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

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.

Primeros Mtodos de Multi-residuos: Extraccin con solventes

Acetonitrilo:

Mtodos CFDA Canadiense.

Acetona:

Metodos Holandeses Luke.

Etil Acetato:

Suiza, Suecia.

Regresando a los 60s (1960)

6/11/2014
4

Eureka.tenemos un metodo
Luke

Canadiense

Primer QuEChers

Based on Canadian Multi-residue

method

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.

QuEChERS: Preparando la muestra para ser procesada

Reduces the heat
produced while grinding

Vamos a ver los beneficios de QuEChERS

Solo 3 pasos

Paso 1: Extraccin

Paso 2: Limpieza SPE dispersiva

Analisis

10

Pasos del metodo .

1

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 estndar interno, agitar

Centrifugar, separacin y extraccion

5
6

3
7

Del Instituto Stuttgart , Micheangelo Anastassiades

Aunque el video sugiere adicionar el IS antes que las sales de
extraccion nosotros lo hacemos antes de adicionar el ACN

11

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

1

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 estndar interno, agitar

Centrifugar, separacin y extraccion

5
6

3
7

Del Instituto Stuttgart , Micheangelo Anastassiades

Aunque el video sugiere adicionar el IS antes que las sales de
extraccion nosotros lo hacemos antes de adicionar el ACN

12

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

2

Transferir una alicuota al tubo d SPE/ agitacin

8

Agitar
Centrifugar
Transferir una alicuota a un vial

Analizar

10

11

Del Instituto Stuttgart , Micheangelo Anastassiades

13

14

15

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

QuEChERS

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

16

Tienes un momento ????

Visite nuestra pagina en internet y vea nuestro video de Quechers

www.agilent.com/chem/quechersvideo

17

#3: QuEChERS: Una vista rpida 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
Metodo Luke, SPE o GPC

QuEChERS

QuEChERS
Benefits!

Tiempo de
procesamiento para 6
muestras (min)

120

30

4x mas rpido

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
cristalera

Embudo, rotavapor,
contenedores de 200mL,.

None

No son necesarios
otros complemento

Ahorro por Muestras

muestra
por da
$72.00
25

Das por
Semana
5

Ao 1
$468,000

El ahorro general $72 USD

por muestra.
Information courtesy of Dr.
Steve Lehotay, USDA

18

Como se relaciona esto con el tema de Seguridad

Alimentaria
QuEChERS
Permite la determinacion simultanea de muchas clases de compuestos

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

Cada contaminante tiene un nivel establecido relacionado, MRL (Limite

Maximo Residual)

As 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)

19

Porque QuEChERS?
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

20

Comparacion MS/SIM versus MS/MS: Seguridad

Alimentaria
SIM - EIC (246)
MRM (2 transitions)
Apple

S/N = 448

Cabbage

S/N = 241

Ginseng

S/N = 446

Orange

S/N = 456

Spinach

S/N = 260

21

Step 1: Comparison of the 3 Extraction Methods

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

22

Basic QuEChERS Extraction Flow Chart

AOAC

EN

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

23

Three Methods: How To Choose

Each works equally well in most cases

Compound stability, pH labile
Determine matrix co-extractions
Matrix effects on quantification
Chemical noise from matrix

24

AOAC Buffered Method

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%

25

EN Buffered Method
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

Acetate buffer system resulted in visibly worse cleanup results compared to

the original QuEChERS method
- M. Anastassiades

26

Matrix Effects: How Do you Choose Best Extraction

Salts for Compound Recovery
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
400000
compound and matrix dependent 350000
Extract: non-buffered = 0.23%
citrate buffered = 0.17%
acetate buffered = 0.13%

Response

300000
250000
200000
150000
Solvent
100000
50000

Matrix

0
-600-500-400-300-200-100 0 100 200 300 400 500 600 700 800 90010001100
ng/mL

27

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

28

QuEChERS Extraction Salts:

Sin y Con Homogenizadores Ceramicos

CH

no CH

CH

no CH

29

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

30

31

QuEChERS
Extraction Matrices and Analysis Examples

32

Excellent Signal to Noise Ratios at Trace Levels

1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.

Abundance

2400
2200
2000

AOAC

1800

4
1600
1400

5
6

Napthalene
Acenaphthylene
Acenaphthene
Fluorene
Phenanthrene
Anthracene
Fluoranthene
Pyrene
Benz[a]anthracene
Chrysene
Benzo[b]fluoranthene
Benzo[k]fluoranthene
Benz[a]pyrene
Indeno[1,2,3-c,d]pyrene
Dibenz[a,h]anthracene
Benzo[g,h,i]perylene

S/N = 11.5
14

1200

1000

8
10

800

600

11

12

14 15

13

16

400
200
4.00

6.00

8.00

10.00

12.00

14.00

16.00

18.00

20.00

Time

GC/MS SIM Chromatogram of 10 ppb PAHs spiked in fish matrix blank extract
GC/MSD:
Column:
Restrictor :
MMInlet:
Carrier:

Oven:
MSD:

7890/5975B with purged ultimate union

DB-5ms UI 20 m 0.18 mm 0.18 m
Siltek 0.7m x 0.15mm ID (Col 2)
0.5L, 320C, splitless , purge flow 50mL/min at 0.8min
gas saver 30mL/min at 2min
Helium, 1.7mL/min cnst flow col 1
PCM 1=3.8 psi cnst pressure, col 2=3.8ml/min flow @ 50C
50C (0.4 min) to 195C (25C/min) hold 1.5 min,
8C/min to 265C 20C/min to 315C (1.25 min)
Postrun backflush 7 min @320C
Transfer line 340C Source 340C Quad 150C

33

Recovery and Repeatability of PAHs in Fortified Red

Snapper Fish with Agilent J&W DB-5ms UI column
25 ng/mL fortified QC
%Recovery RSD (n=6)
Naphthalene
80.35
3.29
Acenaphthylene
95.28
2.30
Acenaphthalene
92.28
2.51
Fluorene
95.98
2.99
Phenanthrene
100.51
3.46
Anthracene
107.38
3.51
Fluoranthene
113.27
3.87
Pyrene
113.55
3.51
Benz[a]anthracene
129.79
3.41
Chrysene
116.75
4.01
Benzo[b]fluoranthene
131.20
3.70
Benzo[k]fluoranthene
139.45
2.52
Benzo[a]pyrene
125.30
3.68
Indeno[1,2,3-cd]pyrene
119.51
3.47
Dibenz[a,h]anthracene
126.35
3.54
Benzo[g,h,i]perylene
114.91
4.93

Analytes

250 ng/mL fortified QC

%Recovery RSD (n=6)
96.77
4.23
103.36
2.80
101.18
2.87
105.94
2.82
104.93
2.71
105.95
3.45
105.76
3.33
103.99
3.24
101.45
3.91
98.55
4.17
98.77
4.08
99.13
3.98
95.33
3.89
94.57
3.23
98.55
3.50
97.30
3.37

500 ng/mL fortified QC

%Recovery RSD (n=6)
98.64
1.88
101.02
2.27
100.69
2.34
105.00
1.28
103.25
1.70
105.38
1.74
103.64
1.81
102.29
1.94
100.61
3.24
95.95
5.61
98.08
3.24
95.31
4.54
96.82
1.80
93.71
2.55
98.85
2.24
95.63
1.83

FDA: PAH in Selected Seafood and Surfactants in Seafood

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

http://www.prnewswire.com/news-releases/noaa-and-fda-announce-chemical-test-for-dispersant-in-gulfseafood-106323493.html

34

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

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
5990-6400EN
35

Recovery and Reproducibility of Pesticides in

Fortified Green Tea with QuEChERS

36

QuEChERS Extraction: Lemon Citrus Oil

After QuEChERS extraction (2 mL hexane)

and 2 d-SPE in Series (modified method)

5990-6432EN
37

Lemon Essential Oil Recovery and RSD

38

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..

39

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 compoundquaternary 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)

40

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

41

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

42

Thank You for Your Time and Attention!

Work of Carl Warner

43

Questions

44