Stock The autosampler sample stability was evaluated by

Stock solution stability wasperformed by comparing the area response of analyte and internal standard inthe stability sample, with the area response of sample prepared from freshstock solution. Stability studies in plasma were performed at the LQC and HQCconcentration level using six replicates at each level. Analyte was consideredstable if the % Change is less than 15% as per US FDA guidelines 17.The stability of spiked human plasma samples stored at room temperature (benchtop stability) was evaluated for 61 h. The stability of spiked human plasmasamples stored at -30 °C in autosampler (autosampler stability) was evaluatedfor 70.0 h.

The autosampler sample stability was evaluated by comparing theextracted plasma samples that were injected immediately (time 0 h), with thesamples that were reinjected after storing in the autosampler at 20°C for 70.0h. The reinjection reproducibility was evaluated by comparing the extractedplasma samples that were injected immediately (time 0 h), with the samples thatwere re-injected after storing in the autosampler at 20°C for 70 h. Thefreeze-thaw stability was conducted by comparing the stability samples that hadbeen frozen at –30 °C and thawed three times, with freshly spiked qualitycontrol samples.

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Six aliquots each of LQC and HQC concentration levels wereused for the freeze-thaw stability evaluation. For long term stabilityevaluation the concentrations obtained after 91 days were compared with initialconcentrations and the results were depicted in Table 14.Mean % accuracy should be 85-115 % and % CV should be ? 15%.Table 13: Stability of Dapagliflozin and Saxagliptin inspiked human plasma samples Stability experiments Storage condition Spiked plasma concentration (pg/ml) Concentration measured (n=6) Mean ± SD CV(%) (n=6)   Accuracy (%) Dapagliflozin (DG) Bench top (Room temperature) RT 61 hr 150.0 148.

3 ± 8.1 5.5 98.9 7000.0 6728.3±206.

3 3.1 81.5 Processed (extracted sample) Autosampler 70 hr 150.0 162.3 ± 2.4 1.5 108.

2 7000.0 7536.7±294.5 3.9 90.4 Freeze & Thaw stability -300C Cycle-3 150.0 156.

5 ± 4.0 2.5 104.3 7000.0 7381.

7±173.4 2.3 90.4 Longterm stability – 300C, 91 days   50.

0 160.3±13.2 8.2 106.9 7000.

0 7450.0±229.1 3.

1 90.5  Saxagliptin (SG) Bench top (Room temperature) RT 61 hr 150.0 156.3 ± 8.

7 5.6 104.2 7000.0 7411.7±213.7 2.

9 92.6 Processed (extracted sample) Autosampler 70 hr 150.0 161.7 ± 4.

9 3.0 107.8 7000.0 7675.0±473.5 6.

2 95.9 Freeze & Thaw stability -300C Cycle-3 150.0 159.7 ± 7.

6 4.7 106.4 7000.0 7540.0±323.

0 4.3 94.3 Longterm stability – 300C,91 days 50.0 159.0 ± 6.3 4.

0 106.0 7000.0 7608.3±297.2 3.9 95.1  5.

12. Short-term Stock Solution Stability of DG,SG, DGd5, SGd2 at Room Temperature and Spiking Solution (Working Conc.) of InternalStandard (DGd5, SGd2) at RefrigeratedConditions (2-8°C)Roomtemperature stock solution stability was determined using standard stocksolutions of DG, SG, DGd5, SGd2 (ST stability samples) were set aside on thebench up to 9.5 h and compared with newly prepared stock solutions (comparisonsamples).

Refrigerated IS (DGd5, SGd2) spiking solution stability (IS stabilitysamples)  was determined using IS spikingsolutions of DGd5, SGd2 was set aside in refrigerator (2-8°C)  up to ten days and compared newly prepared ISstock solution samples (comparison samples). The results were depicted in Table 15 to 17.Table 14:Assessment of short term stock solution stability of DG,SG at room temperature   DG SG Comparison samples peak area (0 h) ST Stability samples  peak area (10.30 h) Comparison samples peak area (0 h) ST Stability samples  peak area (10.30 h) 1233917 1237149 1332152 1237291 1264574 1255305 1346779 1249621 1254554 1285973 1348989 1264690 1291273 1290668 1388859 1267163 1301221 1286153 1292460 1301671 1338241 1293599 1298217 1274475 N 6 6 6 6 Mean 1280630 1274807.83 1334576 1265818 SD(±) 37352.77 23050.27 35790.

79 22088.89 CV (%) 2.92 1.

81 2.68 1.75 Mean % Accuracy   99.55   94.85  Table 15:Assessment of short term stock solution stability of DGd5, SGd2 at roomtemperature   DGd5 SGd2 Comparison samples peak area (0 h) ST Stability samples  peak area (10.30 h) Comparison samples peak area (0 h) ST Stability samples  peak area (10.

30 h) 955509 944548 1244332 1220070 960660 971134 1265453 1260508 954981 954303 1220722 1278003 986337 913571 1281722 1178982 1008136 973937 1328707 1270762 1047670 1057829 1356197 1371250 N 6 6 6 6 Mean 985549 969220 1282856 1263263 SD(±) 36948.83 48591.16 51244.

71 64670.25 CV (%) 3.75 5.01 3.99 5.12 Mean % Accuracy   98.

34   98.47  Conclusion In conclusion, The proposed research work is highly specific due to the inherentselectivity of tandem mass spectrometry and has significant advantages overother described methods in previously. Quantification of Dapagliflozinand Saxagliptin were compared with respective isotope labeled internal standards.Extraction of analyte and IS (Internal Standard)  were achieved by using LLE(Liquid liquidextraction). Linearity range, column, mobile phase, flow rate, injectionvolume, plasma usage volume for analysis was improved. Hence this method hassignificant advantages over previously reported methods in-terms ofSelectivity, sensitivity, Linearity, Reproducibility