QuEChERS 101: El Como, Que, Donde y el Por que y de una Tecnica Rapida de Preparacion de Muestras.

Jose Juarez

Especialista de Producto CSD

Agilent Technologies, Inc, Mexico

Magnificent work of Carl Warner

En los comienzos…

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En 1902…. Un grupo de 12 jovenes en la FDA, conocido con el The Poison Squad fueron los responsables de la

evaluacion de alimentos enriqueciendo los mismos con conservadores desconocidos y que tienen efectos en la

salud y que podrian ser documentados.

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Primeros Métodos de Multi-residuos: Extracción con solventes

Regresando a los 60´s (1960)

Acetonitrilo: Métodos CFDA ó Canadiense.

Acetona: Metodos Holandeses ó Luke.

Etil Acetato: Suiza, Suecia.

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6/11/2014

Eureka….tenemos un metodo

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Based on Canadian Multi-residue

method

Luke Canadiense Primer “QuEChers”

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La Referencia Original del metodo de metodo

Multiresiduos.

QuEChERS (Pronunciado “catchers”)

• Quick, Easy, Cheap, Effective, Rugged, and Safe

• Mezcla de dos o mas palabras

• www.quechers.com

• Presentado en 2003: M. Anastassiades, S.J. Lehotay, D. Stajnbaher, and F.J.

Schenck, J. AOAC Int 86 (2003) 412.

• Validado en el 2005, con subsecuente modificacion en el 2007 AOAC 2007.01 y

en metodo europeo EN 15662.

• El metodo que es mas facil, sencillo y mas barato para la muestreo de residuos

de pesticidas en alimentos.

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QuEChERS: Preparando la muestra para ser procesada

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Reduces the heat

produced while grinding

Vamos a ver los beneficios de QuEChERS

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Solo 3 pasos

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Paso 1: Extracción

Paso 2: Limpieza SPE dispersiva

Analisis

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Pasos del metodo .

Adciona un homogenizador al tubo de muestreo

Adiciona 2 homogenizadores ceramicos (como se muestra abajo).

Adicionar ACN, mezclar

Adicionar las sales empaquetadas

Mezclar, Mezclar, Mezclar

Adicionar el estándar interno, agitar

Centrifugar, separación y extraccion

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2 3

4

3

5 3

6 7

Del Instituto Stuttgart , Micheangelo Anastassiades

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Aunque el video sugiere adicionar el IS antes que las sales de

extraccion nosotros lo hacemos antes de adicionar el ACN

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#1:Procedimiento QuEChERS: Facil, rapido y con menos pasos.

Adciona un homogenizador al tubo de muestreo

Adiciona 2 homogenizadores ceramicos (como se muestra abajo).

Adicionar ACN, mezclar

Adicionar las sales empaquetadas

Mezclar, Mezclar, Mezclar

Adicionar el estándar interno, agitar

Centrifugar, separación y extraccion

1

2 3

4

3

5 3

6 7

Del Instituto Stuttgart , Micheangelo Anastassiades

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Aunque el video sugiere adicionar el IS antes que las sales de

extraccion nosotros lo hacemos antes de adicionar el ACN

#1:Procedimiento QuEChERS: Facil, rapido y con menos pasos.

2 Transferir una alicuota al tubo d –SPE/ agitación

Agitar

Centrifugar

Transferir una alicuota a un vial

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9

10 11

Del Instituto Stuttgart , Micheangelo Anastassiades

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Analizar 3

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#2: Usos de los QuEChERs, menos vidrio y menos uso cristalería

QuEChERS

Slide courtesy of Dr. Jon Wong, FDA, College Park, Maryland

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www.agilent.com/chem/quechersvideo

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Visite nuestra pagina en internet y vea nuestro video de Quechers

Tienes un momento ????

#3: QuEChERS: Una vista rápida de la comparacion en beneficios

•Un metodo alternativo a los existentes: LLE, SPE, GPC

•QuEChERS esta siendo adoptado en todo el mundo

•Menos error y variacion dentro del Analisis

•Disponilibilidad del detector: MS and MS/MS (selectivo/sensitivo)

•QuEChERS decrese substancialmente el costo por muestra

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Metodo Luke, SPE o GPC QuEChERS QuEChERS

Benefits!

Tiempo de

procesamiento para 6

muestras (min)

120 30 4x mas rápido

Solvente usado (mL) 90 mL 10-15 mL 9 x menos solvente

Desperdicio de

clorados (mL) 30 mL none

Seguro, ecologico

y menos costoso

Equipamiento en

cristalería

Embudo, rotavapor,

contenedores de 200mL,. None

No son necesarios

otros complemento

Ahorro por

muestra

Muestras

por día

Días por

Semana Año 1

$72.00 25 5 $468,000

El ahorro general $72 USD

por muestra.

Information courtesy of Dr.

Steve Lehotay, USDA

Como se relaciona esto con el tema de Seguridad Alimentaria

QuEChERS

Permite la determinacion simultanea de muchas clases de compuestos

3 Pasos de análisis: Extraccion, limpieza d-SPE y analizar

Cada contaminante tiene un nivel establecido relacionado, MRL (Limite

Maximo Residual)

Así la tecnica de la preparacion de la muestra (QuEChERS) esta

disponible para alcazar los MRL

Detectores Selectivos y Sensitivos que permiten a los QuEChERS ser

una opcion (MS/MS)

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Forma de transicion GC/FPD, NPD, ECD los cuales ven (observan)

compuestos especificos, e.g. FPD en el modo fosforo para la deteccion

de organofosforados (OP) pesticidas.

To GC/MS to GC/MS/MS: Sensible y selectivo

LC/MS to LC/MS/MS: Sensible y selectivo

Transicion a menos costo y mas complejidad de muestra

Porque MS/MS?

Es acerca de la matrix y la

prepacion de la muestra

Porque QuEChERS?

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Comparacion MS/SIM versus MS/MS: Seguridad Alimentaria

Apple

Orange

Spinach

Ginseng

Cabbage

SIM - EIC (246) MRM (2 transitions)

S/N = 448

S/N = 456

S/N = 260

S/N = 446

S/N = 241

X

X

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• Non-buffered QuEChERS method (Original) (10 or 15 g sample)

• 4 or 6 g MgSO4, 1 or 1.5 g NaCl

• AOAC method 2007.01 (AOAC) (15 g sample)

• 6 g MgSO4, 1.5 g Na Acetate

• EN method 15662 (CEN) (10 g sample)

• 4 g MgSO4, 1 g NaCl, 1 g NaCitrate, 0.5 g disodium citrate

sesquihydrate

Purpose of the first step is to facilitate extraction/partitioning: excess salt and

anhydrous magnesium sulfate

Step 1: Comparison of the 3 Extraction Methods

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Basic QuEChERS Extraction Flow Chart

15 gm homogenized sample, (spike) 10 gm homogenized sample, (spike)

Add 15 mL ACN (1% AA), vortex Add 10 mL ACN, vortex

Add AOAC Extraction Salts, shake Add EN Extraction Salts, shake

Centrifuge Centrifuge

Transfer volume to AOAC d-SPE tube, vortex, centrifuge Transfer volume to EN d-SPE tube, vortex, centrifuge

Transfer to Analysis vial Transfer to Analysis vial

Analyze by GC/MS or LC/MS/MS*

* Requires a dilution or

evaporation/recon prior to analysis

AOAC EN

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• Each works equally well in most cases

• Compound stability, pH labile

• Determine matrix co-extractions

• Matrix effects on quantification

• Chemical noise from matrix

Three Methods: How To Choose

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Higher and more consistent recoveries

pymetrozine, thiabendazole, imazalil

Reason: low recoveries of pymetrozine in citrus, variable recoveries for

pH-dependent pesticides in general

Pymetrozine: AOAC 82% (7% RSD)

EN ~ 30% (51% RSD)

Unbuffered (100% RSD)

Strong acetate buffering at pH 4.8 during extraction gave optimum

balance and acceptable recoveries > 70%

AOAC Buffered Method

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Acetate buffer system resulted in “visibly worse cleanup results compared to

the original QuEChERS method”

- M. Anastassiades

Incorporates weaker citrate buffers to facilitate buffering

system for pH labile compounds, pH 5.0-5.5

Does not require a 1% acetic acid ACN, preferred by GC

users

EN Buffered Method

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• S. Lehotay: J. Chromatography A 1217 (2010), 2548-2560

“Slight differences in color and color intensity: AOAC versus EN”

1) Determine by gravimetric measurements

2) Chromatography

3) Determination of matrix effects on quantitation, matrix effects are

compound and matrix dependent

Extract: non-buffered = 0.23%

citrate buffered = 0.17%

acetate buffered = 0.13%

Matrix Effects: How Do you Choose Best Extraction Salts for Compound Recovery

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0

50000

100000

150000

200000

250000

300000

350000

400000

-600-500-400-300-200-100 0 100 200 300 400 500 600 700 800 90010001100

Solvent

Matrix

ng/mL

Response

How can we Improvement the Extraction of Samples?

Step 1: Extraction salts with comminuted fruit/vegetable or other commodities

• Consistency in shaking, everyone shakes differently

• Variability in QuEChERS applications, recovery and RSDs

Ceramic Homogenizers

• Reduces shaking time from 1 minute to <20 seconds, spiked sample

- Consistent extraction of the sample with the salts

- Breaks up salt agglomerates

- Facilitates homogenization with angle cut

- Increase recovery of pesticides from sample

- Three different sizes: 50 mL extraction tube and two d-SPE tubes: 15 and 2 mL

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QuEChERS Extraction Salts: Sin y Con Homogenizadores Ceramicos

CH no CH CH no CH

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QuEChERS Modifications

• Literature noted extraction solvent variance:

Ethyl acetate

Acetone

Acetone/Ethyl acetate/Cyclohexane

(implemented due to solubility, lipid content, sugar content)

Warning: When extraction solvent is changed from ACN, the chemistries associated with the d-SPE might also change

• Clean-up options:

Freezing out-Lipid removal

Hexane addition-Lipid removal

Different sorbents or sorbent combinations

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Extraction Matrices and Analysis Examples

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QuEChERS

Excellent Signal to Noise Ratios at Trace Levels

GC/MS SIM Chromatogram of 10 ppb PAHs spiked in fish matrix blank extract GC/MSD: 7890/5975B with purged ultimate union

Column: DB-5ms UI 20 m 0.18 mm 0.18 µm

Restrictor : Siltek 0.7m x 0.15mm ID (Col 2)

MMInlet: 0.5µL, 320°C, splitless , purge flow 50mL/min at 0.8min

gas saver 30mL/min at 2min

Carrier: Helium, 1.7mL/min cnst flow col 1

PCM 1=3.8 psi cnst pressure, col 2=3.8ml/min flow @ 50°C

Oven: 50°C (0.4 min) to 195°C (25°C/min) hold 1.5 min,

8°C/min to 265°C 20°C/min to 315°C (1.25 min)

Postrun backflush 7 min @320°C

MSD: Transfer line 340°C Source 340°C Quad 150°C

4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

2400

Time

Abundance

1 2

3

4 5

6

7 8

9

10

11 13 12

14 16

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S/N = 11.5

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1. Napthalene

2. Acenaphthylene

3. Acenaphthene

4. Fluorene

5. Phenanthrene

6. Anthracene

7. Fluoranthene

8. Pyrene

9. Benz[a]anthracene

10. Chrysene

11. Benzo[b]fluoranthene

12. Benzo[k]fluoranthene

13. Benz[a]pyrene

14. Indeno[1,2,3-c,d]pyrene

15. Dibenz[a,h]anthracene

16. Benzo[g,h,i]perylene

AOAC

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25 ng/mL fortified QC 250 ng/mL fortified QC 500 ng/mL fortified QC

Analytes %Recovery RSD (n=6) %Recovery RSD (n=6) %Recovery RSD (n=6)

Naphthalene 80.35 3.29 96.77 4.23 98.64 1.88

Acenaphthylene 95.28 2.30 103.36 2.80 101.02 2.27

Acenaphthalene 92.28 2.51 101.18 2.87 100.69 2.34

Fluorene 95.98 2.99 105.94 2.82 105.00 1.28

Phenanthrene 100.51 3.46 104.93 2.71 103.25 1.70

Anthracene 107.38 3.51 105.95 3.45 105.38 1.74

Fluoranthene 113.27 3.87 105.76 3.33 103.64 1.81

Pyrene 113.55 3.51 103.99 3.24 102.29 1.94

Benz[a]anthracene 129.79 3.41 101.45 3.91 100.61 3.24

Chrysene 116.75 4.01 98.55 4.17 95.95 5.61

Benzo[b]fluoranthene 131.20 3.70 98.77 4.08 98.08 3.24

Benzo[k]fluoranthene 139.45 2.52 99.13 3.98 95.31 4.54

Benzo[a]pyrene 125.30 3.68 95.33 3.89 96.82 1.80

Indeno[1,2,3-cd]pyrene 119.51 3.47 94.57 3.23 93.71 2.55

Dibenz[a,h]anthracene 126.35 3.54 98.55 3.50 98.85 2.24

Benzo[g,h,i]perylene 114.91 4.93 97.30 3.37 95.63 1.83

Recovery and Repeatability of PAHs in Fortified Red

Snapper Fish with Agilent J&W DB-5ms UI column

http://www.fda.gov/downloads/ScienceResearch/UCM220209.pdf

http://www.prnewswire.com/news-releases/noaa-and-fda-announce-chemical-test-for-dispersant-in-gulf-

seafood-106323493.html

FDA: PAH in Selected Seafood and Surfactants in Seafood

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MRM chromatograms of 50 ng/g fortified sample processed by EN method. Peak identification: 1. Acephate, 2. Pymetrozine, 3. Carbendazim, 4. Thiabendazole, 5. Imidacloprid, 6. Imazalil, 7. Propoxur, 8. Carbaryl, 9. Cyprodinil, 10. Ethoprophos, 11. Penconazole, 12. Kresoxim-methyl, IS: TPP

Pesticides in Green Tea (Dry Matrix): LC/MS/MS

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5990-6400EN

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Recovery and Reproducibility of Pesticides in Fortified Green Tea with QuEChERS

QuEChERS Extraction: Lemon Citrus Oil

After QuEChERS extraction (2 mL hexane)

and 2 d-SPE in Series (modified method)

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5990-6432EN

Lemon Essential Oil Recovery and RSD

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QuEChERS: Not Just for Pesticides Anymore Pesticides in other

commodities

PAHs

PCBs

Illegal Dyes

Mycotoxins

Veterinary Drugs

Antibiotics

Pharmaceuticals Drugs in

biologicals

Acrylamides

And More to Come…..

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QuEChERS is not the Holy Grail of Sample Preparation

There are limitations to the QuEChERS

extraction procedure

Not applicable to certain pesticides

and other types of compounds ( e.g.

constantly charged compound-

quaternary amine

The extracts may be too dirty for

certain types of detection

Improvements would be welcome for

very difficult matrices ( e.g. high

fat/lipid)

QuEChERS: How, What, Where and Why

How: By extraction/partitioning (Step 1) and d-SPE (Step 2),

Analysis (step 3)

What: Fruits, vegetables, grains/flours/wheat, dried material,

processed food, meats, oils, seafood….and many more

Where: Any lab, mobile labs, minimal sample preparation

expertise

Why: MS and MS/MS, selectivity and sensitivity

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Conclusion: QuEChERS Advantages

QuEChERS approach is as easy as 1-2-3!

Simple and Attractive Sample Preparation Methodology

Minimal sample preparation expertise required: If you can

weigh, pipette and shake, you can do QuEChERS!

Easy method optimization

Excellent results

Green and very cost-effective technique

Capabilities surprise even the experts

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Work of Carl Warner

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Thank You for Your Time and Attention!

Questions

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