Food Safety Firstwith

Accelerated Solvent Extraction (ASE)

Deepali MohindraDionex Corporation, CA

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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

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● 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● Have temperature fluctuations● 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 countryISO Slide Number

Introduction

This introduces many points for possible contamination

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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

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Agricultural Contaminants

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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

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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

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● 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

Agricultural Chemical Contaminants:Pesticides

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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

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Agricultural Chemical Contaminants:ASE Conditions for Pesticide Extractions

Sample Size 1–20 g

Solvent Acetonitrile

Temperature 100 °C

Pressure 1500 psi

Static Time 5 Min

Flush Volume 40%

Purge Time 100 Sec

Cycles 1–2

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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

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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

OPP= Organophosphorus Pesticides

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Target CompoundsDichlorvos/Naled Mevinphos TEPP

Ethoprophos Sulfotep Phorate

Demeton(–O, –S) Dimethoate Diazinon

Parathion(methyl, ethyl) Disulfoton Fenchlorphos

Malathion Fenthion Chlorpyrifos

Trichloronat Tetrachlorvinphos Prothiofos

Merphos Fensulfothion Sulprofos

EPN Azinphos-methyl Coumaphos

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

OPP= Organophosphorus Pesticides

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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

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● Average recovery—91.0% ● 26 compounds, 12 replicates● 30-g samples

● 11.8% RSD● Recovery range—65 to 128%● Equivalent to Soxhlet

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

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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

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Agricultural Chemical Contaminants:Recovery of Pesticides and Herbicides from Wheat by ASE®

Compound Spike Level (µg/kg) Recovery (%)

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

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Agricultural Chemical Contaminants: ASE Extraction of Portioned Fungicide from Raisins

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

Conditions:

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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

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Agricultural Chemical ContaminantsVeterinary 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 exposure

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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

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Agricultural Chemical Contaminants:Extraction of Antibiotics 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

Antibiotic (ppm) in Animal Feed

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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

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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

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Environmental Contaminants

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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

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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

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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 and T2 are of major concern

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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

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Environmental Contaminants:Aflatoxins

● Aflatoxins conventionally extracted by Soxhlet & solid-phase extraction (SPE) Dionex automates these sample prep steps with the ASEDionex 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 attendanceASE reduces the extraction time to ~ 15 min per sample & Uses only 25–35 mL of solvent

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Environmental Contaminants:Extraction of Fumonisin from Grain

Sample Size10 g of Corn Meal Flakes + 3 g DE 0.5 mL Acetic Acid Spiked onto the Sample in the Cell

Solvent MeOH/Water (75/25)

Temperature 75 °C

Pressure 1500 psi

Static Time 3 Min

Flush Volume 40%

Purge Time 120 Sec

Conditions:

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Environmental Contaminants:Recovery of Fumonisin from Corn Meal

Recovery (ug/kg)

Sample CEN Method ASE®

Corn Meal 1 550600

Corn Meal 2 690738

Corn Meal 3 700806

Corn Meal 4 Non-Detect91

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Environmental Contaminants:Extraction of Aflatoxins from Grain -ASE® 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

Conditions:

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Environmental Contaminants:Recovery* of Spiked Aflatoxins

Matrix

Aflatoxins

G2 G1 B2 B1

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

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Environmental Contaminants:Extraction of Aflatoxins from Peanut SRM

Aflatoxin B1 Certified Value

213.0

ASE® Value 307.8

%RSD (n=3) 6.6

20-g Sample of Defatted Peanut Meal

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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

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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 minsAdditional cleanup steps are not required when in-cell adsorbants are usedExtraction recoveries from fruits, vegetables, and grains range from 89 to 110% for quantities as low as 10 ppb (ng/g).

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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

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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

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

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Environmental Contaminants:Schematic of In-Line Cleanup in ASE® Cell

Plant and Dispersant

Alumina/C18

Extraction Flow

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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

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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

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Food Additive Contaminants

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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

ASE would be ideal for the extraction of food additives from foodstuff

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Food Processing Contaminants

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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

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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)

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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

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

Procedure Water Added? De-Fatted? Acrylamide (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

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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)

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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 foodstuff