3.1. Dapagliflozin (DG) 100.00 µg/mL 25 mL Methanol Saxagliptin

3.1.

Reagents and Solvents PreparationAll the solvents used in this research wereHPLC grade and regents were AR-grade. The volumes and concentrations werementioned in the process are theoretical. The volumes and concentrations couldbe corrected on the basis of actual weights used.Table-1:Reagents and solvents preparation of DG and SG Name of the solution Preparation Extraction solvent Ethyl acetate (HPLC grade) Mobile phase 10mM ammonium acetate: Methanol (20:80 v/v) Autosampler wash Methanol Reconstitution solution 10mM ammonium acetate: Methanol (20:80 v/v) 50 % methanol 50mL of HPLC grade water and 50mL of HPLC grade methanol Extraction buffer 5mM NaH2PO4 solution  Table-2:  Stock solution preparation of DG, SG, DGd5and SGd2 Name of the stock solution Concentration Volume (mL) Diluent Dapagliflozin (DG) 100.

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00 µg/mL 25 mL Methanol Saxagliptin (SG) 100.00 µg/mL 25 mL Methanol Dapagliflozin d5 (DGd5) 100.00 µg/mL 25 mL Methanol Saxagliptin d2 (SGd2) 100.00 µg/mL 25 mL Methanol  3.

2. Standard Calibration (CC) and Quality control(QC) Samples PreparationStandard Stock solutions of DG (100.0 µg.mL-1),DGd5 (100.0 µg.mL-1), SG (100.

0 µg.mL-1), SGd2 (100.0µg.

mL-1) were prepared in methanol. From each stock solution 100.0ng.mL-1 intermediate dilution was prepared in plasma.

Aliquots of100.0 ng.mL-1 were used to spike blank human plasma in order toobtain calibration curve standards of 50.0, 100.

0, 500.0, 1000.0, 2000.0,4000.0, 6000.0, 8000.

0, 10000.0 pg/mL. Four levels of QC concentrations at50.0, 150.0, 3000.0 and 7000.0 pg/mL (LLOQ, LQC, MQC and HQC) were prepared byusing the different plasma.

Spiked calibration curve standards and Qualitycontrol standards were stored at -30oC. Standard stock solutions of DGd5,SGd2 (100.0 µg.mL-1) were prepared in methanol . DGd5 andSGd2 was further diluted to 10.

0 ng.mL-1 (Spikedconcentration of internal standard) using 50 % methanol and stored in therefrigerator 2-8 0C until analysis.4. BIOANALYTICAL METHOD DEVELOPMENTDuring bioanalyticalmethod development HPLC,mass spectrometric and sample extraction parameters were optimized to achievethe best results.4.1. Development of HPLC ParametersDuring the development stage HPLC conditionslike column, mobile phase and internal standard were optimized in a logical andsequential manner. 4.

2. Development of MassParametersThe MS optimization was performed by direct infusion of solutionsof SG, DG, DGd5 and SGd2 into the ESI source of the mass spectrometer. Thevital parameters like ionization type, temperature, voltage, gas parameterssuch as nebulizer and heater gases, compound parameters like DP, EP, FP, CE andCXP were optimized to obtain a better spray shape and ionization to form therespective productions from the protonated SG, DG, DGd5 and SGd2 molecules .The masstransitions were selected as m/z 410.2/250.

6,415.3/250.6, 316.1/272.4 and m/z 318.2/272.3 for quantification of DG,DGd5, SG and SGd2 respectively.4.

3. Development of Extraction Solvent and Buffer Various organic solvents and buffers wereoptimized to extract DG and SG from plasmasamples. After a series of trials, ethyl acetate and 5mM NaH2PO4buffer were selected as appropriate due to high recovery efficiency andmatrix free interference. Sample Extraction and Cleanup Procedure (SamplePreparation)Liquid-liquid extraction was carried out to extract thedrug and IS for this purpose 100 µL of respective concentration of plasmasample was taken into polypropylene tubes and mixed with 50µL of internal standard (10.0 ng.mL-1). Thiswas followed by addition of 100 mL of 5mM NaH2PO4solution and 3.0 mL of ethyl acetateand vortexed for approximately 10 minutes.

Then the Samples were centrifuged at4000 rpm for 10 minutes at 20°C.Further, the supernatant wastransferred into labeled polypropylenetubes and evaporated with nitrogen gas at 40°C.  Then the samples werereconstituted with the mobile phase and vortexed for 2 minutes.  Finally, Sample was transferred into autosampler vials to inject into the LC-MS/MS.4.

4. Development of Calibration Curve and RegressionModel The developed standard curve shows correlationcoefficient (r2) greater than 0.9993 with linearity range of50.00-10000.00 pg/mL using  the linearregression model.y = ax + b; Where, y= Peak arearatio of analyte  ,X = Concentration (pg/mL) ofanalyte in plasma, a =Slope, b = Intercept, r2= Correlation coefficient .5.BIOANALYTICAL METHOD VALIDATION The method was validatedaccording to US food and drug administration bioanalytical method validationguidelines  includes systemsuitability, selectivity and specificity, LOQ (limit of quantification orsensitivity), injector carryover, linearity, precision and accuracy (P&A;Intra day & Inter day), recovery, matrix effect, dilution integrity, re-injectionreproducibility, ruggedness (analyst and column), sample stability studiesinclude auto sampler, freeze-thaw, bench top, long term in plasma and shortterm stock solution stabilities were carried out during method validation.Representativechromatograms of plasma blank, blank with IS, lowerlimit QC, low QC, mid QC, high QC, upper limit QC samples andcalibration curve of the GP and ES were depictedin Figure 5 to .