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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

Analytical Method Validation Report
Saglip Tablet
Document No. CCL-AMVR-170-A

Table of Contents
1

INTRODUCTION...................................................................................................... 3

2

ANALYTICAL PROCEDURE........................................................................................3
2.1

3

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

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precision. Forced degradation studies of finished pharmaceutical product (Saglip tablet) were performed.1 Assay procedure Buffer: Dissolve 2. CCL-AMVR-170-A 1 Introduction 2 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. the resolution between degraded products and principle peak was found to be >2. Mode: LC Detector: UV 213 nm Column: C18 Column temperature: Ambient Flow rate: 1.0% Calculate the percentage of the labeled amount of Saxagliptin in the portion of Tablets taken: Result = (rU/rS) x (CS/CU) x 100/1. Take 10ml from this solution into a 100ml volumetric flask and make up the volume with diluent.1N HCl Mobile phase: Buffer: Acetonitrile (80:20) Standard solution: Weigh on analytical balance. Diluent: 0. make up the volume with diluent and sonicate for 10 minutes. make up the volume with diluent and sonicate for 10 minutes (50ppm).72 grams of Potassium Dihydrogen Phosphate in 1000 mL of distilled water and adjust the pH 4.5 mL/min Injection size: 10 µL System suitability requirements Tailing factor: NMT 2.6 with dilute Sodium Hydroxide solution. Sample solution: Weigh on analytical balance. All the parameters were performed on Agilent 1260 series HPLC equipped with Auto sampler. quaternary low pressure gradient pump and PDA detector for peak purity purposes 2. robustness.0% Page 3 of 25 .0 Relative standard deviation: NMT 2. column oven. and LOQ. accuracy.Analytical Method Validation Report Saglip Tablet Document No. Method was investigated for its range. Peak purity and relative retention times confirmed the specificity of method. LOD and solution stability. working standard of Saxagliptin HCl equivalent to 25mg of Saxagliptin and transfer it into a 50ml volumetric flask.0%–110. linearity. ground powder of tablets equivalent to 5mg of Saxagliptin and transfer it into a 100ml volumetric flask.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.

668. 669. 5 666.2 0 2. of degradation Content Peak n product (µg Area Area (Area. The following degradation conditions were selected: Obtain or prepare solutions of ~0.76 99.05 110.17% 3.95% 3.Analytical Method Validation Report Saglip Tablet Document No. 2 N NaOH ~1% H2O2. 7 608. No.49 5 606.52% 0. 7 667. Repeatability ii. 10% H2O2 and purified water. CCL-AMVR-170-A 3 Validation of Method Following Validation characteristics were considered for analytical method validation: 1) 2) 3) 4) 5) 3. RT) mL−1) 606.4 0 2.1 Specificity (Identification) Linearity Range Accuracy (Recovery) Precision i.48 3 669. 1 231. 1 N NaOH.0 1 2 3 4 0. base.48% 3.15 N NaOH.8 2 Page 4 of 25 10 11 Table 1 FORCED DEGRADATION FOR SAXAGLIPTIN Theoretic al Mea No. 607. 1 N HCL.43 4 671. acid.48 8 673. 2 N HCL ~0. 4 675.17 % -10.04 111. 1 669. The degraded samples were then analyzed using the method to determine if there were interferences with the active or related compound peaks.33% .0 5 5 6 7 1 N HCl 229. 675.8 0 2. 5% H2O2. light.1 0 2. 5 668. Stress Condition Unstress ed sample weight taken (mg) Chrom.95 % -11. and oxidizing agent to produce 10%–30% degradation of the active.33 % Degradat ion -11. Intermediate Precision 6) LOD and LOQ 7) Robustness 8) Solution stability 9) System suitability Specificity Samples of drug product were exposed to heat. Amount recovere d (µg mL−1) Recover y (%) 2.9 4 8 9 2 N HCl 229.1 N HCl 226.1 N HCL.08 111.

04 109. Page 5 of 25 . 6 40.1.47% 3.7. 4 551.3 0 2.5 5 2N NaOH 227.2.3 7 29 30 34 Standard 35 36 37 38 25.8.0 (6) 0.8. 0 550.20% 2. 2 ----553.48 4 3.3.9 2. 601.6 666.03% Acceptance Criteria:  Peak purity should be NLT 990  Resolution should be more than 2.2 1% H2O2 229.74 97.8 (6) 41.00% 100.51% 0.0 0. 2 551.0 2.19 6.0 (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.7 1 1N NaOH 233.8. 8 601.Analytical Method Validation Report Saglip Tablet Document No.2.20 % -9.03 109.1N NaOH 228.45 0.1.3. (2) 667. 0 550.4 4.8 2. 6 443.46 6 2.3.9 9 14 15 16 17 18 19 20 21 22 23 24 25 5% H2O2 231.00 0.3.1.0 2.3.49% 26.8 665. (4) 443.7. 5 665.00% 3.6.2. 0 667.39% 0.7.7.0 Conclusion: Resolution was found to be more than 2.1 2.2 8 26 27 28 10 % H2O2 232. CCL-AMVR-170-A 12 13 0. 0 602. 7 551.50 8 600.9 2.97% 2.49 1 3. 0 555.47 % -9.52 40. (1) 665.0 4.3. 7 443.6. 2.1 444.5 664. 3 667.1. 2.02 73.8.5 0.7.61% 93.3.1 40.6.12 39 Table 1 FORCED DEGRADATION FOR SAXAGLIPTIN 8 677.

Page 6 of 25 . The detection wavelength was 213 nm. peak area was calculated for each dilution.5mg mL−1 saxagliptan) was prepared by dissolving 1000 mg drug in 100 mL Diluent. The mobile phase was filtered through a 0.22-µm membrane filter and delivered at 1. Solutions of different concentration (30--70 µg mL−1) for construction of calibration plots were prepared from this stock solution. the baseline was monitored continuously during this process.50 mL min−1 for column equilibration. and concentration was plotted against peak area.Analytical Method Validation Report Saglip Tablet Document No.2 Linearity A stock solution of Saxagliptan (0. CCL-AMVR-170-A 3. The prepared dilutions were injected at the rate of 10µl in series.

1 0. Calibration data.217% 447.3 223.355% 372. Conclusion: The calibration plot of peak area against concentration was linear in the range investigated (30–70 µg mL−1).3 447.077% Linear plot for Saglip Tablet 600 500 f(x) = 7.1 0. # Chrom.7 447.169% 519.7 519.8 0. CCL-AMVR-170-A Table 2 Linearity study of Saxagliptin Sr.Analytical Method Validation Report Saglip Tablet Document No.41x + 1.1 372.990. with their relative standard deviations.2 0.7 299.2 446.4 298. % Page 7 of 25 .86 R² = 1 400 Peak Area 300 200 100 0 25 30 35 40 45 50 55 60 65 70 75 Concentration Acceptance Criteria:  Coefficient of determination (r2) should be greater than 0.4 0. # 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.  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.5 519.8 518.3 372. (ppm) 30 30 30 40 40 40 50 50 50 60 60 60 70 70 70 Peak Area 222.7 373.5 223.339% 298.6 297.1 MEAN RSD (%) 223.

The detection wavelength was 213 nm.075% .50 mL min−1 for column equilibration.0 MEAN RSD (%) 148 0. CCL-AMVR-170-A RSD and standard error are listed in Table I.4 298. The prepared dilutions were injected at the rate of 10µl in series.Analytical Method Validation Report Saglip Tablet Document No.6 297.3 372.7 373. The linear regression equation was y =1468. peak area was calculated for each dilution.1 600. The linear regression data for the calibration plot are indicative of a good linear relationship between peak area and concentration over a wide range.4 148. the baseline was monitored continuously during this process.339% 298 0.077% 600 0.3 600.4 600.217% 447 0.5 519. Solutions of different concentration (20--100 µg mL−1) for construction of calibration plots were prepared from this stock solution.7 447.7 519.6 222.434% 223 0.1 741. 3. The low values of RSD and standard error show the method is precise.8 518.7 299.3 Range A stock solution of Saxagliptan (0. Table 3 Range study of Sitagliptin Sr.5 223.3 223. and concentration was plotted against peak area.025% 742 0.4 148.2 446.7 742.3 447.6 742.355% 373 0.5x + 40069 and the re-gression coefficient was 0.9999. # 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.1 372.5mg mL−1 saxagliptan) was prepared by dissolving 1000 mg drug in 100 mL Diluent. (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 147.169% 519 0. The mobile phase was filtered through a 0.22-µm membrane filter and delivered at 1.

CCL-AMVR-170-A Concentration (ppm) Line Fit Plot for Range study of Saglip Tablet 800 f(x) = 7. and 12 ml of this solution in three different 100 ml volumetric flasks A. C) 2-A stock solution of Saxagliptan HCl (0.990 3.1N HCl and make up volume 0. Transfer 8.44x + 0. The low values of RSD and standard error show the method is precise. with their relative standard deviations. Calibration data. Acceptance Criteria Regression Coefficient > 0.1N HCl.Analytical Method Validation Report Saglip Tablet Document No.5mg mL−1) was prepared by dissolving 50 mg of Saxagliptan HCl in 100 mL 0. 10.14 R² = 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 µg mL−1).1N HCl. % RSD and standard error are listed in Table I. C respectively containing placebo. The linear regression data for the calibration plot are indicative of a good linear relationship between peak area and concentration over a wide range.1N HCl. Sample preparation: Weight accurately about 195. (A.9999. The linear regression equation was y = 1412.0 mg of placebo in a 100 ml volumetric flask (thre different flasks) and dissolve it in water.1x + 13700 and the regression coefficient was 0. B. B.4 Accuracy Standard preparation: Weight accurately about 50 mg of Saxagliptin in a 100 ml volumetric flask and dissolve it in 0. Make up Page 9 of 25 . Take 10ml from this dilution in 100ml volumetric flask and makeup volume with 0.

4 545.6 550.1 551.89 98.73 Working Standard 50.93 Mean recovery (%) 99.30 8.0 140.74 140.9 537.14 49.95 Mean recovery (%) 99.1 541.70 99.26 12.7 437.59 656 0.34 99.34 99.40 48.11 98.9 554.38 552.00 150mg 50.6 550.4 551.18 539 0.07 59.02 59.3 436.6 656.23 Recovery %age Peak Area 438 438. Accuracy Set 1 Potency of WS: Weight of placebo: 99.6 656.49 652 0.49 39.67 140.91 544 0.9 652.0 Actual Theoretical Content (µg/ml) 39.46 99.5 39.5 655.51 Working Standard 50.9 554.30 8.1 551.4 551.59 59.26 12.10% 195.28 SET 1 Mean Area RSD Content (µg/ml) 434 0.0 49.10 0.26 .10% Prop Wt: Weight of active: 140.0 Actual Theoretical Content (µg/ml) 10.5 ----- Page 10 of 25 SET 2 Mean Area RSD Content (µg/ml) 438 0.04 ----- Recovery %age Peak Area 431.2 543. CCL-AMVR-170-A volume with diluent to get three concentration levels of 80%.23 Table 5 Accuracy Weight of placebo mg Volume of stock solution (ml) 140. 100% and 120% respectively.15 39.1 552.8 538.10 0.0 140.23 552.1 552.Analytical Method Validation Report Saglip Tablet Document No.3 652.00 Prop Wt: Weight of active: 230mg 50.0 652.59 59.9 543.04 Table 4 Accuracy Weight of placebo mg Volume of stock solution (ml) 140.0 49.28 10.26 ---- Accuracy Set 2 Potency of WS: Weight of placebo: 99.9 434.

For intermediate precision same sample was analyzed on two different equipments.98 436.79 543. days and by two different analyst and their standard deviation and relative standard deviations were recorded Page 11 of 25 .2 543.41 97.59 531.Analytical Method Validation Report Saglip Tablet Document No.0% for the average of each set of three weights.39 Recover y Peak Area 437.71 98.79 Table 6 Accuracy SET 3 Weight of placebo mg Volume of Active ml 140.08 39.74 100. Acceptance Criteria:  The percent recovery of the spiked placebos should be within 100±2.10% 140.0 643.01 8.0 533. RSD (%).1 49. For repeatability six replicates of a sample were analyzed on three different days and their RSD was calculated.54 10.62 645 0.  Each individual sample recovery should lie within the range of 98% to 102%. 5 and 6 listed above indicate that the method is accurate. The values of recovery (%). and SEM listed in Table 4.88 532 0.09 543.1 Mean Area RSD Content (w/w %) %age 436. Conclusion: Hence above acceptance criteria for accuracy is met.51 648 542.5 Precision Precision was determined as both repeatability and intermediate precision.07 12.8 644.8 437 0. CCL-AMVR-170-A Accuracy Set 3 Potency of WS: Weight of placebo: 99.3 59.38 58. therefore method is accurate 3.0 Actual Theoretical Content (%) 39.2 543.4 140.17 48.6 140.14 0.4 531. columns.66 Mean recovery (%) 99.00 Prop Wt: Weight of active: 150 mg 49.9 The recovery of the method is determined by spiking a placebo with active pharmaceutical ingredient of known potency.6 Working Standard 49.8 542.

4 mg Proposed weight / Tablet 210 Sample Peak Areas Sr # 1 2 RSD (%) Area 274.6 2 350.6 3 4 5 6 362.6 362.40% .10 % Potency of standard Weight of sample taken 205.4 274.4 3 Average 0.116 Standard Peak Areas Sr # Area 1 349.46 Factor 1.3 302.6 2 361.Analytical Method Validation Report Saglip Tablet Document No.10 % Potency of standard Weight of sample taken 210.8 % Weight of standard taken 48.1 Average 363.06 70. 5 mg Proposed weight / Tablet 210 RSD (%) Sample Peak Areas Sr # 1 2 3 Average 0.3 362.33 RSD (%) m g m g Area 301.8 6 348.2 3 348.3 0 Result 0.9 5 346.57 Factor 1.0 % Weight of standard taken 51.4 302.5 Average Page 12 of 25 348.5 4 348.0 7 Result 0.1 274.6 363.5.51% Tablet 8 Set 2 99. CCL-AMVR-170-A 3.1 Repeatability Tablet 7 Set 1 99.16 74.39 RSD (%) m g m g 274.5 302.116 Standard Peak Areas Sr # Area 1 365.

9 318.7 Average 0.7 4 5 6 376.3 320. 1 210.116 Standard Peak Areas Sr # Area 1 376.1 4 361.0 374.8 0 Result 0.2 3 297. 7 mg Proposed weight / Tablet 210 Sample Peak Areas Sr # 1 2 3 RSD (%) Average 0.1 6 363.66% Tablet 10 Set 4 Potency of standard Weight of sample taken 99.2 2 377.0 Average 361.27 RSD (%) m g m g Area 319.54 73. CCL-AMVR-170-A Tablet 9 Set 3 99.0 3 376.116 Standard Peak Areas Sr # Sample Peak Areas Area 1 360.6 7 Result 0.18 mg Proposed weight / Tablet 210 Factor 1.07 Factor 1.Analytical Method Validation Report Saglip Tablet Document No.5 2 360.5 Average 376.2 316.74% .26 RSD (%) 299.3 Page 13 of 25 m g m g RSD (%) Sr # Area 1 299.7 375.6 5 361.77 73.10 % % Weight of standard taken 50.10 % Potency of standard Weight of sample taken 209.0 % Weight of standard taken 48.1 2 302.5 3 361.

06 mg Proposed weight / Tablet 210 Factor 1.3 2 365. CCL-AMVR-170-A Tablet 11 Set 5 Potency of standard Weight of sample taken 99.8 6 364.92% .0 6 361.88 72.8 4 210.5 Average 0.91% Tablet 12 Set 6 Potency of standard Weight of sample taken 99.40% RSD (%) 298.4 7 Result 0.2 Average 362.10 % % Weight of standard taken 50.30 RSD (%) 302.8 m g m g RSD (%) Sr # Area 1 302.0 Average 0.3 5 363.10 % % Weight of standard taken 49.19% 1.02 73.4 5 362.2 Average RSD Page 14 of 25 m g m g RSD (%) Sr # Area 1 301.Analytical Method Validation Report Saglip Tablet Document No.6 2 364.9 3 297.4 3 363.5 3 Result 0.4 4 366.5 2 296.6 3 302.7 4 360.1 3 365.42 73.5 2 302.1 9 210.116 Standard Peak Areas Sr # Sample Peak Areas Area 1 363.7 Average 364.06 mg Proposed weight / Tablet 210 Factor 1.116 Standard Peak Areas Sr # Sample Peak Areas Area 1 361.

0%.0 0 Result 0.40% (i.41% RSD (%) Absolute difference m g m g Area 304.10 % Potency of standard Weight of sample taken 210.95% 0.16% 0.28 366.116 Standard Peak Areas Sr # Area 1 360. CCL-AMVR-170-A Acceptance Criteria: The % RSD of the assay values should not be greater than 2.e.6 5 361.Analytical Method Validation Report Saglip Tablet Document No.5.0 Average % Weight of standard taken 50.34 74.2 305.0%) hence repeatability is accepted 3.58 .26 RSD (%) 361.5 2 360.1 5 366.1 4 361.1 302.3 Average Average RSD 15 of 25 Page % Weight of standard taken 50.6 7 Result 0.3 3 mg Proposed weight / Tablet 210 RSD (%) Sample Peak Areas Sr # 1 2 3 Average 0.3 m g m g Area 299.2 Intermediate precision DIFFERENT DAYS Tablet 13 Day 1 99. Conclusion: % RSD for assay repeatability is 1.5 304.4 6 366.77 73.3 306.74% Tablet 14 DAY 2 99.5 3 361. less than 2.6 73.2 297.6 4 368.5 taken 6 Factor 1.7 3 367.116 Standard Peak Areas Sr # Area 1 365.7 299.18 Factor 1.1 6 363.10 Potency of standard % Weight of sample 210. 1 mg Proposed weight / Tablet 210 Sample Peak Areas Sr # 1 2 3 RSD (%) Average 0.6 2 365.

0 3 4 5 6 550.116 Standard Peak Areas Sr # Area 1 555.69 RSD (%) Average RSD m g m g Area 640.21 101.2 3 Result 0.0 taken 7 Factor 1.8 0 Result 0.Analytical Method Validation Report Saglip Tablet Document No.26% 99.7 608.1 2 mg Proposed weight / Tablet 230 Sample Peak Areas Sr # 1 2 RSD (%) Area 606.9 % Weight of standard taken 25.5 639.8 3 626.18 97.2 553.0 taken 1 Factor 1.0 DIFFERENT EQUIPMENTS Tablet 15 Agilent (QA-0129) 99.0 % Weight of standard taken 57.10 Potency of standard % Weight of sample 231.54 NMT 4.5 607.3 4 631.3 640.6 642.50% .2 Average 551.9 4 mg Proposed weight / Tablet 230 RSD (%) Sample Peak Areas Sr # 1 2 3 Average 0.116 Standard Peak Areas Sr # Area 1 621. CCL-AMVR-170-A Acceptance Value NMT 4.74% Tablet 16 AGILENT(QA-095) 99.35 RSD (%) m g m g 606.10 Potency of standard % Weight of sample 478.50% 2.0 550.7 551.6 Absolute difference Acceptance Value Page 16 of 25 3.3 Average 626.3 5 630.4 2 551.5 3 Average 0.1 2 623.

4 274.0 -3.6 3 362.0 6 361.Analytical Method Validation Report Saglip Tablet Document No.0 % Weight of standard taken 51.10 Potency of standard % Weight of sample 210.116 Standard Peak Areas Sr # Area 1 365.88 72.0 298.116 Standard Peak Areas Sr # Area 1 361.7 4 360.2 Average % Weight of standard taken 49. 4 mg Proposed weight / Tablet 210 RSD (%) Sample Peak Areas Sr # 1 2 3 Average 0.51% 71.35 NMT 4.42 RSD (%) 362.71% 2.10 % Potency of standard Weight of sample taken 205. CCL-AMVR-170-A DIFFERENT ANALYST Tablet 17 Kashif Mumtaz 99.37% .3 0 Result 0.6 4 363.4 274.06 70.2 m g m g Area 301.6 2 364.9 297.8 4 mg Proposed weight / Tablet 210 Sample Peak Areas Sr # 1 2 3 RSD (%) Average 0.1 274.4 3 363.6 6 362.3 5 362.39 RSD (%) Average RSD m g m g Area 274.4 7 Result 0.46 Factor 1.5 296.4 5 362.0 taken 6 Factor 1.1 Average Absolute difference Acceptance Value Page 17 of 25 363.6 2 361.92% Tablet 18 Arslan Khalid 99.

over both days should not be greater than 3.20687992 7. Robustness was determined by changing the mobile phase pH + 0. 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. Conclusion: Repeatability has been performed as per protocol and values for % RSDs and Absolute difference is with acceptance limit hence repeatability is accepted.  The % RSD of the combined assay/recovery values generated by both analysts.999871 2.0.3* 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.2.0%.444561404 1.15 mL min−1 and the concentration of acetonitrile in the mobile phase to 57 and 63% Page 18 of 25 . flow rate to 0. CCL-AMVR-170-A Acceptance Criteria:  The % RSD of the assay/recovery values generated by a single analyst should not be greater than 2.0 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.20688 24 2. LOQ was determined by the residual standard deviation and slop of the line in range study.Analytical Method Validation Report Saglip Tablet Document No.85and 1.7 (ppm) (ppm) 0.0%. 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 3.0 3. The relationship for Detection Limit is given below Detection Limit= 3. Absolute difference should not be more than 4.

6 2 366.6 2 365.8 3 366.6 4 366.17% pH 4.2 3 mg Proposed weight / Tablet 210 m g m g pH 4.7 3 367.8 Standard Peak Areas Sr # Sample Peak Areas Area 1 367.1 2 299.27 RSD (%) 303.9 7 Result 1.5 2 304.1 4 371.34 74.7 Average 367.2 Average 0.5 5 365.3 3 302. CCL-AMVR-170-A 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 99.24% pH 4.0 0 Result 0.9 6 368.11 73.0 Page 19 of 25 Sr # RSD (%) Area 1 307.3 Average 366.10 % 210.4 6 366.9 2 369.Analytical Method Validation Report Saglip Tablet Document No.1 5 366.116 % Weight of standard taken 50.6 4 368.6 Standard Peak Areas Sr # Sample Peak Areas Area 1 365.9 2 302.6 Sr # RSD (%) Area 1 304.5 Average 368.1 3 Factor 1.1 Average 0.8 Average 0.3 3 306.4 Sr # RSD (%) Area 1 302.6 6 365.7 3 305.84% .6 7 Result 0.28 RSD (%) 305.2 5 366.4 Standard Peak Areas Sr # Sample Peak Areas Area 1 367.26 73.2 3 370.59 RSD (%) 302.

8 Page 20 of 25 RSD (%) Sample Peak Areas Sr # 2 3 Average Area 288.9 3 375.0 293.8 Average 376.23 RSD (%) Area 296.4 RSD (%) Sample Peak Areas Sr # 1 2 3 Average 0.39% Acetonitrile 20 % Sr # Standard Peak Areas Area 1 372.2 288.2 0 Result 0.8 0 Result 0.9 Average 373.Analytical Method Validation Report Saglip Tablet Document No.5 293.116 % Weight of standard taken 50.1 3 1.75% RSD 0.23 66.7 289.71 70.35 RSD (%) Area 294.7 5 375.10 % 210.5 4 373.2 2 377.8 295.1 Average 373.7 6 373.0 3 4 5 6 375.7 378.32 RSD (%) . CCL-AMVR-170-A Average 73.9 RSD (%) Sample Peak Areas Sr # 1 2 3 Average 0.1 296.6 0 Result 0.5 2 373.7 6 374.15% Acetonitrile 23 % Sr # Standard Peak Areas Area 1 375.5 3 374.2 3 mg Proposed weight / Tablet 210 m g m g Acetonitrile 17 % Standard Peak Areas Sr # Area 1 373.9 293.7 292.8 4 372.26 70.9 2 372.2 376.1 5 372.9 378.28% 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.

15% 1.2 3 mg Proposed weight / Tablet 210 m g m g 1.9 3 375.5 1 2 3 Average 0.5 6 416.4 265.16% 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 99.4 0 Result 0.35 RSD (%) 293.15 69.5 ml Sr # Standard Peak Areas Area 1 372.0 1 2 3 RSD (%) 265.0 325.8 2 415.87% 0.Analytical Method Validation Report Saglip Tablet Document No.0 Average 339.3 5 338.1 3 1.1 5 372.5 293.25% Area 265.0 324.35 ml Standard Peak Areas Sr # Area 1 415.7 5 415.9 6 339.0 3 339.9 2 372.8 0 Result 0.1 4 339. CCL-AMVR-170-A Average 69.65 ml Sr # Standard Peak Areas Area 1 338.79% .0 3 415.9 4 415.04 69.7 6 373.8 4 372.10 % 210.61% RSD 1.116 % Weight of standard taken 50.4 Sample Peak Areas RSD (%) Sr # Area 294.67% 1.9 2 339.26 70.8 265.9 293.0 Average RSD Page 21 of 25 Sample Peak Areas RSD (%) Sr # Average 0.0 Average 415.7 Sample Peak Areas RSD (%) Sr # Area 325.8 3 Result 0.0 1 2 3 Average 0.09 RSD (%) 324.9 Average 373.09 69.

8 4 564.1 540.83 0.33 RSD (%) 564.5 538.18 Result 85.2 4 566.4 5 563.7 538.9 2 559.2 3 563.27 RSD (%) 537.8 Area 543.0 3 563.8 3 559.03 0.0 543.0 85.4 538.5 2 562.116 20 ° C Standard Peak Areas Sr # Area 1 565.13 Factor % Weight of standard taken mg Proposed weight / Tablet 50.1 539.23 mg 210 mg 1.49% RSD (%) Area 538.56% 25 ° C Standard Peak Areas Sr # Area 1 558.8 Average Sample Peak Areas Sr # 1 2 3 RSD (%) Average 0.3 4 561.93 0.6 5 565.0 542.3 6 564. CCL-AMVR-170-A Acceptable NMT 3% Results from testing of the robustness of the method by changing temperature Table 23 Temperature Change Potency of standard 99.15% 30 ° C Standard Peak Areas Sr # Area 1 565.31 Result 86.17% .7 Sample Peak Areas Sr # 1 2 RSD (%) Area 540.0 2 563.24 564.6 Average 557.Analytical Method Validation Report Saglip Tablet Document No.3 6 543.33 Result 85.9 5 566.9 538.8 Average Average RSD Page 22 of 25 RSD (%) Sample Peak Areas Sr # 1 2 3 Average 0.63% 0.10% Weight of sample taken 210.6 6 561.7 3 Average 1.

46% 0.3 Average 366.7 Average 364.30 RSD (%) 302.37% NMT 3% System Suitability SYSTEM SUITABILITY Page 23 of 25 Area 304.116 0 Hour Standard Peak Areas Sr # Area 1 363.6 2 365.3 2 3 4 5 6 365.1 5 366.34 Result 74.6 4 368.28 RSD (%) 74.02 Result 73.46% .5 304.3 363.2 305.4 6 366.8 364. CCL-AMVR-170-A Acceptable NMT 3% Table 24 Solution stability Potency of standard 99.56 Factor % Weight of standard taken mg Proposed weight / Tablet 50.5 302.00 0.6 302.Analytical Method Validation Report Saglip Tablet Document No.10% Weight of sample taken 210.8 Sample Peak Areas Sr # 1 RSD (%) Area 302.94% 24 Hour Standard Peak Areas Sr # Area 1 365.8 RSD (%) Sample Peak Areas Sr # 1 2 3 Average 0.4 366.7 3 367.53 0.33 mg 210 mg 1.1 365.3 306.6 Average RSD Acceptable 3.5 2 3 Average 0.

9482% 0.5900 0.0 0. P n=6 Mean=362 SD=1.Analytical Method Validation Report Saglip Tablet Document No.626 87. F k S/N 6 6 < 1.0 95.6 364.4 362 Theoretical plates 5518. # 1 2 3 4 5 Peak Area 361.8 > 2.1N HCl)and method was VALIDATED thereof (see section 2.0 5390.777 18.600 14.000 ----- 0.0000% ----- 3.59 ----- 18 97 105.4 363.2 Conclusion It is obvious from all the above mentioned results that this method is valid for the analysis of Saglip tablet for intermediate.584 17.777 0.532 RSD %=0.4 89.2 5404.1027% Peak Area T.717 1. finished and stability stages.0 > 2000 0.5900 Resolution ---------------- Capacity Factor k 17.0 5480.1 Results 6 6 Resolution 6 5430 0.9 T. 3.4 6 361.5900 0.1).5 97.0 5250. Page 24 of 25 .3249% 15. CCL-AMVR-170-A Table 25 Sr.0 5535.3 89.918 18.5900 ---- 18.5900 0.4228% Acceptance Criteria Relative Standard deviation Theoretical plates Tailing Factor 3.7 360.397 17.5900 0. It complies with all the required parameters and hence declared as validated method for Saglip tablet.0 Symmetry 0. Diluent was changed (0.055 S/N 126.10 Deviation The Analytical Method mentioned in protocol was found fit for Saglip Tablet.

CCL-AMVR-170-A Author. Quality Assurance (Validation). CCL Pharmaceuticals Pvt. Ltd Date Page 25 of 25 . CCL Pharmaceuticals Pvt. CCL Pharmaceuticals (Pvt. Deputy Manager Quality Assurance (Validation). CCL Pharmaceuticals Pvt.Analytical Method Validation Report Saglip Tablet Document No. Ltd Date Reviewed by. Ltd Date Approved by. Head of QA&R.) Ltd Date Reviewed by. QCM.