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Chemical Engineering

Halal Testing and Gelatin Screening using LC-MS/MS

By: Tan Shin Jowi, PhD

SCIEX has 50 years of innovation which is shown in the timetable below.

SCIEX present in all around the world which divided into four types of workspace;
headquarter, research and development, manufacturing, and key sales offices.
 Things that Mass Spectrometry can analyze is molecules, such as pesticides, antibiotics,
hormones, petro oils, explosive, proteins, RNA/DNA, pharmaceuticals, sugars, lipids, fats,
drugs of abuse, and residues. Matrices, such as plasma, hair, urine, blood, soil, makeup, bread,
meat, cookies, cannot be analyzed using LC-MS, but the compound of those substances can be
analyzed. Below are the applications of Mass Spectrometry:
• Forensic (Opiates, benzodiazepines, THC)
• Food testing (pesticides, nitrofuranes, mycotoxins, amino acids, vitamins)
• Environmental (pesticides, perchlorate, HAAs, PPCPs)
• Pharma applications (DMPK, ADME, Microdosing, impurity)
• Clinical research (studying inborn errors of metabolism, research into therapeutic
drug efficacy)
• Chemistry application

The advantages of LC-MS/MS analysis:

• Specificity (Mass based (m/z) vs non-specific (UV-VIS, fluorescence), ELS)
• Selectivity (ability to discriminate against matrix, background, other chemical
species)
• Sensitivity
• Speed
• Throughput (short analysis times and multiple compounds in single analysis)

How Mass Spectrometry works:

1. Molecules are ionized
2. The ions are sorted by m/z
3. The signal is detected and counted
4. The results are displayed

Before the molecules ionized in the Mass Spectrometry, it needs to be converted. Here’s
how it converted.
1. Formation of changed droplets
2. Evaporation
3. Ionic repulsion with cluster formation
4. Entry of ions into the mass analyzer
 Types of Mass Spectrometry Systems:
• Nominal Mass (Target known screening)
1. Triple Quad
a. Quantitation
b. ID with MRM ratio
2. Q TRAP (Target known screening, known unknown)
a. Quantitation
b. ID with MRM ratio
c. ID with MS/MS library searching

• High Resolution
1. X500R QTOF (Target known screening, known unknown, unknown screening)
a. Quantitation
b. ID with accurate mass
c. ID with MS/MS library searching
d. ID true unknowns
e. Retrospective data processing

Qualitative LC-MS/MS Analysis for Meat Speciation in Raw and Processed Meat
Methods for meat speciation:
1. PCR (Only able to perform 1 test per species, fail to detect processed meat)
2. ELISA (not multi-target friendly, poor specificity)
3. LC-MS/MS (highly specific, good sensitivity, processed and unprocessed meat
speciation, and multiple meat detection in a single analysis)

Selection of signature meat markers:

1. Specificity for each meat species
2. Uniqueness compared to background proteins
3. Sensitivity of detection
4. Ability to be detected in both raw and processed meat samples

Markers discovery:
1. Protein digestion and peptide mapping analysis
 2. ProteinPilot database search
3. BLASTp/skyline (algorithm to check the database more thoroughly)
4. Unique/signature peptide selection

Meat preparation before being analyzed:

1. Buy the meat
2. Grind the meat
3. Homogenization and protein extraction (1 gr meat with 5 mL buffer, 5
mins/sample)
4. Reduction and cysteines blocking (TCEP – 60℃ for 1 h)
5. Protein digestion (add trypsin and incubate at 37℃ for 4 hours or overnight)
6. Sample clean-up (60 mg SPE cartridge)
7. LC-MS/MS analysis

LC-MS/MS Analysis for Gelatin

LC-MS/MS Analysis for Gelatin can be done for capsule, yogurt, ice cream,
marshmallow, jelly, etc.
Methods:
1. PCR (Only able to perform 1 test per species, fail to detect processed meat)
2. ELISA (not multi-target friendly, poor specificity)
3. LC-MS/MS (highly specific, good sensitivity, and multiple gelatin detection in a
single analysis)

Steps:
1. Dissolve gelatin at 37
2. Microwave for 5 mins
3. LC-MS/MS detection
4. Analysis using SCIEX OS Software