1. Precipitation of proteins
2. Extraction of liquids
3. Extraction in solid phase

• Analyte extraction with a suitable solvent
• Removal of compounds which are interfered
• Concentration of analyte before.

Chemical Structure:

Synonym: (2S, 3R, 4R, 5S, 6R)-2-(4-chloro-3-(4-ethoxybenzyl) phenyl)-6- (hydroxymethyl) tetrahydro-2H-pyran-3, 4, 5-triol

IUPAC name: (2S, 3R, 4R, 5S, 6R)-2-{4-chloro-3-[(4-ethoxyphenyl) methyl] phenyl}-6-(hydroxymethyl) oxane-3, 4, 5-triol.

Solubility: Soluble in water

Description: Drug remains pale yellow in color. It prevents glucose re-absorption in kidney.

Storage: Stored at 20˚C. Kept in a firmly locked container.

Molecular Weight: 413.9 g/mol

Dapagliflozin D₅ [5]

Molecular Formula: C21H20CID5O6

Chemical Structure:

Synonym: (1S)-1, 5-anhydro-1-C- [4-chloro-3- [[4-(Ethoxy-1, 1,2,2,2, d5) Phenyl] methyl] phenyl]-D-glucitol

Chemical Name: Dapagliflozin D5

Solubility: To some extent Solvable in methanol also DMSO.

Description: White solid

Storage: Stored at -20^oC in inert condition

Preparation of reagents: Dissolve 1:1 ratio of methanol and water in appropriate container. Label and store at room temperature.

Mobile Phase Buffer:

Weigh about 315.5 mg of NH₄CO₂ to 1000 ml volumetric flask with a small amount of HPLC grade water and mix well. Then 1 ml of NH₄OH was added and finally make up with the same solvent.

Reconstitution Solution [Acetonitrile/Mobile Phase Buffer (70:30 v/v): 7:3 ratio of Acetonitile and mobile phase buffer to 1000 ml volumetric flask was prepared and kept at room temperature.

Auto sampler Rinsing solution: 1:1 ratio of Acetonitrile and HPLC grade water was mixed and stored at room temperature.

Internal Standard Solution: 1 mg of Dapagliflozin into 1ml DMSO and methanol. Store at 2-8^oC. Protected from light.

ISTD Working solution (2ng/ml): 20 ul of ISTD stock (1 mg/ml) into 100ml volumetric flask with diluent. Store at 2-8^oC.

Linearity determination:

Preparation of stock and spiking solution (1mg/ml)

10 mg of Dapagliflozin is transferred into 10 ml vol. flask, add 2.5ml of DMSO and make up to mark with methanol. Store at 2-8^oC (Table 1).

Preparation of Quality Control and Stabilization Samples Preparation of QC stock solution (1.000 mg/ml):

Weighed and transferred 10 mg of Dapagliflozin into 10 ml vol. flask, dissolved in 2.5 ml of DMSO, and made up through methanol. It was mixed well, labelled and kept at 2-8°C, protected from light.

Preparation of QC Spiking Solutions

Quality Control spiking solutions was set (1.000 mg/ml) as designated in the following Table 3.

Optimized chromatographic condition (Table 4)



Compound dependent parameters (Table 6)

Mass spectra’s (Figures 1 & 2)

Extracted Sample Preparation:

Extraction Technique: SPE

Arranged the pre-labeled unfilled tubes as per the lot order and added 50 µl of ISTD working solution, except in STD Blank. Transferred 300.000 µl of plasma from Step-1 into altogether tubes and vortexed for about 10 sec. Then added 200.000 µl of HPLC grade water into all the tubes and vortexed for around 05 sec. Arranged the mandatory number of OASIS HLB cartridges (30 mg/1ml, 1CC) on solid phase extraction manifolds. Conditioned the cartridges with methanol (1ml), 1ml HPLC grade water at low manifold pressure. Loaded about 550.000 µl of prepared samples on conditioned cartridges carefully then washed the cartridges with 1ml HPLC grade water. Dried at high manifold pressure for 2 min eluted the contents with 1.000 ml of Acetonitrile and collected the elution into prelabelled vials. Evaporated all the samples to aridness beneath nitrogen gas at 40 ± 5^oC. Then 200 ul of reconstituted solution is injected into LC-MS/MS.

Method Validation (Figures 3 & 4)


Linearity (Table 7)

Calibration curve (Figure 5)

Sensitivity

The concentration was kept at 1.010 min for Dapagliflozin was found to be 8.13 % and 102.59 % (Table 8).

Precision and Accuracy

Batch Precision and Accuracy

Precision of Dapaglifozin within for LLOQ QC, QCL, QCM-1, QCM-2, QCM-3 and QCH extended from 2.59% to 5.11%, 3.98% to 7.54%, 5.7% to 6.79%, 5.57% to 5.69%, 2.19% to 2.92%, 1.90% to 3.02% respectively. Accuracy within batch for LLOQ QC, QCL, QCM-1, QCM-2, QCM-3 and QCH extended from 100.47% to 106.01%, 95.49% to 103.59%, 96.01% to 98.16%, 99.88 % to 100.32%, 107.10% to 110.26%, 98.23% to 102.81% respectively.

Stability studies

Extended period stock solution stability for Analyte and ISTD (2-8ºC)

It was done for 7 days 14 h with 19958.425 ng/ml of Dapagliflozin and 101.365ng/ml internal standard (Table 10).

Long term spiking solution stability for Analyte and ISTD (2-8ºC)

Study was carried at 6 days 19 (Table 11).

Reinjection stability

For this study, six sets of QC samples (QCL, QCM 2 and QCH) were analyzed. The % stability at 48 h extended from 98.14 % to 101.33% and CV extended from 1.18% to 8.30% correspondingly (Table 12).

Interday Precision and Accuracy (Table 9)

Recovery

Six groups of QCL, QCM-1 and QCH samples were treated and injected (extracted samples). The overall mean recovery of Dapagliflozin was 85.75% with CV extending from 1.31 % to 4.81%. The overall mean recovery of internal standard was 84.14 % with CV extending from 0.87% to 2.89% respectively (Tables 13 & 14).


Carryover Test (Figures 6-10)

RESULTS AND DISCUSSION

The study is to develop and validate a rapid LC-MS/MS assay of Dapaglifozin in human plasma. The technique was evaluated on the basis of precision, linearity, accuracy, recovery, selectivity and carry over test. The final chromatographic separation was done on a mixture of acetonitrile: buffer (70:30 v/v) with 1.2ml flow rate per mts. The time period for separation is about 1.7 min for analyte and internal standard.

CONCLUSION

A rapid, small volume LC-MS/MS was developed validated, with high precision, accuracy for plasma quantitation in Dapagliflozin. The simple and high quality easy automated clinically proven studies are carried. The results prove that Dapagliflozin and ISTD continue in autosampler for 67 h 15mts without any loss and the sample is analyzed within the time.

ACKNOWLEDGEMENT

I am very grateful to SIMS College of Pharmacy management for supporting me and providing everything without which it is not possible.

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  Table 1
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  Table 2
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  Table 3
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  Table 4
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  Table 5
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  Table 6
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  Table 7
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  Table 8
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  Table 9
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  Table 10
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  Table 11
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  Table 12
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  Table 13
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  Table 14