Separation of Metformin and Voglibose Using a Thermo Scientific ...

2 Experimental DetailsConsumablesPart NumberFisher Scientific Optima LC/MS grade acetonitrile A955-4Water, from TKA Water Purification SystemFisher Scientific Optima LC/MS Ammonium formate A115-50Metformin hydrochlorideAkums PharmaVogliboseAkums PharmaThermo Scientific Micro+ Vial 300 µL, Fused Insert 60180-507Thermo Scientific 9mm Screw Top Cap W/ PTFE/Silicone septa 60180-516Preparation of SamplesSeparate stock solutions of metformin hydrochloride and voglibose were prepared in 50:50 (v/v) methanol/water at aconcentration of 1 mg/mL. A working standard, containing 1 µg/mL metformin and 2 µg/mL voglibose, was prepared byserial dilution of the above stock solutions in 85:15:0.1 (v/v/v) acetonitrile/water/formic acid.Separation ConditionsInstrumentation:Thermo Scientific Accela 1250 pump, interfaced toboth a Thermo Scientific Accela Open Autosampler,and, a Thermo Scientific TSQ Vantage triple stagequadrupole mass spectrometer.Part NumberColumn: Accucore HILIC 2.6 µm, 50 x 2.1 mm 17526-052130Mobile phase:Flow rate:Column temperature:17:83 (v/v) 100 mM ammonium formate, adjust pHto 3.2 with formic acid/acetonitrile0.4 mL/minAmbientInjection volume: 5 µLSyringe volume: 100 µLLoop Size: 20 µLSyringe flush:Cool stack temperature: 10 °CDetection:Column backpressure:Wash 1: 90:10 (v/v) acetonitrile/waterWash 2: 50:50 (v/v) methanol/waterMS57 barMS ConditionsThermo Scientific TSQ VantageIon Source TypeHESI-2PolarityPositiveSpray Voltage (V) 3000Vaporizer Temperature (°C) 275Sheath Gas Pressure (Arb) 35Ion Sweep Gas Pressure (Arb) 1.0Auxiliary Gas Pressure (Arb) 10Capillary Temperature (°C) 350Declustering Voltage (V) 0Collision Pressure (mTorr) 1.5Table 1: TSQ Vantage ionisation parameters

MS Acquisition ParametersIdentification was performed by selected reaction monitoring (SRM) using the precursor-to-productcombinations shown below:3Compound [M+1] m/z Product m/z Collision energy S-LensMetformin 130.089 71.090 21 56Voglibose 268.157 92.070 21 87Table 2: TSQ Vantage acquisition parametersScan type:Peak width:Scan width:Scan time:MS acquisition time:SRMQ1 - 0.7 (FWHM)Q3 - 0.7 (FWHM)0.02 m/z0.1 s3.5 minutesData ProcessingAll data were processed using Thermo Scientific LCQuan (v. 2.6) software. Algorithm for integration - ICISResultsThe analysis was performed on an Accucore HILIC 2.6 µm, 50 x 2.1 mm HPLC column.As shown in Figure 1, metformin and voglibose were analyzed in less than 3.5 minutes.Table 3 shows the results from six replicate injections.MetforminVogliboseRetention time (minutes) 1.05 (K'=2.9) 1.96 (K'=6.3)%RSD on retention time 0.42 0.52Table 3: Retention time results for metformin and voglibose120120110100RT: 1.05Metformin110100RT: 1.96Voglibose9090Relative Abundance8070605040Relative Abundance807060504030302020101000.00.5 1.0 1.5 2.0 2.5 3.0Time (min)00.00.5 1.0 1.5 2.0 2.5 3.0Time (min)Figure 1: SRM chromatograms of metformin and voglibose on Accucore HILIC 2.6 µm, 50 x 2.1 mm column