Chemicals

HPLC grade Qualigens (Mumbai, India) trifluoroacetic acid, methanol and acetonitrile, (Pune, India).

Instrumentation

Waters (USA) HPLC2105 system, Waters (USA) photodiode array detector 2487 system, Waters (USA) Empower version 2 software, Scaletec (Vadodara, India) SAB224CL electronic balance, Enertech (Mumbai, India) SE60USultrasonicator, Smis (New Delhi, India) PH-7000 pH meter and Merck (Bangalore, India) Millipore 0.45 microns filter paper.

Conditions of chromatography

Xbridge Phenyl C18 column, 250 × 4.6 mm, 5 μm was used at room temperature with gradient elution occurring at flow rate of 1 ml per min. 0.05% aqueous trifluoroacetic acid (Phase I) and acetonitrile made up the mobile phase (Phase II). The gradient programme was: 0 min (80% Phase I and 20% Phase II), 3 min (50% Phase I and 50% Phase II), 10 min (30% Phase I and 70% Phase II), 13 min (30% Phase I and 70% Phase II), 19 min (80% Phase I and 20% Phase II) and 25 min (80% Phase I and 20% Phase II). In detection and evaluation of 5-BC and 4-BC impurities in DPF, sample volume of 10 µl and wavelength of 240 nm were utilised. For LC–MS study, the same methodology as for HPLC was used. To achieve high sensitivity and a good signal, the ESI source conditions were also tuned. Various conditions such as drying gas flow, nebulizing gas flow, capillary voltage, and spray voltage were tuned to increase sensitivity at low concentrations in order to identify and characterise degradation products.

LC MS study:

1.

Optimised MS Conditions:

Collision energy: 15 V.

Ion spray voltage: 5500 V.

Source temperature: 550 °C.

Drying gas temperature: 120–250 °C.

Collision gas: nitrogen.

Drying gas flow stream: 5 L/min.

Declustering potential: 40 V.

Entrance potential: 10 V.

Exit potential: 7 V.

Dwell time: 1 s.

2.

LC–MS/MS instrument details: LC–MS: Waters Alliance e2695 HPLC coupled to SCIEX QTRAP 5500 mass spectrometer equipped with electrospray ionisation (ESI).

3.

Software: SCIEX.

The mass spectrometer was managed in positive ion electrospray ionisation interface mode.

Solutions of 5-BC and 4-BC impurities

In methanol, a mixed impurity stock solution (0.5%w/v) for 5-BC and 4-BC was made. A series of working solutions were created by adding methanol to the suitable aliquots of the mixed impurity stock solution (0.5% w/v), which ranged in concentration from 0.01 to 0.225% for 5-BC and 0.03 to 0.225% for 4-BC. By combining a suitable amount of mixed impurity stock solution (0.15%w/v concentration) with methanol, a working solution of 5-BC and 4-BC with 0.15%w/v concentration was also created.

PF sample

This methanol-prepared solution had a concentration of 0.5 mg/ml. The DPF sample was sonicated for 20 min and filtered through Millipore 0.45µ microns.

Procedure to evaluate 5-BC and 4-BC impurities in DFP sample

Diluent solution, working solutions of 5-BC and 4-BC, and DPF solution were infused (10 µl) after column equilibration for 30 min. Chromatograms were then recorded using the recommended HPLC procedure. The peak area of 5-BC and 4-BCimpurities in DFP solution and in working solution were documented.

Trails

Inertsil ODS 3 V (150 mm × 4.6 mm, 5 µm), Inertsil ODS (250 mm × 4.6 mm, 5 µm), and Inertsil C8 column (150 mm × 4.6 mm, 5 µm) with isocratic elution using solvents combinations like 0.1% aqueous formic acid/MeOH (methanol) (50:50 ratio), 0.1% aqueous formic acid/MeOH (methanol) (40:60 ratio), and 0.1% trifluoroacetic acid/acetonitrile (40:60 ratio) were tried during trail experiments. A Xbridge Phenyl C18 (250 mm × 4.6 mm, 5 μm) column with gradient elution with 0.05% trifluoroacetic acid/AcN (acetonitrile) combination as mobile phase was also tried. The sample volume for analysis, temperature, and flow rate were all held constant during trials at 10 µl, room temperature, and 1.0 ml/min, respectively. Xbridge Phenyl C18 (250 mm 4.6 mm, 5 m) column with gradient elution using mobile phase 0.05% trifluoroacetic acid (Phase I)/acetonitrile (Phase II) was chosen as the best conditions to identify and estimate 5-BC and 4-BC impurities concurrently in DPF based on resolution, peak shape, and sensitivity values attained during trials. The gradient programme opted was: 0 min (80% Phase I and 20% Phase II), 3 min (50% Phase I and 50% Phase II), 10 min (30% Phase I and 70% Phase II), 13 min (30% Phase I and 70% Phase II), 19 min (80% Phase I and 20% Phase II), and 25 min (80% Phase I and 20% Phase II). 5-BC and 4-BC impurities were studied at 210 nm because this is the wavelength at which they were most sensitive.

Validation

The method for 5-BC and 4-BC impurities evaluation in DPF was proved in harmony through ICH approaches [14].

System suitability

To check suitability of the HPLC system, DPF samples spiked at 0.15% concentration with 5-BC and 4-BC impurities were analysed six times by way of suggested HPLC method.

Specificity

To confirm that the DPF and diluent did not interfere with the analysis of 5-BC and 4-BC impurities, the specificity of this procedure was examined. The recommended HPLC method was used to prepare and analyse the DPF sample (0.5%), each impurity solution (0.15%), solution of DPF spiked with 5-BC and 4-BC impurities (0.15%), and diluent blank and proves that DPF shows no effect on analysis of 5-BC and 4-BC impurities is unaffected by DPF. The blank peak, in contrast, did not overlap the impurity peaks of 5-BC and 4-BC. It is therefore a very selective procedure.

Quantification and detection limits

The quantification and detection limits for 4-BC and 5-BC impurities at concentrations that result in S/N fractions ≥ 10 and ≥ 3, respectively, were confirmed. The quantification limit for 4-BC and 5-BC impurities was 0.00016 ppm and 0.00005 ppm respectively. The detection limits were 0.000053 ppm and 0.0000165 ppm for 4-BC and 5-BC impurities, respectively.

Linearity

The quantification limit level (0.03% for 4-BC impurity and 0.01% for 5-BC impurity) to 150% of the specification quantity limit (0.225% for both 4-BC and 5-BC impurities) was used to confirm the linear quantity range for 4-BC and 5-BC impurities.

Accuracy

Replicates (n = 3) of the DFP sample were spiked with the appropriate concentrations of 4-BC and 5-BC impurities at LOQ levels, 50%, 100%, and 150% of the specified quantity limit.

Robustness

To validate robustness, DPF sample solution spiked with 5-BC (0.15%) and 4-BC (0.15%) impurities was analysed by way of recommended HPLC method with slight dissimilarities in wavelength (± 2 nm) and flowrate (± 0.1 ml per min) and column temperature (± 2 °C).

Precision

The system and method precision were verified by analysing the working solution (0.15% of 5-BC and 0.15% of 4-BC) and DFP sample spiked with 5-BC (0.15%) and 4-BC (0.15%), respectively. The system precision was expressed as mean area response and RSD of six peak area responses of 4-BC impurity and 5-BC impurity. The mean concentration quantified and RSD of six quantified values of the 4-BC impurity and 5-BC impurity were used to express the method’s precision (Fig. 1).

Fig. 1

Typical chromatogram of DFP, 5-BC impurity, and 4-BC impurity

Stability studies

According to ICH guidelines, stress degradation tests of Dapagliflozin were performed in the presence of heat, oxidation, photolytic, and hydrolysis (acid, base, and neutral). Acidic and basic hydrolysis were executed for 24 h in 2N HCl and 2N NaOH, respectively (Figs. 2, 3). Oxidative degradation was carried out at ambient temperature using 30% H2O2 (Fig. 4), and in the photostability chamber, solid drug in the form of thin layer taken in a Petri dish was exposed to a UV lamp (200-Wh/m2) at 240 nm for 7 days (Fig. 5). Sample is thermally degraded by placing the sample in an oven at 105 °C for 6 h (Fig. 6).

Fig. 2

Chromatogram for acid degradation

Fig. 3

Chromatogram for base degradation

Fig. 4

Chromatogram for H2O2 degradation

Fig. 5

Chromatogram for photodegradation

Fig. 6

Chromatogram for thermal degradation