Analysis of Beer, Wine and Components for Pesticides …files.ctctcdn.com/11265e29001/3e0576a2-99da-45af-97cb-b...Components for Pesticides using High Throughput Techniques & QuEChERS

© SPEX CertiPrep, Inc. 2014

Patricia Atkins Applications Specialist, SPEX CertiPrep

Analysis of Beer, Wine and Components for Pesticides using High Throughput Techniques & QuEChERS


Housekeeping

Everyone will receive a copy of the presentation slides

The webinar is being recorded and will be posted to our YouTube account

Questions will be answered at the end of the presentation

– Type any questions you may have into the question box and we will answer them during the Q&A session

One lucky attendee will win a giveaway at the end of the presentation!


World of QuEChERS Studies


Our Newest QuEChERS Study

Benjamin Franklin


Agricultural Products in Wine & Beer

Wine Grapes

Yeast

Preservatives

Sugar (some varieties)

Finishing components

Beer Grains (Barley)

Hops

Yeast

Added flavors


Wine Making Process

Grapes Crushing Grapes

Punching Down the

Must

Pressing the

Grapes

Casking and Fermentation Filtering & Fining Bottling

10 oz of grapes needed to create a 6 oz glass of wine!

5.25 lbs of grapes needed to create 750 mL bottle!


Beer Making Process

Just over 1 lb of barley is needed to create a six pack of beer!


Chemistry in a Glass

1-3% Pectins, Proteins, Acids

1% Vitamins & Minerals

1% Polyphenols, Flavenoids, Tannins & trace flavor compounds

Low PPM & PPB levels pesticides in finished product

Water Ethanol

4-8% Carbohydrates Hops Barley Rice Malt Sugar

<2% Aroma & Flavor components

PPM & PPB pesticides in finished product

Type ABV Table Wine 8-14%

Claret 6-10% Shiraz 10% Rose 10%

White, medium 11% White, dry 11%

Red, medium 11.5% White, sparkling 12%

White, sweet 12% Cabernet, Pinot

Noir 11–14% Dessert Wine 14-20%

Zinfandel 17-22% Vermouth 17-22%

Syrah 17-23% Port 20%

Type ABV Light 0-3%

Pilsners 3-6% ESB (Bitter) 3-6%

Lager 4-5% Porter 4-5%

Brown Ale 4-6% IPA (India Pale Ale) 6-7%

Stout 5-10%

Fortified 11-57%

Alcohol content limited to 18% w/o sucrose addition

Fermentation: >9% Alcohol -prevent Acetobacter Target alcohol = 13% Alcohol (24 Brix) Final Ethanol Content varies by variety

Wine 70-90% 6-23%

Beer 90%

2-16*%


Chemical Anatomy of the Grape Peripheral Zone

3rd Pressing

• Potassium

• Sugars

• Oxidases

• Acids

• Astringents

• Aroma & Flavor compounds

Skin

• Anthocyanins • Quercetin • Resveratrol • Tannins • Catechins • Aroma & Flavor

compounds

Intermediate Zone 1st Pressing

• Tartaric Acid

• Sugars

Central Zone 2nd Pressing

• Malic Acid

• Sugars

Seeds & Stems (Optional inclusion)

• Tannins


Flavor & Aroma Chemistry of Wine Varietals

Compound Examples Odor Varietals

Methoxypyrazines

2-methoxy-3-isobutylpyrazine (IBMP) Earthy, Grassy, Pepper

Bell Peppers

Cabernet Sauvignon and

Sauvignon blanc. 2-methoxy-3-isopropylpyrazine (IPMP) Asparagus, Peas, Earth

Terpenes & Terpenoids

geraniol, linalool and nerol Floral Gewürztraminer, Muscat, Riesling

megastigmatrienone Spice Chardonnay zingerone Spice

damascenone Rose Oil Pinot noir vanillin Vanilla

Thiols

Mercaptans Onion, Garlic

Faults, Cabernet Sauvignon,

Gewürztraminer, Merlot, Muscat, Petit Manseng,

Pinot blanc, Pinot gris, Riesling, Scheurebe,

Semillon and Sylvaner

4-mercapto-4-methylpentan-2-one (4MMP), 4-mercapto-4-methylpentan-2-ol, 3-

mercaptohexanol, 3-mercaptohexyl acetate Sauvignon blanc

Glycosides Chocolate, Tobacco Ports


Chemical Anatomy of the Hops

Bract

Bracteole

Lupulin Glands • Organic Acids • Flavor & Aroma

Compounds


Flavor & Aroma Chemistry of Beer Hops

Use Fragrance Flavor Amount Highest Varieties Beer examples Alpha Acids (humulone, cis/trans isohumulone,

adhumulone, cohumulone, posthumulone, and

prehumulone)

No contribution Bittering 2.0-18.0% Warrior, Tomahawk Craft beers, high punch.

Beta Acids (lupulone, colupulone, and adlupulone) May produce faults May produce faults 0.0-9.0% Pacific Gem,

Galena, Satus Ales, Porters, Stouts

Cohumulone (of Alpha Acid) No contribution In higher levels presents a harsh, unpleasant bitterness. 2-50% US Brewers Gold

Lambic, Pilsners, English Ale, Pale Ale, German

Lagers

Total Oil (mg/100g) - - 0.2-5.0 Galaxy, CitraHops, Satus IPA

Myrcene (of total oil) Grapes, peaches, vanilla, wine

Matches its fragrance citrus with an earthy flavor. 0-70% Horizon & Amarillo

Red Ale, Light Ale, Pumpkin Ale,

Oktoberfest, Wheat

Humulene (of total oil) Robust, woody, earthy, and herbal

Woody organic flavor, spice like in coriander. 0-45%

Vanguard, Northdown, Hallertauer

Lager, Pilsner, Bock, Kölsch, Wheat, Munich

Helles, Belgian-Style Ales

Caryophyllene (of total oil) Dry woody, spicy, earthy bouquet, ‘hoppy’ aroma.

Strong dry wood, pepper, and earthy spice flavor. 0-20% Golding, Apollo IPA, Irish Stout

Farnesene (of total oil) Magnolia flowers, citrusy notes with green, woody,

with hints of lavender

Essence of herbs and organic wood-like - a great

supplement in teas and juices.

0-19% German Tettnang,

Spalt Select, Santium US

Lagers, Pilsners, and other German-Style

beers: Hefeweizen, Pale Ale, Kolsch, Bocks


Terpenes

Organic compounds, Aromatic hydrocarbons Derived from units of isoprene Produced by a variety of plants (conifers) Strong-smelling Terpenoids – terpenes with additional functional groups

~40 terpenes/terpenoids – Geraniol – Linalool – Nerol

Skin and inner layer of grape Boosted by fungal growth < Fraction of 1% of compounds

>200 terpenes/terpenoids – Myrcene – Humulene – Linalool – Limonene – Caryophyllene

Lupulin Glands of the Bracts (buds) 1-5% of total compounds Can be >50% of oil compounds


Common Terpenes & Terpenoids

Geraniol (Roses & Wine Grapes)

Pinene (Pines)

Myrcene (Myrtles & Cannabis)

Limonene (Citrus Lemon)

Linalool (Mints, Lavender)

Terpineols (Junipers, Orange Peel)

Humulene (Hops)

Caryophyllene (Peppercorns)

Carene (Cedar, Rosemary)

Nerol (Lemon Grass)


Pesticide Use in Wine Grapes & Beer Hops

Name Name Name 1,3-Dichloropropene Endosulfan Sulfate Myclobutanil

Abamectin Ethion Oryzalin Azoxystrobin Fenarimol Oxyfluorfen

Befenthrin Fenhexamid Paraquat dichloride Bifenate Fenpyroximate Pendimethalin

Bifenazate Fluazifop-p-butyl Procymidone Bifenthrin Fludioxonil Propargite Boscalid Flumioxazin Pymetrozine

Bromopropylate Folpet Pyraclostrobin Buprofezin Fosetyl-aluminum Pyridaben Carbaryl Glufosinate-ammonium Pyrimethanil

Carfentrazone-ethyl Haloxyfop Quinoxyfen Chlorpyrifos Hexythiazox Simazine

Chlorpyrifos methyl Imidacloprid Tebuconazole Cinidon-ethyl Iprodione Tebufenozide Cyhalothrin Iprovalicarb Tebufenpyrad Cymoxanil Kresoxim-methyl Tetradifon Cyprodinil Mefenoxam Tolylfluanid Deiquat Metalaxyl Triadimenol Diazinon Metalaxyl-M Trifloxystrobin Dicofol Metam-sodium Triflumizole

Dimethomorph Methoxyfenozide Dithianon Methyl soyate

Wine Grapes: Oils most abundantly applied

Imidacloprid

2nd most abundant in wine grapes

1st most used in world

Linked to bee hive collapse

Carbaryl (Sevin)

3rd most used in world

Hops Imidacloprid

Carbaryl (Sevin)

Bifenthrin


Extraction of Agricultural Products Pesticide Extraction Methods

– Soxhlet

– SPE

– SPME

– Supercritical Fluid Extraction

– Microwave Extraction

Issues with Methods

– Contamination

– Poor recovery

– Solvent waste

– Specialized cartridges and equipment

– High volumes of sample

QuEChERS – Quick Easy Cheap Efficient Rugged and Safe = Increased TEMPO!


TEMPO

Characteristic rate, rhythm, or pattern of work, activity or music

In Chess:

Gain/Loss of Time & Effectiveness

Due to one's mobility or developing position

In respect to the number of moves

Required to gain an Objective or Outcome

Dictionary.com


Examining the Laboratory TEMPO

Time

Efficiency

Money

Process

Output

© SPEX CertiPrep, Inc. 2014 Decant for dSPE

Centrifuge

Shake

Add IS & Spikes

Make Standards

Add salts

Weigh out salts

Add solvent

Weigh sample

Initial Sample Prep

TEMPO of the QuEChERS Extraction & dSPE Process

4 to 14 hrs – Extraction for 50 samples $1300 - $1500 Materials $400 - $1400 Manpower

$110 - $220 Materials $200 - $800 Manpower

2 to >8 Hours – Last 6 Steps


Examining TEMPO of QuEChERS

Time

Efficiency

Money

Process

Output

$1400-$1700 Materials $600-$2200 Manpower Total = $2000 - $4000

16 steps 2-3 h a step

1-4 days 50 samples

6 to 22 hrs


Examining the TEMPO of One of our first QuEChERS Studies

Time:

Efficiency

Money

Process

Output

5 Days

15-37% GG

16 Steps 2-3 hrs

$1200 Materials $3000 Manpower Total = $4200

50 sample/week

Manual Prep:

Salts, Solvent & Samples

Mixing:

Manual & Grinder


Maximizing Our Laboratory TEMPO in a New QuEChERS Study

Time

Efficiency

Money

Process

Output

Prepackaged Kits Prepared Standards ShaQer, HT Reactitherm

Time = Money Prepacked Materials Automation

Eliminated 3 Steps


Wine Samples

Two wine grape samples

– Malbec and Syrah

– Urban Vintner

Six red wine samples

– Two varietals: Malbec & Syrah

– Locations: South America, US, Europe and Australia

– Wines correspond to grape varieties

Purpose:

– Scan for pesticide residues

– Compare components to finished product

– Examine the effect of High Throughput processes on TEMPO


Beer Samples

Two grain samples

– Organic and non-organic

Dark malt sample

Four beer hop samples

– Cascade, Magnum, Centennial, CitraHops

Six craft beer samples

– All from US breweries


Materials

SPEX CertiPrep AOAC QuEChERS Kits

– Extraction

– 15 mL - dSPE

Acetonitrile w/ 10% GAA

SPEX CertiPrep Standards

– QUECR-AOAC-IS:

– 2 IS compounds

– 40 ug/mL

– QUECR-AOAC-QC: Spiking Solution

– 40 ug/mL

– 23 Pesticides

– CLPS-I90

– 6 deuterated IS compounds


SPEX CertiPrep AOAC Spiking Solution

27 Pesticides

40 ug/mL Concentration

Spiked Amounts

– Grapes: 10 mL = 1.9 ppm

– Wines & Beers:

12 – 15 mL = 1.3 – 1.5 ppm

– Hops & Grains:

20 mL = 1 ppm

Components CAS# Atrazine 1912-24-9

Azoxystrobin 131860-33-8 Bifenthrin 82657-04-3 Carbaryl 63-25-2

Chlorothalonil 1897-45-6 Chlorpyrifos 2921-88-2

Chlorpyrifos-methyl 5598-13-0

cis-Permethrin 61949-76-6 Cyprodinil 121552-61-2 Dichlorvos 62-73-7

Endosulfan sulfate 1031-07-8 Ethion 563-12-2 Folicur 107534-96-3 Imazalil 35554-44-0

Imidacloprid 138261-41-3 Kresoxim-Methyl 143390-89-0

lambda-Cyhalothrin 91465-08-6

Linuron 330-55-2 Methamidophos 10265-92-6

Methomyl 16752-77-5 o,p'-DDD 53-19-0

Procymidone 32809-16-8 Pymetrozin 123312-89-0

Thiabendazole 148-79-8 Tolylfluanid 731-27-1

trans-Permethrin 51877-74-8


Equipment SPEX SamplePrep Freezer/Mill

• Plant & Animal Tissue

• Seeds, Foodstuffs, Pharma

• Polymers, Electronics

• Textiles, Forensic materials

– Liquid N2 = fine powder

– Study: 10 g samples

SPEX SamplePrep ShaQer 1500

– QuEChERS analysis

– 6 samples

– Increased pesticide recovery

– Eliminates variation

– Grinding media


Equipment & Instrumentation Centrifuge:

– Capacity 4 – 50 mL tubes, 6 – 15 mL tubes

Reacti-Therm:

– Concentrate samples

– Solvent exchange to DCM

– Concentrated to 1mL

Agilent 5975 GCMS

– DB-5 (eq): 30m x 0.25 mm, 0.25 film

– Scan mode


Pesticides Response Challenges Some pesticides had little to no

response

– Destroyed or captured

• Chlorothalanil

• Thiabendazole

Lower responses

– Trifuralin

– Imalizlid

Interference with components

– Linuron & n-Hexadecanoic Acid

• Fatty acid

• Found in wine, yeast, beer

0

5

10

15

20

25

30

35

40

45

50

Wine Grapes Dark Malt Briess Hops Beer

N-Hexadecanoic Acid (ppm)


Wine & Beer

– Carbaryl seen higher than standard

• 1-Naphthol: breakdown peak to Carbaryl – Seen highest in wine, then beer

– Lowest in solid materials

Beer & Hops

– Folicur peak was actually Diketopiperazine compound

– Used as bittering compound

– Not found in wine, wine grapes or grains

Easily misidentified compounds


Wine & Grapes

Wine Grapes:

– Imidacloprid (0.9 ppm)

– Increase only in ShaQer samples.

Wines:

– Carbaryl (Sevin) – 3.7 ppm (most wines)

– Imidacloprid: 2.8 ppm (most wines)

– Pesticides – up to 1.5 ppm (1-2 wines)

– Ethion

– Bifenthrin

– Chlorpyrifos methyl

– Endosulfan sulfate

– Lamba-Cyhalothrin

– Increases recovery using ShaQer 5-15%


Comparison of Components & Product

Wine & Wine Grapes

– Imidacloprid – found in both grapes (1 ppm) and wine (2.5 ppm)

– Wine: Carbaryl (3 ppm), Lambda Cyhalothrin (1.5 ppm) & Endosulfan sulfate (1.5 ppm)

0

0.5

1

1.5

2

2.5

3

3.5

4

WineGrape


Grains & Malt Barley Bittering agent: not seen in Malt & Grain

– Bittering agent from other components

Grains: Organic Breiss < Regular Breiss

– Bifenthrin: Organic and Regular Grain at concentrations <0.5 ppm

– Ethion: 3.6 ppm (Reg) & 1.4 ppm (Org)

Recovery 10-25% higher using ShaQer

Malt Imidacloprid - 0.75 ppm

Less than 0.5 ppm: Bifenthrin & Endosulfan sulfate

Studies show decrease in pesticide levels from barley to malt stage.


Hops

Bittering agent (seen as Folicur) = 18 ppm

Needed more clean-up prior to analysis

Large organic compounds including:

Terpenes: beta-Pinene, Caryophyllene, .alpha-Humulene, Lupulon,

Vitamin E

Phytosterols

Interferences: Imidacloprid, Ethion, Bifenthrin & Endosulfan sulfate seen in other beer components.

2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.0010.0011.0012.0013.0014.0015.00

1e+07

2e+07

3e+07

4e+07

5e+07

6e+07

Time-->

TIC: R13072646.D\data.ms


Cannabis & Beer (Hops)

Myrcene (Hops & Cannabis)

Family: Cannabaceae >100-200 Terpenes/Terpenoids Many common aroma & flavor compounds to Hops!


Terpenes in Wine, Beer & Cannabis

Limonene (Wine, Beer &

Cannabis)

Terpenes/Terpenoids Wine Hops Cannabis

Myrcene x X X

Limonene x X x

Linalool X X x

Geraniol X x

Terpineol X x

Nerol X x

Pinene x x

Humulene X x

Caryophyllene X x

Carene x x

Cymene X

Farnesene x

Terpinoline x

Borneol x

Eucalyptol x

Humulene (Hops & Cannabis)

Pinene (Hops & Cannabis)

Linalool (Wine, Beer &

Cannabis)

Myrcene (Wine, Beer &

Cannabis) Terpineols

(Wine & Cannabis)


Hops

Ethion: 1.6 to 9.0 ppm in most hop varieties

Imidacloprid: up 8.5 ppm (2 varieties)

Carbaryl: up to 6.5 ppm (2 varieties)

0

1

2

3

4

5

6

7

8

9

10

Hop 1

Hop 2

Hop 3

Hop 4


Beer Bittering agent (Folicur) = 18 ppm

Pesticides found

– Imidacloprid – 5.25 ppm

– Carbaryl – 3 ppm

– Ethion – 2.5 ppm

Up to 35% increased recovery with ShaQer

0

50

100

150

200

250

300

Chlorpyrifosmethyl

Chlorpyrifos Imidacloprid

108 102

255

75 71

182

ShaqerHand



Beer, Hops, Grains

– Beer & hops samples = Diketopiperazine compound

– Recovery increased 10-35% by using ShaQer

– Pesticide in all/most beer components: Imidacloprid, Ethion, Bifenthrin (hops??)

0

1

2

3

4

5

6

7

8

9

10

Malt Organic Grain Grain Hops Beer

Imidacloprid

Ethion

Bifenthrin



Beer, Hops, Grains

– Pesticides in beer not found a high levels in products:

– Endosulfan sulfate, Chlorpyrifos (Hidden in hops??)

– Carbaryl, Chlorpyrifos methyl found in Hops

0

1

2

3

4

5

6

7

Malt OrganicGrain

Grain Hops Beer

CarbarylChlorpyrifos methyllambda.-CyhalothrinEndosulfan sulfateChlorpyrifos


Final Look at our New TEMPO

Time

Efficiency

Money

Process

Output

2 days

$1400 Materials $1400 Manpower Total = $1800 ($2400 Savings)

Eliminated 3 steps Reduced time of step by 1 hr

2 days 50 samples

ShaQer increased Recovery 5-35%


Wine & Beer Pesticide Final Snapshot of Conclusions

Wine: <3.5 ppm (Carbaryl)

Grapes: <1ppm (Imidacloprid)

Wine & Grapes

Beer: <5.25 ppm (Imidacloprid)

(most <1.5 ppm)

Grains & Malts: <<1.5 ppm

Organic Grain < Regular

Hops: Highest all products

7-9 ppm (Ethion, Imidacloprid &

Carbaryl)

Beer, Grain &

Malt

Imidacloprid

Carbaryl

Pesticides

Improved Time, Efficiency, Money, Process & Output!


What is in the future for QuEChERS?

Environmental

Phthalates

Vet Residues

Untraditional matrices

Previously Untested Products


So what is next for SPEX CertiPrep?


Thank You! Questions?

Analysis of Beer, Wine and Components for Pesticides …files.ctctcdn.com/11265e29001/3e0576a2-99da-45af-97cb-b...Components for Pesticides using High Throughput Techniques & QuEChERS

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