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