Dionex - Accelerated Solvent Extraction (ase)

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

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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

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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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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

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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 Solvent Temperat ure Pressure Static Time Flush Volume Purge Time Cycles

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1–20 g Acetonitrile 100 °C 1500 psi 5 Min 40% 100 Sec 1–2

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

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

Prothiofos

Merphos

Tetrachlorvinpho s Fensulfothion

EPN

Azinphos-methyl

Coumaphos

P= Organophosphorus Pesticides 13

Sulprofos

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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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

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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® Spike Level (µg/kg)

Recovery (%)

Trifluralin

44

99.6

Demeton-S

38

96.7

Atrazine

14

92.8

8

115

Imazalil

40

109

Endosulfan-beta

68

93.3

Methooxychlor

50

115

Compound

Chlorpyrifos-methyl

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

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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

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 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

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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

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 Antibiotic (ppm) in Animal Feed

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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

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 24

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, 28

Zearalenone Vomitoxin, Fumosinin, Zearalenone 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

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Environmental Contaminants: Aflatoxins ● Aflatoxins conventionally extracted by Soxhlet & solidphase 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

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Pressure

1500 psi

Static Time

3 Min

Flush Volume

40%

Purge Time

120 Sec

Environmental Contaminants: Recovery of Fumonisin from Corn Meal

Recovery (ug/kg) Sample

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CEN Method ASE®

Corn Meal 1

550 600

Corn Meal 2

690 738

Corn Meal 3

700 806

Corn Meal 4

Non-Detect 91

Environmental Contaminants: Extraction of Aflatoxins from Grain -ASE® Conditions Conditions: Sample Size Solvent Temperature

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20 g Corn, Oats, or Peanuts Acetonitrile (MeCN) 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

Environmental Contaminants: Recovery* of Spiked Aflatoxins Aflatoxins Matrix

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 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).

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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

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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

Extraction Flow

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Plant and Dispersant

Alumina/C 18

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 vegetationAddition 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

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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 44

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

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 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)

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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)

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Procedu re

Water Added?

DeFatted?

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

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

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ASE would be ideal for the extraction of PAHs from foodstuff