HPLC - TANDEM MASS SPECTROMETRY

High Performance Liquid Chromotography

Two HPLC systems were used in the analytical process, namely the LTQ Orbitrap XL system and AB SCIEX TripleTOF 5600 system. The mobile phase used is ACN/H2O (3:97; 0.1% FA).

Column Specifications (Orbitrap):

Phenomenex Kinetex (C18, 2.6 μm, 100 Å 100 × 2.10 mm)

Column Specifications (TripleTOF):

Thermo Scientific (C18, 2 μm, 250 mm × 75 μm)

To further enhance the sensitivity of the analytical method, the Eksigent ekspert microLC 200 system was used with two differing columns to replace the existing HPLC systems.

Column Specifications (Agilent):

ZORBAX XDB (C18, 3.5 μm, 0.5 × 150 mm) - Preferred because of lower backpressure and better retention of relevant peptides

Column Specifications (Eksigent):

HALO (C18, 2.7 μm, 90 Å, 0.5 × 100 mm)

Electrospray Ionisation Source

• Analyte solution is usually introduced to the electrospray source at a flow rate of 1µl min^-1.
• Due to the high potential difference, the analyte solution is vaporized and the droplets are repelled from the needle to the sampling cone on the counter electrode.
• The charged analyte are than sorted based on the mass-to-charge (m/z) ratio.

Overview of Tandem Mass Spectrometry

Myofibrillar and sarcoplasmic fractions were extracted from meat samples derived from cattle, pig, wild boar, horse, chicken and lamb. Beef, lamb and chicken (halal) meat samples were used as negative controls. The meat samples were treated with tryptic digest and analysed.

The results were evaluated with database entries. Several biomarker peptides for horse, beef and pork were identified and used. In addition, a marker peptide specific for beef was used as a positive control in spiking experiments, where horse or pork was spiked into beef matrix.