APPLICATION NOTEBOOK FOR AFFINIMIP®SPE - Life …v~affinimip... · APPLICATION NOTEBOOK FOR AFFINIMIP®SPE Q3 2014 Selective Solid Phase Extraction Molecularly Imprinted Polymers

APPLICATION NOTEBOOK FOR

AFFINIMIP®SPE

Q3 2014

Selective Solid Phase ExtractionMolecularly Imprinted Polymers for the Selective

Extraction of Trace Analytes from Complex Matrices

2

INTRODUCTION

AFFINISEP offers a comprehensive range of sorbents for the challenging fields of sample preparation,sample clean-up and extraction, from conventional to more sophisticated sorbents. So, for veryspecific and challenging applications, AFFINISEP has developed AFFINIMIP®SPE products, SPEcartridges based on Molecularly Imprinted Polymers (MIP) which require ready-to-use protocols.

To make you enjoy of this background and for an wealthy, very instructive and easily-accessedoverview of AFFINIMIP®SPE performances, we are pleased to introduce you this ApplicationNotebook which collects a good abstract of AFFINISEP’s experience on AFFINIMIP®SPE.

This Application notebook will be an essential tool to address your technical issues.

QUALITY POLICY

In addition, to ensure the best quality of its products, the performance is checked by followingseveral QC tests according to each product’s quality control procedure. After passing all these tests,the products receive a certificate of analysis which proved the compliance with the defined criterion.

To develop a long term and durable partnership with itscustomers, AFFINISEP ensures the best quality of its productsand services.As an ISO9001:2008 certified company, AFFINISEP hasimplemented Quality management system requirements toshow its commitment to quality, customers, and a willingnessto work towards improving efficiency.

http://www.polyintell.com/

mailto:[email protected]

mailto:[email protected]

AFFINIMIP® SPE VS IMMUNOAFFINITY – COMPARATIVE STUDY 5

AFFINIMIP® SPE FOR MYCOTOXINS ANALYSES 7

AFFINIMIP® SPE Multimyco10

Simultaneous determination of Aflatoxins, Fumonisins, Ochratoxin A, HT-2, T-2, Zearalenone in Wheat 8

AFFINIMIP® SPE Patulin

Patulin in Baby food apple juice 9

Patulin in Apple juice 10

Patulin in Baby food apple puree 11

Patulin in Apple puree 12

Patulin in Apple – Fruit puree 13

Patulin in WHOLE apple 14

Patulin in Cider 15

Patulin in Alcohol Pommeau and Liquor 16

Patulin in Tomato Ketchup and Tomato Powder 17

Patulin in Blueberry juice 18

Patulin in Thick Juice and Concentrate Juice 19

Patulin in Apple puree 20

Patulin in Dried apple 21

AFFINIMIP® SPE Ochratoxin A

Ochratoxin A in Cereals 22

Ochratoxin A in Paprika 23

Ochratoxin A in Red and White wine 24

AFFINIMIP® SPE Zearalenone

Zearalenone in Maize and Rice 25

Zearalenone in Cereal-based Baby food 26

Zearalenone in Edible corn oil 27

AFFINIMIP® SPE Deoxynivalenol

Deoxynivalenol in cereals for food (Water extraction) 28

Deoxynivalenol in babyfood cereals 29

Deoxynivalenol, 3-AcetylDON and 15-AcetylDON in cereals (Hydro-organic extraction) 30

Deoxynivalenol in cereals for animal feeds 31

AFFINIMIP® SPE FumoZON

Fumonisins B1 + B2 and Zearalenone in Maize Flour 32

Fumonisins B1 + B2 and Zearalenone in Maize-based baby food 33

AFFINIMIP® SPE Estrogens 34

Determination of estrogens in plasma 35

Protocol comparative - AFFINIMIP® SPE Estrogens vs usual protocol 36

TABLE OF CONTENTS

3

AFFINIMIP® SPE Bisphenol A 37

Bisphenol A in Liquid infant formula 38

Bisphenol A in powdered infant formula 39

PROTOCOL COMPARATIVE WITH A COMPETITOR – POWDERED INFANT FORMULA 40

Bisphenol A in canned food (liquid form) 41

Bisphenol A in canned food (Vegetable) 42

Bisphenol A in Beer 43

Bisphenol A in White/Red wines 44

Bisphenol A in Cola drinks 45

Bisphenol A and BADGE in Milk 46

Total Bisphenol A in Human Urine 47

AFFINIMIP® SPE Chloramphenicol 48

Chloramphenicol in Honey 49

Chloramphenicol in Bovine urine 50

Chloramphenicol in Shrimp 51

AFFINIMIP® SPE Amphetamines 52

Amphetamines in Human urine 53

Amphetamines in Human serum 54

AFFINIMIP® SPE Tetracyclines 55

Tetracyclines (Oxytetracycline, Chlortetracycline, Tetracycline), theirs epimers and Doxycycline in Milk and Salmon 56

AFFINIMIP® SPE Metanephrines 57

Methanephrines in Plasma – comparison with WCX cartridges 58

AFFINIMIP® SPE Phenolics 59

Parabens in cosmetic products 60

Guaïacol 61

Carnosic acid in meat 62

AFFINIMIP® SPE PRODUCT LIST FOR MYCOTOXIN ANALYSES 63

AFFINIMIP® SPE PRODUCT LIST (MISCELLANEOUS) 64

LIST OF PUBLICATIONS AND POSTERS 66

TABLE OF CONTENTS

4

Solid phase extraction is a simple tool to selectively extract analytes from complex matrices and quantify

concentrations lower and lower. The major disadvantage of conventional SPE sorbents, such as C18 is a lack of

selectivity and interference matrix components are co-extracted with the target analytes. To solve this

problem, affinity-based SPE sorbents have been developed to be selective in extracting the target analytes like

molecularly imprinted polymer (MIP) and immunoaffinity sorbent.

Immunoaffinity columns (IAC) are biological sorbents based on the use of antibodies that are specific to the

target analytes.

Molecularly imprinted polymer is a synthetic material with artificially generated three-dimensional network

able to specifically rebind a target molecule.

Based on molecularly imprinted polymers, AFFINISEP’s AFFINIMIP® SPE cartridges have the advantages to be

highly selective and specific. Contrary to IAC, AFFINIMIP® SPE cartridges are chemically and thermally stable,

compatible with all solvents as well as cost effective.

AFFINIMIP® SPE VS IMMUNOAFFINITY – COMPARATIVE STUDY

Feature IACAFFINIMIP®

SPE

Selectivity High High

Capacity 6µmol/g10-

100µmol/g

Analyte recognition

in waterGood Variable

Analyte recognition

in OrganicsPoor Good

Stability Poor Very High

Reproducibility Variable Good

Cost Expensive Inexpensive

PROPERTIES OF MIP AND IAC

PROTOCOL: Ochratoxin A (OTA) from wheatflour

Step Vicam IACAFFINIMIP®SPE

OTA

Extraction of

target analyte

50g sample in 100mL 60/40

ACN/water

Blender 1 minute + filtration

Preparation

loading

solution

10mL extract +

40mL PBS

10mL extract +

10mL HCl 0.1M

pH=1

Loading10mL Loading

solution

4mL Loading

solution

Washing10mL PBS

10mL Water

7mL 60/40 HCl

0.1M pH=1/ACN

Elution 1.5mL Methanol

2mL 98/2

Methanol/Acetic

acid

Protocol time 30min 20min

PROTOCOL: Zearalenone (ZON) from maizeflour

Step Vicam IACAFFINIMIP®SPE

ZON

Extraction

of target

analyte

25g sample in

100mL 90/10

Methanol/water

Blender 3 minutes

+ filtration

25g sample in

100mL 75/25

ACN/water

Blender 3

minutes +

filtration

Preparation

loading

solution

4mL extract +

96mL water

10mL extract +

10mL Water

Loading100mL Loading

solution

8mL Loading

solution

Washing 20mL Water

4mL 2/58/40

Acetic acid /

water / ACN

Elution 1.5mL Methanol

2mL 98/2

Methanol/Acetic

acid

Protocol

time55min 30min

Compared to IAC, AFFINIMIP® SPE provides:Easier and faster protocolLower dilutionEasier automatisation

(Cf. Automated method for the selective SPE ofOchratoxin A from wheat Using MolecularlyImprinted Polymer; Gilson Application NotesHandbook 2011; volume 1 Issue 4)

Minutes

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mV

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52Fluo

elution B06

Fluo

elution immuno B02

IAC AFFINIMIP® SPE

Figure 1. Chromatogram of Maize sample spiked with Zearalenone at 85 µg/kg obtained after cleanup byAFFINIMIP®SPE Zearalenone (red) or Vicam IAC (blue).

Figure 2. Chromatogram of wheat sample spikedwith Ochratoxin A obtained after cleanup byAFFINIMIP®SPE Zearalenone (red, spiked at 10ng/g)or Vicam IAC (blue, spiked at 6ng/g).

Equivalent chromatograms

98 97

8579

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MIP IAC MIP IAC

Recovery %

OTAZON

RECOVERIES

Figure 3. Recovery of Ochratoxin A or Zearalenoneobtained after cleanup by AFFINIMIP®SPE or VicamIAC.

Higher Recoveriesobtained withAFFINIMIP® SPE

Figure 4. Comparison of capacity betweenAFFINIMIP®SPE Zearalenone (red) and VicamIAC (blue).

AFFINIMIP® SPE

IAC

Figure 5. Comparison of capacity betweenAFFINIMIP®SPE OTA (red) and Vicam IAC (blue).

Capacity MIP > Capacity IAC

10ng/g OTA85% Recovery

6ng/g OTA79% Recovery

AFFINIMIP® SPE CARTRIDGE VS IMMUNOAFFINITY COLUMN

CAPACITY

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

0 1000 2000 3000 4000 5000 6000

Leve

l fo

un

d n

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Spiking level ng/g

0,00E+00

5,00E+07

1,00E+08

1,50E+08

2,00E+08

2,50E+08

3,00E+08

3,50E+08

0 2000 4000 6000 8000 10000

Are

a

Spiking level ng/g

AFFINIMIP® SPE

IAC

CHROMATOGRAM ASPECT

AFFINIMIP® SPE

IAC

AFFINIMIP® SPE for mycotoxins analyses

7

Mycotoxins are toxic secondary metabolites produced by different fungi present in

agricultural commodities. They are regulated in food and feed due to nephrotoxic,

neurotoxic, carcinogenic, estrogenics, and immunosuppressive effects.

AFFINISEP has developed two sets of product for mycotoxins analyses:

For single mycotoxin extraction

Designed for the analysis of one specific mycotoxin or for mycotoxins analyzed by

fluorescence detection

AFFINISEP has developed the following products

AFFINIMIP® SPE Patulin (PI-FS102)

AFFINIMIP® SPE Ochratoxin A (PI-FS101)

AFFINIMIP® SPE Zearalenone (PI-FS100)

AFFINIMIP® SPE Deoxynivalenol (PI-FS117)

For multimycotoxins extraction

Designed for the simultaneous extraction of several mycotoxins which are present in a

same matrix prior to LC-MS/MS analyses

These mycotoxins are all present in the same matrix to be analyzed. Their extraction is done

all at once by SPE. Then the elution solution containing all these mycotoxins is evaporated,

reconstituted and analyzed by LC-MS/MS.

The protocol is short and efficient

AFFINISEP has developed the following products

AFFINIMIP® SPE FumoZON (PI-FS109) for the analyses of

Fumonisins

Zearalenone

AFFINIMIP® SPE Multimyco10 (PI-FS114) for the analyses of

Fumonisins

Aflatoxins

Ochratoxin A

T-2 and HT-2

Zearalenone

AFFINIMIP® SPE Multimyco10

AFFINIMIP® SPE Multimyco10 are Multimycotoxins solid-phase extraction cartridges that

selectively and SIMULTANEOUSLY clean-up and concentrate Fumonisins, Aflatoxins,

Ochratoxin A, T-2, HT-2 and Zearalenone prior to analysis by LC-MS/MS from complex

matrices such as cereals.

FS114

Catalog number: PI-FS114-03 for 50 cartridges, 3mLPI-FS114-04 for 100 cartridges, 3mL

Recovery of multimycotoxins extracted from wheat

and analyzed after AFFINIMIP® SPE Multimyco10

cleanup

Recovery yield

8

Compound name

C°µg/k

g

Mean µg/kg

R%

Aflatoxin B1 2 1.6 85

Fumonisin B1 1000 937 94

HT-2 100 119 119

T-2 50 56.5 113

Zearalenone 50 54 108

Ochratoxin A 3 2.6 88

WHEAT

SIMULTANEOUS DETERMINATION OF MULTIMYCOTOXINS IN WHEAT

PROTOCOL OF CLEANUP

Sample preparation

25g of ground wheat were extracted with

100mL of Acetonitrile/Water (50/50, v/v/v) for

2 min using a blender. The extract was filtered

through a folded filter paper and 4mL of the

filtrate were diluted with 16mL of water. Then,

this solution was filtered through a filter paper.

Cleanup with a 3mL/60mg AFFINIMIP® SPE

Multimyco10 cartridge

Equilibration

•2mL MeOH -2% Formic acid

•3mL Acetonitrile

•2mL water

Loading

•3mL of loading solution

Washing of interferents (W1)

•3mL Water

•3mL Water/Acetonitrile (85/15 v/v)

Drying by applying vacuum 3 minutes

Elution (E)

•3mL Methanol/ACN/Formic acid

(48.5/48.5/3, v/v/v)

The elution fraction was then evaporated and

dissolved in water containing 0.1% acetic acid

before HPLC analysis.

Compound name RT MRM transition

Aflatoxin B1Positive ion mode

1.42

312.8 > 285.0

Fumonisin B1Positive ion mode

1.71

722.4 > 141.1 722.4 > 334.5722.4 > 352.3

HT-2Positive ion mode

1.76

441.9 > 104.9441.9 > 214.7441.9 > 263.2

T-2Positive ion mode

1.89

484.0 > 105.0484.0 > 185.1484.0 > 305.1

ZearalenoneNegative ion mode

2.05

316.8 > 131.1316.8 > 174.9316.8 > 273.1

Ochratoxin APositive ion mode

2.13

403.8 > 221.1403.8 > 239.2403.8 > 356.1

MRM parameters for mycotoxins analyses

UFLC MethodColumn: Phenomenex Kinetix XB-C18Detection: LC-MS/MS with ESI source - MRM modeInjection volume: 20µL.

DETERMINATION OF PATULIN IN BABY FOOD APPLE JUICE

Minutes

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mA

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Patulin

Chromatograms obtained after AFFINIMIP® SPEPatulin Clean-up of an apple juice spiked at10µk/kg with Patulin (Green and blue) or notspiked (Red)

Recovery of Patulin (n=9) at a contamination level of10µg/kg in apple Juice after AFFINIMIP® SPE PatulinClean-up.

Recoveries % (n=9) % RSDR

97.9 11

HPLC MethodColumn: Atlantis T3 column, 150mm x 2.1mmMobile phase: Deionized water/ACN (95/5, v/v)Flow rate: 0.2mL/minDetection: UV - 276nmInjection volume: 100µL.

PROTOCOL OF CLEANUP

Sample preparation

Loading solution: 2.5mL apple juice and 2.5mL

of water-2% acetic acid are mixed.


Patulin cartridge

Equilibration

•2mL Acetonitrile

•1mL water

Loading



•1mL NaHCO3

•2mL Water

Drying by applying vacuum 10 seconds


•1mL Diethyl Ether

Elution (E)

•2mL Ethyl Acetate




Regulations for apple juice: Europe (EC 1881/2006) : 50µg/Kg USA (FDA CPG Sec.510.150) : 50µg/KgRegulations for apple juice for infants and young children:Europe (EC 1881/2006) : 10µg/Kg

RESULTS

Catalog number:3mL-100mg sorbent for apple juice and pureePI-FS102-02 for 25 cartridgesPI-FS102-03 for 50 cartridges6mL-200mg sorbent for a apple-based products, fruit juice and concentrate PI-FS102-02B -200mg for 25 cartridgesPI-FS102-03B -200mg for 50 cartridges

9


DETERMINATION OF PATULIN IN APPLE JUICE

Chromatograms obtained after AFFINIMIP® SPEPatulin Clean-up of an apple juice spiked at40µg/kg (tested twice, red) or at 10µg/kg (testedtwice, blue) with Patulin or not spiked (orange)

Recovery of Patulin in apple juice after AFFINIMIP®

SPE Patulin Clean-up and relative standarddeviation calculated from results generated underreproducibility conditions.


PROTOCOL OF CLEANUP

Sample preparation

Loading solution: 2.5mL apple juice and 2.5mL

of water-2% acetic acid are mixed.

Clean-up with a 3mL/100mg AFFINIMIP® SPE

Patulin cartridge

Equilibration

•2mL Acetonitrile

•1mL water

Loading



•1mL NaHCO3 in Water

•2mL Water

Drying by applying vacuum 10 seconds


•1mL Diethyl Ether

Elution (E)

•2mL Ethyl Acetate




Regulations for apple juice: Europe (EC 1881/2006) : 50µg/Kg USA (FDA CPG Sec.510.150) : 50µg/KgRegulations for apple juice for infants and young children:Europe (EC 1881/2006) : 10µg/Kg

RESULTS

10

Minutes

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UV 6000-276nm

Brut carrefour.dat

UV6000-276nm

C1 C05

Patulin

Before clean-up

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HMF

Chromatograms of apple juice containing 25µg/kgof Patulin before (Red) and after (Blue) AFFINIMIP®

SPE Patulin Clean-up

Concentration of Patulin (ng/mL)

Recoveries % % RSDR

10 97.9 11 (n=9)

40 90.6 11 (n=41)


Catalog number:3mL-100mg sorbent for apple juice and pureePI-FS102-02 for 25 cartridgesPI-FS102-03 for 50 cartridges6mL-200mg sorbent for a apple-based products, fruit juice and concentrate PI-FS102-02B -200mg for 25 cartridgesPI--FS102-03B -200mg for 50 cartridges

DETERMINATION OF PATULIN IN APPLE PUREE

PROTOCOL OF CLEANUPSample preparation10g of apple puree, 150µL of a pectinaseenzyme solution and 10mL water are mixed.Leave solution at room temperature overnightor for 2h at 40°C. Centrifuge at 4500g for 5minand then filter the solution with a 0.2µm filter.This solution is used as the loading solution.

Cleanup with a 3mL/100mg AFFINIMIP® SPEPatulin cartridge

Equilibration•2mL Acetonitrile•1mL Water

Loading•5mL of loading solution

Washing of interferents (W1)•4mL Water -1%Acetic acid•1mL NaHCO3 1% solution•3mL Water

Drying by applying vacuum 10 secondsWashing of interferents (W2)

•500µL Diethyl EtherElution (E)

•2mL Ethyl AcetateThe elution fraction was then evaporated anddissolved in water containing 0.1% acetic acidbefore HPLC analysis.

RESULTS

Catalog number:3mL-100mg sorbent PI-FS102-02 for 25 cartridgesPI-FS102-03 for 50 cartridgesPI-FS102-02K for a kit of 25 cartridges + 50mLPectinasePI-FS102-03K for a kit of 50 cartridges + 50mLPectinasePI-REA-001-50mL for 50mL Pectinase solution

11

Time (min) % water % ACN

0 98 2

20 98 2

21 50 50

25 50 50

26 98 2

HPLC MethodColumn: Atlantis T3 column, 150mm x 2.1mmMobile phase: gradient

Flow rate: 0.2mL/minDetection: UV - 276nmInjection volume: 100µL.

Regulations for apple puree: Europe (EC 1881/2006) : 25µg/Kg Regulations for apple juice for infants and young children:Europe (EC 1881/2006) : 10µg/Kg

Minutes5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

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UV 6000-276nm

C07

UV 6000-276nm

C10

UV 6000-276nm

C15

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Minutes5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

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UV 6000-276nm

C15

Patulin

Chromatograms of apple puree containing 40µg/kgor 80µg/kg of Patulin before (Red) and after (Blue)AFFINIMIP® SPE Patulin Clean-up

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C4-E-bis C21

UV 6000-276nm

C06

UV 6000-276nm

C11

Chromatograms of apple puree containing 0µg/kg(blue) or 20µg/kg (tested twice, green and red)) ofPatulin after AFFINIMIP® SPE Patulin Clean-up

Patulin

Recovery and repeatability of Patulin (n=3) at acontamination level of 20µg/kg in apple puree afterAFFINIMIP® SPE Patulin Clean-up.

Concentration of Patulin (µg/kg)

Recoveries %(n=3)

% RSDr

20 84 4.5


DETERMINATION OF PATULIN IN BABY FOOD APPLE PUREE

Patulin

Chromatograms obtained after AFFINIMIP® SPEPatulin Clean-up of different apple puree.

In the lower part, clean-up of an apple puree froma well-known brand spiked at 25µg/kg (orange),10µk/kg with Patulin (pink, tested twice) or notspiked (red).In the top part, clean-up of an apple puree secondwell known brand spiked at 25µg/kg (green),10µk/kg with Patulin (blue, tested twice) or notspiked (turquoise).









RESULTS


12


0 98 2

20 98 2

21 50 50

25 50 50

26 98 2


Recoveries % (n=4) % RSDR

81.2 2.1



Regulations for apple puree: Europe (EC 1881/2006) : 25µg/Kg Regulations for apple puree for infants and young children:Europe (EC 1881/2006) : 10µg/Kg


DETERMINATION OF PATULIN IN APPLE – FRUIT PUREE









RESULTS


13


0 98 2

20 98 2

21 50 50

25 50 50

26 98 2



Regulations for apple puree: Europe (EC 1881/2006) : 25µg/Kg Regulations for apple puree for infants and young children:Europe (EC 1881/2006) : 10µg/Kg

Minutes

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UV 6000-276nm

C4-E-bis C21

UV 6000-276nm

C06

UV 6000-276nm

C11

Patulin

Chromatograms of apple puree containing 0µg/kg(blue) or 20µg/kg (tested twice, green and red) ofPatulin after AFFINIMIP® SPE Patulin Clean-up.

Minutes

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Chromatograms obtained after AFFINIMIP® SPEPatulin Clean-up of different purees.

Apple-pear puree with Patulin (25µg/kg)

Apple puree with Patulin (40µg/kg)

Apple-strawberry puree with Patulin (40µg/kg)

Recovery and reproducibility of Patulin withdifferent levels of contamination for all testedapple-fruit puree after AFFINIMIP® SPE PatulinClean-up.


Recoveries %

% RSDR

10 (n=9) 77.4 8.1

25 (n=8) 90.9 11.4

40 (n=6) 86.0 11.9

Patulin


DETERMINATION OF PATULIN IN WHOLE APPLE

PROTOCOL OF CLEANUPSample preparationPreparation with microwaveWhole apple is cut into pieces and put in amicrowave for 90s before crushing the pieces.15g sample and 7.5mL water are mixed with150µL pectinase solution and put overnight atroom temperature or for 2h at 40°C before afiltration with filter 4-7µm to obtain the loadingsolution.Preparation with a blenderWhole apple is cut into pieces, put in a blenderwith Water (2:1 Apple: Water) and mix for1min. 15g sample and 300µL pectinase solutionare put overnight at room temperature or for2h at 40°C before a filtration with filter 4-7µmto obtain the loading solution.Cleanup with a 3mL/100mg AFFINIMIP® SPEPatulin cartridge



Washing of interferents (W1)•3mL Water-2% Acetic Acid


•250µL Diethyl EtherDrying by applying vacuum 10 secondsElution (E)


RESULTS

14

Regulations for solid apple products: Europe (EC 1881/2006) : 25µg/Kg

HPLC MethodColumn: Atlantis T3 column, 150mm x 2.1mmMobile phase: gradient profile



0 98 2

20 98 2

21 50 50

25 50 50

26 98 2

Chromatograms obtained after AFFINIMIP® SPEPatulin Clean-up of whole apple spiked at 40µg/kgwith Patulin (dark colors) or not spiked (light colors).

Whole apple prepared with blender

Whole apple prepared with microwave

96 96 95 88

Recovery yields obtained after AFFINIMIP® SPEPatulin Clean-up of spiked whole apple with40µg/kg of Patulin. Whole apples are preparedaccording to 2 different methods

Minutes5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

mA

U

-30

-20

-10

0

10

20

30

40

50

60

70

80

90

-30

-20

-10

0

10

20

30

40

50

60

70

80

90

Patulin

BLENDER PREPARATION

MICROWAVEPREPARATION



DETERMINATION OF PATULIN IN CIDER

PROTOCOL OF CLEANUPSample preparationThe cider is degassed by sonicating sample for1 hour. Then the degas cider is diluted by 2with water containing 2% of acetic acid. Thissolution is mixed and used as the loadingsolution.




Washing of interferents (W1)•1mL NaHCO3 1% in Water•2mL Water




RESULTS

Catalog number:3mL-100mg sorbentPI-FS102-02 for 25 cartridgesPI-FS102-03 for 50 cartridgesPI-FS102-02K for a kit of 25 cartridges + 50mLPectinasePI-FS102-03K for a kit of 50 cartridges + 50mLPectinasePI-REA-001-50mL for 50mL Pectinase solution

15

Regulations for cider: Europe (EC 1881/2006) : 50µg/Kg


Patulin

Chromatograms obtained after AFFINIMIP® SPEPatulin Clean-up of a cider spiked at 40µg/kg (testedtwice, pink) or at 10µg/kg (tested twice, blue) withPatulin or not spiked (red).

Recovery of Patulin at a contamination level of10µg/kg and 40µg/kg in cider after AFFINIMIP® SPEPatulin Clean-up and relative standard deviationcalculated from results generated underreproducibility conditions.

Concentration of Patulin (ng/mL)

Recoveries % % RSDR

10 87.5 (n=2) -

40 80.5 (n=5) 7.5


DETERMINATION OF PATULIN IN ALCOHOL POMMEAU AND LIQUOR

Manzella liquor contains 20% alcohol and 2.1%of concentrated apple juice.Alcohol Pommeau is a mixture of Calvados andApple Juice. It contains 17% Alcohol.

PROTOCOL OF CLEANUPSample preparationTo 1mL of Manzella Liquor or AlcoholPommeau, add 2mL Water to obtain theloading solution.




Washing of interferents (W1)•3mL Water (containing 2% Acetic Acidfor AA W1 protocol)


•250µL Diethyl EtherDrying by applying vacuum 10 secondsElution (E)


RESULTS

16


) Flow rate: 0.2mL/minDetection: UV - 276nmInjection volume: 100µL.


0 98 2

20 98 2

21 50 50

25 50 50

26 98 2

Chromatograms obtained after AFFINIMIP® SPEPatulin Clean-up of Manzella liquor spiked at40µg/L with Patulin (dark blue for Water in W1 andred for Water –AA in W1) or not spiked (light blueand pink). Washing with Acetic acid is moreefficient.


Regulations for apple based beverage : Europe (EC 1881/2006) : 50µg/Kg

Minutes5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

mA

U

25

50

75

100

125

150

175

200

225

250

275

300

m

25

50

75

100

125

150

175

200

225

250

275

300

Minutes8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

mA

U

0

20

40

60

80

100

120

140

160

0

20

40

60

80

100

120

140

160

UV 6000-276nmC10

UV 6000-276nmC12

UV 6000-276nmC11

UV 6000-276nmC13

UV 6000-276nmC15

UV 6000-276nmC14

Water for W1 Water-AA for W1

Pommeau 101 101 90 93

Manzella 102 106 87 90

Patulin

Patulin

Chromatograms obtained after AFFINIMIP® SPEPatulin Clean-up of Pommeau spiked at 40µg/L withPatulin (dark blue for Water in W1 and red forWater –AA in W1) or not spiked (light blue andpink). Washing with Acetic acid is more efficient.

Recovery yields obtained for Pommeau andManzella after AFFINIMIP® SPE Patulin Clean-up.W1 with water or Water -2%Acetic acid


DETERMINATION OF PATULIN IN TOMATO KETCHUP AND TOMATO POWDER

PROTOCOL OF CLEANUPSample preparationPreparation OF TOMATO KETCHUP10g tomato ketchup and 10mL water are mixedwith 150µL pectinase solution and leftovernight at RT before a filtration with filter0.2µm to obtain the loading solution.

Preparation OF TOMATO POWDER10g tomato ketchup and 20mL water aremixed. 10g of the mixture, 10mL water and150µL pectinase solution are left overnight atRT before a centrifugation at 4500rpm during 5min. Then the mixture is filtered with filter0.2µm to obtain the loading solution.



Loading•5mL of loading solution from tomatoketchup or 2mL from tomato powder

Washing of interferents (W1)•4mL Water-1% Acetic Acid•4mL Water




RESULTS

17




0 98 2

20 98 2

21 50 50

25 50 50

26 98 2

Chromatograms obtained after AFFINIMIP® SPEPatulin Clean-up of TOMATO KETCHUP spiked at40µg/kg with Patulin (red) or not spiked (light blue).

Patulin


Minutes

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28

mA

U

0

100

200

300

400

500

600

700

800

900

1000

1100

mA

U

0

100

200

300

400

500

600

700

800

900

1000

1100

UV 6000-276nm

B14

UV 6000-276nm

B15

Minutes

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

mA

U

0

10

20

30

40

50

60

70

80

90

100

110

mA

U

0

10

20

30

40

50

60

70

80

90

100

110

UV 6000-276nm

B14

UV 6000-276nm

B15

Patulin

Recovery yield80%

Minutes

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

mA

U

-100

0

100

200

300

400

500

600

700

800

900

1000

1100

mA

U

-100

0

100

200

300

400

500

600

700

800

900

1000

1100

UV 6000-276nm

B16

UV 6000-276nm

B17

TOMATO KETCHUP

TOMATO POWDER

Chromatograms obtained after AFFINIMIP® SPEPatulin Clean-up of TOMATO POWDER spiked at36µg/kg with Patulin (red) or not spiked (light blue).

Minutes

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

mA

U

0

10

20

30

40

50

60

70

80

90

100

110

mA

U

0

10

20

30

40

50

60

70

80

90

100

110UV 6000-276nm

B16

UV 6000-276nm

B17

Patulin

Recovery yield 70%


DETERMINATION OF PATULIN IN BLUEBERRY JUICE

PROTOCOL OF CLEANUPSample preparation5mL Blueberry juice is diluted with 5mL watercontaining 2% of acetic acid to obtain theloading solution.








RESULTS


18

Chromatograms obtained after AFFINIMIP® SPEPatulin Clean-up of Blueberry juice spiked at 40µg/Lwith Patulin (red) or not spiked (light blue).




0 98 2

20 98 2

21 50 50

25 50 50

26 98 2

Minutes0 2 4 6 8 10 12 14 16 18 20 22 24 26 28

m A U

-20

-10

0

10

20

30

40

50

60

70

-20

-10

0

10

20

30

40

50

60

70

Recovery yields: 90 and 96%

Patulin


DETERMINATION OF PATULIN IN CONCENTRATE JUICE AND THICK JUICE

PROTOCOL OF CLEANUPPreparation of fruit juice concentrate samples2.5g of fruit juice concentrate are mixed with10mL water and 100µL Pectinase. (REA-001-50mL). Leave the solution at room temperatureovernight or for 2h at 40°C. Centrifuge at4000g for 10min and collect the supernatant.Dilute the supernatant by 2 with Acetic Acid 2%in water. This solution is used as the loadingsolution.

Preparation of thick fruit juice samples15mL of thick fruit juice are mixed with 120µLPectinase (REA-001-50mL). Leave the solutionat room temperature overnight or for 2h at40°C. Centrifuge at 4000g for 10min and collectthe supernatant. Dilute the supernatant by 2with acetic acid 2% in water. This solution isused as the loading solution.



Loading•4 to 6mL of loading solution



•1mL Diethyl EtherElution (E)


RESULTS

Catalog number:6mL-200mg sorbentPI-FS102-02B-200mg for 25 cartridgesPI-FS102-03B-200mg for 50 cartridgesPI-FS102-02KB-200mg for a kit of 25 cartridges+ 50mL PectinasePI-FS102-03KB-200mg for a kit of 50 cartridges+ 50mL PectinasePI-REA-001-50mL for 50mL Pectinase solution

19

Chromatograms obtained after AFFINIMIP® SPEPatulin clean-up of apple mango juice spiked at20µg/kg (blue) with Patulin or not spiked (red). Ingreen, Patulin solution at 50ng/mL. prepared bydilution of a 100µg/mL Patulin standard solution(REA-PAT-1mL) in mobile phase.




0 98 2

20 98 2

21 50 50

25 50 50

26 98 2


Minutes0 2 4 6 8 10 12 14 16 18 20 22 24 26 28

mA

U

0

50

100

150

200

250

300

350

400

450

Patulin

THICK JUICE

Minutes

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28

mA

U

-20

0

20

40

60

80

100

120

140

160

mA

U

-20

0

20

40

60

80

100

120

140

160

CONCENTRATE JUICE

Chromatograms obtained after AFFINIMIP® SPEPatulin Clean-up of grapefruit juice concentratespiked at 10µg/kg (blue) with Patulin or not spiked(red).

DETERMINATION OF PATULIN IN APPLE PUREE









RESULTS

Catalog number:6mL - 200mg sorbent PI-FS102-02B-200mg for 25 cartridgesPI-FS102-03B-200mg for 50 cartridgesPI-FS102-02KB-200mg for a kit of 25 cartridges+ 50mL PectinasePI-FS102-03KB-200mg for a kit of 50 cartridges+ 50mL PectinasePI-REA-001-50mL for 50mL Pectinase solution


0 98 2

20 98 2

21 50 50

25 50 50

26 98 2



Regulations for apple puree: Europe (EC 1881/2006) : 25µg/Kg Regulations for apple juice for infants and young children:Europe (EC 1881/2006) : 10µg/Kg

Chromatograms of apple puree spiked with 20µg/kgof Patulin (Red) and not spiked (blue) afterAFFINIMIP® SPE Patulin Clean-up



Recoveries % % RSDr

10 (n=6) 90 9

20 (n=3) 92 11


Minutes

4 5 6 7 8 9 10 11 12 13 14 15

mA

U

100

200

300

400

500

600

700

800

mA

U

100

200

300

400

500

600

700

800

Patulin

A format tailored for the larger liquid volume required for apple puree protocol

20

DETERMINATION OF PATULIN IN DRIED APPLE

PROTOCOL OF CLEANUPSample preparation3g of dried apple dices, 30mL of water and150µL of pectinase are mixed and left at roomtemperature overnight. Then, they arecentrifuged at 4500rpm during 5min andfiltered with 0.2µm filter to obtain the loadingsolution.




Washing of interferents (W1)•5mL Water-2% Acetic Acid•5mL Water




RESULTS

21

Regulations for solid apple products: Europe (EC 1881/2006) : 25µg/Kg




0 98 2

20 98 2

21 50 50

25 50 50

26 98 2

Minutes

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

mA

U

-50

-40

-30

-20

-10

0

10

20

30

40

50

60

70

80

mA

U

-50

-40

-30

-20

-10

0

10

20

30

40

50

60

70

80

UV6000-276nm

B16

UV6000-276nm

B17

UV6000-276nm

B13

Chromatograms obtained after AFFINIMIP® SPEPatulin Clean-up of dried apple dices spiked at20µg/kg (red) or at 10µg/kg (blue) with Patulin ornot spiked (green).

Patulin

Recovery >90%


Catalog number:6mL - 200mg sorbent for apple-based products and fruit juice, concentratePI-FS102-02B-200mg for 25 cartridgesPI-FS102-03B-200mg for 50 cartridgesPI-FS102-02KB-200mg for a kit of 25 cartridges+ 50mL PectinasePI-FS102-03KB-200mg for a kit of 50 cartridges+ 50mL PectinasePI-REA-001-50mL for 50mL Pectinase solution

DETERMINATION OF OCHRATOXIN A IN CEREALS


PROTOCOL OF CLEANUPSample preparation50g of finely ground wheat are mixed during 1minute in a blender with 100mL of extractionsolvent (60/40 Acetonitrile/deionized Water).The extract is filtered through a filter paper.Then, 5mL of the extract is diluted with 5mL ofHCl solution pH=1, 0.1M. After a filtrationthrough a filter paper, this solution is used asthe loading solution.

Cleanup with a 3mL/100mg AFFINIMIP® SPEOchratoxin A cartridge


Loading•4mL of loading solution (eq. 1g wheat)

Washing of interferents•6mL 60/40 HCl solution pH 1, 0.1M/ACN

Elution (E)•2mL Methanol – 2% Acetic acid

The elution fraction was then evaporated anddissolved in water before HPLC analysis.

RESULTS

Catalog number:PI-FS101-02 for 25 cartridgesPI-FS101-03 for 50 cartridges

22

Regulations for umprocessed cereals: Europe (EC 1881/2006) : 5µg/KgCodex Alimentarius Standard: 5µg/Kg for raw wheat

HPLC Method with Fluorescence detectionColumn: Hypersil Gold C18 column 150mm x 2.1mmMobile phase: water/acetic acid/MeOH (39/1/60,v/v)Flow rate: 0.2mL/minFluorescence detection: excitation/emissionwavelengths: 333 / 460nmInjection volume: 20µL.

0,0 2,0 4,0 6,0 8,0 10,0 12,0 14,0 16,0 18,0

0

2000

4000

6000

Inte

nsi

ty

0,0 2,0 4,0 6,0 8,0 10,0 12,0 14,0 16,0 18,0

0

2000

4000

6000

Inte

nsi

ty

Chromatogram obtained after Cleanup of wheat(spiked at 5µg / kg (pink) or not contaminated(orange)) with AFFINIMIP® SPE Ochratoxin A

C° (µg/kg) Recoveries % % RSD

5 96.3 7.7

Recoveries of Ochratoxin A after AFFINIMIP® SPEOchratoxin A Clean-up in wheat (n=6)

Ochratoxin A

DETERMINATION OF OCHRATOXIN A IN PAPRIKA

PROTOCOL OF CLEANUPSample preparation10g of paprika are shaken during 30 minuteswith 100mL of NaHCO3 1% in water. Theextract is centrifuged for 30 minutes at 4000rpm at room temperature then filtered througha filter paper.25mL of the extract is diluted with 25mL of HClsolution pH=1, 0.1M. After a filtration througha filter paper, this solution is used as theloading solution.



Loading•4mL of loading solution (eq. 1g sample)




RESULTS


23

Regulations for paprika: Europe (EC 594/2012) : 30µg/Kg until 31.12.14 then 15µg/Kg


0,0 2,0 4,0 6,0 8,0 10,0 12,0 14,0 16,0 18,0

0

20000

Intensity

0,0 2,0 4,0 6,0 8,0 10,0 12,0 14,0 16,0 18,0

0

20000

Intensity

Ochratoxin A

Chromatogram obtained after Cleanup of paprika(spiked at 30µg / kg (pink) or not contaminated(orange)) with AFFINIMIP® SPE Ochratoxin A

C° (µg/kg) Recoveries % % RSD

30 93.3 3.4

Recoveries of Ochratoxin A after AFFINIMIP® SPEOchratoxin A Clean-up in paprika (n=4).


DETERMINATION OF OCHRATOXIN A IN RED AND WHITE WINE

PROTOCOL OF CLEANUPSample preparation10mL of wine is diluted with 10mL of HClsolution pH=1, 0.1M. This solution is used asthe loading solution.



Loading•2 to 10mL of loading solution (eq. 1 to5mL sample)




RESULTS


24

Regulations for wine: Europe (EC 1881/2006) : 2µg/L


Ochratoxin A

0,0 2,0 4,0 6,0 8,0 10,0 12,0 14,0 16,0

0

5000

10000

Inte

nsi

ty

0,0 2,0 4,0 6,0 8,0 10,0 12,0 14,0 16,0

0

5000

10000

Inte

nsi

ty

0,0 2,0 4,0 6,0 8,0 10,0 12,0 14,0 16,0

0

5000

10000

Inte

nsi

ty

Chromatograms obtained after Cleanup of whitewine spiked at 2µg/kg (loading with 5mL (blue);loading with 10mL (pink)) and after a loading of 5mLof not contaminated white wine (orange) withAFFINIMIP® SPE Ochratoxin A

0,0 2,0 4,0 6,0 8,0 10,0 12,0 14,0 16,0

0

2000

4000

6000

Inte

nsi

ty

0,0 2,0 4,0 6,0 8,0 10,0 12,0 14,0 16,0 18,0 0

20000

40000

60000

Inte

nsi

ty

0,0 2,0 4,0 6,0 8,0 10,0 12,0 14,0 16,0

0

2000

4000

6000

Inte

nsi

ty

0,0 2,0 4,0 6,0 8,0 10,0 12,0 14,0 16,0

0

2000

4000

6000

Inte

nsi

ty

0,0 2,0 4,0 6,0 8,0 10,0 12,0 14,0 16,0 18,0 0

20000

40000

60000

Inte

nsi

ty0,0 2,0 4,0 6,0 8,0 10,0 12,0 14,0 16,0 18,0

0

20000

40000

60000

Inte

nsi

ty

Chromatograms obtained after Cleanup of red winespiked at 2µg / kg (loading with 2mL (orange);loading with 5mL (blue); loading with 10mL (pink))and after a loading of 2mL of not contaminated redwine (grey) with AFFINIMIP® SPE Ochratoxin A

Ochratoxin A

Ochratoxin A

Matrix

C°

(µg/kg

)

Recoveries

%

%

RSD

White wine

(n=10)2 91.3 6.2

Red wine (n=4) 2 78.8 2.8

Recoveries of Ochratoxin A after AFFINIMIP® SPEOchratoxin A Clean-up in wine (white and red).


DETERMINATION OF ZEARALENONE IN MAIZE AND RICE


PROTOCOL OF CLEANUPSample preparation

Cleanup with a 3mL/100mg AFFINIMIP® SPEZearalenone cartridge25g of ground cereal-based samples wereextracted with 100 mL ofacetonitrile/deionized water (75/25, v/v) for 3min. The extract was filtered through a foldedfilter paper and 10 mL of the filtrate werediluted with 10 mL of deionized water. Then,this solution was filtered through a filter paper.This solution was used as the loading solution.


Loading•12mL of loading solution (eq. 1.5gsample)

Washing of interferents (W1)•3mL 58/2/40 Water/Acetic Acid/ACN

Elution (E)•2mL Methanol – 2% Acetic Acid


RESULTS

Catalog number:3mL-100mg sorbentPI-FS100-02 for 25 cartridgesPI-FS100-03 for 50 cartridges

25

Regulations for unprocessed cereal except maize: Europe (EC 1126/2007) : 100µg/Kg Regulations for maize:Europe (EC 1126/2007) : 350µg/Kg

HPLC Method with Fluorescence detectionColumn: Hypersil Gold C18 column 150mm x 4.6mmMobile phase: water/MeOH (40/60, v/v)Flow rate: 1mL/minFluorescence detection: excitation/emissionwavelengths: 275 / 450nmInjection volume: 100µL.

Chromatogram obtained after Cleanup of Maize(contamined at 41 µg / kg) with AFFINIMIP® SPEZearalenone

Chromatogram obtained after Cleanup of Rice(contamined at 41 µg / kg) with AFFINIMIP® SPEZearalenone .

Recoveries % % RSD

86 8

Recoveries of Zearalenone at a contamination levelof 41µg / kg after AFFINIMIP® SPE Zearalenone .Clean-up in Maize (n=9)

ZON

ZON

DETERMINATION OF ZEARALENONE IN CEREAL-BASED BABY FOOD


Cleanup with a 3mL/100mg AFFINIMIP® SPEZearalenone cartridge25g of ground cereal-based samples wereextracted with 100 mL ofacetonitrile/deionized water (75/25, v/v) for 3min. The extract was filtered through a foldedfilter paper and 10 mL of the filtrate werediluted with 10 mL of deionized water. Then,this solution was filtered through a filter paper.This solution was used as the loading solution.


Loading•12mL of loading solution (eq. 1.5gsample)

Washing of interferents (W1)•3mL 58/2/40 Water/Acetic Acid/ACN



RESULTS


26


Recoveries % % RSD

80 3

Recoveries of Zearalenone at a contamination levelof 41µg / kg after AFFINIMIP® SPE Zearalenone .Clean-up in Cereal – based baby food (n=5)

Chromatogram obtained after Cleanup ofCereal-based babyfood (contamined at 41µg /kg) AFFINIMIP® SPE Zearalenone (after dilutionby 2 of the elution fraction with water).

Chromatograms obtained after Cleanup of Cereal-based babyfood (contamined at 10µg/kg (blue) or0µg/kg (red)) with AFFINIMIP® SPE Zearalenone(after evaporation of the elution fraction anddissolution in 1mL of the mobile phase).

Regulations for processed cereal based food for baby food: Europe (EC 1126/2007) : 20µg/Kg


ZONZON

ZON

DETERMINATION OF ZEARALENONE IN EDIBLE CORN OIL

RESULTS


27


Recoveries of Zearalenone in Corn Oil at variouscontamination levels after AFFINIMIP®SPEZearalenone cleanup.

Regulations for processed cereal based food for baby food: Europe (EC 1126/2007) : 20µg/Kg


Cleanup with a 3mL/100mg AFFINIMIP® SPEZearalenone cartridgeCorn oil is diluted 1/3 in Diethyl Ether to obtain the loading solution.

Equilibration•3mL Diethyl Ether

Loading•3mL of loading solution (eq. 1mL of cornoil)

Washing of interferents (W1)•6mL Diethyl ether

Drying 30 secondsWashing of interferents (W2)

•6mL 58/2/40 Water/Acetic Acid/ACNElution (E)

•4mL Methanol – 2% Acetic AcidThe elution fraction was then evaporated anddissolved in water before HPLC analysis.

C° (µg/L) Mean C° (µg/L) Recoveries %

200 230 115

400 440 110

600 678 113

Chromatograms obtained after cleanup byAFFINIMIP®SPE Zearalenone of Corn Oil spiked withZearalenone at 200µg/L (red), 400µg/L (green), 600µg/L (blue) or not spiked (purple).

Minutes

0 2 4 6 8 1

0

1

2

1

4

1

6

1

8

2

0

2

2

2

4

2

6

2

8

3

0

mV

olt

s

0

5

1

0

1

5

2

0

2

5

3

0

3

5

4

0

4

5

Zearalenone

Chromatograms of Corn Oil spiked withZearalenone at 400µg/L (blue) or not spiked(orange) obtained after cleanup by AFFINIMIP®SPEZearalenone.


Zearalenone

DETERMINATION OF DEOXYNIVALENOL IN CEREALS FORFOOD (Water extraction)

RESULTS

28

HPLC Method with MS or UV detectionColumn: Hypersil Gold C18 column 50mm x 2,1mmMobile phase: water with 0,1% formic acid/ACN(95/5, v/v)Flow rate: 0,2mL/minMS detection: m/z 265 (ESI-)UV detection: 220nmInjection volume: 20µL.

Recovery of Deoxynivalenol after AFFINIMIP®SPEDeoxynivalenol clean-up and relative standarddeviation (repeatability conditions).

Regulations for unprocessed corn or durum wheat for food: Europe (EC 1126/2007) : 1750µg/Kg

PROTOCOL OF CLEANUPSample preparation with EXTRACTION WITHWATER20g of cereals were ground in a blender for 1minute. Then, 80 ml of deionized water wereadded. This mixture was then ground for 2additional minutes. After grinding the mixturewas placed in a beaker and left stirred undermagnetic agitation for 30 minutes.Then the whole mixture was transferred in acentrifuge vial and centrifuged at 2500 rpm for15 minutes. After centrifugation thesupernatant was filtered through filter paper.This solution was then diluted 5 times usingdeionized water.

Cleanup with a 6mL/100mg AFFINIMIP® SPEDeoxynivalenol cartridge.



Washing of interferents (W1)•3mL NaHCO3 1% in water


•1mL DiethyletherElution (E)

•4mL Ethyl AcetateThe elution fraction was then evaporated anddissolved in water -0,1% HCOOH before HPLCanalysis.


Recovery of Deoxynivalenol after AFFINIMIP®SPEDeoxynivalenol clean-up and relative standarddeviation (reproducibility conditions).

MatrixDetect

ion

Mean

µg/kgR% %RSDR

Corn (n=6) UV 658.7 82.3 7.5

Corn (n=6) MS 659.5 82.4 7.8

Deoxynivalenol (DON)

MatrixDetec

tion

Mean

µg/kgR% %RSDr

Corn

(800µg/kg)UV 623.4 78.0

1.4

(n=6)

Corn

(800µg/kg)MS 642.7 80.3

3.4

(n=6)

Wheat (n=3)

(600µg/kg)MS 540.0 90.0

9.8

(n=3)

UV chromatograms obtained after WATERextraction of DON from corn and clean-up withAFFINIMIP®SPE Deoxynivalenol :• black, red and green spiked with DON at

800µg/kg• dark yellow not spiked• blue, a standard solution of DON at 200ng/mL is

prepared by dilution of a 100µg/mLDeoxynivalenol standard solution (reference :REA-DON-1mL) in mobile phase

Catalog number:6mL-100mg sorbent for (baby)foodPI-FS117-02B for 25 cartridgesPI-FS117-03B for 50 cartridges6mL-200mg sorbent for feedPI-FS117-02B-200mg for 25 cartridgesPI-FS117-03B-200mg for 50 cartridges

DETERMINATION OF DEOXYNIVALENOL IN BABYFOOD CEREALS

RESULTS


29

HPLC Method with MS detectionColumn: Hypersil Gold C18 column 50mm x 2,1mmMobile phase: water with 0,1% formic acid/ACN(95/5, v/v)Flow rate: 0,2mL/minMS detection: m/z 265 (ESI-)Injection volume: 20µL.

Recovery of Deoxynivalenol after AFFINIMIP®SPEDeoxynivalenol clean-up and relative standarddeviation calculated from results generated underrepeatability conditions.

Regulations for cereal based food for baby food: Europe (EC 1126/2007) : 200µg/Kg

PROTOCOL OF CLEANUPSample preparation150 ml of deionized water were added to 20gof cereals - based babyfood. This mixture wasthen placed in a beaker and left stirring undermagnetic agitation for 30 minutes.Then, the whole mixture was centrifuged at2500 g for 15 minutes. After centrifugation, thesupernatant was filtered through filter paper.








MatrixC°

µg/kg

Mean

µg/kgR% %RSDr

Babyfood

(n=3)150 136.5 91 0.4


MS chromatograms obtained after water extractionof Deoxynivalenol from cereals - based babyfoodsand clean-up with AFFINIMIP® SPE Deoxynivalenol:• black, red and green spiked with Deoxynivalenol

at 150µg/kg• dark yellow not spiked• blue, a standard solution of Deoxynivalenol at

200ng/mL is prepared by dilution of a 100µg/mLDeoxynivalenol standard solution (reference :REA-DON-1mL) in mobile phase

Recovery of Deoxynivalenol after AFFINIMIP®SPEDeoxynivalenol clean-up and relative standarddeviation calculated from results generated underreproducibility conditions.

MatrixC°

µg/kg

Mean

µg/kgR% %RSDR

Babyfood

(n=3)150 136.5 91 0.4


DETERMINATION OF DEOXYNIVALENOL, 3-AcetylDON AND15-AcetylDON IN CEREALS (Hydro-organic extraction)

RESULTS

30

HPLC Method with MS detectionColumn: Hypersil Gold C18 column 50mm x 2,1mmMobile phase for DON analyses: water with 0,1%formic acid/ACN (95/5, v/v)Mobile phase for 3-AcDON and 15-AcDON analyses:water with 0.1% formic acid/ACN (90/10, v/v)Flow rate: 0.2mL/minMS detection: m/z 265 (ESI-)Injection volume: 20µL.

Recovery obtained for DON, 3-acetylDON and 15-acetylDON after AFFINIMIP®SPE Deoxynivalenolclean-up of Corn and relative standard deviation -repeatability conditions (n=3).

Regulations for unprocessed corn or durum wheat for food: Europe (EC 1126/2007) : 1750µg/Kg

PROTOCOL OF CLEANUPSample preparation WITH HYDROORGANICEXTRACTION20g of cereals were ground in a blender for 1minute. Then, a solution of deionized water:acetonitrile (50:50) was added. This mixturewas then ground for 2 additional minutes. Aftergrinding, the mixture was placed in a beakerand left stirred under magnetic agitation for 30minutes.Then the mixture was centrifuged at 2500 g for15 minutes. After centrifugation, thesupernatant was filtered through filter paper.This solution was then diluted 10 times usingdeionized water.







•4mL Ethyl AcetateThe elution fraction was then evaporated anddissolved in water-0.1% formic acid beforeHPLC analysis.


3-AcetylDeoxynivalenol (3-AcDON)

Compound C° µg/kgMean

µg/kgR% %RSDr

DON 800 653.7 81.7 0.3

3-AcetylDON 800 601.0 75.1 2.3

15-

AcetylDON800 641.8 80.2 3.4

MS chromatograms obtained after hydro-organicextraction of 3-acetylDON from corn and clean-upwith AFFINIMIP®SPE Deoxynivalenol :-black, red and green: spiked with Deoxynivalenolat 800µg/kg-dark yellow: not spiked-blue: a standard solution of 3-AcetylDON at200ng/mL is prepared by dilution of a 100µg/mL 3-AcetylDeoxynivalenol standard solution (reference: REA-3AcDON-1mL) in mobile phase

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DETERMINATION OF DEOXYNIVALENOL IN CEREALS FORANIMAL FEED

RESULTS

31

HPLC Method with UV detectionColumn: Hypersil Gold C18 column 50mm x 2,1mmMobile phase: water with 0,1% formic acid/ACN(95/5, v/v)Flow rate: 0,2mL/minUV detection: 220nmInjection volume: 20µL.

Recovery of Deoxynivalenol after AFFINIMIP®SPEDeoxynivalenol clean-up and relative standarddeviation - repeatability conditions (n=3).

Regulations for DON in animal feed: Europe (EC 576/2006) :8mg/Kg for cereals and cereals products12mg/Kg for maize by-products

PROTOCOL OF CLEANUPSample preparation with EXTRACTION WITHWATER20g of animal feed were ground in a blenderfor 1 minute. Then, 80 ml of deionized waterwere added. This mixture was then ground for2 additional minutes. After grinding themixture was placed in a beaker and left stirredunder magnetic agitation for 30 minutes.Then, the whole mixture was centrifuged at2500 g for 15 minutes. After centrifugation thesupernatant was filtered through filter paper.This solution was then diluted 5 times usingdeionized water.










UV chromatograms obtained after WATERextraction of DON from wheat (animal feed) andclean-up with AFFINIMIP®SPE Deoxynivalenol :• black, red and green spiked with DON at 6mg/kg• dark yellow not spiked• blue, a standard solution of DON at 1µg/mL is

prepared by dilution of a 100µg/mLDeoxynivalenol standard solution (reference :REA-DON-1mL) in mobile phase


a b c

Analysis of Whiskas:

a. Extraction solution with water

b. Loading solution

c. Elution solution

Feed

Matrices

C°

mg/kg

Mean

mg/kgR% %RSDr

Wheat 6 5.7 94 0.1

Whiskas 0.8 0.73 91 2.4

DETERMINATION OF FUMONISINS B1 / B2 ANDZEARALENONE IN MAIZE FLOUR



Cleanup with a 3mL/100mg AFFINIMIP® SPEFumoZON cartridge25g of ground samples were extracted with100 mL of Acetonitrile/Methanol/deionizedWater (25/25/50, v/v/v) for 3 min using ablender. The extract was filtered through afolded filter paper and 10 mL of the filtratewere diluted with 10 mL of deionized water.Then, this solution was filtered through a filterpaper.This solution was used as the loading solution.



Washing of interferents•6mL 60/40 Water/ACN



RESULTS

32

HPLC Method with MS detectionColumn: Hypersil Gold C18 column 50mm x 2.1mmMobile phase ZON AND FB1: Water-Formic Acid0.1%/ACN (73/27)Mobile phase FB2: Water-Formic Acid 0.1%/ACN(65/35)Flow rate: 0.2mL/minMS detection: m/z 722 for Fumonisin B1 (ESI+)m/z 706 for Fumonisin B2 (ESI+)m/z 317 for Zearalenone (ESI-)Injection volume: 20µL.

Chromatograms obtained after AFFINIMIP® SPEFumoZON Clean-up of a maize flour spiked at38µg/kg with Zearalenone, 2408µk/kg withFumonisin B1 and 630µg/kg with Fumonisin B2.

SampleC°

µg/kgMeanµg/kg

Recoveries %

% RSDR

Zearalenone

38 39.2 103.28.5

(n=8)

Fumonisin B1

2408 2002.2 83.110.3 (n=8)

Fumonisin B1

400 401.0 100.2-

(n=2)

Fumonisin B2

630 684.6 108.711.5(n=3)

Recovery of Zearalenone, Fumonisins B1 and B2 inmaize flour after AFFINIMIP® SPE FumoZON clean-up and relative standard deviation calculated fromresults generated under reproducibility conditions

Catalog number:3mL-100mg sorbent PI-FS109-02 for 25 cartridgesPI-FS109-03 for 50 cartridges

Fumonisin B2

Fumonisin B1

Zearalenone

Regulations for cereal flour: ZearalenoneEurope (EC 1126/2007) : 75µg/Kg Fumonisins Europe (EC 1126/2007) : 1000µg/Kg for maize flourUSA: FDA advisory 2000µg/Kg RT:0.08 - 30.00

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Cleanup with a 3mL/100mg AFFINIMIP® SPEFumoZON cartridge25g of ground samples were extracted with100 mL of Acetonitrile/Methanol/deionizedWater (25/25/50, v/v/v) for 3 min using ablender. The extract was filtered through afolded filter paper and 10 mL of the filtratewere diluted with 10 mL of deionized water.Then, this solution was filtered through a filterpaper.This solution was used as the loading solution.



Washing of interferents•6mL 60/40 Water/ACN



RESULTS

33

HPLC Method with MS detectionColumn: Hypersil Gold C18 column 50mm x 2.1mmMobile phase ZON AND FB1: Water-Formic Acid0.1%/ACN (73/27)Mobile phase FB2: Water-Formic Acid 0.1%/ACN(65/35)Flow rate: 0.2mL/minMS detection: m/z 722 for Fumonisin B1 (ESI+)m/z 706 for Fumonisin B2 (ESI+)m/z 317 for Zearalenone (ESI-)Injection volume: 20µL.

Catalog number:3mL-100mg sorbent PI-FS109-02 for 25 cartridgesPI-FS109-03 for 50 cartridges

Recovery of Zearalenone, Fumonisins B1 and B2 inmaize-based baby food after AFFINIMIP® SPEFumoZON clean-up and relative standard deviationcalculated from results generated underreproducibility conditions.

SampleC°

µg/kgMeanµg/kg

Recoveries %

% RSDR

Zearalenone 20 16.9 84.41.6

(n=4)

Fumonisin B1 200 168.6 84.31.4

(n=3)

Fumonisin B2 200 185.6 92.81.9

(n=3)

DETERMINATION OF FUMONISINS B1 / B2 AND ZEARALENONE

IN MAIZE-BASED BABY FOOD

ION SUPPRESSION EVALUATION

Ion suppression phenomenon can induce anerroneous quantification. To evaluate the ion-suppression, blank maize-based baby foodsamples were cleaned up with AFFINIMIP® SPEFumoZON . The SPE extracts were spiked witha mixture of Fumonisin B1 and Zearalenone at2 different concentrations. The standardcalibration curves were compared to the matrixSPE extracts. The use of AFFINIMIP® SPEFumoZON strongly reduces ion-suppressionphenomena with a maximum of 15% observedfor Fumonisins.

Ion suppression percentage obtained in Maize-based baby food (tested twice).

AnalyteC°

µg/kg

Ion suppression

%

Zearalenone 10 1% and 5%

Zearalenone 50 0% and 5%

Fumonisin B1 100 8% and 11%

Fumonisin B1 500 12% and 14%

Regulations for maize-based baby food: ZearalenoneEurope (EC 1126/2007) : 20µg/Kg Fumonisins Europe (EC 1126/2007) : 200µg/Kg


Estrogens are a group of compounds which play an importantrole in the estrous cycle. They are either natural (Estrone,Estriol, 17α- and 17β-Estradiol) or synthetic compounds (17α-EthinylEstradiol, Dienestrol, Diethylstilbestrol).Estrogens play a key role in developmental and reproductivefunctions. They also affect a diversity of biological processesinvolved in coronary artery disease, immunocompetence andcancer susceptibility. When they are present in wastewater,these endocrine disrupting chemicals (EDC) have adverseeffects on endocrine systems of human beings and animals.

Catalog number:PI-100-02 for 25 cartridges, 3mLPI-100-03 for 50 cartridges, 3mLPI-100-1.96W for 1 96-well plate

AFFINIMIP® SPE ESTROGENSPH100

OH

OH

H

HH

17β- Estradiol

In addition, because of their anabolic effects, estrogens have been used in animal fattening.Steroid hormones are used in animal fattening because of their capacity to increase weightgain and to reduce the feed conversion ratio which is the average feed intake in relation tothe weight gain. For several years now, the use of anabolic steroids in animal fattening isprohibited in the European Community because of their possible toxic effects on publichealth (96/22/EC). Nevertheless, they are still offered on the ‘black’ market for animalfattening purposes.

AFFINIMIP® SPE Estrogens are selective solid phase extraction cartridges that selectivelyclean and concentrate Estrogens prior to analysis by HPLC.AFFINIMIP® SPE Estrogens areselective solid-phase extraction cartridges that selectively clean and concentrate the naturalor synthetic estrogens family prior to further analysis from complex matrices such as Water,Plasma or Serum

DETERMINATION OF ESTROGENS IN PLASMA

AFFINIMIP® SPE Estrogens

PROTOCOL OF CLEANUPSample preparation2mL serum samples spiked with 40pg 17β-Estradiol-d3. Then 2mL of Acetate buffer(0.8M, pH 6.8) and 100µL β-glucuronidasewere added. Hydrolysis performed overnight at37°C and samples centrifuged at 4000 rpm for10min. Upper layer was used as loadingsolution.Cleanup with a 3mL/100mg AFFINIMIP® SPEEstrogens cartridge

Equilibration•3mL Methanol•3mL Acetonitrile•3mL Water

Loading solution from sample preparationWashing of interferents

•3mL Water•3mL Water/Acetonitrile (60/40)

Elution (E)3mL Methanol

The elution fraction was then evaporated andestrogens were derivatised 40min at 60°C withBSTFA before GC-MS/MS analysis.

RESULTS

35

GC-MS/MS AnalysisColumn: RTX-1614 Resteck 15m x 0.25mm x 0.10µmGradient temperature: 80 to 320°C (15°C/min)

Blank plasma 10ppt 40ppt 100ppt

17β-Estradiol-d3

419>285

17 α/β-Estradiol416>129

17 α/β-Estradiol416>285

Data extracted from Quantification of estrogens atppt levels in bovine plasma by MolecularlyImprinted Solid Phase Extraction and GC-MS/MSanalysis, Emmanuelle Bichon et al. (LABERCA)Poster session, HTSP-2 and HTC 2012

MRM chromatograms from GC-MS/MS analysis offortified calves’ plasma samples at 0, 10, 40 and 100pg.mL-1 with 17α-estradiol, 17β-estradiol and estrone.Chromatograms obtained after a clean-up withAFFINIMIP® SPE Estrogens (Courtesy of EmmanuelleBichon - LABERCA)

Regulations for Estrogens: Europe (EC directive) : 40pg/mL of plasma or serum of bovine animals


PROTOCOL COMPARISON –AFFINIMIP® SPE ESTROGENS vs usual protocol

36

AFFINISEP method

2mL Bovine plasma

Enzymatic hydrolysis

AFFINIMIP® SPE Estrogens 3cc

Usual method

Liquid - Liquid Extraction with Ether

HPLC analysis : GC-MS/MS

Preparative HPLC dimethylaminopropyle

Liquid - Liquid Extraction with Pentane

•Data extracted from Quantification of estrogensat ppt levels in bovine plasma by MolecularlyImprinted Solid Phase Extraction and GC-MS/MSanalysis, Emmanuelle Bichon et al. (LABERCA)Poster session, HTSP-2 and HTC 2012

Performance. Save your time.

Copolymeric SPE

Silica SPE

Derivatisation PFB/TMS


AFFINIMIP® SPE Estrogens

AFFINIMIP® SPE Bisphenol AFS106

37

CH3

CH3

OH OH

Bisphenol A (or BPA) is a molecule widely used in industryfor the synthesis of polycarbonate plastics and epoxyresins. Polycarbonate plastics are used to make a varietyof common products including baby and water bottles.Epoxy resins are used as coatings on the inside of almostall food and beverage cans.

The migration of this endocrine disruptor compound from the packaging to food is the mainsource of consumers’ exposure to BPA. Its consumption is critical for babies. So, a Europeandirective prohibits the use of BPA to manufacture infant feeding bottles (Directive 2011/8/EUof 28 January 2011). Progressively, countries become more and more restrictive on BPA usefor food packaging. So, in October 2011, French parliament voted a law for banning BPA fromcanned foods and plastic boxes applicable in 2013 for infants and for all consumers on 1st

January 2014. In the same way,Sweden bans BPA in food packaging for under-threes (2012) and in Denmark since July 2010,it has been illegal to sell infant feeding bottles and cups, and packaging for baby food,containing BPA.

So, BPA is a topical issue with a worldwide regulation going to still lower concentrations ofBPA allowed in food. Highly sensitive and reliable detection methods are required forroutine analysis of BPA in food samples, particularly for baby food.

In these application notes, we described protocols enabling the determination of very lowconcentration of BPA in liquid and powdered infant formula, and several other matrices.using AFFINIMIP® SPE Bisphenol A cartridge.These methods show the determination of very low concentration of Bisphenol A with afluorescence detector. Therefore, the use of AFFINIMIP® SPE Bisphenol A enables toeliminate the tedious derivatization step required by gas chromatography.This method is also perfectly suitable for clean-up before GC-MS/MS or LC-MS/MS.

Catalog number:3mL-100mg sorbent in a PP cartridgePI-FS106-02 for 25 cartridgesPI-FS106-03 for 50 cartridges6mL-100mg sorbent in a PP cartridgePI-FS106-02B for 25 cartridgesPI-FS106-03B for 50 cartridges6mL-100mg sorbent in a glass cartridgePI-FS106-02G for 25 cartridgesPI-FS106-03G for 50 cartridges

DETERMINATION OF BISPHENOL A IN LIQUID INFANT FORMULA

AFFINIMIP® SPE Bisphenol A


Cleanup with a 3mLor 6mL/100mgAFFINIMIP® SPE Bisphenol A cartridge

Equilibration•3mL Methanol -2% Acetic Acid•3mL Acetonitrile•3mL Water

LoadingUp to 15mL of infant formula

Washing of interferents•9mL Water•6mL Water/Acetonitrile (60/40)

Drying 30 secondsElution (E)

3mL MethanolThe elution fraction was then evaporated anddissolved in the mobile phase before HPLCanalysis.

RESULTS

38

HPLC Method with Fluorescence detectionColumn: Hypersil Gold C18 column 150mm x 4.6mmMobile phase: gradient profile

Flow rate: 1mL/minFluorescence detection: excitation/emissionwavelengths: 230 / 315nmInjection volume: 50µL.


0 65 35

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Injection 50µL of Infant Formula before treatment

Bisphenol A after treatment of 15mL of Infant Formula

Chromatograms of Infant Formula containing 1µg/Lof Bisphenol A before clean-up (Red) and afterclean-up (Blue) with AFFINIMIP® SPE Bisphenol A.

Minutes

9,0 9,2 9,4 9,6 9,8 10,0 10,2 10,4 10,6 10,8 11,0 11,2 11,4 11,6 11,8 12,0 12,2 12,4 12,6 12,8 13,0

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Chromatograms obtained after clean-up withAFFINIMIP® SPE Bisphenol A of 15mL of InfantFormula spiked with Bisphenol A at 2µg/L (testedtwice, blue) or at 1µg/L (tested twice, red) or notspiked (pink).

Recovery of Bisphenol A in 15mL of infant formula afterAFFINIMIP® SPE Bisphenol A clean-up and relativestandard deviation calculated from results generated:

- under repeatability conditions (n=3, % RSDr)

C° (µg/L) Mean (µg/L) Recoveries % % RSDr

1.0 0.9 88.4 1.5

2.0 1.7 85.7 2.7

C° (µg/L) Mean (µg/L) Recoveries % % RSDR

1.0 0.8 84.4 7.4

2.0 1.7 85.8 5.3

- under reproducibility conditions ( % RSDR).

Regulations for Bisphenol A: Europe (directive 2011/8/EU) : forbiden in infant feeding bottles


DETERMINATION OF BISPHENOL A IN POWDERED INFANT FORMULA

PROTOCOL OF CLEANUPSample preparation4.4g powdered infant milk was reconstituted in30 mL of water and warmed up at ~ 50°Cduring 20 seconds using microwaves. Then 20mL of acetonitrile were added to 20 mL ofwarm milk and centrifuged at 4000 rpm during10 minutes. The supernatant was collected andfiltered on filter paper (4-7µm). This extractwas diluted 1:1 with water to form the loadingsolution.Cleanup with a 3mL or 6mL/100mgAFFINIMIP® SPE Bisphenol A cartridge


LoadingUp to 40mL of infant formula




RESULTS

39




0 65 35

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20 50 50

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35 65 35

Concentration of

BPA in

reconstituted

milk (µg/L)

Mean

concentration

(µg/L)

Recoveries

% RSDr %

2.1 2.3 (n=5) 108 8.7

4.3 4.0 (n=4) 95 3.7

Recovery of Bisphenol A spiked at differentconcentrations after 3mL/100mg AFFINIMIP® SPEBisphenol A clean-up of 40mL of loading solution(equivalent to 10mL of reconstituted Infant milk)and relative standard deviation calculated fromresults generated under repeatability conditions

Minutes

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2-MD1.dat

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4-md1.dat

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4-md2.dat

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mnd.dat

Chromatograms obtained after clean-up withAFFINIMIP® SPE Bisphenol A of equivalent at 10mLof Infant Formula spiked with Bisphenol A at4.3µg/L (tested twice, red) or at 2.1µg/L (testedtwice, blue) or not spiked (pink).

Regulations for Bisphenol A: Europe (directive 2011/8/EU) : forbiden in infant feeding bottles

Bisphenol A



PROTOCOL COMPARISON –AFFINIMIP® SPE Bisphenol A vs competitor

FS106

40

AFFINISEP method

Precipitation (15g sample + 15mL ACN)

Centrifuge and collect supernatant

Dilute 20mL supernatant with 20mL H20

Load on AFFINIMIP® SPE Bisphenol A

3cc (40mL)

Wash 10mL H20 and 6mL 40% ACN ;

Elute 3mL 100% MeOH

HPLC analysis :

LC Fluorescence detector

or GC-MS/MS or LC-MS/MS

WATERS method*

Precipitation (10g sample + 10mL ACN)

Centrifuge and collect supernatant

Quecher 1

Add contents from DisQue tube 1.

Shake. Centrifuge and collect 10mL

HPLC analysis : LC-MS/MS

Dilute supernatant with 70mL H20

Load on SPE cartridge OASIS HLB 3cc

(70mL)

Wash 2mL 40% Methanol ;

Elute 1mL 100% MeOH

Quecher 2

Add contents from DisQue tube 2.

Shake. Centrifuge and collect

supernatant

•Extract from Waters application note,

published 2012 : Rapid analysis of Bisphenol A

POWDERED INFANT FORMULA ANALYSIS

Performance. Save your time.



DETERMINATION OF BISPHENOL A IN CANNED FOOD (Liquid form)


Cleanup with a 3mL or 6mL/100mgAFFINIMIP® SPE Bisphenol A cartridge


Loading10mL liquid from canned food after filterpaper filtration




RESULTS

Catalog number:3mL-100mg sorbent in a PP cartridgePI-FS106-02 for 25 cartridgesPI-FS106-03 for 50 cartridges6mL-100mg sorbent in a glass cartridgePI-FS106-02G for 25 cartridgesPI-FS106-03G for 50 cartridges

41




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180

200

220

Bisphenol A

Chromatograms after clean-up with AFFINIMIP® SPEBisphenol A of 10mL liquid form of canned Peas andcarrots spiked with Bisphenol A at 1µg/L (testedtwice, blue) or not spiked (green).


1.0 1.05 105.1 5

Recovery of Bisphenol A after AFFINIMIP® SPEBisphenol A clean-up of 10mL of canned peas andcarrots (liquid) spiked at 1µg/L and relative standarddeviation calculated from results generated- under reproducibility conditions (n=4).

Minutes

3,5 4,0 4,5 5,0 5,5 6,0 6,5 7,0 7,5 8,0 8,5 9,0 9,5 10,0 10,5 11,0 11,5 12,0

mV

olts

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-50

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Minutes

3,5 4,0 4,5 5,0 5,5 6,0 6,5 7,0 7,5 8,0 8,5 9,0 9,5 10,0 10,5 11,0 11,5 12,0

mV

olts

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-50

0

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300

350

400

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BPA : 2µg/L

BPA : 0.5 µg/L

BPA : 0.3 µg/L

Bisphenol A

Chromatograms after clean-up with AFFINIMIP® SPEBisphenol A of 10mL of canned salmon and tuna(liquid form).Blue: 1st price canned salmon; Green: middle gradecanned salmon: no BPA was detected; Red:premium canned salmon; Pink: canned tuna

EVALUATION OF BPA IN COMMERCIAL CANNED FOODS

- under reproducibility conditions (n=4).


1.0 1.04 104.3 10

Regulations for Bisphenol A: Europe (directive 2011/8/EU) : Specific migration limit in food from packaging of 0.6mg/kg


Minutes

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

mVo

lts

-50

0

50

100

150

200

250

300

350

400

450

500

550 Fluo

MD1.dat

Fluo

md2.dat

Fluo

mnd.dat

DETERMINATION OF BISPHENOL A IN CANNED FOOD (Vegetable)

PROTOCOL OF CLEANUPSample preparation150g of drained canned peas - carrots and200mL of Water /ACN (50/50) are blendedduring 2 min and centrifuged during 10min at4000rpm. The supernatant solution is collected, filtered (4-7µm) and diluted ½ with water togive the loading solution



Loading20mL loading solution




RESULTS

42




0 65 35

2 65 35

12 50 50

20 50 50

20.5 65 35

35 65 35

Chromatograms after clean-up with AFFINIMIP® SPEBisphenol A of 20mL loading solution of extract ofcanned Peas- carrots spiked with Bisphenol A at2µg/L (tested twice, blue and red) or not spiked(green).


Minutes

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40

mV

olts

-50

0

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300

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400

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550

600

650

Fluo

MD1.dat

Fluo

md2.dat

Fluo

mnd.dat

Bisphenol A

Recovery yield : 97-99%

ZOOM

Bisphenol A



DETERMINATION OF BISPHENOL A IN BEER

PROTOCOL OF CLEANUPSample preparationThe beer is degassed by sonication for 1 hour.



Loading10mL of degassed beer




RESULTS


43




0 65 35

2 65 35

12 50 50

12.5 65 35

22 65 35

Injection 50µL of beer before treatment

Bisphenol A after treatment of 10mL of Beer

Chromatograms of beer containing 1µg/L ofBisphenol A before (Red) and after (Blue)AFFINIMIP® SPE Bisphenol A Clean-up.

Bisphenol A

Chromatograms obtained after AFFINIMIP® SPEBisphenol A Clean-up of 10mL of beer spiked at2µg/L (tested 3 times, orange) or at 1µg/L (tested 3times, blue) with Bisphenol A or not spiked (red)

C° (µg/L) Mean µg/L Recoveries % % RSDr

1.0 1.1 106.9 1.0

2.0 1.9 93.4 1.0

C° (µg/L) Mean µg/L Recoveries % % RSDR

1.0 1.0 99.3 8.9

2.0 1.8 90.6 6.0

Recovery of Bisphenol A in spiked beer after AFFINIMIP®SPE Bisphenol A clean-up and relative standard deviationcalculated from results generated:





DETERMINATION OF BISPHENOL A IN RED/WHITE WINES

PROTOCOL OF CLEANUP



LoadingUp to 10mL of wine


Drying 1 minuteElution (E)


RESULTS


44




0 65 35

2 65 35

12 50 50

12.5 65 35

22 65 35

Recovery of Bisphenol A spiked at 2µg/kg afterAFFINIMIP® SPE Bisphenol A clean-up of 6mL of redwine or 10mL of white wine.

Chromatograms obtained after clean-up with AFFINIMIP®SPE Bisphenol A of 10mL of white wine spiked withBisphenol A at 2µg/kg (tested three times, blue) or notspiked (red). The white wine naturally contained 2µg/kgof BPA


Matrice

Spiked at 2µg/kg

Mean

concentration

(µg/kg)

Recoveries

%

Red wine 1 1.93 (n=2) 96.6

Red wine 2 2.13 (n=2) 106.5

Red wine 3 1.66 (n=2) 83.0

White wine 1.60 (n=3) 80.0

Minutes

5,0 5,5 6,0 6,5 7,0 7,5 8,0 8,5 9,0 9,5 10,0 10,5 11,0 11,5 12,0 12,5 13,0

mV

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Fluo

MND.dat

Fluo

md1.dat

Fluo

md2.dat

Fluo

md3.dat

Bisphenol A


DETERMINATION OF BISPHENOL A IN COLA DRINKS

PROTOCOL OF CLEAUNP



Loading6mL of Cola drinks after 30min degassingwith ultrasounds




RESULTS


45

Recovery of Bisphenol A spiked at 5µg/kg afterAFFINIMIP® SPE Bisphenol A clean-up of 6mL of Coladrinks

Chromatograms obtained after clean-up with AFFINIMIP®SPE Bisphenol A of 10mL of white wine spiked withBisphenol A at 2µg/kg (tested three times, blue) or notspiked (red). The white wine naturally contained 2µg/kgof BPA


Mean

concentration

(µg/kg)

Recoveries

% RSDr %

1.93 (n=2) 96.6 1.0

Bisphenol A

Comparison of the solution obtained before and after using AFFINIMIP® SPE Bisphenol A




0 65 35

2 65 35

12 50 50

20 50 50

20.5 65 35

40 65 35

Minutes

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PROTOCOL OF CLEANUP


Equilibration•3mL Methanol -2% Formic Acid•3mL Acetonitrile•3mL Water

Loading9mL of Milk



3mL Methanol (E1)3mL Acetonitrile (E2)

The elution fractions E1 and E2 were gathered,evaporated and dissolved in the mobile phasebefore HPLC analysis.

RESULTS


46

Recovery of Bisphenol A and BADGE spiked at10ng/mL after AFFINIMIP® SPE Bisphenol A clean-up of9mL of milk.

Fluorescence chromatograms obtained after clean-upwith AFFINIMIP® SPE Bisphenol A of 9mL of milk spikedwith 10µg/kg Bisphenol A and 10µg/kg BADGE (testedtwice, blue) or not spiked (red).


Matrice

Spiked at

10ng/mL

Mean

concentration

(µg/kg)

Recoveries

%

BPA 10.85 108.5

BADGE 7.5 75

Bisphenol A BADGE




0 65 35

2 65 35

12 50 50

20 20 80

25 20 80

30 65 35

40 65 35

DETERMINATION OF BISPHENOL A AND BADGE IN MILK

Bisphenol A Diglycidyl Ether (BADGE)

Bisphenol A

DETERMINATION OF TOTAL BISPHENOL A IN HUMAN URINE


PROTOCOL OF CLEANUPSample preparation3mL urine sample, 1mL of sodium acetatebuffer 0.1M at pH 5.0 and 20µL of β-glucuronidase/sulfatase Helix pomatia enzymesolution at 1.0mg/mL in the same buffer weremixed thoroughly by vortex. The enzymaticreaction was carried out for 2h at 37°C toobtain the loading solution.

Cleanup with a 6mL/100mg AFFINIMIP® SPEBisphenol A glass cartridge


Loading solutionUp to 12mL of loading solution (Equivalentto around 9mL of urine)

Washing of interferences•4mL Water•4mL Water/Acetonitrile (60/40)

Elution (E)3mL Methanol

The elution fraction was then concentrated anddiluted to 1mL before HPLC analysis.

RESULTS


47

HPLC Method with LC-MS/MSHPLC Column: Kinetex 2.6µm PFP 100mm x 4.6mmMobile phase: gradient profile

Flow rate: 0.5mL/minInjection volume: 20µL.Detector: ESI-MS/MS

Time (min) % water % Methanol

0 70 30

1 70 30

2 5 95

5 5 95

6 70 30

9 70 30

LC-MS/MS Chromatograms obtained after clean-up withAFFINIMIP® SPE Bisphenol A

(a) of children urine at 0.38ng/mL BPA, signal to noise(S/N) 13.9

(b) for the blank sample (neither urine nor BPA), S/N=1.9

Mean percentage recoveries of Bisphenol A spiked atdifferent concentrations in 3mL of urine afterAFFINIMIP® SPE Bisphenol A clean-up:

C° (ng/mL) 1 10 100

Recoveries % 102.6 94.7 97.6

By courtesy of Nadia Diano, Dept. of ExperimentalMedicine, Second University of Naples (Italy)More details in the following articleC. Nicolucci, S. Rossi, C. Menale, E. Giudice, P. Miraglia delGiudice, L. Perrone, P. Gallo, D. Mita, N. Diano, Analyticaland Bioanalytical Chemistry, 1618-2642, 2013.

Inte

nsi

ty,

cps

Inte

nsi

ty,

cps

m/z 227.1 → m/z 212.1m/z 227.1 → m/z 133.2(a)

(b)

AFFINIMIP® SPE ChloramphenicolFS110

48

Catalog number:1mL-50mg sorbentPI-FS110-02A for 25 cartridgesPI-FS110-03A for 50 cartridges

Chloramphenicol: a major concern for human health and achallenge in food safety analysisChloramphenicol is a broad-spectrum antibiotic widely used in theworld in the past. Several health problems are related to its use. As aconsequence, several countries (e.g. U.S.A, E.U, Canada…) haveprohibited its use for food-producing animals. As no permitted limithas been established, E.U. has defined a Minimum RequiredPerformance Limits (MRPLs) of 0.3µg/kg for product of animal origin(Commission decision 2003/181/EC).

However, due to its broad spectrum of activity and its availability, Chloramphenicol is stillused in several countries to treat food-producing animals. Therefore, chloramphenicolanalysis is still a current affair.In addition, food matrices are very complexes and induce ion-suppression phenomena whichdistort analysis results. For such a low MRPL threshold, a clean-up step is crucial in order toimprove the sensitivity, the reliability and the specificity before analysis. It is therefore criticalto develop a highly selective and sensitive analytical assay to control and monitorChloramphenicol residues in difficult matrices such as food stuffs.AFFINISEP has developed AFFINIMIP® SPE Chloramphenicol cartridge, a simple, fast,sensitive and selective tool for the extraction of Chloramphenicol from complex matrices.

We demonstrate in these application notes that a reliable quantification ofChloramphenicol from honey and bovine urine at low concentrations using AFFINIMIP®SPE Chloramphenicol and a single quadrupole mass detection is possible.In a very complex matrix such as honey, we obtained a high recovery yield (> 90%) with a lowbackground, even with UV detection. The tests carried out on several kinds of honeydemonstrated a good reproducibility, proving the efficiency of AFFINIMIP® SPEChloramphenicol cleanup.

DETERMINATION OF CHLORAMPHENICOL IN HONEY

AFFINIMIP® SPE Chloramphenicol

PROTOCOL OF CLEANUPSample preparation10g of honey and 10mL Water were mixedunder magnetic stirring during 10 minutes andused as the loading solution.Cleanup with a 1mL/50mg AFFINIMIP® SPEChloramphenicol cartridge


Loading1mL of loading solution for 15µg/kg (or10mL for 0.3µg/Kg)

Washing of interferents (W1)•1mL Water•1mL (Water - 0.5% AA)/ACN (95/5)•2mL of Ammonia (1%) in Water•2mL (Water-1% Ammonia)/ACN (80/20)

Drying 1 minWashing of interferents (W2)

0.25mL Diethyl etherElution (E)


RESULTS


49

Regulations for Chloramphenicol inresidues in food of animal origin:Europe 2003/181/EC prohibited with aminimum required performance limits of0.3µg/Kg

HPLC Method with MS detectionColumn: Thermo Accucore C18 column 50mm x2.1mmMobile phase: Ammonium acetate (10mM) inwater /Methanol (75/25)flow rate: 0.2mL/minMS detection: m/z 322 (ESI-)Injection volume: 20µL.

2 3 4 5 6 7 8 9 10

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nsity

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Chloramphenicol

Honey spiked at 15.7µg/kg

Not spiked

SIM Chromatograms obtained after clean-up withAFFINIMIP® SPE Chloramphenicol of 1g of Honeyspiked with Chloramphenicol at 15.7µg/kg (red) ornot spiked (blue).

SIM Chromatograms obtained after clean-up withAFFINIMIP® SPE Chloramphenicol of 1g of Honeyspiked with Chloramphenicol at 15.7µg/kg (red) ornot spiked (blue).

Recovery of Chloramphenicol spiked at 16µg/kgafter AFFINIMIP® SPE Chloramphenicol clean-up of1g of Honey and relative standard deviationcalculated from results generated:.


C° (µg/kg) Mean (µg/kg) Recoveries % % RSDr

16.0 15.4 96.1 3.3


C°

(µg/kg)

Mean (µg/kg) Recoveries % % RSDR

15.7 16.9 108.1 6.5 (n=6)

18.2 16.6 91.4 11.4 (n=12)

SIM Chromatogram obtained after clean-up withAFFINIMIP® SPE Chloramphenicol of 10g ofHoney spiked with Chloramphenicol at 0.3µg/kg.

Chloramphenicol

0.3µg/Kg Chloramphenicol

DETERMINATION OF CHLORAMPHENICOL IN BOVINE URINE

PROTOCOL OF CLEANUPSample preparation10 mL of urine were adjusted at pH 7 with

Ammonia 1%. This solution was mixed andused as the loading solution.

Cleanup with a 1mL/50mg AFFINIMIP® SPEChloramphenicol cartridge


Loading1mL of loading solution

Washing of interferents (W1)•1mL (Water - 0.5% AceticAcid)/Acetonitrile (95/5)•2mL of Ammonia (1%) in Water•2mL (Water-1% Ammonia)/Acetonitrile(80/20))




RESULTS


50

Regulations for Chloramphenicol in residues in food of animal origin: Europe (2003/181/EC) : prohibited with a Minimum Required Performance Limits of 0.3µg/KgUSA FDA: prohibited


Chloramphenicol

SIM Chromatograms obtained after clean-up with AFFINIMIP® SPE Chloramphenicol of 1 mL of Urine spiked with Chloramphenicol at 17.6µg/kg (red and blue) or not spiked (green).

UV Chromatograms of Urine spiked withChloramphenicol at 17.6 µg/kg (red and black) ornot spiked (green) after clean-up with AFFINIMIP®SPE Chloramphenicol

C° (µg/kg) Mean (µg/kg) Recovery %

17.6 16.7 90

Recovery of Chloramphenicol spiked at17.6µg/kg after AFFINIMIP® SPEChloramphenicol clean-up of 1 mL of Urine.

UV: VERY LOW BACKGROUND

RT: 0.00 - 30.00

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28

Time (min)

0

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uAU

NL:1.32E5

Channel A UV UrineD1-E

NL:1.32E5

Channel A UV urined2-e

NL:1.32E5

Channel A UV UrineND-E


DETERMINATION OF CHLORAMPHENICOL IN SHRIMP

PROTOCOL OF CLEANUPSample preparation5g peeled shrimp were homogenized 2min

with a vortex in 20mL of ethyl acetate. Thenthe solution was filtered on filter paper(25µm). The supernatant was evaporated todryness and reconstituted in 10mL of Water toobtain the loading solution.

Cleanup with a 1mL/50mg AFFINIMIP® SPEChloramphenicol cartridge


Loading1 or 2mL of loading solution

Washing of interferents (W1)•1mL Water•1mL (Water - 0.5% AceticAcid)/Acetonitrile (95/5)•2mL of Ammonia (1%) in Water•2mL (Water-1% Ammonia)/Acetonitrile(80/20))




RESULTS


51


Chloramphenicol

SIM Chromatograms obtained after clean-up withAFFINIMIP® SPE Chloramphenicol of Shrimp spikedwith Chloramphenicol at 38µg/kg. Loading of 1mL(spiked in green and not spiked in black) and of 2mL(spiked in red and not spiked in blue)

Recovery of Chloramphenicol spiked at 38µg/kgafter AFFINIMIP® SPE Chloramphenicol clean-up ofShrimp.

UV: VERY LOW BACKGROUND

C° (µg/kg)Loading volume

Mean (µg/kg)

Recovery %

38 1mL 38.7 101.7

38 2mL 36.4 95.8

UV Chromatograms obtained after clean-up withAFFINIMIP® SPE Chloramphenicol of Shrimp spikedwith Chloramphenicol at 38µg/kg. Loading of 1mL(spiked in green and not spiked in black) and of 2mL(spiked in red and not spiked in blue)

Chloramphenicol

Regulations for Chloramphenicol in residues in food of animal origin: Europe (2003/181/EC) : prohibited with a Minimum Required Performance Limits of 0.3µg/KgUSA FDA: prohibited

0 2 4 6 8 10 12 14 16

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AFFINIMIP® SPE AmphetaminesDG102

52

Catalog number:3mL-50mg cartridgePI-DG102-02 for 25 cartridgesPI-DG102-03 for 50 cartridges

Amphetamines are a class of illegal drugs that exhibit strong central nervous systemstimulant effect. So, to determine if drivers are under influence of (Meth)amphetamine,several US and European states have set up a cut-off value in urine or blood [e.g. France andVirginia (respectively 50ng/mL and 100ng/mL of blood)].For such low concentrations, a clean up step is crucial in order to improve the sensitivity, thereliability and the specificity before LC analysis.

NH2

NH

NH2

O

ONH

O

O

NH

O

O

Amphetamine Methamphetamine

3,4-Methylenedioxyamphetamine MDA

3,4-Methylenedioxymethamphetamine MDMA

3,4-methylenedioxy-N-ethylamphetamine MDEA

To do so, we have developed a AFFINIMIP® SPE Amphetamines cartridge, a powerfultechnique for clean-up and pre-concentration applications of Amphetamines. Theseapplication notes describe the solid phase extraction of Amphetamines from human urineand human serum using AFFINIMIP® SPE Amphetamines.

DETERMINATION OF AMPHETAMINES IN HUMAN URINE

AFFINIMIP® SPE Amphetamines

PROTOCOL OF CLEANUPSample preparationHuman urine is diluted by 2 with an ammoniumacetate buffer (13mM, pH 8.5). The pH of thediluted urine is adjusted with NH3 or CH3COOHat pH 8.5.

Cleanup with a 3mL AFFINIMIP® SPEAmphetamines cartridge


Loading5mL of diluted urine

Washing of interferents (W1)•3mL Water•3mL Water/Acetonitrile (60/40)


1.5mL Methanol – 2% Formic acidThe elution fraction was then evaporated anddissolved in the mobile phase before HPLCanalysis.

RESULTS


53

Example of Regulations: France : prohibited cut-off limit of 1µg/mL in urine and 50ng/mL of bloodVirginia (USA): 100ng/mL of blood

HPLC Method with MS detectionColumn: Syncronis Aq column 150mm x 2.1mmMobile phase: gradient profile with A (Water –Ammonium Acetate 10mM) and B (Acetonitrile –Ammonium Acetate 1mM)

flow rate: 0.4mL/minMS detection (ESI+) : m/z 136 (Amphetamine) ; 180(MDA); 150 (Methamphetamine); 194 (MDMA); 208(MDEA)Injection volume: 20µL.

Time (min) % A % B

0 95 5

2 95 5

12 60 40

12.1 95 5

Recovery of Amphetamines in human urine spikedat 20ng/mL after AFFINIMIP® SPE Amphetaminesclean-up and relative standard deviation calculatedfrom results generated under reproducibilityconditions.

SampleMean

ng/mL

Recoveries

%% RSDR

Amphetamine 17.5 87.5 8.9 (n=8)

MDA 18.6 93.1 9.6 (n=8)

Methamphetamine 18.6 93.2 9.2 (n=8)

MDMA 21.1 105.4 1.5 (n=4)

MDEA 20.3 101.7 12.4 (n=8)

Quantity loaded

µg

Quantity obtained in the elution

fraction

µg

1.0 0.90

2.5 2.41

5.0 3.51

Capacity: different concentrations of Amphetaminein urine were applied on AFFINIMIP® SPEAmphetamines cartridge (25mg) to measure thecapacity of the product.

2 4 6 8 10 12 14 16 18 20 22 24

Time (min)

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50

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85

Amphetamine

MDA

Methamphetamine

MDMA

MDEA

2 4 6 8 10 12 14 16 18 20 22 24

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Amphetamine

MDA

Methamphetamine

MDMA

MDEA

Mass Chromatogram (SIM) obtained afterAFFINIMIP® SPE Amphetamines clean-up of ahuman urine sample spiked at 20ng/mL withAmphetamine and its derivatives.

DETERMINATION OF AMPHETAMINES IN HUMAN SERUM

PROTOCOL OF CLEANUPSample preparationHuman serum is diluted by 5 with anammonium acetate buffer (13mM, pH 8.5). ThepH of the diluted urine is adjusted with NH3 orCH3COOH at pH 8.5.

Cleanup with a 3mL AFFINIMIP® SPEAmphetamines cartridge


Loading2.5mL of diluted serum

Washing of interferents (W1)•3mL Water•3mL Water/Acetonitrile (60/40)


1.5mL Methanol – 2% Formic acidThe elution fraction was then evaporated anddissolved in the mobile phase before HPLCanalysis.

RESULTS


54

HPLC Method with MS detectionColumn: Syncronis Aq column 150mm x 2.1mmMobile phase: gradient profile with A (Water –Ammonium Acetate 10mM) and B (Acetonitrile –Ammonium Acetate 1mM)

flow rate: 0.4mL/minMS detection (ESI+) : m/z 136 (Amphetamine) ; 180(MDA); 150 (Methamphetamine); 194 (MDMA); 208(MDEA)Injection volume: 20µL.

Time (min) % A % B

0 95 5

2 95 5

12 60 40

12.1 95 5

Recovery of Amphetamines in human serum spikedat 100ng/mL after AFFINIMIP® SPE Amphetaminesclean-up and relative standard deviation calculatedfrom results generated under reproducibilityconditions (n=4).

SampleMean

ng/mL

Recoveries

%% RSDR

Amphetamine 87.9 87.9 5.0

MDA 94.4 94.4 3.7

Methamphetamine 90.7 90.7 2.2

MDMA 106.2 106.2 2.5

MDEA 111.0 111.0 4.9

2 4 6 8 10 12 14 16 18 20 22 24Time (min)

0

10

20

30

40

50

60

70

80

90

Amphetamine

Methamphetamine

MDA

MDMA

MDEA

2 4 6 8 10 12 14 16 18 20 22 24Time (min)

0

10

20

30

40

50

60

70

80

90

Amphetamine

Methamphetamine

MDA

MDMA

MDEA

Mass Chromatogram (SIM) obtained afterAFFINIMIP® SPE Amphetamines clean-up of ahuman serum sample spiked at 100ng/mL withAmphetamine and its derivatives.

Example of Regulations: France : prohibited cut-off limit of 1µg/mL in urine and 50ng/mL of bloodVirginia (USA): 100ng/mL of blood

AFFINIMIP® SPE Amphetamines

AFFINIMIP® SPE TetracyclinesFS112

55

Tetracyclines (TCs) like oxytetracycline (OTC), tetracycline (TC) and chlortetracycline (CTC) arebroad-spectrum antibiotics and are widely used as veterinary medicines and feed additives.These residues can cause toxic or allergic reactions in hypersensitive individuals and alsotransfer drug-resistant bacteria from food to humans.In response to these concerns and to prevent harmful effects of residual antibiotics in milkon the human health, various international health organizations have established themaximum residual limit (MRL) of TCs in all circulating milk in their countries. Worldwidemaximum residue levels (MRL) for tetracycline antibiotics are 100ppb (µg/L).For such low concentrations, a clean up step is crucial in order to improve the sensitivity, thereliability and the specificity before LC analysis.

To do so, we have developed a AFFINIMIP® SPE Tetracyclines cartridge, a powerful techniquefor clean-up and pre-concentration applications of Tetracyclines.

Catalog number:1mL-10mg sorbentPI-FS112-02A for 25 cartridges 1mLPI-FS112-03A for 50 cartridges 1mLkit of 12 reservoirs 15ml and adapters for use with 1, 3 & 6 mL columnsPI-ACC-AR2

Tetracycline Oxytetracycline

Chlortetracycline

DETERMINATION OF TETRACYCLINES, THEIR EPIMERS ANDDOXYCYCLINE IN MILK AND SALMON

PROTOCOL OF CLEANUP

Sample preparation for Milk

Mix 1.5mL of Milk with 6mL of EDTA/Mc Ilvaine’s

Buffer and centrifuge at 4000rpm for 10 min at a

temperature below 15°C. Collect the supernatant

and add 750µL 1N NaOH solution. Adjust to pH 10

with a NaOH solution (this mixture was the loading

solution).

Sample Preparation for Salmon based on AOAC

995.09 method

Blend 10g Salmon with 40mL of EDTA/Mc Ilvaine’s

Buffer during 30 s and stir during 10min with a

magnetic stirrer. Centrifuge the mixture at 2500g

for 10 min at a temperature < 15°C. Collect the

supernatant

Repeat this operation with 40mL buffer and again

with 20mL of buffer. Then, gather all the

supernatants and centrifuge during 20min at 2500g,

filter on Buchner. Add 750µL 1N NaOH solution to

the filtrate and adjust to pH 10 with a NaOH

solution (this mixture was the loading solution).


Tetracyclines cartridge

Equilibration

•1mL Acetonitrile

•1mL Water

Loading

Loading solution (7.5mL)

Washing of interferents

•1mL Water

•2mL Water/Acetonitrile (60/40)

Drying 3 minutes

Elution (E)

2mL Methanol with 2% Formic acid


dissolved in the mobile phase before HPLC analysis.

RESULTS

56

HPLC Method with UV detectionColumn: Hypersil Gold C18 column 150mm x2.1mm, 3µmMobile phase: gradient profile

Flow rate: 0.2mL/minUV detection: 355nmInjection volume: 100µL.

Time (min)

% 10mM Oxalic Acid

Water

% 10mM Oxalic Acid

ACN% MeOH

0 90 5 5

20 90 5 5

21 80 10 10

40 80 10 10

41 90 5 5

UV Chromatograms (355nm) obtained after clean-up with AFFINIMIP® SPE Tetracyclines of 1.5mL ofMilk spiked with Tetracycline, Chlortetracycline and4-epioxytetracycline (4-epiOTC) at 50µg/L (blue) ornot spiked (red) or of 1.5mL of water spiked withTetracycline, Chlortetracycline and 4-epioxytetracycline at 50µg/L (pink)

AFFINIMIP® SPE Tetracyclines

Catalog number:1mL-10mg sorbentPI-FS112-02A for 25 cartridges 1mLPI-FS112-03A for 50 cartridges 1mLkit of 12 reservoirs 15ml and adapters foruse with 1, 3 & 6 mL columnsPI-ACC-AR2

Recovery of Tetracyclines after AFFINIMIP® SPETetracyclines clean-up of Salmon or milk spiked at50 or 100µg/L and relative standard deviationcalculated from results generated underrepeatability conditions (n=3).

MoleculesMean

(µg/L)

Milk Salmon

R % % RSDr R%

Tetracycline 49.6 99.4 4.9 113

Oxytetracycline 45.6 91.3 7.1 -

Chlortetracycline 37.2 74.4 6.3 74

4-epitetracycline

(4-epiTC)47.9 95.9 5.1 -

4-

epichlortetracycline108.4 108.4 15.0 97

4-

epioxytetracycline 43.7 87.4 9.1 71

Doxycycline (DOX) 43.8 88.0 2.9 89

Minutes

6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50

mA

U

-10

0

10

20

30

40

50

60

70

80

mA

U

-10

0

10

20

30

40

50

60

70

80

4-epiOTC TC CTC

AFFINIMIP® SPE MetanephrinesDG101

57

Catalog number:1mL cartridgePI-DG101-02A for 25 cartridgesPI-DG101-03A for 50 cartridges3mL cartridgePI-DG101-02 for 25 cartridgesPI-DG101-03 for 50 cartridges

3-Methoxytyramine (3-MT)

Normetanephrine (NMN)Metanephrine (MN)

NH2

OH

OH

MeO

NH

OH

OH

Me

MeO

NH2

OH

MeO

Quantification of free Metanephrines inPlasma is considered to be the most accuratetest for the clinical chemical diagnosis ofPheocromocytoma.The concentrations of these endogenousmolecules are very low in serum and plasma(lower than 1nM). A clean up step is crucial inorder to improve the sensitivity and thespecificity before LC analysis.

Current method involves non specific sample preparation. To propose an accurate solution, we have developed AFFINIMIP® SPE Metanephrinescartridges, a powerful technique for clean-up and pre-concentration applications ofMetanephrines.

This study describes the solid phase extraction of Metanephrines from plasma sample usingAFFINIMIP® SPE Metanephrines. In addition, a comparison with a Weak Cation Exchange SPEcartridge is shown.

DETERMINATION OF METANEFHRINES IN PLASMA COMPARISON WITH WCX CARTRIDGES

AFFINIMIP® SPE Metanephrines

PROTOCOL OF CLEANUPSample preparationThe plasma or serum is diluted by 5 with water. This solution is used as the loading solution.

Cleanup with a 1mL AFFINIMIP® SPEMetanephrines cartridge

Equilibration•1mL of phosphate buffer pH 7•2mL Water

Loading1.5mL of loading solution

Washing of interferents (W1)•1mL Water•500µL Water/Methanol (60/40)


•500µL MethanolElution (E)

1mL Methanol – 5% Acetic acidThe elution fraction was then evaporated anddissolved in the mobile phase before HPLCanalysis.

RESULTS

Catalog number:1mL cartridgePI-DG101-02A for 25 cartridgesPI-DG101-03A for 50 cartridges3mL cartridgePI-DG101-02 for 25 cartridgesPI-DG101-03 for 50 cartridges

58

HPLC Method with LC-MS/MS detectionColumn: Syncronis aQ column 150mm x 2.1mmMobile phase: Water – 0.1% Formic Acidflow rate: 0.2mL/minMS detection: m/z 322 (ESI+)Injection volume: 20µL.

Analysis by LC-MS/MS: Total Ion Current of a calfserum after Cleanup by AFFINIMIP® SPEMetanephrines. The sample naturally containedMetanephrine. Concentration of MN found: 30nM.In parallel, a SPE was performed on a protocoldeveloped for the analysis of MN using WCXcartridges: the concentration obtained was 7nM forthe same sample.

WCXRecovery : 33%

AFFINIMIP SPERecovery: 101%

NMN

Analysis by LC-MS/MS: Selected ion monitoring ofNormetanephrine (m/z 180). Chromatogramsobtained after Cleanup by AFFINIMIP® SPEMetanephrines or by WCX of a calf serum spiked at27nM with Normetanephrine.

Recoveries of MN and NMN at a contaminationlevel of 500nM in rabbit plasma after AFFINIMIP®

SPE Metanephrines Clean-up and relative standarddeviation calculated from results generated underreproducibility conditions (Analysis by LC-MS).

WCX AFFINIMIP® SPE

Analytes Recoveries % % RSDR

Metanephrine 79.4 6.3

Normetanephrine 109 11

AFFINIMIP® SPE PhenolicsFS103

59

Class-selective extraction of Phenolic compounds from a variety samples: water, cosmectics, biological or food matrices.

Phenolic compounds are important families of products found as natural substances in plantsand life sciences or as synthetic products such as drugs.AFFINIMIP® SPE Phenolics are class-selective solid phase extraction cartridges thatselectively clean and concentrate a broad range of phenolic compounds prior to analysis.This treatment strongly reduces the amount of interferents and the matrix effects.

Benefits•Extraction of a broad family of phenolic compounds•Reduction of matrix effects and of most interferents•Several protocols proposed to purify your product


Minutes 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

mAU

0

200

400

600

800

1000

1200

1400

1600

1800

mAU

0

200

400

600

800

1000

1200

1400

1600

1800

DETERMINATION OF PARABENS IN COSMETIC PRODUCTS


1g of Lotion was mixed 1minute with 1mL ofH2SO4 2M and 50mL of 90/10 Ethanol/Water.The mixture was heated during 5min at 60°C.Then the solution is filtered on filter paper (4-7µm). This extract was diluted by 3 with water.The solution was spiked with methylparaben tosimulate a concentration of paraben in thelotion at 0.2%, 0.4% and 0.8%.

Cleanup with a 3mL/100mg AFFINIMIP® SPEPhenolics cartridge


Loading•Up to 5mL of loading solution

Washing of interferents•3mL Water / Acetonitrile (75/25 v/v)

Elution (E)•3mL MethanolThe elution fraction was diluted by 2 withwater prior to analysis.

RESULTS

60

HPLC-UV MethodColumn: Thermo Hypersil gold, 150mm x 2.1mmMobile phase: 60/40 (v/v) Water/MethanolFlow rate: 0.2mL/minDetection: UV - 254nmInjection volume: 20µL.

Chromatograms of a cream containing 0.2% ofmethylparaben before clean-up (blue) and afterclean-up (Red) with AFFINIMIP® SPE Phenolics.


AFFINIMIP® SPE Phenolics

Chromatograms obtained after clean-up withAFFINIMIP® SPE Phenolics of a cream (withoutparabens) spiked with different concentrations ofmethylparaben

Recovery yields and reproducibility afterAFFINIMIP® SPE Phenolics Clean-up.

Recoveries % (n=6) RSDR%

101.1 8

General structure of Parabens

Minutes 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

mAU

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

mAU

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

Before

After

Cream spiked at 0.8%, yield 91% (n=2)

Cream spiked at 0.4%, yield 106 (n=2)

Cream spiked at 0.2%, yield 106 (n=2)

DETERMINATION OF GUAIACOL

PROTOCOL OF CLEANUP



Loading•Up to 2mL of red or white wine


Elution (E)•2mL Methanol

RESULTS

61

HPLC-UV MethodColumn: Thermo Hypersil gold, 150mm x 4.6mmMobile phase: 15/85 (v/v) Acetonitrile WaterFlow rate: 1mL/minDetection: UV - 272nmInjection volume: 100µL.

Chromatograms obtained after clean-up withAFFINIMIP® SPE Phenolics of red wine spiked withGuaïacol (0.1µM) (red) or not spiked (blue).


Chromatograms obtained before (red) and after(blue) clean-up with AFFINIMIP® SPE Phenolics ofred wine spiked with Guaïacol (0.1µM)

Recovery yields and reproducibility evaluated with 3cartridges and 3 different batches of AFFINIMIP®SPE Phenolics by matrix (n=9)

General structure of Guaîacol

Minutes 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

mAU

- 50 0

50 100 150 200 250 300 350

mAU

- 50 0 50 100 150 200 250 300 350

Guaïacol

Minutes 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

mAU

- 200 0

200 400 600 800

1000 1200 1400 1600 1800 2000 2200 2400 2600

200

Guaïacol

C° (µM) Recoveries % RSDR%

Red wine 1 0.1 88.1 3.9

Red wine 2 0.1 93.1 3.7

White wine 1 0.02 96.8 1.7

White wine 2 0.02 93.5 2.6


DETERMINATION OF CARNOSIC ACID IN MEAT


25g of turkey was mixed with 200mL of74.5/25/0.5 ACN/H2O/H3PO4 or Ethanol-0.5%H3PO4 using a blender during 3 minutes. After,the mixture was mixed during 30 minutes withmagnetic stirrer. The mixture was filtered onfilter paper (4-7µm). Then the mixture wasdiluted by 2 with water.



Loading•Up to 80mL of loading solution


Elution (E)•2mL Methanol -1% H3PO4

RESULTS

62

HPLC-UV MethodColumn: Thermo Hypersil gold, 150mm x 4.6mmMobile phase: 65/35 (v/v) ACN/Water-0.5% H3PO4

Flow rate: 1mL/minDetection: UV - 230nmInjection volume: 5µL.

Chromatogram of a turkey containing 50ppm ofCarnosic acid after clean-up with AFFINIMIP® SPEPhenolics. Extraction of the turkey with Ethanol-0.5% H3PO4


Chromatogram of a turkey containing 50ppm ofCarnosic acid after clean-up with AFFINIMIP® SPEPhenolics. Extraction of the turkey with 74.5/25/0.5ACN/H2O/H3PO4

Recovery yields obtained by both extraction solventafter AFFINIMIP® SPE Phenolics Clean-up.

Minutes

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

mA

U

0

50

100

150

200

250

300

350

400

450

mA

U

0

50

100

150

200

250

300

350

400

450

UV 6000-230nm

E ZON ethanol.dat

Carnosic acid

Minutes

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

mA

U

0

50

100

150

200

250

300

350

400

450

500

mA

U

0

50

100

150

200

250

300

350

400

450

500UV 6000-230nm

E ZON 75-25.dat

Carnosic acid

Extraction solvent Recoveries %

74.5/25/0.5 ACN/H2O/H3PO4 >85%

Ethanol-0.5% H3PO4 >80%

structure of Carnosic acid


AFFINIMIP® SPE PRODUCT LIST FOR MYCOTOXINS ANALYSES

63

Products Designation Definition ReferenceNber of

cartridges

Zearalenone and Fumonisins

AFFINIMIP® SPEFumoZON

3mL Selective SPE cartridges for Zearalenone and Fumonisins

PI-FS109-02 25

PI-FS109-03 50

MultimycotoxinsAFFINIMIP® SPE

Multimyco10

3mL Selective SPE cartridges for analyses ofAflatoxins, Zearalenone, Ochratoxin A, T-2,HT-2, Fumonisins

PI-FS114-03 50

PI-FS114-04 100

Patulin


3mL – 100mg Selective SPE cartridges forPatulin

PI-FS102-02 25

PI-FS102-03 50

6mL – 200mg Selective SPE cartridges forPatulin (for DRIED APPLE and higherenrichment with apple juice)

PI-FS102-02B-200mg 25

PI-FS102-03B-200mg 50

AFFINIMIP® SPE Patulin & Pectinase kit

Kit of 3mL selective SPE cartridges forPatulin + 50mL Pectinase enzyme solution

PI-FS102-02K 25

PI-FS102-03K 50

Kit of 6mL - 200mg selective SPE cartridgesfor Patulin in dried apple + 50mL Pectinaseenzyme solution

PI-FS102-02KB-200mg 25

PI-FS102-03KB-200mg 50

Ochratoxin AAFFINIMIP® SPE

Ochratoxin A

3mL Selective SPE cartridges for OchratoxinA

PI-FS101-02 25

PI-FS101-03 50

6mL Selective SPE cartridges for OchratoxinA

PI-FS101-02B 25

PI-FS101-03B 50

DeoxynivalenolAFFINIMIP® SPEDeoxynivalenol

6mL -100mg Selective SPE cartridges forDeoxynivalenol in FOOD

PI-FS117-02B 25

PI-FS117-03B 50

6mL -200mg Selective SPE cartridges forDeoxynivalenol in ANIMAL FEED

PI-FS101-02B-200mg 25

PI-FS101-03B-200mg 50

Pectinase 50 mL Pectinase enzyme solution PI-REA-001-50mL

ZearalenoneAFFINIMIP® SPE

Zearalenone3mL Selective SPE cartridges for ZON

PI-FS100-02 25

PI-FS100-03 50

For other formats, please contact usAFFINISEP can provide you with other formats than the one described in the product list. Other

formats available on demand and with an adapted protocol can be :

• different volumes of SPE cartridges (1mL, 3mL, LRC, 60mL, etc… )

• 96 well plates,

• HPLC columns,

• Preparative HPLC columns

• the format adapted to your application and your automate

AFFINIMIP® SPE PRODUCT LIST (MISCELLANEOUS)

Products Designation Definition ReferenceNber of

cartridges

AmphetaminesAFFINIMIP® SPEAmphetamines

3mL Selective SPE cartridges forAmphetamines derivatives

PI-DG102-02 25

PI-DG102-03 50

Bisphenol AAFFINIMIP® SPE

Bisphenol A

3mL Selective SPE cartridges forBisphenol A (PP)

PI-FS106-02 25

PI-FS106-03 50

6mL Selective SPE cartridges forBisphenol A (PP)

PI-FS106-02B 25

PI-FS106-03B 50

6mL Selective SPE cartridges forBisphenol A (Glass)

PI-FS106-02G 25

PI-FS106-03G 50

CatecholaminesAFFINIMIP® SPECatecholamines

3mL Selective SPE cartridges forCatecholamines

PI-DG100-02 25

PI-DG100-03 50

1mL Selective SPE cartridges forCatecholamines

PI-DG100-02A 25

PI-DG100-03A 50

ChloramphenicolAFFINIMIP® SPE

Chloramphenicol1mL Selective SPE cartridges forChloramphenicol

PI-FS110-02A 25

PI-FS110-03A 50

EstrogensAFFINIMIP® SPE

Estrogens

1mL Selective SPE cartridges forEstrogens

PI-FS104-02A 25

PI-FS104-03A 50

3mL Selective SPE cartridges forEstrogens

PI-FS104-02 25

PI-FS104-03 50

Picolinic acid based herbicides

AFFINIMIP® SPEPicolinic

Herbicides

Selective SPE cartridges for Picolinicacid based herbicides

PI-FS115-02 25

PI-FS115-03 50

Metanephrines

AFFINIMIP® SPEMetanephrines

3mL Selective SPE cartridges forMetanephrines

PI-DG101-02 25

PI-DG101-03 50

1mL Selective SPE cartridges forMetanephrines

PI-DG101-02A 25

PI-DG101-03A 50

PhenolicsAFFINIMIP® SPE

Phenolics3mL Selective SPE cartridges for Phenolic compounds

PI-FS103-03 50

TamoxifenAFFINIMIP® SPE

Tamoxifen3mL Selective SPE cartridges for Tamoxifen

PI-PH101-02 25

TetracyclinesAFFINIMIP® SPE

Tetracyclines1mL Selective SPE cartridges for Tetracyclines

PI-FS112-02A 25

PI-FS112-03A 50

Zeranol ResiduesAFFINIMIP® SPE

Zeranol Residues3mL Selective SPE cartridges for Zeranol Residues

PI-FS105-02 25

PI-FS105-03 50

clsmac

Sticky Note

Marked set by clsmac

Solid-phase extraction using molecularly imprinted polymers for selective extraction of a

mycotoxin in cereals, J. Chrom. A., 1217, 6668-6673, 2010.

Molecularly imprinted polymer solid-phase extraction for detection of zearalenone in cereal

sample extracts detection, Analytica Chimica Acta, 672, 15–19, 2010.

Molecularly imprinted polymer as sorbent in micro-solid phase extraction of ochratoxin A in

coffee, grape juice and urine, Tien Ping Lee, Bahruddin Saad, Wejdan Shakir Khayoon, Baharuddin

Salleh, Talanta, 88, 129–135, 2012.

Molecularly imprinted polymer-based solid phase clean-up for analysis of ochratoxin A in ginger

and LC-MS/MS confirmation, J. Cao, S. Zhou, W. Kong, M. Yang, L. Wan, S. Yang, Food control, 33(2),

337-343, 2013.

Molecularly imprinted polymer-based solid phase clean-up for analysis of ochratoxin A in beer,

red wine, and grape juice, J. Cao, W. Kong, S. Zhou, L. Yin, L. Wan, M. Yang, J. Sep. Sci., 36(7), 1291-

1297, 2013.

Application of molecularly imprinted polymers to determine B1, B2, and B3 fumonisins in cereal

products, M. Bryła, R. Jedrzejczak, M. Roszko, K. Szymczyk, M. W. Obiedziński, J. Sękul, M.

Rzepkowska, J. Sep. Sci., 36(3), 578-584, 2013.

Breakthrough innovation in rapid detection kits using MOLECULARLY IMPRINTED POLYMERS SPE

for an early quantification of PATULIN from apple-based food matrices, D. Derrien, C. Pérollier, O.

Lépine, K. Naraghi, S. Bayoudh, poster at 7th World Mycotoxin Forum, Rotterdam, The Netherlands,

November 5-9, 2012.

Molecularly Imprinted Polymer for Solid Phase Extraction of Patulin mycotoxin, D. Derrien, M.

Mulet, F. Alix, C. Pérollier, O. Lépine, K. Naraghi, S. Bayoudh, 33rd Mycotoxin Workshop, Freising,

Germany, 30 May - 1 June 2011

Interest of molecularly imprinted polymers in the fight against doping. Extraction of tamoxifen

and its main metabolite from urine followed by high-performance liquid chromatography with UV

detection. J. Chrom. A, 1196–1197, 81–88, 2008.

Analysis of urinary neurotransmitters by capillary electrophoresis: Sensitivity enhancement using

field-amplified sample injection and molecular imprinted polymer solid phase extraction, B. Claude

, R. Nehmé, P. Morin, Analytica Chimica Acta, 699 (2), 242–248, 2011.

Solid-phase extraction using molecularly imprinted polymer for selective extraction of natural

and synthetic estrogens from aqueous samples, P. Lucci, O. Núñez, M.T. Galceran, J. Chrom. A, 1218,

4828-4833, 2011.

On-line molecularly imprinted solid-phase extraction coupled to liquid chromatography-tandem

mass spectrometry for the determination of hormones in water and sediment samples, D.

Matějíček, J. Vlček, A. Burešová, P. Pelcová, J. Sep. Sci., 36(9-10), 1509-1515, 2013.

Quantification of estrogens at ppt levels in bovine plasma by Molecularly Imprinted Solid Phase

Extraction and GC-MS/MS analysis, S. Rochereau, E. Bichon, F. Courant, F. Monteau, S. Prévost, F.

Hanganu, N. Cesbron, G. Dervilly-Pinel, B. Le Bizec (LABERCA), Poster Euroresidues VIIth conference,

2012 .

The use of molecularly imprinted polymers for the multicomponent determination of endocrine-

disrupting compounds in water and sediment, D. Matějíček, A. Grycová, J. Vlček, J. Sep. Sci., 36(6),

1097-1103, 2013.

LIST OF PUBLICATIONS AND POSTERS

66

Molecularly imprinted polymer applied to the selective isolation of urinary steroid hormones: An

efficient tool in the control of natural steroid hormones abuse in cattle, M. Doué, E. Bichon, G.

Dervilly-Pinel, V. Pichon, F. Chapuis-Hugon, E. Lesellier, C. West, F. Monteau, B. Le Bizec, J. Chrom A,

1270, 51-56, 2012.

High efficiency of semi-preparative Supercritical Fluid Chromatography with Molecularly

Imprinted Polymer as stationary phase (SFC-MIP). Application on urinary steroids purification for

IRMS analysis, M. Doué, E. Bichon, F. Monteau, B. Le Bizec (LABERCA), poster 2nd International

Symposium on HTSP 2012.

New technological tools for isolating and measuring growth promoting agents in edible tissues

and biological fluids, E. Bichon, S. Rochereau, L. Seree, S. Prevost, F. Monteau, B. Le Bizec (LABERCA)

Poster session RAFA 2011.

Utilisation de la spectrométrie de masse pour le dosage du Bisphénol A dans les matrices

alimentaires, Y. DECEUNINCK, Z. ZENDONG, E. BICHON, J.-P. ANTIGNAC, B. LE BIZEC (LABERCA)

poster SMAP 2011, Avignon, France, 19-22 sept. 2011.

Quantitative analysis of Bisphenol A in all liquid or solid food matrices were carried out by using

AFFINIMIP® SPE Bisphenol A (p132, Annex 12 of Annexes to the report on the assessment of the risks

associated with bisphenol A (BPA) for human health, and on toxicological data and data on the use

of bisphenols S, F, M, B, AP, AF, and BADGE (In French)), ANSES April 2013.

A high selective and sensitive liquid chromatography–tandem mass spectrometry method for

quantization of BPA urinary levels in children, C. Nicolucci, S. Rossi, C. Menale, E. Giudice, P. Miraglia

del Giudice, L. Perrone, P. Gallo, D. Mita, N. Diano, Analytical and Bioanalytical Chemistry, 1618-

2642, 2013.

Molecularly imprinted polymers-liquid chromatography/fluorescence for the selective clean-up

of hydroxylated polycyclic aromatic hydrocarbons in soils, O. Baltrons, M. Lopez-Mesas, C. Palet,

F. Lederf and F. Portet-Koltalo, Anal. Methods, 2013,5, 6297-6305

Fast and Selective Purification of a Fluorous Radiotracer using AFFINIMIP® SPE 18F based on

Molecularly Imprinted Polymers SPE, C. Pérollier, F. Alix, S. Bayoudh, K. Naraghi, Poster 19th ISRS

2011.

Rapid Purification of Fluorine-18 Containing Synthons Using Molecularly Imprinted Polymer

Cartridges, G. Smith, S. Bayoudh, C. Pérollier, R. Bhalla, International Symposium on

Radiopharmaceutical Sciences (ISRS), Jeju, Korea, 12-17 May 2013. Works carried out in collaboration

with GE Healthcare.

Selective solid phase extraction of catecholamines and metanephrines from serum using a new

molecularly imprinted polymer, B. Claude, P. Morin and L. Denoroy, J. Liquid Chromatography &

Related Technologies, 2014, 37 (18), 2624-2638.

18F-Anti-MMR-nanobodies for PET imaging of tumor-associated macrophage subtypes as

surrogate markers for tumor hypoxia. A. Blykers, C. Xavier, I. Vaneycken, D. Laoui, N. Devoogdt, M.

D’huyvetter, T. Lahoutte, J. A. Van Ginderachter and V. Caveliers, poster at European Molecular

Imaging Meeting in Turin (2013).

A developmental hepatotoxicity study of dietary bisphenol A in Sparus aurata juveniles, F.

Maradonna, V. Nozzi, L. Dalla Valle, I. Traversi, G. Gioacchini, F. Benato, E. Colletti, P. Gallo, I. Di

Marco Pisciottano, D. G. Mita, G. Hardiman, A. Mandich, O. Carnevali, Comparative Biochemistry and

Physiology Part C: Toxicology & Pharmacology, 2014, 166, 1–13.

LIST OF PUBLICATIONS AND POSTERS

67

Documents

APPLICATION NOTEBOOK FOR AFFINIMIP®SPE - Life …v~affinimip... · APPLICATION NOTEBOOK FOR AFFINIMIP®SPE Q3 2014 Selective Solid Phase Extraction Molecularly Imprinted Polymers