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 A sensitive and rapid liquid chromatography–tandem mass spectrometry (LC-ESI-MS/MS) method was developed for thesimultaneous determination of dapagliflozin and saxagliptin in human plasmausing their deutereated internal standards.

Sample pre-treatment involved liquid–liquid  extraction on waters oasis HLBcartridges using 100µL of plasma, followed by liquid chromatography on hypersilGold C 18 (50mmx3.0mm,5µm)column.The analytes were eluted within 1.

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62 minutes dapagliflozin (DG), 0.56 minutes deutereateddapagliflozin (DGd2) and 1.94 minutes saxagliptin (SG), 1.93 minutes deutereated saxagliptin (SGd5).usingthe optimized mobile phase 10mM ammoniumacetate: methanol (20:80 v/v) as the mobile phase, at a flow-rate of 0.5mL/minutes and injection volume of 20 µL. The analytes and internal standardswere analyzed in the positive ionization mode and quantified using multiplereaction monitoring.

The method showed excellent linearity over theconcentration range  of 50.00-10000.00pg/mL for both the analytes.The intra-batch and inter batch  precision (%CV)was ?4.5% and theirextraction recoveries were in the range of 95.13% -99.67%.

Matrix effect wasevaluated in terms of internal standard normalized matrix factors (%CV) was 1.27%,1.20% .for both the analytes.The validated method wassuccessfully applied to determine the plasma concentration of the drugs using 5mgsaxagliptin and 10mg dapagliflozin. Key words: saxagliptin, dapagliflozin, LC-ESI-MS/MS, liquid–liquid extraction, Human plasma1. Introduction Type 2 diabetes  is a chronic problem in which blood glucose(sugar) can no longer be regulated. There are two reasons for this.

First, thecells of the body become resistant to insulin (insulin resistant). Insulinworks like a key to let glucose (blood sugar) move out of the blood and intothe cells where it is used as fuel for energy. When the cells become insulinresistant, it requires more and more insulin to move sugar into the cells, andtoo much sugar stays in the blood. Over time, if the cells require more andmore insulin, the pancreas can’t make enough insulin to keep up and begins tofail.

1. Dapagliflozin is indicated for the management of diabetes mellitustype 2, and functions to improve glycemic control in adults when combined withdiet and exercise. Dapagliflozin is a sodium-glucose co transporter 2inhibitor, which prevents glucose reabsorption in the kidney. Chemical name Dapagliflozinis(2S,3R,4R,5S,6R)-2-{4-chloro 3(4ethoxyphenyl)methylphenyl}-(hydroxymethyl)oxane-3,4,5-triol.It has a molecular formula of C21H25ClO6 and a molecular weight of 408.

13. Saxagliptinis an orally active hypoglycemic of the new dipeptidyl peptidase-4 (DPP-4)inhibitor class of drugs. Chemical name for Saxagliptin is(1S,3S,5S)-2-(2S)-Amino(3-hydroxytricyclo3.3.1.1(3,7)dec-1-yl)acetyl-2-azabicyclo3.1.0hexane-3-carbonitrilemono hydrochloride.

It has a molecular formula of C18H26ClN3O2 and a molecularweight of 351.87. 2.Literature survey revealed that no method have beenreported for the simultaneous determination of Dapagliflozin and Saxagliptin inhuman plasma by LC-ESI-MS/MS.  Fewmethods reported are Development and stability indicating HPLC method fordapagliflozin in API and pharmaceutical dosage form.3.

Validated LC-MS/MSmethods for the determination of dapagliflozin, a sodium-glucose co-transporter2 inhibitor in normal and ZDF rat plasma.4. simultaneous estimation ofdapagliflozin in api and pharmaceutical dos age form by development andstability indicating hplc method.

5. A validated stability indicatinghigh-performance liquid chromatographic method for simultaneous determinationof metformin HCL and dapagliflozin in bulk drug and tablet dosage form6.LC-MS/MS analysis of metformin, saxagliptin and 5-hydroxy saxagliptin in humanplasma and its pharmacokinetic study with a fixed-dose formulation in healthyIndian subjects: Quantitation of metformin, saxagliptin and 5-hydroxysaxagliptin 7.

Stability indicating RP-LC-PDA method for the quantitativeanalysis of saxagliptin in pharmaceutical dosage form. 8. Development of arapid UPLC-MS/MS method for quantification of saxagliptin in rat plasma andapplication to pharmacokinetic study.9. Bioanalytical method validationincludes all of the procedures that demonstrate that a particular method usedfor quantitative measurement of analytes in a given biological matrix, such asblood, plasma, serum, or urine, is reliable and reproducible for the intendeduse. 10. The main goal ofthe present study is to develop and validate the novel simple, sensitive,selective, rapid, rugged and reproducible analytical LC-MS/MS method inpositive ionization mode method for quantitative determination of Dapagliflozin andSaxagliptin in human plasma by LC-MS/MS with a small amount of sample volume.

Themethod offers the advantage of  simpleextraction method, with a highly sensitive, good linear method with the smallamount of plasma usage.2.Experimental2.1.Chemicals and materialsThe drugs Dapagliflozin, Saxagliptin, Dapagliflozin d5 and Saxagliptin d2 were procured as a gift samples from Symed labs,Hyderabad, India and Toronto research chemicals, Canada. Ethyl acetate, HPLCgrade methanol and acetonitrile were purchased from J.

T. Baker USA. Sodiumdihydrogen phosphate (NaH2PO4, reagent grade), Ammoniumacetate (reagent grade) was purchased from Merck Limited, Worli, Mumbai. Humanplasma was obtained from Doctors labs, Hyderabad, India. Ultra pure water fromMilliQ-system (Millipore) was used through the study.

2.2. Liquid chromatography and massspectrometry conditionsAn API 4000HPLC-ESI-MS/MS system (Applied Biosystems), 1200 Series HPLC system (AgilentTechnologies, Waldron,Germany), data acquisition and processing were accomplished using Analyst® Software 1.4.1. Thechromatographic separation of the analytes was carried out at 30ºc using HypersilGold C18 (50mm x 3.

0 mm, 5µm) column.A mixture of 10mM ammonium acetate: Methanol (20:80 v/v) was used as mobile phase. 0.5 mL/minutes and injection volume of20 µL. Liquid-liquid extraction was chosen to optimize the drug and internal standardtriple quadrapole mass spectrometer equipped with electro spray ionization andoperated in positive ionization mode was used for detection and quantificationof analytes and internal standards.

The optimization of the source and compoundparameters were optimized as Declustering potential: 40V, entrance potential: 10V,exit Potential: 7, collision energy: 15V for Dapagliflozin and16V for Saxagliptin .The source parameters were optimized as collision gas:5, ion spray voltage: 5500V and temperature: 550ºC.Themass transitions are shown in (Fig.1-4).

Literature survey reveals that only limited methods were developed forQuantification of Dapagliflozin and Saxagliptin by using LC-MS/MS Bulk Drug andTablet Dosageform. It is important to develop the good bio analytical methodwith proper deuterated or analogue based internal standards in terms of matrixeffect and reproducibility. Moreover, Should not consider the runtime always tominimize the analysis rather than reproducibility and stability for longanalytical batches. The reportedmethodswerenotcosteffectiveduetouse of costlysolventsand highly volatile solvents and the retention timewas foundto be more.

So inthe present work is a novel, simple, sensitive andstabilityindicating method developed by using low-cost solvent 10mM ammonium acetate:Methanol (20:80 v/v),which was highly sensitive to detect ata lowerconcentration andlesser retention time of drugs.


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