
443.
6
443.
(4)
443.9
2.46
6
2.2,3.6,3.8,7.1
444.
6
40.8
(6)
41.1
40.8
2.2,3.6,3.8,7.1,1.7,
2.52
40.4
4.5
0.0
(6)
0.0
0.0
2.2,3.6,3.8,7.1,1.7,
2.45
0.0
4.5
664.
5
665.
(1)
665.0
2.48
4
3.8
665.
0
667.
3
667.
(2)
667.1
2.49
1
3.8,3.6
666.
8
601.
0
602.
601.3
0
2.50
8
600.
0
555.
4
551.
0
550.
2
----553.
0
550.
7
551.
2
551.9
2.02
73.49%
26.51%
0.19
6.61%
93.39%
0.00
0.00%
100.00%
3.03
109.47
%
-9.47%
3.04
109.20
%
-9.20%
2.74
97.97%
2.03%
Acceptance Criteria:
Peak purity should be NLT 990
Resolution should be more than 2.0
Conclusion:
Resolution was found to be more than 2.0 (See chromatograms)
Peak purity was found to be more than 990 (See chromatograms)
Relative retention times are mentioned in table and are different for degraded products
from active.
Page 5 of 25

Saglip Tablet
3.2
Linearity
A stock solution of Saxagliptan (0.5mg mL1 saxagliptan) was prepared by dissolving

1000 mg drug in 100 mL Diluent. Solutions of different concentration (30--70 g mL1) for
construction of calibration plots were prepared from this stock solution. The mobile phase
was filtered through a 0.22-m membrane filter and delivered at 1.50 mL min1 for
column equilibration; the baseline was monitored continuously during this process. The
detection wavelength was 213 nm. The prepared dilutions were injected at the rate of 10l
in series, peak area was calculated for each dilution, and concentration was plotted
against peak area.
Page 6 of 25

Saglip Tablet
Table 2
Linearity study of Saxagliptin

Sr. #
Chrom. #
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Conc. (ppm)
30
30
30
40
40
40
50
50
50
60
60
60
70
70
70
Peak Area
222.3
223.5
223.7
299.4
298.6
297.3
372.1
372.7
373.7
447.2
446.3
447.8
518.7
519.5
519.1
MEAN
RSD (%)
223.2
0.339%
298.4
0.355%
372.8
0.217%
447.1
0.169%
519.1
0.077%
Linear plot for Saglip Tablet

600
500
f(x) = 7.41x + 1.86

R = 1
400
Peak Area
300
200
100
0
25
30
35
40
45
50
55
60
65
70
75
Concentration
Coefficient of determination (r2) should be greater than 0.990.
The y intercept should not significantly depart from zero
Area response of y intercept should be less than 5% of the response of the
midrange concentration value.
Conclusion:
The calibration plot of peak area against concentration was linear in the range
investigated (3070 g mL1). Calibration data, with their relative standard deviations, %
Page 7 of 25

Saglip Tablet
RSD and standard error are listed in Table I. The low values of RSD and standard error
show the method is precise. The linear regression data for the calibration plot are
indicative of a good linear relationship between peak area and concentration over a wide
range. The linear regression equation was y =1468.5x + 40069 and the re-gression
coefficient was 0.9999.
3.3
Range
A stock solution of Saxagliptan (0.5mg mL1 saxagliptan) was prepared by dissolving

1000 mg drug in 100 mL Diluent. Solutions of different concentration (20--100 g mL1)
for construction of calibration plots were prepared from this stock solution. The mobile
phase was filtered through a 0.22-m membrane filter and delivered at 1.50 mL min1 for
column equilibration; the baseline was monitored continuously during this process. The
detection wavelength was 213 nm. The prepared dilutions were injected at the rate of 10l
in series, peak area was calculated for each dilution, and concentration was plotted
against peak area.
Table 3
Range study of Sitagliptin
Sr. #
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Page 8 of 25
Conc. (ppm)
20
20
20
30
30
30
40
40
40
50
50
50
60
60
60
70
70
70
80
80
80
100
100
100
Peak Area
147.4
148.4
148.6
222.3
223.5
223.7
299.4
298.6
297.3
372.1
372.7
373.7
447.2
446.3
447.8
518.7
519.5
519.1
600.3
600.4
600.1
741.6
742.7
742.0
MEAN
RSD (%)
148
0.434%
223
0.339%
298
0.355%
373
0.217%
447
0.169%
519
0.077%
600
0.025%
742
0.075%

Saglip Tablet
Concentration (ppm) Line Fit Plot for Range study of Saglip Tablet
800
f(x) = 7.44x + 0.14
R = 1
700
600
500
Peak Area
400
300
200
100
0
10
20
30
40
50
60
70
80
90
100
110
Concentration (ppm)
Conc
lusion
The calibration plot of peak area against concentration was linear in the range
investigated (20-100 g mL1). Calibration data, with their relative standard deviations,
% RSD and standard error are listed in Table I. The low values of RSD and standard error
show the method is precise. The linear regression data for the calibration plot are
indicative of a good linear relationship between peak area and concentration over a wide
range. The linear regression equation was y = 1412.1x + 13700 and the regression
coefficient was 0.9999.
Acceptance Criteria
Regression Coefficient
> 0.990
3.4
Accuracy
Standard preparation:
Weight accurately about 50 mg of Saxagliptin in a 100 ml volumetric flask and dissolve it
in 0.1N HCl and make up volume 0.1N HCl. Take 10ml from this dilution in 100ml
volumetric flask and makeup volume with 0.1N HCl.
Sample preparation:
Weight accurately about 195.0 mg of placebo in a 100 ml volumetric flask (thre different
flasks) and dissolve it in water. (A, B, C)
2-A stock solution of Saxagliptan HCl (0.5mg mL1) was prepared by dissolving 50 mg of
Saxagliptan HCl in 100 mL 0.1N HCl. Transfer 8, 10, and 12 ml of this solution in three
different 100 ml volumetric flasks A, B, C respectively containing placebo. Make up
Page 9 of 25

Saglip Tablet
volume with diluent to get three concentration levels of 80%, 100% and 120%
respectively.
Accuracy Set 1
Potency of WS:
Weight of placebo:
99.10%
195.00
Prop Wt:
Weight of active:
230mg
50.04
Table 4
Accuracy
Weight of
placebo
mg
Volume of
stock
solution
(ml)
140.30
8.0
Actual
Theoretical
Content
(g/ml)
10.0
140.26
12.0
49.59
59.51
Working Standard
50.04
-----
Recovery
%age
Peak
Area
431.9
434.3
436.1
541.9
537.8
538.6
656.5
655.6
656.1
551.1
552.6
550.9
554.4
551.5
39.67
140.28
SET 1
Mean Area
RSD
Content (g/ml)
434
0.49
39.34
99.18
539
0.40
48.89
98.59
656
0.07
59.46
99.93
Mean recovery (%)
99.23
552.10
0.26
----
Accuracy Set 2
Potency of WS:
Weight of placebo:
99.10%
Prop Wt:
Weight of
active:
140.00
150mg
50.23
Table 5
Accuracy
Weight of
placebo
mg
Volume of
stock
solution
(ml)
140.30
8.0
Actual
Theoretical
Content
(g/ml)
39.74
140.28
10.0
140.26
12.0
49.59
59.73
Working Standard
50.23
Recovery
%age
Peak Area
438
438.7
437.4
545.2
543.9
543.9
652.0
652.3
652.1
551.1
552.6
550.9
554.4
551.5
-----
Page 10 of 25
SET 2
Mean Area
RSD
Content (g/ml)
438
0.15
39.70
99.91
544
0.14
49.34
99.49
652
0.02
59.11
98.95
Mean recovery (%)
99.38
552.10
0.26

Saglip Tablet
Accuracy Set 3
Potency of WS:
Weight of placebo:
99.10%
140.00
Prop Wt:
Weight of active:
150
mg
49.79
Table 6
Accuracy
SET 3
Weight of
placebo
mg
Volume of
Active
ml
140.01
8.0
Actual
Theoretical
Content (%)
39.39
Recover
y
Peak
Area
437.1
Mean Area
RSD
Content (w/w
%)
%age
436.8
437
0.08
39.74
100.88
532
0.17
48.41
97.62
645
0.38
58.71
98.66
Mean recovery
(%)
99.98
436.4
531.4
140.54
10.0
533.1
49.59
531.8
644.6
140.07
12.0
643.3
59.51
648
542.8
542.6
Working
Standard
49.79
543.2
543.2
543.14
0.09
543.9
The recovery of the method is determined by spiking a placebo with active pharmaceutical
ingredient of known potency. The values of recovery (%), RSD (%), and SEM listed in Table
4, 5 and 6 listed above indicate that the method is accurate.
The percent recovery of the spiked placebos should be within 1002.0% for the
average of each set of three weights.
Each individual sample recovery should lie within the range of 98% to 102%.
Conclusion:
Hence above acceptance criteria for accuracy is met, therefore method is accurate
3.5
Precision
Precision was determined as both repeatability and intermediate precision. For

repeatability six replicates of a sample were analyzed on three different days and their
RSD was calculated. For intermediate precision same sample was analyzed on two
different equipments, columns, days and by two different analyst and their standard
deviation and relative standard deviations were recorded
Page 11 of 25

Saglip Tablet
3.5.1
Repeatability
Tablet 7
Set 1
99.10
%
Potency of standard
Weight of sample
taken
205.46
Factor
1.116
Standard Peak Areas
Sr #
Area
1
365.6
2
361.6
3
4
5
6
362.6
363.3
362.6
362.1
Average
363.0
Weight of standard taken
51.
4
mg
Proposed weight / Tablet
210
Sample Peak Areas

Sr #
1
2
RSD
(%)
Area
274.1
274.4
3
Average
0.39
RSD (%)
m
g
m
g
274.4
274.3
0
Result
0.06
70.51%
Tablet 8
Set 2
99.10
%
Potency of standard
Weight of sample
taken
210.57
Factor
1.116
Standard Peak Areas
Sr #
Area
1
349.6
2
350.2
3
348.5
4
348.9
5
346.8
6
348.5
Average
Page 12 of 25
348.8
48.
5
mg
210
RSD
(%)
Sample Peak Areas

Sr #
1
2
3
Average
0.33
RSD (%)
m
g
m
g
Area
301.5
302.3
302.4
302.0
7
Result
0.16
74.40%

Saglip Tablet
Tablet 9
Set 3
99.10
%
Potency of standard
Weight of sample
taken
209.07
Factor
1.116
Standard Peak Areas
Sr #
Area
1
376.2
2
377.0
3
376.7
4
5
6
376.7
375.0
374.5
Average
376.0
48.
7
mg
210
Sample Peak Areas

Sr #
1
2
3
RSD
(%)
Average
0.27
RSD (%)
m
g
m
g
Area
319.3
320.2
316.9
318.8
0
Result
0.54
73.66%
Tablet 10
Set 4
Potency of standard
Weight of sample
taken
99.10
%
50.
1
210.18
mg
210
Factor
1.116
Standard Peak Areas
Sr #
Sample Peak Areas
Area
1
360.5
360.5
361.1
361.6
361.1
363.0
Average
361.3
Page 13 of 25
m
g
m
g
RSD
(%)
Sr #
Area
299.1
302.2
297.7
Average
0.26
RSD (%)
299.6
7
Result
0.77
73.74%

Saglip Tablet
Tablet 11
Set 5
Potency of standard
Weight of sample
taken
99.10
%
50.1
9
210.06
mg
210
Factor
1.116
Standard Peak Areas
Sr #
Sample Peak Areas
Area
1
363.3
365.1
365.4
366.3
363.8
364.7
Average
364.8
m
g
m
g
RSD
(%)
Sr #
Area
302.5
302.6
302.5
Average
0.30
RSD (%)
302.5
3
Result
0.02
73.91%
Tablet 12
Set 6
Potency of standard
Weight of sample
taken
99.10
%
49.8
4
210.06
mg
210
Factor
1.116
Standard Peak Areas
Sr #
Sample Peak Areas
Area
1
361.6
364.4
363.7
360.4
362.0
361.2
Average
362.2
Average
RSD
Page 14 of 25
m
g
m
g
RSD
(%)
Sr #
Area
301.5
296.9
297.0
Average
0.42
73.19%
1.40%
RSD (%)
298.4
7
Result
0.88
72.92%

Saglip Tablet
The % RSD of the assay values should not be greater than 2.0%.
Conclusion:
% RSD for assay repeatability is 1.40% (i.e. less than 2.0%) hence repeatability is
accepted
3.5.2
Intermediate precision
DIFFERENT DAYS
Tablet 13
Day 1
99.10
%
Potency of standard
Weight of sample
taken
210.18
Factor
1.116
Standard Peak Areas
Sr #
Area
1
360.5
2
360.5
3
361.1
4
361.6
5
361.1
6
363.0
Average
50.
1
mg
210
Sample Peak Areas

Sr #
1
2
3
RSD
(%)
Average
0.26
RSD (%)
361.3
m
g
m
g
Area
299.1
302.2
297.7
299.6
7
Result
0.77
73.74%
Tablet 14
DAY 2
99.10
Potency of standard
%
Weight of sample
210.5
taken
6
Factor
1.116
Standard Peak Areas
Sr #
Area
1
365.6
2
365.7
3
367.6
4
368.1
5
366.4
6
366.3
Average
Average
RSD 15 of 25
Page
50.3
3
mg
210
RSD
(%)
Sample Peak Areas

Sr #
1
2
3
Average
0.28
366.6
73.95%
0.41%
RSD (%)
Absolute difference
m
g
m
g
Area
304.5
304.3
306.2
305.0
0
Result
0.34
74.16%
0.58

Saglip Tablet
Acceptance Value
NMT 4.0
DIFFERENT EQUIPMENTS
Tablet 15
Agilent (QA-0129)
99.10
Potency of standard
%
Weight of sample
231.0
taken
1
Factor
1.116
Standard Peak Areas
Sr #
Area
1
555.4
2
551.0
3
4
5
6
550.2
553.0
550.7
551.2
Average
551.9
25.1
2
mg
230
Sample Peak Areas

Sr #
1
2
RSD
(%)
Area
606.7
608.5
3
Average
0.35
RSD (%)
m
g
m
g
606.5
607.2
3
Result
0.18
97.74%
Tablet 16
AGILENT(QA-095)
99.10
Potency of standard
%
Weight of sample
478.0
taken
7
Factor
1.116
Standard Peak Areas
Sr #
Area
1
621.1
2
623.8
3
626.3
4
631.3
5
630.3
Average
626.6
Absolute
difference
Acceptance Value
Page 16 of 25
3.54
NMT 4.0
57.9
4
mg
230
RSD
(%)
Sample Peak Areas

Sr #
1
2
3
Average
0.69
RSD (%)
Average
RSD
m
g
m
g
Area
640.5
639.6
642.3
640.8
0
Result
0.21
101.26%
99.50%
2.50%

Saglip Tablet
DIFFERENT ANALYST
Tablet 17
Kashif Mumtaz
99.10
Potency of standard
%
Weight of sample
210.0
taken
6
Factor
1.116
Standard Peak Areas
Sr #
Area
1
361.6
2
364.4
3
363.7
4
360.4
5
362.0
6
361.2
Average
49.8
4
mg
210
Sample Peak Areas

Sr #
1
2
3
RSD
(%)
Average
0.42
RSD (%)
362.2
m
g
m
g
Area
301.5
296.9
297.0
298.4
7
Result
0.88
72.92%
Tablet 18
Arslan Khalid
99.10
%
Potency of standard
Weight of sample
taken
205.46
Factor
1.116
Standard Peak Areas
Sr #
Area
1
365.6
2
361.6
3
362.6
4
363.3
5
362.6
6
362.1
Average
Absolute
difference
Acceptance Value
Page 17 of 25
363.0
-3.35
NMT 4.0
51.
4
mg
210
RSD
(%)
Sample Peak Areas

Sr #
1
2
3
Average
0.39
RSD (%)
Average
RSD
m
g
m
g
Area
274.1
274.4
274.4
274.3
0
Result
0.06
70.51%
71.71%
2.37%

Saglip Tablet
The % RSD of the assay/recovery values generated by a single analyst should not
be greater than 2.0%.
The % RSD of the combined assay/recovery values generated by both analysts, over
both days should not be greater than 3.0%.
Absolute difference should not be more than 4.0.
Conclusion:
Repeatability has been performed as per protocol and values for % RSDs and Absolute
difference is with acceptance limit hence repeatability is accepted.
3.6
Limit of Detection (LOD) and Limit of Quantitation (LOQ)
LOD was determined by the residual standard deviation and slop of the line in range study.
The relationship for Detection Limit is given below
Detection Limit= 3.3* RSD/ m
Where m is the slop of line in linear relation
RSD is the residual standard deviation of the y intercept in the linear regression.
LOQ was determined by the residual standard deviation and slop of the line in range study.
The relationship for Quantitation Limit is given below
Quantitation Limit= 10* RSD/ m
Where m is the slop of line in linear relation
RSD is the residual standard deviation of the y intercept in the linear regression
Regression Statistics
R Square
Standard Error
Observations
Sum of residual
squares
Slop
Limit of detection
Limit of
quantitation
3.7
(ppm)
(ppm)
0.999871
2.20688
24
2.20687992
7.444561404
1.0
3.0
Robustness
The robustness of the method was determined to assess the effect of small but deliberate
variation of the chromatographic conditions on the determination of Saxagliptin.
Robustness was determined by changing the mobile phase pH + 0.2, flow rate to 0.85and
1.15 mL min1 and the concentration of acetonitrile in the mobile phase to 57 and 63%
Page 18 of 25

Saglip Tablet
Results from testing of the robustness of the method by changing the pH of the
mobile phase
Table 20
Mobile phase pH
Potency of standard
Weight of sample
taken
99.10
%
210.1
3
Factor
1.116
50.2
3
mg
210
m
g
m
g
pH 4.4
Standard Peak Areas
Sr #
Sample Peak Areas
Area
1
367.9
369.2
370.1
371.2
366.6
365.5
Average
368.4
Sr #
RSD
(%)
Area
1
302.9
302.3
302.8
Average
0.59
RSD (%)
302.6
7
Result
0.11
73.24%
pH 4.6
Standard Peak Areas
Sr #
Sample Peak Areas
Area
1
365.6
365.7
367.6
368.1
366.4
366.3
Average
366.6
Sr #
RSD
(%)
Area
1
304.5
304.3
306.2
Average
0.28
RSD (%)
305.0
0
Result
0.34
74.17%
pH 4.8
Standard Peak Areas
Sr #
Sample Peak Areas
Area
1
367.6
366.8
366.6
366.5
365.9
368.7
Average
367.0
Page 19 of 25
Sr #
RSD
(%)
Area
1
307.1
299.7
305.1
Average
0.27
RSD (%)
303.9
7
Result
1.26
73.84%

Saglip Tablet
Average
73.75%
RSD
0.47%
Acceptable
NMT 3%
Results from testing of the robustness of the method by changing the

composition of the mobile phase
Table 21
Mobile phase Composition
Potency of standard
Weight of sample
taken
Factor
99.10
%
210.1
3
1.116
50.2
3
mg
210
m
g
m
g
Acetonitrile 17 %
Standard Peak Areas
Sr #
Area
1
373.5
2
373.5
3
374.5
4
373.7
5
375.7
6
374.1
Average
373.9
RSD
(%)
Sample Peak Areas

Sr #
1
2
3
Average
0.23
RSD (%)
Area
296.1
296.7
292.8
295.2
0
Result
0.71
70.39%
Acetonitrile 20 %
Sr #
Standard Peak Areas

Area
1
372.9
2
372.9
3
375.8
4
372.1
5
372.7
6
373.9
Average
373.4
RSD
(%)
Sample Peak Areas

Sr #
1
2
3
Average
0.35
RSD (%)
Area
294.5
293.9
293.0
293.8
0
Result
0.26
70.15%
Acetonitrile 23 %
Sr #
Standard Peak Areas

Area
1
375.2
2
377.0
3
4
5
6
375.7
378.2
376.9
378.8
Average
376.8
Page 20 of 25
RSD
(%)
Sample Peak Areas

Sr #
2
3
Average
Area
288.7
289.2
288.6
0
Result
0.23
66.28%
0.32
RSD (%)

Saglip Tablet
Average
69.61%
RSD
1.16%
NMT 3%
Acceptable
Results from testing of the robustness of the method by changing flow rate of
the mobile phase
Table 22
Mobile phase Flow rate
Potency of standard
Weight of sample
taken
Factor
99.10
%
210.1
3
1.116
50.2
3
mg
210
m
g
m
g
1.35 ml
Standard Peak Areas
Sr #
Area
1
415.8
2
415.0
3
415.9
4
415.7
5
415.5
6
416.0
Average
415.7
Sample Peak Areas

RSD
(%)
Sr #
Area
325.0
325.0
324.5
1
2
3
Average
0.09
RSD (%)
324.8
3
Result
0.09
69.67%
1.5 ml
Sr #
Standard Peak Areas

Area
1
372.9
2
372.9
3
375.8
4
372.1
5
372.7
6
373.9
Average
373.4
Sample Peak Areas

RSD
(%)
Sr #
Area
294.5
293.9
293.0
1
2
3
Average
0.35
RSD (%)
293.8
0
Result
0.26
70.15%
1.65 ml
Sr #
Standard Peak Areas

Area
1
338.9
2
339.0
3
339.1
4
339.3
5
338.9
6
339.0
Average
339.0
Average
RSD
Page 21 of 25
Sample Peak Areas

RSD
(%)
Sr #
Average
0.04
69.87%
0.25%
Area
265.8
265.4
265.0
1
2
3
RSD (%)
265.4
0
Result
0.15
69.79%

Saglip Tablet
Acceptable
NMT 3%
Results from testing of the robustness of the method by changing temperature

Table 23
Temperature Change
Potency of standard 99.10%
Weight of sample
taken
210.13
Factor
mg
50.23 mg
210
mg
1.116
20 C
Standard Peak Areas

Sr #
Area
1
565.5
2
562.0
3
563.2
4
566.9
5
566.3
6
564.8
Average
Sample Peak Areas

Sr #
1
2
3
RSD
(%)
Average
0.33
RSD (%)
564.8
Area
543.1
540.0
543.0
542.03
0.33
Result
85.56%
25 C
Standard Peak Areas
Sr #
Area
1
558.9
2
559.8
3
559.3
561.4
563.3
543.6
Average
557.7
Sample Peak Areas

Sr #
1
2
RSD
(%)
Area
540.7
538.7
3
Average
1.27
RSD (%)
537.4
538.93
0.31
Result
86.15%
30 C
Standard Peak Areas
Sr #
Area
1
565.0
2
563.2
3
563.8
4
564.6
5
565.6
6
561.8
Average
Average
RSD
Page 22 of 25
RSD
(%)
Sample Peak Areas

Sr #
1
2
3
Average
0.24
564.0
85.63%
0.49%
RSD (%)
Area
538.5
538.1
539.9
538.83
0.18
Result
85.17%

Saglip Tablet
Acceptable
NMT 3%
Table 24
Solution stability
Potency of standard 99.10%
Weight of sample
taken
210.56
Factor
mg
50.33 mg
210
mg
1.116
0 Hour
Standard Peak Areas
Sr #
Area
1
363.3
2
3
4
5
6
365.1
365.4
366.3
363.8
364.7
Average
364.8
Sample Peak Areas

Sr #
1
RSD
(%)
Area
302.5
2
3
Average
0.30
RSD (%)
302.6
302.5
302.53
0.02
Result
73.94%
24 Hour
Standard Peak Areas
Sr #
Area
1
365.6
2
365.7
3
367.6
4
368.1
366.4
366.3
Average
366.6
Average
RSD
Acceptable
3.8
RSD
(%)
Sample Peak Areas

Sr #
1
2
3
Average
0.28
RSD (%)
74.46%
0.37%
NMT 3%
System Suitability
SYSTEM SUITABILITY
Page 23 of 25
Area
304.5
304.3
306.2
305.00
0.34
Result
74.46%

Saglip Tablet
Table 25
Sr. #
1
2
3
4
5
Peak Area
361.6
364.4
363.7
360.4
362
Theoretical plates
5518.0
5480.0
5535.0
5250.0
5390.0
Symmetry
0.5900
0.5900
0.5900
0.5900
0.5900
Resolution
----------------
Capacity
Factor k
17.584
17.397
17.777
18.918
18.055
S/N
126.5
97.0
95.3
89.4
89.4
361.2
5404.0
0.5900
----
18.626
87.1
Results
Resolution
6
5430
0.59
-----
18
97
105.777
0.000
-----
0.600
14.717
1.9482%
0.0000%
-----
3.3249%
15.1027%
Peak Area T. P
n=6
Mean=362
SD=1.532
RSD %=0.4228%
Acceptance Criteria
Relative Standard
deviation
Theoretical plates
Tailing Factor
3.9
T. F
S/N
< 1.0
> 2000
0.8 > 2.2
Conclusion
It is obvious from all the above mentioned results that this method is valid for the analysis
of Saglip tablet for intermediate, finished and stability stages. It complies with all the
required parameters and hence declared as validated method for Saglip tablet.
3.10 Deviation
The Analytical Method mentioned in protocol was found fit for Saglip Tablet. Diluent was
changed (0.1N HCl)and method was VALIDATED thereof (see section 2.1).
Page 24 of 25

Saglip Tablet
Author, Quality Assurance (Validation), CCL Pharmaceuticals (Pvt.) Ltd
Date
Reviewed by, Deputy Manager Quality Assurance (Validation), CCL Pharmaceuticals Pvt. Ltd Date
Reviewed by, QCM, CCL Pharmaceuticals Pvt. Ltd
Date
Approved by, Head of QA&R, CCL Pharmaceuticals Pvt. Ltd
Date
Page 25 of 25

Analytical Method Validation Report of Saxagliptin Tablet

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Analytical Method Validation Report of Saxagliptin Tablet

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Analytical Method Validation Report