Extraction of melamine from milk (infant formula) using EVOLUTE® EXPRESS CX solid phase extraction cartridges with analysis by LC-MS/MS

Figure 1: Structure of melamine 

Introduction

This application note describes the extraction of melamine from infant formula using EVOLUTE® EXPRESS CX polymeric mixed-mode strong cation exchange SPE cartridges.
Melamine is traditionally used in making plastics, however, it’s low cost and high nitrogen content has led to exploitation in various sections of the food industry, most notably involving dairy products. The standard test for estimating protein content is based on measurement of nitrogen levels, therefore, addition of melamine to sub standard or watered down milk results in the protein levels appearing higher. Sustained melamine exposure can result in kidney stones and renal failure, with the young being most susceptible.

EVOLUTE® EXPRESS CX SPE cartridges and plates extract a wide range of basic analytes from biological fluids and other aqueous matrices. EVOLUTE® EXPRESS CX removes matrix components such as proteins, salts, non-ionizable interferences and phospholipids, delivering cleaner extracts with reproducible recoveries for accurate quantitation.

Analytes

Melamine

Sample preparation procedure

Format

EVOLUTE® EXPRESS CX 60 mg/ 3 mL (Tables) SPE cartridges, part number 611-0006-BXG.
[Note: This application was originally developed using EVOLUTE® CX 50 mg/3 mL SPE cartridges, p/n 611-0005-B. This format has now been replaced by the EXPRESS format, which provides improved flow characteristics].

Sample pre-treatment

For powdered formula: make up as per manufacturer’s instructions, and cool prior to extraction. Dilute all milk samples (1 mL) with ammonium acetate buffer (1:1, v/v, 50 mM, pH 5).

Cartridge conditioning

Condition cartridge with methanol (3 mL).

Cartridge equilibration

Equilibrate cartridge with ammonium acetate buffer (50 mM, pH 5, 3 mL).

Sample load

Load pre-treated sample (2 mL) at a flow rate of 3 mL /min.

Interference elution 1

Remove polar and ionic interferences with ammonium acetate buffer (50 mM, pH 5, 3 mL).

Interference elution 2

Remove non-polar interferences with methanol (3 mL).

Analyte elution

Elute melamine with methanol containing ammonium hydroxide (95:5, v/v, 3 mL).

Post extraction

Evaporate to dryness at 40°C and reconstitute in acetonitrile/ water (90:10, v/v, 500 μL) prior to analysis.

HPLC conditions

Instrument

Waters 2795 Liquid Handling System

Cartridge

Phenomenex Luna HILIC 3 μm analytical cartridge (100 x 2.0 mm id)

Guard cartridge

Phenomenex Luna Phenyl-Hexyl security guard cartridge

Mobile phase

Isocratic, acetonitrile:20mM Ammonium Formate pH 3.2 (75:25, v/v), flow rate of 0.3 mL/min.

Injection volume

20 μL

Temperature

Ambient

Mass spectrometry conditions

Instrument

Waters Ultima Pt triple quadrupole mass spectrometer equipped with an electrospray interface

Desolvation temperature

350 °C

Ion source temperature

100 °C

Collision gas pressure

2.4 x 10-³ mbar
Positive ions were acquired in the multiple reaction monitoring mode (MRM).

Quantifier ion transition

127 >85 (collision energy 12 eV)

Qualifier ion transition:

127 >68 (collision energy 14 eV)

Results

From the results it can be seen that recoveries from a range of infant formula products were above 85% with <10% relative standard deviations below 10.

References

This application note is based on the poster ‘Extraction of melamine from various matrices using resin based mixed-mode cation exchange SPE and analysis with LC-MS/MS’, L Williams et al, presented at ASMS, Philadelphia, May 31st June 4th, 2009.

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