• Application Note

Oasis 2x4 Method: Proof of Concept

Oasis 2x4 Method: Proof of Concept
  • Waters Corporation
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Blood plasma in gloved hands for PRP therapy

This is an Application Brief and does not contain a detailed Experimental section.

Abstract

This application brief highlights Oasis 2x4 Method which is a viable approach to SPE sorbent and protocol selection.

Introduction

In order to prove that the Oasis 2x4 Method is a viable approach to SPE sorbent and protocol selection, a group of representative small molecules was spiked into rat plasma. The molecules include a base, a quaternary ammonium salt, a neutral, an acid and a strong acid. The spiked plasma samples were then extracted following the Oasis 2x4 Method. All molecules elute in the correct Elute 1 or Elute 2 fractions according to what theory predicted, proving that the method is viable.

Follow the simple steps outlined in this flow chart to achieve high recoveries and the cleanest extracts:

  • Characterize your analyte [Neutral, Acid or Base, pKa].
  • Select one of the four Oasis sorbents.
  • Apply the indicated Protocol [1 or 2].
  • Determine SPE recoveries by LC analysis.

Experimental

10 mg Oasis 96-well Plates

Protocol 1

Condition:

500 μL MeOH

Equilibrate:

500 μL H2O

Load:

500 μL sample (250 μL plasma diluted 1:1 with 4% H3PO4 in H2O)

Wash 1:

500 μL 2% HCOOH in H2O

Elution 1:

2 x 125 μL MeOH

Elution 2:

2 x 125 μL 5% NH4OH in MeOH

Dilution:

250 μL water

Protocol 2

Condition:

500 μL MeOH

Equilibrate:

500 μL H2O

Load:

500 μL sample (250 μL plasma diluted 1:1 with 4% H3PO4 in H2O)

Wash 1:

5% NH4OH in H2O

Elution 1:

2 x 125 μL MeOH

Elution 2:

2 x 125 μL 2% HCOOH in MeOH

Dilution:

5% NH4OH in H2O (To neutralize acid for high pH LC)

Column:

XBridge C18 2.1 x 20 mm IS, 3.5 μm

Mobile Phase A:

10 mM NH4HCO3, pH 10

Mobile Phase B:

10 mM NH4H

Injection Volume:

10.0 μ L

Column Temperature:

Ambient

Detection:

UV @ 254 nm (Prednisone)

Instrumentation:

2777 Sample Manager, 1525μ Binary HPLC Pump, Quattro Premier and 2996 PDA

Gradient

Time (min)

Profile

%A

%B

0

95

5

3.0

5

95

4.8

5

95

5.0

95

5

7.0

95

5

Quattro Premier

Capillary:

3.4 kV

Source Temp.:

120 °C

Desolvation Temp.:

350 °C

Cone Gas Flow:

50 L /Hr

Desolvation Gas Flow:

700 L /Hr

Collision Cell Pressure:

2.59e-3 mbar

MRM Transitions:

Imipramine 281.2 > 85.95 ESI+

Decanesulfonic Acid 220.9 > 79.7 ESI -

Ibuprofen 205.2 > 161 ESI -

Valethamate 306.3 > 162.8 ESI+

Results and Discussion

Elute 2, Primary Analyte Data

Primary Analyte Data

Prednisone SPE Recovery Data

Prednisone SPE Recovery Data

Elute 1, Counter Analyte Data

Elute 1, Counter Analyte Data

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