TODAY FOOD TESTING IN VIETNAM -
DIFFICULTIES AND CHALLENGES
Chu Pham Ngoc Son
Chemical Society of Ho Chi Minh City
Chu Pham Ngoc Son, Pham thi Anh, Diep ngoc Suong
Chemical Society of Vietnam,
Sac Ky Hai Dang Science-Technology Service Company
S.1
I. INTRODUCTION
II. HARMFUL CHEMICALS POSSIBLY FOUND IN FOODS
I. DIFFICULTIES AND CHALLENGES IN FOOD AND FEED
TESTING
III.1. Sample preparation
III.2. Detection and quantification
III.3. Issues related to actual local food production
IV. CONCLUSIONS
S.2
Food analysis, an area in continuous evolution:
* increasing demand of consumers for food safety and quality,
* food authority desire to ensure safe food with highest nutritional quality,
* effort of producers to meet these demands.
Globalization of safe food supply and continuous improvement of human
living standards require rigorous regulations and intensive testing ( food safety
and authenticity assessment, detection of molecules with possible beneficial or toxic
effect on human health)
Risk may be quite acute for food and feed being produced in countries with
less stringent monitoring and less advanced infrastructure (water, electricity,
sanitation, storage, transport, cold chains…).
I. INTRODUCTION
S. 3
Misslabeling and deliberate substitution of permitted food additives by
cheaper and possibly harmful alternatives for illegal profit were detected in
local food:
*melamine in milk and milk products,
*sugar syrup in honey, roasted maize or soybean in coffee,
*di-ethylhexyl phthalate DEHP as a clouding agent,
*non-food azo dyes ( Sudan dyes, 2,4-diaminoazobenzene, Orange II… added
to various types of food to give a more golden or more orange or bright red
attractive aspect
*fluorescent whitening Tinopal in rice noodles
Rapid urbanization and industrialization in developing countries, wastes
from various food industries and farms without appropriate management
adversely affect food safety and the quality of the natural environment
leaving people somewhat distrustful of their food.
In the last few years, Vietnam food quality for exportation was improved
because of more severe management and efficient control. Local food safety
however is still an issue to be solved.
S.4
For food safety management, two issues remain to be solved: *large quantities of chemicals and foods of unknown source continuously
smuggled into the country
*Inside the country, food and industry grade chemicals still freely sold
together in local markets, without proper management, leading to easy abuse
of possibly hazardous chemicals for illegal profit
S.5
Food safety issues:
*necessity of continuous improvement and development of powerful analytical techniques
for food analysis;
*Intensive food testing may produce negative effects on health and environment ( use of
large quantity of toxic solvents and reagents, uncontrolled disposal of chemical waste).
*Safer and more efficient analytical methods have to be devised to ensure food safety while
complying with the urgent need of environmental preservation and health protection, the
outcome is:
- professional achievement:
+identification and quantitation of all contaminants in food samples
+low MRL for contaminants in complex food and feed matrices
- environmental benefit:
+substantial reduction or elimination of dangerous solvents and reagents,
+less energy and less time expense,
+large decrease of waste, preventing pollution at the source rather than dealing with costly
remediation afterwards (eco-friendliness).
THIS IS GREEN ANALYTICAL CHEMISTRY, BEING MORE
AND MORE DEVELOPED IN FOOD SAFETY CONTROL
LABORATORIES
S.6
II.1.PATHOGENIC MICROORGANISMS
THERMOTOLERANT COLIFORMS
SALMONELLA SHIGELLA
E. COLI STAPHYLOCOCCUS AUREUS
CLOSTRIDIUM PERFRINGENS
LISTERIA MONOCYTOGENES VIBRIO CHOLERA
VIBRIO PARAHAEMOLYTICUS
BACILLUS CEREUS
FUNGI
VIRUSES
BACTERIA
S.7
Street foods most easily
threathened
II. POSSIBLE CAUSES OF FOOD INTOXICATION
II.2.HARMFUL CHEMICALS POSSIBLY FOUND IN FOODS
II.2.1 Natural toxins (cyanogenic glycosides in bamboo shoots, manioc;
tetrodotoxin in puffer fish and some other sea products; marine biotoxins in
bivalves such as ASP, DSP, PSP; mycotoxins ; histamine and other biogenic
amines...).
II.2.2. Pesticides being forbidden or used beyond the permitted limits (organochlorides, organophosphates, carbamates, pyrethroids, triazines,
trifluralin…) Plant growth promoters (giberellic acid, cytokinin,α-naphthalene
acetic acid…)
tetrodotoxin
aflatoxins
Cyanogenic
glycosides
pesticides
Aflatoxins
Tetrodotoxin
S.8
fungicides
II.2.3. Veterinary drugs being forbidden or used beyond the
permitted limits: ( tetracyclines, sulfonamides, aminoglycosides, chloramphenicol, malachite
green, crystal violet, nitrofurans, fluoroquinolones, nitroimidazoles, β-
agonists such as clenbuterol, salbutamol, ractopamine...).
II.2.4. Food additives used beyond the permitted limits (preservatives, food colors, synthetic sweeteners such as aspartame,
saccharin, acesulfame K, sodium cyclamate..).
II.2.5. Toxic substances produced during food preparation
(acrylamide, 3-chloro-1,2- propandiol, 1,3-dichloro-2-propanol…).
ACRYLAMIDE
3-MCPD
ANTIBIOTICS
ANTIBIOTICS BROMINATED
VEGETBLE OIL ACRYLAMIDE
S.9
II.2.6. Environmental pollutants (heavy metals, alkylphenols, PAH,
PCB, organotin compounds, dioxins and furans...).
II.2.7. Toxic chemicals released from packaging materials (styrene,
dialkyl phthalates, bisphenol A, DEHA…).
II.2.8. Food adulterants (melamine in milk, high fructose corn
syrup in honey, roasted maize or soybean in coffee, DEHP as
cloudy agent, toxic non-food colors, Tinopal as fluorescent
whitening agent for rice noodles,...).
DEHP MELAMINE BIS PHENOL A
S.10
Chemicals Packaging Test on
materials
Max
permitted
limit
Specific
migration
test
MRL
Styrene,
ethylbenzene,
xylenes, toluene
Polystyrene boxes 83-215
mg/kg
2000
mg/kg*
Bisphenol A Baby bottles 0.5-1.5 µg/L 2500 µg/L
DEHP Plastic coated paper
for butter & cheese
15-218 µg/kg 1500 µg/kg
DEHA Plastic films (DEHA
positive in 5/6
samples)
7.2-28.1%
DEHA
Plastic films (DEHA
positive in 10/12
samples)
0.12 – 1.52
mg/L (EtOH
20%, 60oC,
30 minutes)
18 mg/L**
33 – 117
mg/L
(heptane,
25oC, 1 hr)
18 mg/L**
Results of chemicals leaching from food packaging (2013)
.
* QCVN 12-1:2011/BYT, **EU Regulation 10/2011 of 1/14/2011
S 11
Growth regulators for bean sprouts
+ Cl OCH2COONa
6-benzylaminopurine Sodium 4-chlorophenoxyacetate
S 12
Non-Food Dyes
N N
H2N
NH2
2,4-diaminoazobenzene Orange II para Red Rhodamine B
S.13
Melamine
Day 1 Day 3 Day 20
Sample without Sample with
melamine 10 ppm Mel.
melamine
Sample without Sample with
melamine 10 ppm Mel.
Sample without Sample with
melamine 10 ppm Mel.
MRL= 1 mg/kg in milk
2.5 mg/kg in food
and feed
Added to milk to cause it to have apparently a higher
protein content. To increase by adulteration protein
nitrogen index in milk by one unit, 1.5 g melamine/kg
(1500 ppm) is needed
Out of several thousands of samples being analyzed
in different Vietnam laboratories, just 2 samples
contained more than 1000 ppm, others had only 100
ppb to few ppm. Other role of melamine???
S.14
MELAMINE
By the beginning of July 2013, Tinopal CBS-X, identified by LC-MSMS in our lab, was
found in “bun” (fresh rice vermicelli), “banh canh”(rice spaghetti) , “banh cuon” (rice
rolls), “banh hoi” (fine fresh rice vermicelli), the traditional Vietnam “Pho” …
Tinopal CBS-X, a fluorescent whitening agent, normally used in detergent, textile and
paper industry, was recently added to rice noodles as well as in other foods made from
rice for a better looking appearance. Tinopal CBS-X was not listed as additive for foods
and therefore must be controlled.
TINOPAL IN RICE NOODLES AND OTHER RICE PRODUCTS S.15
FRAGMENTATION MECHANISM OF TINOPAL CBS-X
IN ESI(-) MODE
S.16
III. DIFFICULTIES AND CHALLENGES IN FOOD TESTING
Trace organic analysis: a very difficult task when dealing with complex
matrices, especially with the biological ones.
Chromatography with detection by mass spectrometry appears to be the
most relevant technique, with quite high separation power and sensitivity of
detection provided by actual advanced GC/MS and LC/MS equipments. For
many contaminants, low or zero tolerance of residues presents real challenge to
detector response..
Careful sample preparation always needed:
*to extract all the analytes while eliminating all sources of contamination,
* to minimize matrix effects due to co-extractives which may cause accuracy,
precision and robustness issues besides possible equipment contamination and
plugging.
Green analytical chemistry intervenes in both sample
preparation and instrument analysis.
S.17
*SPE with its improved selectivity and sensitivity,
*Solid supported liquid extraction (SLE) with inert diatomaceous earth sorbent
cartridges
*Microwave and Ultrasound assisted extraction
*Immunoaffinity columns for isolation of mycotoxins, filtration with Captiva
ND Lipid cartridges for removal of particles, residual proteins and lipids
*QuEChERS technique for efficient extraction and clean-up of a wide range
of analytes at trace levels in food and feed matrices in compliance with
green sample preparation techniques while ensuring the quality of
analytical results.
*Solventless technique: HS-GC, HS-GCMS, SPME-GC, SPME-
GCMS (limited applications however)
Advantages of these techniques: Good analytical results, Cost effectiveness,
Health Protection, Eco-Friendliness
III.1 Sample preparation Goal: to extract from food matrices all the target analytes while minimizing co-
extracted interferences
Techniques of sample preparation in compliance with green analytical chemistry
being fully exploited in Vietnam Quality Control Laboratories:
S 18
QuEChERS sample preparation followed by GC-MS or LC-MS,
LC-MS/MS confirmation and quantitation at our laboratory Sac Ky
Hai Dang: trifluralin herbicide in basa catfish by GC-MS (LOD: 0.02 µg/kg, recovery better
than 91% at 0.25 µg/kg, MRL: 1 µg/kg ),
ethoxyquin in shrimp by LC-MSMS (LOD: 0.21 µg/kg, recovery better than 95%
at 5 µg/kg, MRL: 10 µg/kg ),
2,4-diaminoazobenzene dying chicken and duck skin by HPLC (LOD: 46 µg/kg,
recovery better than 97% at 450 µg/kg), Orange II dying roast pork skin by HPLC
(LOD: 42 µg/kg, recovery better than 94% at 115 µg/kg),
di-ethylhexyl phthalate DEHP in fatty foods by GC-MS (for cheese and butter,
LOD: 0.38-0.43 µg/kg, recovery better than 91% at 10-200 µg/kg, MRL:
1500µg/kg.
In these cases: insignificant matrix effects detected
Difficulties: nature of analytes and samples-strong interaction between
analytes and samples(DEHP in fatty foods, Tinopal and rice noodles)
Challenges: choice of sample prep techniques to ensure clean and quantitative
recovery of analytes.
S.19
Fast GC, UPLC with all their continuously
improved resolution and sensitivity and more
efficient monitoring.
UPLC advantages: -shorter run times, high throughput
-solvent saving by the use of short column with smaller
diameter and particle size reduced down to sub- 2µm
Multidimensional chromatography
GC x GC and LC x LC help analyzing very
complex multicomponent mixtures with close
retention times.
III.2 Detection and Quantification III.2.1 Improvement in chromatographic separation
New column technologies for method
development
- bonded phase change in column for better selectivity
and sensitivity,
- HILIC columns for polar compounds , mixed mode
columns for simultaneous analysis of polar and non-
polar, ionizable and neutral compounds
- fused core columns for higher efficiency,
S. 20
III.2.2 Improvement in detection technique Recent trends toward multiresidue, multiclass analysis in most cases require
advanced MS detection.
*Challenges
-Unequivocal identification and quantitation of
multi- targets with wide polarity and hydrophobicity
range in one single run;
-Very low LOD for many zero tolerance contaminants :
real difficult but challenging task for analytical chemists.
*Solutions Advanced LC-MS, GC-MS techniques with excellent resolution , sensitivity and high
troughput being continuously improved at a fantastic speed through:
*enhancement of ion generation
*better ion transmission
*better fragmentation and elimination of neutral noise
*better ion detection
*Tandem mass spectrometry with high accuracy and precision (MRM mode),
presently available in all Vietnam well known quality control laboratories, permit
to solve numerous problems of food safety for exportation, importation and local
consumption.
S.21
Shimadzu and Agilent LC-MS/MS improvement in sensitivity
SHIMADZU AGILENT
Launching
year 2010 2012 2013 2015 2006 2008 2010 2014
Equipment LCMS
8030
LCMS
8040
LCMS
8050
LCMS
8060
LCMS
6410
LCMS
6460
LCMS
6490
LCMS
6495
S/N for 1 pg
reserpine 200 1000 60000 250000 100 1000 50000
150000
(393000)
S 22
S.23
(June 2014)
Matrix effects Coelution of residual matrix components with the analyte in many cases badly affect
the ionization efficiency of the target (suppression or less frequently enhancement of the
analyte response, especially with ESI mode) thus compromising severely the accuracy
and precision when analyzing compounds at trace level. This occurs so often that an
exhaustive matrix effect evaluation should be included in the method of validation to
ensure the correctness and robustness of the analytical method.
TWO MAIN WEAKNESSES
Overcoming matrix effects especially in LC-MS-ESI mode constitutes in fact a real
challenge in trace organic analysis for quality control laboratories. In this context,
QuEChERS technique was found helpful in a number of cases such as the analysis of
ethoxyquin, trifluralin in shrimp where no matrix effect was detected
To overcome matrix effects: *smaller sample
*more selective sample prep technique
(immunoaffinty column, mixed mode SPE
MIPSPE,Agilent Captiva ND lipids
cartridge…)
*equipment with higher resolution
*matrix matched standard calibration curve
*Standard addition method
*Stable isotope labeled compounds as internal
standard
S. 24
S. 25
Target and non-target compounds
Present tandem MS equipments in our laboratories allow to analyze
quantitatively with good accuracy and precision target analytes but not the
non-target ones.
GC-QTOF, LC-QTOF and the very high resolution orbitrap technology
allowing unambiguous mass spectrometry identification of contaminants are
not yet available in most Vietnam quality control laboratories. High resolution
accurate mass tandem MS would allow screening of virtually unlimited
number of unknown target additives in food samples, thus assessing better
food safety issues in Vietnam.
In recent years, more performing tandem MS equipments permit with the
aid of expensive database, not only to quantify the aimed targets but also to
screen a number of non-target ones.
In our laboratory, database was created for screening around 40 compounds
being frequently detected by LC-MS/MS
S.26
Screening of fungicides in a grape sample (carbaryl analysis requested)
S.27
III.3 Issues related to actual local food production Through mass media, better information and education about food and
food safety issues, consumers are now more aware of food choices
However, from time to time, worldwide food safety incidents and sometimes
conflicting, somewhat exaggerated informations by local newspaper provoke
growing public concern about food contamination and adulteration.
Thus, besides the regular food analytical services, laboratories have to deal
with delicate problems, such as to prove that dried shredded squid is real and
not plastic shreds, mooncake contains nutritive shark fin and not nylon
filaments, fresh meat is authentic and not transformed from rotten meat.
S. 28
Moon cake Dried Shredded squid
No Real shark fin Filament from
mooncake
Aminoacids g/100 g g/100 g
1 Alanine 3.06 3.28
2 Glycine 12.78 12.31
3 Valine 2.11 1.93
4 Leucine 2.84 1.88
5 Isoleucine 1.29 1.29
6 Threonine 1.38 1.47
7 Serine 2.00 2.36
8 Proline 7.74 7.34
9 Aspartic acid 3.02 3.18
10 Methionine 1.92 1.24
11 4-hydroxyproline 1.84 5.09
12 Glutamic acid 6.46 6.67
13 Phenylalanine 2.52 1.53
14 Lysine 3.98 2.45
15 Histidine 1.84 1.20
16 Hydroxylysine 1.18 0.61
17 Tyrosine 9.96 5.47
18 Cysteine 0.32 0.09
Total 66.24 59.38
Amino acid analysis
helps to show that the so
called nylon filaments in
mooncake are really
shark fin
S.29
In fact, analytical results on antibiotic and pesticide residues in aquatic
products, meat and feedstuffs, rice and vegetables in at Sac Ky Hai Dang
laboratory though absolutely not representative of Vietnam food safety
situation, showed substantial improvement from year to year since 2013.
To really get a reasonably good picture of food safety, food inspection
campaign should be conducted regularly
With forbidden hazardous chemicals providing quite important illegal
profit to producers, severe food safety management is the only way to
ensure consumers’ safety. An example is the use of clenbuterol and
salbutamol in feed as illegal growth promoters for pigs and chickens.
S. 30
Parameters P1 P2 P3 P4 P5 Remarks
Initial weight of pig (Kg) 18.63 18.57 16.56 16.8 17.48 P1. Control
P2, P3: Case of
feed containing
500ppb clen
P4, P5: Case of
feed containing
500ppb sal
Weight after 18 weeks (Kg) 65.84 100.8 112.79 115.41 114.69
Weight increase (Kg) 47.21 82.23 96.23 98.61 97.21
Weight of consumed feed(Kg) 172.9 175.8 179.9 172.9 170.1
Feed Conversion Ratio FCR 3.66 2.14 1.86 1.75 1.74
Average FCR 3.66 2.00 1.745
Weight increase of pigs when using clenbuterol and salbutamol in feed
S.31
First semester of 2014 First semester of 2015
# of analyzed
feed samples
# of contaminated
samples (%)
# of analyzed
feed samples
# of contaminated
samples (%)
Salbutamol 49 6(12.2) 0.73-9.8 ppb 153 3 (2) 1.38-1.98 ppb
Clenbuterol 45 0 33 0
Ractopamine 40 1(2.5) 31.1 ppb 167 0
IV. CONCLUSION In view of intensive activities in testing laboratories at present time, efforts in
greening analytical methods must be deployed to reduce significantly the use of
solvents and reagents with high ecotoxicity and human toxicity. The result is cost
effectiveness, health protection, eco-friendliness, thus preventing pollution at the
source rather than dealing with costly remediation afterwards… Greening testing
laboratories is therefore not only a logic step toward sustainable development, it is
a MUST for quality control laboratories dealing with incessantly increasing
number of samples, thus contributing to the strengthening of food safety
management, while effectively ensuring environmental preservation.
FOR A BETTER FUTURE
THANK YOU
S.32