the necessity of laboratory analysis to verify the ......the necessity of laboratory analysis to...
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The Necessity of Laboratory Analysis to
Verify the Authenticity of Halal Products
Dr. Shuhaimi MustafaDeputy Director, Putra University
Malaysia
The Second Gulf Conference on Halal Industry and its Services22-24 January, 2013
Farwaniyah, State of Kuwait-Crown Plaza Hotel, Al
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�����ق ا����ر� �رورة ا������ل �ن �����ت ا���ل
�ط��. د� ��������ب ا��د�ر، ���� �ورا، ����ز��
�ل و�د���ا����� �ؤ�ر ا����� � ا����������2013ر 22-24
دو� ا�#و�ت– ا�$روا�� –#روان ��زا!�دق
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Contents
� Background � Current issues in halal products� Halal analysis and authentication� Conclusion
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◌ً ��!ً ط���
Halal – permissible by Shariah Law.
Thoyyib – Good and wholesome (quality , safe, nutritious, pure).
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The needs for analysis
� Religious beliefs� Allergic� Fair-trade� Laidan (2011) stated that a particular Halal-certified
product cannot be genuinely guaranteed halal withoutbeing tested in halal laboratory.
� The following examples justify the need for analysis:� From 902 meat products tested; 15.9% cases for raw product
containing undeclared animal species and 22.9% cases forcooked products containing undeclared animal species.
� ~50% animal feeds contain undeclared animal species.
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Challenges in halal analyses
� No pressure/requirement by relevant authorities.
� Industry wants faster halal certification process
� Complex analytical techniques.
� Lack of competent analysts.
� Undeclared ingredients and processing aids.
� Lack of biomarkers:
- oil/fat-based
- protein-based
- DNA-based
- metabolites-based
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Some methods developed specifically for halal
Technique Target gene/sequence Reference
Conventional PCR 12S rRNA Dalmasso et al. (2004)
Taqman Real-time PCR Genomic Cai et al. (2012)
Molecular Beacon real time PCR Mitochondrial cytochrome b Mohd Yusop et al. (2012)
Commercial Real-time PCR Kit Unknown Demirhan et al. (2012)
Technique Sample Reference
FTIR Shortening Syahariza et al. (2005)
FTIR Gelatin Hashim et al. (2010)
FTIR + PLS Meatball Rohman et al. (2011)
FTIR + chemometrics Ham sausages Xu et al.(2012)
Technique Sample(s) Reference
DSC Cooking oils + lard Mansor et al. (2012)
Technique Sample(s) Reference
MALDI-TOF-MS, LC-MS Porcine gelatine Zhang et al. (2009)
HS-SPME-GC-MS Alcoholic beverage Garcia-Martin et al. (2010)
GCxGC-TOF-MS Lard Indrasti et al. (2010)
GC-MS Pork Nurjuliana et al. (2011)
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Halal Products Analyses
Towards ISO 17025
Accreditation
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Halal products analysis using Real-
Time PCR
� Reliable
� Sensitive
� 0.001ng DNA - SYBR Green
� 0.0001ng DNA - TaqMan
� 0.001% in actual sample
� Fast, specific and cheap.
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1 step, 1 button, ~ 1 hour
Collect Sample
Place in
Test Module
Insert into Analyzer
On one touch
Yes/No Results
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Red line : Internal control; blue line : beef sample (Negative Result)
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Red line : Internal control; blue line : Cat food containing pork (Positive Result)
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Red line : Internal control; blue line : no -template control
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Animal feeds
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PIG HAIR BRUSH-HIGH RESOLUTION STEREOSCOPIC ZOOM
MICROSCOPE
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HIGH RESOLUTION STEREOSCOPIC ZOOM MICROSCOPE
ANIMAL BRUSH
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FTIR SPECTRUM OF
DIFFERENT SYNTHETIC
BRUSH
FTIR SPECTRUM OF
DIFFERENT PIG HAIR
BRUSH
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Amino acid analysis of gelatin using
HPLC and Chemometric approaches
Stages in analysis
Explanation
Acid hydrolysis � hydrochloric acid is the most widely used agent for hydrolyzing
proteins.
� the hydrolysis process occurred in 110OC within 24hrs
Pre-column derivatization
(Using AccQ tag Fluor reagent )
� using 6-aminoquinolyl-N- hydroxysuccinimidyl carbamate derivatives
Quantification separations by HPLC-FD
� All amino acid derivative separations required a gradient
chromatography due to the wide hydrophobicity range of the amino
acid side chains
� use fluorescence detection with excitation at 248nm and emission at
395nm.
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PCA VIEW – SCORES PLOT
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Projection of unknown samples
Gelatin capsules – Nitta (Japan), Gelita (india), Sterling (brazil) & Rousoullet (china).
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Porcine samples
� Porcine (Merck)� Porcine (Fluka)� Porcine skin gelatin� Porcine(Dr.Oetker)� Porcine (Tessenderlon)
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Alcohol in products
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Static Headspace parameters
Parameter Value
Thermostat temperature (ºC) 70
Needle Temperature (ºC) 80
Transfer Line Temperature (ºC) 90
Vial Equilibration Time (min) 10.0
Sample Amount (ml) 5.0
Injection time (min) 0.6
Pressure (psi) 10.0
Pressurise time (min) 1.0
Loop Fill time (min) 0.3
Shaking high
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GC/MS parameters
ParametersChromatographic column DB-WAX, Agilent – J&W122-7032
Carrier gas Helium, 1ml/min
Injector Split type, 250ºCSplit 10 :1
Oven temp. programming 40 ºC (4 min) - 20 ºC/min to 65 ºC (3 min)- 50 ºC/min up to 220 ºC (1 min).
DetectorMS SourceMS QuadElectron EnergyScan modeMS delay parameter
Mass Spectrometry230ºC180ºC70eVmz 30-4501.90 min
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Standard and Sample preparation
Standard preparation GC Standard/ Sample preparation
Sample dilution (if required) 5mL of liquid std/sample was
pipetted into 20mL of headspace vial
5µL of internal standard was pipetted into the vials and capped
immediately
Run sample
Weighting accurately 1 gram of ethanol (purity> 99.9 %) in 100-
mL volumetric flask
Topped up with deionised water, mix well
A series of 7 dilutions (1 ppm –1000 ppm)
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y = 0,0069x + 0,0696
R² = 0,9989
0,0000
1,0000
2,0000
3,0000
4,0000
5,0000
6,0000
7,0000
8,0000
0 200 400 600 800 1000 1200
ET
OH
/AC
N P
EA
K A
RE
A
ETHANOL CONCENTRATION (PPM)
LOW ETHANOL CALIBRATION CURVE
(1 - 1000 PPM)
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y = 0,0001x - 0,0019
R² = 0,9982
0,0000
0,2000
0,4000
0,6000
0,8000
1,0000
1,2000
1,4000
1,6000
0 1000 2000 3000 4000 5000 6000 7000 8000 9000 1000011000
ET
OH
/AC
N P
EA
K A
RE
A
ETHANOL CONCENTRATION (PPM)
HIGH ETHANOL CALIBRATION CURVE
(1000 - 10 000 PPM)
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Calculation of Ethanol Content
� By taking the equation y = 0.0001x - 0.0019, theamount of ethanol present in sample (ex : Sushidipping sauce) can be calculated.
Y = Peak area of Ethanol / Peak area ofAcetonitrile
x = Ethanol Concentration (ppm)� When y = 0.6204, x = 0.6204 + 0.0019/0.0001
= 6222.916 ppm or 0.6223%� Multiply by 10 (1 gm of original sample was diluted
with deionized water up to 10 ml )� So, original sample contains 6.223% w/v ethanol
(considered as HARAM).
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Conclusion
�Halal should take into account beyond just
trust, social, spiritual, environmental, and
sustainability issues through document audit-
based and site visit approaches as practiced
by almost all halal certification bodies, but
must also be verified through laboratory
analysis.
�Are we ready?
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