Fast Extraction of Metanephrine and Normetanephrine from Urine Using EVOLUTE® EXPRESS CX Prior to LC–MS–MS Analysis

Frank Kero and Victor Vandell, Biotage LLC

A new solid-phase extraction (SPE) technology has been developed to speed up sample preparation by eliminating steps in the extraction workflow. This application note describes the extraction of metanephrine and normetanephrine from synthetic urine using this 'load-wash-elute' approach with EVOLUTE EXPRESS CX 96-well SPE plates. Recoveries exceeding 80% and excellent linearity over the range 10–70 ng/mL are achieved.

Extraction Conditions

Plate configuration: EVOLUTE EXPRESS CX 30 mg plate p/n 601-0030-PX01

Sample pre-treatment: Dilute synthetic urine sample with water (1:1, v/v).

Sample loading: Load pre-treated sample (1 mL) at a flow rate of 1 mL/min using positive pressure (PRESSURE + 96 Positive Pressure Manifold, p/n PPM-96). Note that plate conditioning and equilibration steps are not required.

Interference wash 1: Water (1 mL)

Interference wash 2: Methanol (1 mL)

Analyte elution: Methanol–concentrated ammonium hydroxide (95:5, v/v, 1 mL).

Post extraction: Evaporate extracts to dryness (SPE Dry Dual Sample Concentrator, p/n SD-9600-DHS) and reconstitute in 80/20 (v/v) mobile phase A–mobile phase B (500 μL).

HPLC Conditions

Instrument: Agilent 1200 Liquid Handling System (Agilent Technologies, Berkshire, UK)

Column: Organic Acids 150 mm × 4.6 mm (5 μm) (Restek)

Mobile phase A: 1% (v/v) formic acid in water

Mobile phase B: Acetonitrile

Gradient:

Mass Spectrometry Conditions

Instrument: Applied Biosystems/MDS Sciex 4000 Q-Trap triple quadrupole mass spectrometer (Applied Biosystems, Foster City, California, USA) equipped with a Turbo Ionspray interface.

Monitored transitions: Metanephrine 198>165; normetanephrine 185>166.

Results

Figure 1 shows the extracted ion chromatogram for metanephrine and normetanephrine from a 20 ng/mL fortified synthetic urine sample. The method demonstrated high (>80%), reproducible recoveries of both analytes, with excellent linearity (r² >0.99) over the range 10–70 ng/mL.

Figure 1: Extracted ion chromatogram for metanephrine and normetanephrine (20 ng/mL fortified urine sample).

Conclusions

The load-wash-elute procedure utilizing EVOLUTE EXPRESS CX plates described in this application note proved fast, simple, and effective for the extraction of the catecholamine metabolites, metanephrine and normetanephrine, from synthetic urine samples. Elimination of the traditional conditioning and equilibration steps speeds up the sample preparation process, without compromising on analytical quality.

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