High Throughput Extraction of Melamine using EVOLUTE CX Mixed-mode SPE Plates

Lee Williams and Joanna Caulfield, Biotage GB Ltd, Uppsala, Sweden.

Introduction

The use of mixed-mode solid-phase extraction (SPE) provides significant selectivity advantages compared with non-polar resin-based SPE. Resin-based mixed-mode cation exchange SPE sorbents provide a dual retention mechanism, allowing for the use of a rigorous interference elution regime. This significantly improves extract cleanliness and reduces matrix effects in LC–MS–MS analysis.

This application note from Biotage describes the high-throughput extraction of melamine from biological fluids and various infant formula products using EVOLUTE CX mixed-mode resin-based SPE.

Sample Preparation Procedure

EVOLUTE CX configuration

EVOLUTE CX 25 mg fixed-well plate (part number 601-0025-P01)

EVOLUTE CX procedure

Sample: Sample spiked at 100 ng/mL

Sample pre-treatment: Dilute samples [1:3 (v/v), 100 μL] with 0.05 M NH₄OAc, pH 5 (total analyte load 10 ng). Make up powdered milk according to manufacturer's instructions.

Column conditioning: Methanol (1 mL)

Column equilibration: 0.05 M NH4OAc, pH 5 (1 mL)

Sample application: Apply diluted sample (400 μL)

Interference elution 1: Rinse with 0.05 M NH4OAc, pH 5 (1 mL)

Interference elution 2: Rinse with methanol (1 mL)

Analyte elution: Methanol/NH4OH (95:5 (v/v), 500 μL)

Post extraction: Evaporate to dryness and reconstitute in ACN/water (90:10, 500 μL) for subsequent LC–MS–MS analysis

For general guidelines on the use of EVOLUTE CX SPE plates and columns, request chemistry data sheet

TN139 EVOLUTE CX columns for solid-phase extraction in bioanalysis.

HPLC conditions

Instrument: Waters 2795 liquid handling system (Waters Assoc., Milford, Massachusetts, USA)

Column: LUNA HILIC LC column (100 × 2.0 mm, 3 μ)

Mobile phase: Isocratic 75:25 ACN/0.02 M ammonium formate v/v at pH 3.2 at a flow-rate of 0.3 mL/min

Injection volume: 25 μL

Temperature: Ambient

Mass spectrometry

Instrument: Ultima Pt triple quadrupole mass spectrometer (Waters Assoc., Manchester, UK) equipped with an electrospray interface for mass analysis. Positive ions were acquired in the multiple reaction monitoring mode (MRM) 127.0 > 85.0 and 127.0 > 68.0.

Desolvation temperature: 350 °C

Ion source temperature: 100 °C

Collision gas pressure: 2.4 × 10^–3 mbar

Results

Figure 1

Table 1: Summary of recoveries and RSDs for the extraction of melamine from various sample matrices.

Conclusions

The use of EVOLUTE CX mixed-mode SPE plates provides high recoveries and low RSDs for high throughput analysis of melamine in biological fluids and milk-based infant formula products.

For higher sample volumes or more viscous samples EVOLUTE CX 50 μm is available in larger SPE column formats.

EVOLUTE is a registered trademark of Biotage.

Biotage GB Ltd

Kungsgatan 76, SE-753 18 Uppsala, Sweden

tel. +46 18 56 57 10 fax +46 18 56 57 05

Website: www.biotage.com