INTRODUCTION

The detection of horse/porcine meat in halal beef is an important aspect in food quality control as it concerns consumers against certain species of animals such as horse/porcine. The accidental or fraudulent contamination of horse/porcine meat in halal beef is an issue which many scientific and religious institutions have started tackling. Some common analytical methods to detect horse/porcine meat in halal beef are two dimensional sodium dodecyl sulfate - polyacrylamide electrophoresis (2D SDS-PAGE), polymerase chain reaction (PCR), enzyme-linked immunoabsorbent assay (ELISA) and mass spectrometry (MS). An alternative approach as identified by the researchers is the utilization of tandem mass spectrometry (TMS) as a more efficient method in detecting horse/porcine meat in halal beef. The effectiveness of the current analytical methods versus the method in question is determined using the limit of detection (LOD).

CURRENT ANALYTICAL METHODS

There are several well characterized methods for use in detecting foreign meat contamination in halal products, and this includes (but not exclusive to) 2D SDS-PAGE, PCR, ELISA and MS.

Fig. 1 An example of a 2D SDS-PAGE result on chloroplast cyclophilin functions

Fig. 4 HPLC-LC chromatograms depicting traces of horse meat in beef samples

The current LOD values for these methods with reference to the research paper are listed below:

LOD for 2D SDS-PAGE ~ 5%
LOD for PCR ~ 0.5%
LOD for ELISA ~ 0.1%
LOD for HPLC MS/MS ~ 0.13%

MATERIALS

Reagents:

1. 5% Methanol
2. 1% Formic Acid
3. Acetonitrile
4. HPLC Grade Water
5. 6 M Urea Solution
6. Trypsin Digest
7. Promega Sequencing Grade Modified Trypsin
8. Dithiothreitol
9. Dimethyl Sulfoxide
10. Iodoacetamide
11. Liquid Nitrogen
12. Extraction Buffer (0.3 M KCl, 0.15 M KH2PO4, 0.15 M K2HPO4 at pH 6.5)

Tubes:

1. Corning 15 mL Plastic Centrifuge Tube
2. Corning 50 mL Plastic Centrifuge Tube
3. 1.5 mL Eppendorf Reaction Tube

Equipment

1. Thermoshaker
2. Fritsch Pulverisette 14 Motor Mill with 2 mm Sieve
3. GFL 3005 Rotary Lab Shaker
4. Phenomenex Strata-X 33u Polymeric Reversed-Phase Cartridges
5. S-Concentrator BA-VC-300H
6. LTQ Orbitrap XL
7. AB SCIEX TripleTOF 5600
8. Accela HPLC
9. Eksigent nanoLC
10. VWR Hitachi HPLC
11. Turbo V Electrospray Ionization Source
12. Eksigent Ekspert MicroLC 200 System
13. Phenomenex Kinetex C18 (2.6 μm, 100 mm × 2.10 mm) column
14. Thermo Scientific C18 (2.0 μm, 250 mm × 75 μm) column
15. Agilent ZORBAX XDB C18 (3.5 μm, 0.5 × 150 mm) column
16. Eksigent HALO C18 (2.7 μm, 0.5 × 100 mm) column

RISK ASSESSMENT

Risk Assessment

Setting:

Laboratory setting

Process:

Handling and Processing of Various Chemicals in High Performance Liquid Chromatography – Tandem Mass Spectrometry

SAMPLES

Samples Used

Horse (Equus caballus)

Wild Boar (Sus scrofa)

Chicken (Gallus gallus)

Lamb (Ovis aries)

Cattle (Bos primigenius)

Sample Preparation

1. Meat samples were diced and frozen with liquid nitrogen.
2. Upon removal from liquid nitrogen, the meat samples were grounded in the rotor mill.
3. 300 mg of grounded meat samples were weighed and transferred to a tube.
4. 4 mL of extraction buffer was added to the tube.
5. The tube was vortexed and extraction was conducted on the rotary lab shaker.
6. The extracted samples were then centrifuged.
7. 100 μL aliquot of the supernatant was transferred to a 1.5 mL Eppendorf tube.
8. The extraction buffer was removed.
9. The air dried pellet was redissolved in urea solution and trypsinized.
10. The samples undergo reduction and alkylation during this process in which they are then incubated overnight.
11. The digested samples were diluted with HPLC grade water and desalted using polymeric reverse-phase cartridges.
12. The samples were loaded onto the cartridges and washed with methanol and formic acid, followed by acetonitrile and HPLC grade water.
13. The remaining eluate was collected in an Eppendorf tube and DMSO was added.

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.

ANALYTICAL RESULTS

Marker peptide 1 (YDIINLR) is found in both horse and porcine, and is absent in the negative controls, hence it could be used as a combined marker both species. The estimated LOD for porcine meat in beef matrix when detecting marker 1 is about 2% when using HPLC and could be reduced by a factor of 3−4 using micro-LC.

The 3 most intense marker peptides (1 - 3) were used for analytical testing of the meat samples.

Spiking of the commercial beef sample was done to stimulate a contamination of halal meat. In sample G, 0.24% porcine meat was mixed into the beef sample. In sample F, 0.55% of horse meat was mixed into the beef sample.

Multiple Reaction Monitoring (MRM) method was then utilised to break down the sample into smaller fragments to increase sensitivity and enhance signal-to-noise ratio. Both porcine and horse meat contamination could be detected down to a maximum LOD of 0.13%.

DISCUSSION AND CONCLUSION

Previously, separation and analysis of proteins in different species was conducted using 2D gel electrophoresis. However, this method is expensive, tedious and labour-intensive.

Through the use of the 12 biomarker peptides specific for pork/horse meat, HPLC MS/MS was able to detect pork/horse meat contamination in halal beef. This method shows sensitivity that are comparable to current analytical methods such as PCR and ELISA. In addition, the identification of specific biomarker peptides allows for a sensitive and rapid MRM-based method that could be regularly used in QC laboratories.

To conclude, HPLC MS/MS has huge potential to be utilised as an alternative analytical method to PCR and ELISA for the detection of different meat contaminations.