Stock The autosampler sample stability was evaluated by

Stock solution stability was
performed by comparing the area response of analyte and internal standard in
the stability sample, with the area response of sample prepared from fresh
stock solution. Stability studies in plasma were performed at the LQC and HQC
concentration level using six replicates at each level. Analyte was considered
stable if the % Change is less than 15% as per US FDA guidelines 17.
The stability of spiked human plasma samples stored at room temperature (bench
top stability) was evaluated for 61 h. The stability of spiked human plasma
samples stored at -30 °C in autosampler (autosampler stability) was evaluated
for 70.0 h. The autosampler sample stability was evaluated by comparing the
extracted plasma samples that were injected immediately (time 0 h), with the
samples that were reinjected after storing in the autosampler at 20°C for 70.0
h. The reinjection reproducibility was evaluated by comparing the extracted
plasma samples that were injected immediately (time 0 h), with the samples that
were re-injected after storing in the autosampler at 20°C for 70 h. The
freeze-thaw stability was conducted by comparing the stability samples that had
been frozen at –30 °C and thawed three times, with freshly spiked quality
control samples. Six aliquots each of LQC and HQC concentration levels were
used for the freeze-thaw stability evaluation. For long term stability
evaluation the concentrations obtained after 91 days were compared with initial
concentrations 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 in
spiked human plasma samples

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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 Internal
Standard (DGd5, SGd2) at Refrigerated
Conditions (2-8°C)

Room
temperature stock solution stability was determined using standard stock
solutions of DG, SG, DGd5, SGd2 (ST stability samples) were set aside on the
bench up to 9.5 h and compared with newly prepared stock solutions (comparison
samples).Refrigerated IS (DGd5, SGd2) spiking solution stability (IS stability
samples)  was determined using IS spiking
solutions of DGd5, SGd2 was set aside in refrigerator (2-8°C)  up to ten days and compared newly prepared IS
stock 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 room
temperature

 

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 inherent
selectivity of tandem mass spectrometry and has significant advantages over
other described methods in previously. Quantification of Dapagliflozin
and Saxagliptin were compared with respective isotope labeled internal standards.
Extraction of analyte and IS (Internal Standard)  were achieved by using LLE(Liquid liquid
extraction). Linearity range, column, mobile phase, flow rate, injection
volume, plasma usage volume for analysis was improved. Hence this method has
significant advantages over previously reported methods in-terms of
Selectivity, sensitivity, Linearity, Reproducibility