Food Safety First

with
Accelerated Solvent Extraction
(ASE)

Deepali Mohindra
Dionex Corporation, CA
 Agenda

● Overview on Food Safety

● Different Categories of Contaminants
● Agricultural Chemical
● Environmental
● Food Additive
● Food Processing
● Example of each Type of Contaminant
● Dionex ASE Solution for each Contaminant

2
 Introduction

● We now have a global food supply chain making food

safety a global concern
● The US imports ~$65 billion of food products

● Food is traveling thousands of miles

● Kept in various storage conditions
This introduces many points for
● Have temperature fluctuations
possible contamination
● Handled by many people

● The US Food and Drug Administration (FDA) inspects

about 1% of the imported foods
● No one global screening standard
● Each country has to verify that the imported food is
adhering to the regulations of the country
ISO Slide Number

3
 Types of Contaminants and Health Risks

● Contaminants may originate from

● Agricultural Sources
● Environmental Sources
● Food Additives Sources
● Food Processing Sources
● Food Contaminants can cause a host of problems
● Acute Effects : Mild Rash, Light Headache, Vomiting
● Serious Effects: Cancer, Auto-immune diseases, Thyroid
problems, Death
● Health effects of exposure to contaminants may become
apparent after years of prolonged exposure

4
 Agricultural Contaminants

5
 Agricultural Chemical Contaminants

● Agricultural chemicals are used to:

● Increase crop yields
● Control weeds and pests
● Prevent disease to crops and livestock
● These chemicals include:
● Pesticides
● Herbicides
● Plant growth regulators
● Growth Hormones for livestock
● Veterinary drugs/Antibiotics for livestock

6
 Agricultural Chemical Contaminants:
Pesticides

● Some pesticides that pose severe health and

environmental risks have been banned by many
countries but are still used in others
● Due to the global supply chain the banned
pesticides can appear in our food supply
● The build up of legal pesticides can also pose
health risk to humans
● The health risk depends on the type of pesticide
and the length of exposure

7
 Agricultural Chemical Contaminants:
Pesticides

●Dionex ASE systems can be used for the

extraction of:
●Organophosphates pesticides
●Organochlorides pesticides
●Carbamates pesticides
●From solid and semisolid food samples
rapidly, and with far less solvent than
Soxhlet or sonication

8
 Agricultural Chemical Contaminants:
Pesticide Residue in Grain

● Pesticides residue are found in wheat, corn, and

barley
● Organochlorine Pesticide (OCP) & Organophosphorous
Pesticide (OPP)
● Conventional techniques are
● Labor intensive and lots of solvent
● ASE extractions
● 20 min and 50 mLs of solvent

9
 Agricultural Chemical Contaminants:
ASE Conditions for Pesticide Extractions

Sample
1–20 g
Size
Solvent Acetonitrile
Temperat
100 °C
ure
Pressure 1500 psi
Static
5 Min
Time
Flush
40%
Volume
Purge
100 Sec
Time
Cycles 1–2

10
 Agricultural Chemical Contaminants:
Results of Pesticides Extraction Using ASE

● 29 Organophosphorus pesticides (OPPs)

● Spiked at 8–102 ppb
● Recoveries for ASE ranged from 54.1–115.7%
● Samples had higher recoveries when extracted
by ASE as compared to wrist action shaker

11
 Agricultural Chemical Contaminants:
ASE Extraction of OPPs from Fruits & Vegetables

● 30 g of apples and carrots mixed with 20 g

diatomaceous earth (baby food, 100-mL cells)
● 7.5 µL spike of standard (26 compounds)
● Ethyl acetate/cyclohexane (1:1, v/v), 100 °C,
1500 psi
● 17–20 min/sample, 135–145 mL/sample
● GPC clean up followed by GC-FPD

P= Organophosphorus Pesticides

12
 Agricultural Chemical Contaminants:
ASE Extraction of OPPs Pesticides from Fruits &
Vegetables

Target Compounds
Dichlorvos/Naled Mevinphos TEPP

Ethoprophos Sulfotep Phorate

Demeton(–O, –S) Dimethoate Diazinon

Parathion(methyl, ethyl) Disulfoton Fenchlorphos

Malathion Fenthion Chlorpyrifos

Trichloronat Tetrachlorvinpho Prothiofos
s
Merphos Fensulfothion Sulprofos
EPN Azinphos-methyl Coumaphos
P= Organophosphorus Pesticides
13
Agricultural Chemical Contaminants:
ASE Extraction of OPP Pesticides from Carrots (50
µg/kg)

● Average recovery: 89.7%

● 26 compounds, 12 replicates
● 30-g samples
● 8.7% RSD
● Recovery range: 82 to 125%
● Equivalent to Soxhlet

14
Agricultural Chemical Contaminants:
ASE Extraction of OPP Pesticides from Carrots (50
µg/kg)

● Average recovery—91.0%
● 26 compounds, 12 replicates
● 30-g samples
● 11.8% RSD
● Recovery range—65 to 128%
● Equivalent to Soxhlet

15
Agricultural Chemical Contaminants:
Extraction of Pesticides & Herbicides from Wheat by ASE®

Conditions:
● 3- to 20-g samples
● Acetonitrile or acetone
● 2000 psi
● 100 °C
● 5-min heat-up, 5-min static
● 12-min total time, 14-mL solvent
● GC or GC-MS analysis

16
 Agricultural Chemical Contaminants:
Recovery of Pesticides and Herbicides from Wheat
by ASE®
Spike Level
Compound Recovery (%)
(µg/kg)
Trifluralin 44 99.6

Demeton-S 38 96.7

Atrazine 14 92.8

Chlorpyrifos-methyl 8 115

Imazalil 40 109

Endosulfan-beta 68 93.3

Methooxychlor 50 115

17
 Agricultural Chemical Contaminants:
ASE Extraction of Portioned Fungicide from Raisins

Conditions:
Sample Size 10 g Raisins + 10 g Diatomaceous Earth

Solvent Ethyl Acetate l

Temperature 55 °C

Pressure 1500 psi

Static Time 5 Min

Flush Volume 60%

Total Time 13 Min per Sample

Total Volume 30 mL per Sample

18
 Agricultural Chemical Contaminants:
ASE Extraction of Iprodione Fungicide from Raisins

Results:
● 20 samples extracted by both ASE and shaking
techniques
● Two samples were ND by both techniques
● For 18 remaining samples, ASE recovery
averaged
126% of the shaking method
● MDL = 0.02 mg/kg for both techniques

19
 Agricultural Chemical Contaminants
Veterinary Drugs/Antibiotics

● Veterinary drugs and Antibiotics are used in animal

breeding industry:
● To treat, prevent and control diseases
● Used to stimulate growth and produce leaner meat
● In humans these drugs can cause
● Allergic reaction
● Bacterial resistance to antibiotics
● Cancer
● US and EU Regulatory agencies have established
which drugs can be used and established
maximum residue levels (MRLs) and banned
others in meats and milk products
● Testing is essential to protect humans from
20 exposure
 Agricultural Chemical Contaminants:
Extraction of Antibiotics in Animal Feed

● Solvent: methanol with 0.3% acetic acid

● Temperature: 80 °C
● Pressure: 10 MPa (1500 psi)
● 5-min heat-up time; 5-min static time
● 60% flush
● <30-mL total solvent, < 15-min total time
● Extracts analyzed by HPLC; 10-g samples

21
 Agricultural Chemical Contaminants:
Extraction of Antibiotics in Animal Feed

Antibiotic (ppm) in Animal Feed

Sample ASE® Sonication

1 80.0 77.5

2 82.2 78.4

3 81.4 79.4

4 82.0 78.7

5 89.5 78.2

6 85.5 81.2

22
 Agricultural Chemical Contaminants:
Sulfonamides from Meat

● Sample size
● 2 g mixed with 4 g C18 resin placed
in an 11-mL cell with glass fiber filter
● ASE® conditions
● HPLC grade water
● 160 ºC, 1500 PSI
● One 5-min static cycle
● Analysis by LC/MS-MS

23
Agricultural Chemical Contaminants:
Recoveries (%) of Sulfonamides from Meat Matrices
Spiked at 100 ppb
Analytes Bovine Meat Porcine Meat Poultry Meat
  N=4–6 N=4–6 N=4–6
SPD 89 92 91
SDZ 92 93 94
SMX 92 94 96
SMR 99 98 98
SMO 70 72 76
SMT 86 90 91
SIM 94 98 99
SMZ 88 90 92
SMP 85 85 85
SMM 90 92 94
SCP 79 85 82
SQX 81 82 85
SDM 85 90 91
Data id courtesy of Alessandra Gentili University of La Sapeinza, Department of Chemistry, Rome, Italy

24
 Environmental Contaminants

25
 Environmental Contaminants

● Can be natural or man-made

● Are present in water, air & soil
● Some are biological byproducts of bacteria or
fungi that come into contact with food
● During growth
● During storage

26
 Environmental Contaminants:
Different Types
● Types of environmental contaminants
● Mycotoxins
● Biogenic Amines
● Perchlorate
● Nitrate
● Polycyclic aromatic hydrocarbons (PAH)
● Polychlorinated biphenyls (PCB)
● Polybrominated diphenyl ether (PBDE)
● Dioxins
● Lead
● Arsenic
● Mercury
● BADGE

27
 Environmental Contaminants:
Mycotoxins

● Mycotoxins are mild to fatal poisons produced by

fungi
● Develop in crops during growth or storage
● Symptoms of exposure include
● Allergic reaction
● Diarrehea
● Nausea
● Kidney damage
● Immunosupression
● Nerve damage
● Death
● Many of these toxins are carcinogens
● Over 400 mycotoxins have been identified
● Based on toxicity and occurrence – Aflatoxins,
Zearalenone Vomitoxin, Fumosinin, Zearalenone
28 and T2 are of major concern
 Environmental Contaminants:
Aflatoxins

● Mycotoxins are mild to fatal poisons produced by fungi

● Aflatoxins are produced by the fungus Aspergillus that
grows in soil and decaying vegetation
● Aflatoxins B1, B2, G1 and G2 are highly carcinogenic and
regulated in many countries
● Zearalenone can be found in wide variety of plants and
soils
● Can have negative health effects in humans and animals

29
 Environmental Contaminants:
Aflatoxins

● Aflatoxins conventionally extracted by Soxhlet & solid-

phase extraction (SPE)
Dionex automates these sample prep steps with the
ASE

● Traditional extraction of Zearalenone from animal feed

is done by wrist shaking or blending
● These methods take 30–60 min per sample
● Constant lab technician attendance
ASE reduces the extraction time to ~ 15 min per
sample &
Uses only 25–35 mL of solvent

30
 Environmental Contaminants:
Extraction of Fumonisin from Grain

Conditions:
10 g of Corn Meal Flakes + 3 g DE 0.5
Sample Size mL Acetic Acid
Spiked onto the Sample in the Cell
Solvent MeOH/Water (75/25)
Temperatur
75 °C
e
Pressure 1500 psi

Static Time 3 Min

Flush
40%
Volume
Purge Time 120 Sec

31
 Environmental Contaminants:
Recovery of Fumonisin from Corn Meal

Recovery (ug/kg)

Sample CEN Method ASE®

550
Corn Meal 1
600
690
Corn Meal 2
738
700
Corn Meal 3
806
Non-Detect
Corn Meal 4
91

32
 Environmental Contaminants:
Extraction of Aflatoxins from Grain -ASE® Conditions

Conditions:
Sample Size 20 g Corn, Oats, or Peanuts
Solvent Acetonitrile (MeCN)
Temperature 75 °C
Pressure 1500 psi
Static Time 5 Min

Flush Volume 75%

Purge Time 100 Sec

Total Time 17 Min per Sample
Analysis HPLC/Fluorescence after Filtering

33
 Environmental Contaminants:
Recovery* of Spiked Aflatoxins

Aflatoxins
Matrix
G2 G1 B2 B1

100.1
Sand 90.7 (4.8) 93.8 (6.3) 73.1 (3.8)
(11.6)

Oats 79.4 (3.1) 90.7 (7.6) 86.7 (4.9) 87.9 (6.0)

Corn 104.4 (7.3) 92.9 (1.3) 78.3 (14.9) 91.9 (3.2)

* %Recovery (%RSD), n=3; Spiking Level at 1 ppm

34
 Environmental Contaminants:
Extraction of Aflatoxins from Peanut SRM

Aflatoxin B1
213.0
Certified Value

ASE® Value 307.8

%RSD (n=3) 6.6

20-g Sample of Defatted Peanut Meal

35
 Environmental Contaminants:
Perchlorate

● Perchlorate is a naturally occurring mineral found

in nitrate fertilizers
● Also a manmade chemical used as an ingredient
for
● Rocket propellant
● Explosives
● Industrial processed like rubber manufacturing
● Perchlorate in irrigation water can build up in
crops (lettuce, spinach, melons)
● Health Risks
● Interferes with the iodide uptake into the thyroid gland
● Can disrupt neurological development in fetuses and
children
36
 Environmental Contaminants:
Perchlorate

●Extraction of perchlorate typically involves high

speed blending and ultrasonication extraction
● This is not efficient enough to extract tightly bound ions
such as perchlorate from complex vegetation
● Additional cleanup steps, such as solid phase extraction, are
also usually required

An ASE system can extract perchlorate from most

samples in ~15 mins
Additional cleanup steps are not required when in-cell
adsorbants are used
Extraction recoveries from fruits, vegetables, and
grains range from 89 to 110% for quantities as low as
10 ppb (ng/g).
37
 Environmental Contaminants:
Extraction of Perchlorate from Vegetation

● Challenging for sample preparation

● Many interferences can be coextracted
● EPA method labor intensive and time consuming*
● 20 h for extraction
● 20+ h for cleanup
● The Accelerated Solvent Extraction can be used
to address labor and time

38
 Environmental Contaminants:
Perchlorate Sample Prep Options
Off-line Cleanup
● Oven-dried samples (5 g)
mixed with diatomaceous
earth
● Extraction with standard ASE®
conditions
● D. I. water, 125 °C, 1500
psi, 17-min total time
● SPE cleanup of extracts with
alumina and C18
39
In-line Cleanup
● Oven-dried samples (5 g)
mixed with diatomaceous
earth
● Samples loaded into cell
containing alumina/C18 in
outlet
● Extracted with standard ASE
conditions
● D. I. water, 125 °C, 1500
psi, 17min total time
● Extracts analyzed without
cleanup
 Environmental Contaminants:
Schematic of In-Line Cleanup in ASE® Cell

Plant and

Extraction
Flow Dispersant

Alumina/C
18

40
 Environmental Contaminants:
Perchlorate Conclusion

● ASE® can be used as a sample extraction/cleanup

procedure prior to the determination of perchlorate
in
soils and plant materials
● Alumina/C18 in ASE cell (in-line) can provide clean
extracts ready for analysis
● Total time is less than 20 min for extraction and cleanup
● Automation of ASE improves the precision of the
analytical
scheme and increases sample capacity
● Same ASE instrument can be used for both ionic
and nonpolar contaminants

41
 Environmental Contaminants:
Perchlorate

● Sample prep typically involves high speed blending &

ultrasonication
● These techniques are not efficient to extract tightly
bound ions like Perchlorate from complex vegetation-
Addition SPE is required

● ASE system can extract Perchlorate in 15 mins

● Additional clean up steps NOT required with in-cell
absorbents usage
● Extraction recoveries from fruits, vegetables & grains
are from 89-110% as low as 10ppb (ng/g)
● Analysis done on the IC

42
 Food Additive Contaminants

43
 Food Additive Contaminants

● Chemicals intentionally added to products during

the manufacturing process
● Many chemical are added for valid reasons and
need to be monitored
● To assure that the specifications are being met
● To detect for contamination
● Labeling purposes due to potential allergic reactions for
consumers
● Examples: Nitrate, Nitrite, Sulfite, Benzoate
● Adulteration: dangerous or banned substance used
for financial gains
● Examples: Melamine in pet food

44 ASE would be ideal for the extraction of food

additives from foodstuff
 Food Processing Contaminants

45
 Food Processing Contaminants

● Processing contaminants are generated during the

processing of foodstuff
● Formed in processes like:
● Disinfection
● Fermentation
● Heating
● Canning
● Grilling
● Examples include:
● Acrylamide
● PAHs
● Oxyhalides
● Haloacetic acids

46
 Food Processing Contaminants:
Acrylamide

● Acrylamide is a genotoxic compound found in

fried or baked goods
● Produced when asparagine reacts with reducing
sugars or carbonyl compounds
● Produced in browning and overcooking
● The Acrylamide content in some samples like
hash browns and French fries can be very high
(several mg/kg)

47
 Food Processing Contaminants:
ASE Extraction of Acrylamide

● Extraction conditions:
● Sample size: 10–15 g (33-mL cell)
● Oven temperature: 80 °C
● Pressure: 1500 psi
● Solvent: Acetonitrile (Hexane for defatting)
● 3 x 5 min extraction cycles
● 40% flush volume
● Total extract volume: about 40 mL
● Total extraction time: 23 min

48
 Food Processing Contaminants:
ASE Extraction of Acrylamide from Hash Brown (shredded
fried potatoes)

Procedu Water De- Acrylamide

re Added? Fatted? (µg/g)
1 No Yes 0.01
1 No No 0.02
2 Yes Yes 0.67
2 Yes No 0.54
3 Yes Yes 0.70
3 Yes No 0.75

49
 Food Processing Contaminants:
Keys to Successful Extraction of Acrylamide

● Addition of water prior to extraction is important

● ASE® extraction streamlines the sample
preparation process
● Defatting with hexane prior to extraction does not
appear to remove acrylamide from sample
● Use of IonPac® ICE-AS1 column eliminates need
for SPE
● HPLC/MS method much easier than GC/MSD
method
● Up to 24 samples unattended (12 samples with
defatting step)

50
 Food Processing Contaminants:
ASE Extraction of PAHs

● PAH: Polycyclic Aromatic Hydrocarbons

● Carcinogenic
● Occur in charbroiled and dried foods & edible oils by
pyrolytic processes
such as incomplete combustion of organic
substances
● PAHs result in foods from baking, frying, grilling,
carmelizing
● PAHs in food can also occur by environmental
contamination

ASE would be ideal for the extraction of PAHs from

51 foodstuff