• Application Note

Simultaneous Extraction of Metformin, Linagliptin and Empagliflozin From Human Plasma Using Mixed-Mode SPE with Oasis (WCX) and Analysis by LC-MS/MS

Simultaneous Extraction of Metformin, Linagliptin and Empagliflozin From Human Plasma Using Mixed-Mode SPE with Oasis (WCX) and Analysis by LC-MS/MS
  • P.M.N. Rajesh
  • Padmakar Wagh
  • Waters Corporation
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This is an Application Brief and does not contain a detailed Experimental section.

Abstract

This application highlights simultaneous analysis of Metformin, Linagliptin, and Empagliflozin from plasma using a single SPE extraction method with Oasis mixed-mode WCX Cartridges and subsequent LC-MS/MS analysis.

Benefits

Efficient extraction for LC-MS/MS analysis.

Introduction

The combination of Metformin, Linagliptin, and Empagliflozin is available as tablets formulation for oral use in diabetes. Metformin introduced in 1950 as glucose-lowering agents to treat non-insulin-dependent diabetes mellitus. It reduces elevated blood glucose concentration in diabetic patients, but it does not increase insulin secretion. Empagliflozin is an oral , potent, and selective inhibitors  of sodium glucose contranspoter 2, inhibition of which reduces renal glucose cotransporter 2, inhibition of which reduces renal glucose reabsorption, and results in increased urinary reabsorption and increased urinary glucose extraction. Linagliptin is an oral inhibitor of dipeptidyl peptidase-4 approved for treatment of type 2 diabetes Metformin, Linagliptin, and Empagliflozin. These drugs are generally prescribed in multi-component dosage forms, which are available in the market. In view of this, a simple, precise, and accurate LC-MS/MS method for the simultaneous estimation in pharmaceutical dosage forms by reverse phase high performance liquid chromatography with mass spectrometry has been developed.

Experimental

Experiment details

System:

ACQUITY UPLC I-Class with Xevo TQ-S micro Mass Spectrometer

Column:

ACQUITY UPLC HSS Cyano (CN) 1.8 μm, 2.1 mm x 50 mm

Mobile phase A:

2 mM Ammonium acetate

Mobile phase B:

Acetonitrile

Flow rate:

0.4 mL/min

Column temp.:

40 °C

Injection volume:

2 μL

Ionization mode:

ESI+

Gradient

Time (min)

%A

%B

0

90

10

0.4

90

10

1.5

5

95

2.5

5

95

3

50

50

4

50

50

4.5

90

10
Table 1. MRM transitions for metformin, linagliptin, and empagliflozin.

Sample preparation

For the extraction of Metformin, Linagliptin, and Empagliflozin from plasma, a mixed-mode  solid phase extraction (SPE) clean-up strategy with Oasis  WCX Cartridges was employed. Spiked plasma samples (200 µL) were precipitated with diluted ammonia solution (400 µL) and the sample supernatant (500 µL) was loaded  onto a pre-conditioned Oasis WCX Cartridge using the SPE extraction protocol shown below.

Table 2. SPE protocol for sample extraction.
Table 3. Sample extraction recovery.
Figure 1. Extracted chromatograms of Metformin (A), Linagliptin (B) and Empagliflozin (C).

Conclusion

Successful simultaneous analysis of Metformin, Linagliptin, and Empagliflozin from plasma was achieved using a single  SPE extraction method with Oasis mixed-mode WCX Cartridges and subsequent LC-MS/MS analysis. 

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