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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.
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
2 4 6 8 10 12 14 16 18 20 22 24 26
mV
olts
41
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mV
olts
41
42
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44
45
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47
48
49
50
51
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
0
10
20
30
40
50
60
70
80
90
100
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
g/g
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
9 10 11 12 13 14 15 16 17 18 19 20
mA
U
-25
-20
-15
-10
-5
0
5
10
15
20
25
30
35
40
mA
U
-25
-20
-15
-10
-5
0
5
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25
30
35
40
Minutes
9 10 11 12 13 14 15 16 17 18 19 20
mA
U
-25
-20
-15
-10
-5
0
5
10
15
Minutes
9 10 11 12 13 14 15 16 17 18 19 20
mA
U
-25
-20
-15
-10
-5
0
5
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35
40
mA
U
-25
-20
-15
-10
-5
0
5
10
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20
25
30
35
40
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.
Cleanup with a 3mL/100mg AFFINIMIP® SPE
Patulin cartridge
Equilibration
•2mL Acetonitrile
•1mL water
Loading
•4mL of loading solution
Washing of interferents (W1)
•1mL NaHCO3
•2mL Water
Drying by applying vacuum 10 seconds
Washing of interferents (W2)
•1mL Diethyl Ether
Elution (E)
•2mL Ethyl Acetate
The elution fraction was then evaporated and
dissolved in water containing 0.1% acetic acid
before HPLC analysis.
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
AFFINIMIP® SPE Patulin
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.
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.
Clean-up with a 3mL/100mg AFFINIMIP® SPE
Patulin cartridge
Equilibration
•2mL Acetonitrile
•1mL water
Loading
•4mL of loading solution
Washing of interferents (W1)
•1mL NaHCO3 in Water
•2mL Water
Drying by applying vacuum 10 seconds
Washing of interferents (W2)
•1mL Diethyl Ether
Elution (E)
•2mL Ethyl Acetate
The elution fraction was then evaporated and
dissolved in water containing 0.1% acetic acid
before HPLC analysis.
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
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
mA
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1400
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2000
mA
U
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200
400
600
800
1000
1200
1400
1600
1800
2000
UV 6000-276nm
Brut carrefour.dat
UV6000-276nm
C1 C05
Patulin
Before clean-up
Minutes
9 10 11 12 13 14 15 16 17 18 19
mA
U
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-10
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60
70
mA
U
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Minutes
9 10 11 12 13 14 15 16 17 18 19
mA
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Minutes
9 10 11 12 13 14 15 16 17 18 19
mA
U
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-10
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mA
U
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70
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)
AFFINIMIP® SPE Patulin
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
mA
U
0
20
40
60
80
100
120
140
mA
U
0
20
40
60
80
100
120
140
UV 6000-276nm
C07
UV 6000-276nm
C10
UV 6000-276nm
C15
Minutes5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Minutes5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
mA
U
0
20
40
60
80
100
120
140
mA
U
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20
40
60
80
100
22 23
mA
U
0
20
40
60
80
100
120
140
mA
U
0
20
40
60
80
100
120
140
UV 6000-276nm
C07
UV 6000-276nm
C10
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
Minutes
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
mA
U
0
20
40
60
80
100
120
mA
U
0
20
40
60
80
100
120
UV 6000-276nm
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
AFFINIMIP® SPE Patulin
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).
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
12
Time (min) % water % ACN
0 98 2
20 98 2
21 50 50
25 50 50
26 98 2
Recovery and repeatability of Patulin (n=4) at acontamination level of 10µg/kg in apple puree afterAFFINIMIP® SPE Patulin Clean-up.
Recoveries % (n=4) % RSDR
81.2 2.1
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 puree for infants and young children:Europe (EC 1881/2006) : 10µg/Kg
AFFINIMIP® SPE Patulin
DETERMINATION OF PATULIN IN APPLE – FRUIT 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
13
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 puree for infants and young children:Europe (EC 1881/2006) : 10µg/Kg
Minutes
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
mA
U
0
20
40
60
80
100
120
mA
U
0
20
40
60
80
100
120
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
12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
mA
U
-75
-50
-25
0
25
50
75
100
125
150
175
200
225
250
mA
U
-75
-50
-25
0
25
50
75
100
125
150
175
200
225
250
Minutes
12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
mA
U
-75
-50
Minutes
12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
mA
U
-75
-50
-25
0
25
50
75
100
125
150
175
200
225
250
mA
U
-75
-50
-25
0
25
50
75
100
125
150
175
200
225
250
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.
Concentration of Patulin (µg/kg)
Recoveries %
% RSDR
10 (n=9) 77.4 8.1
25 (n=8) 90.9 11.4
40 (n=6) 86.0 11.9
Patulin
AFFINIMIP® SPE 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
Equilibration•2mL Acetonitrile•1mL Water
Loading•3mL of loading solution
Washing of interferents (W1)•3mL Water-2% Acetic Acid
Drying by applying vacuum 10 secondsWashing of interferents (W2)
•250µL Diethyl EtherDrying by applying vacuum 10 secondsElution (E)
•1mL Ethyl AcetateThe elution fraction was then evaporated anddissolved in water containing 0.1% acetic acidbefore HPLC analysis.
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
Flow rate: 0.2mL/minDetection: UV - 276nmInjection volume: 100µL.
Time (min) % water % ACN
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
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
AFFINIMIP® SPE Patulin
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.
Cleanup with a 3mL/100mg AFFINIMIP® SPEPatulin cartridge
Equilibration•2mL Acetonitrile•1mL Water
Loading•4mL of loading solution
Washing of interferents (W1)•1mL NaHCO3 1% in Water•2mL 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 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
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.
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
AFFINIMIP® SPE Patulin
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.
Cleanup with a 3mL/100mg AFFINIMIP® SPEPatulin cartridge
Equilibration•2mL Acetonitrile•1mL Water
Loading•3mL of loading solution
Washing of interferents (W1)•3mL Water (containing 2% Acetic Acidfor AA W1 protocol)
Drying by applying vacuum 10 secondsWashing of interferents (W2)
•250µL Diethyl EtherDrying by applying vacuum 10 secondsElution (E)
•2mL Ethyl AcetateThe elution fraction was then evaporated anddissolved in water containing 0.1% acetic acidbefore HPLC analysis.
RESULTS
16
HPLC MethodColumn: Atlantis T3 column, 150mm x 2.1mmMobile phase: gradient profile
) Flow rate: 0.2mL/minDetection: UV - 276nmInjection volume: 100µL.
Time (min) % water % ACN
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.
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
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
AFFINIMIP® SPE Patulin
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.
Cleanup with a 3mL/100mg AFFINIMIP® SPEPatulin cartridge
Equilibration•2mL Acetonitrile•1mL Water
Loading•5mL of loading solution from tomatoketchup or 2mL from tomato powder
Washing of interferents (W1)•4mL Water-1% Acetic Acid•4mL 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
17
HPLC MethodColumn: Atlantis T3 column, 150mm x 2.1mmMobile phase: gradient profile
Flow rate: 0.2mL/minDetection: UV - 276nmInjection volume: 100µL.
Time (min) % water % ACN
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
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
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%
AFFINIMIP® SPE Patulin
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.
Cleanup with a 3mL/100mg AFFINIMIP® SPEPatulin cartridge
Equilibration•2mL Acetonitrile•1mL Water
Loading•4mL of loading solution
Washing of interferents (W1)•1mL NaHCO3 1% in Water•2mL 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 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
18
Chromatograms obtained after AFFINIMIP® SPEPatulin Clean-up of Blueberry juice spiked at 40µg/Lwith Patulin (red) or not spiked (light blue).
HPLC MethodColumn: Atlantis T3 column, 150mm x 2.1mmMobile phase: gradient profile
) Flow rate: 0.2mL/minDetection: UV - 276nmInjection volume: 100µL.
Time (min) % water % ACN
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
AFFINIMIP® SPE 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.
Cleanup with a 6mL/200mg AFFINIMIP® SPEPatulin cartridge
Equilibration•4mL Acetonitrile•4mL Water
Loading•4 to 6mL of loading solution
Washing of interferents (W1)•2mL NaHCO3 1% in Water•4mL Water
Drying by applying vacuum 30 secondsWashing of interferents (W2)
•1mL Diethyl EtherElution (E)
•2mL Ethyl AcetateThe elution fraction was then evaporated anddissolved in water containing 0.1% acetic acidbefore HPLC analysis.
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.
HPLC MethodColumn: Atlantis T3 column, 150mm x 2.1mmMobile phase: gradient profile
) Flow rate: 0.2mL/minDetection: UV - 276nmInjection volume: 100µL.
Time (min) % water % ACN
0 98 2
20 98 2
21 50 50
25 50 50
26 98 2
AFFINIMIP® SPE Patulin
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
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 6mL/200mg 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: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
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
Chromatograms of apple puree spiked with 20µg/kgof Patulin (Red) and not spiked (blue) afterAFFINIMIP® SPE Patulin Clean-up
Recovery and repeatability of Patulin (n=6) at acontamination level of 10µg/kg in apple puree afterAFFINIMIP® SPE Patulin Clean-up.
Concentration of Patulin (µg/kg)
Recoveries % % RSDr
10 (n=6) 90 9
20 (n=3) 92 11
AFFINIMIP® SPE Patulin
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.
Cleanup with a 6mL/200mg AFFINIMIP® SPEPatulin cartridge
Equilibration•4mL Acetonitrile•2mL Water
Loading•10mL of loading solution
Washing of interferents (W1)•5mL Water-2% Acetic Acid•5mL Water
Drying by applying vacuum 30 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
21
Regulations for solid apple products: Europe (EC 1881/2006) : 25µg/Kg
HPLC MethodColumn: Atlantis T3 column, 150mm x 2.1mmMobile phase: gradient profile
Flow rate: 0.2mL/minDetection: UV - 276nmInjection volume: 100µL.
Time (min) % water % ACN
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%
AFFINIMIP® SPE Patulin
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
AFFINIMIP® SPE Ochratoxin A
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
Equilibration•3mL Acetonitrile•3mL Water
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.
Cleanup with a 3mL/100mg AFFINIMIP® SPEOchratoxin A cartridge
Equilibration•3mL Acetonitrile•3mL Water
Loading•4mL of loading solution (eq. 1g sample)
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
23
Regulations for paprika: Europe (EC 594/2012) : 30µg/Kg until 31.12.14 then 15µg/Kg
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
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).
AFFINIMIP® SPE Ochratoxin A
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.
Cleanup with a 3mL/100mg AFFINIMIP® SPEOchratoxin A cartridge
Equilibration•3mL Acetonitrile•3mL Water
Loading•2 to 10mL of loading solution (eq. 1 to5mL sample)
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
24
Regulations for wine: Europe (EC 1881/2006) : 2µg/L
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.
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
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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).
AFFINIMIP® SPE Ochratoxin A
DETERMINATION OF ZEARALENONE IN MAIZE AND RICE
AFFINIMIP® SPE Zearalenone
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.
Equilibration•3mL Acetonitrile•3mL Water
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
The elution fraction was then evaporated anddissolved in water before HPLC analysis.
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
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.
Equilibration•3mL Acetonitrile•3mL Water
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
The elution fraction was then evaporated anddissolved in water before HPLC analysis.
RESULTS
Catalog number:3mL-100mg sorbentPI-FS100-02 for 25 cartridgesPI-FS100-03 for 50 cartridges
26
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.
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
AFFINIMIP® SPE Zearalenone
ZONZON
ZON
DETERMINATION OF ZEARALENONE IN EDIBLE CORN OIL
RESULTS
Catalog number:3mL-100mg sorbentPI-FS100-02 for 25 cartridgesPI-FS100-03 for 50 cartridges
27
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.
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
PROTOCOL OF CLEANUPSample preparation
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
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Zearalenone
Chromatograms of Corn Oil spiked withZearalenone at 400µg/L (blue) or not spiked(orange) obtained after cleanup by AFFINIMIP®SPEZearalenone.
AFFINIMIP® SPE Zearalenone
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.
Equilibration•2mL Acetonitrile•2mL Water
Loading•6mL of loading solution
Washing of interferents (W1)•3mL NaHCO3 1% in water
Drying 30 secondsWashing of interferents (W2)
•1mL DiethyletherElution (E)
•4mL Ethyl AcetateThe elution fraction was then evaporated anddissolved in water -0,1% HCOOH before HPLCanalysis.
AFFINIMIP® SPE Deoxynivalenol
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
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
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.
Cleanup with a 6mL/100mg AFFINIMIP® SPEDeoxynivalenol cartridge.
Equilibration•2mL Acetonitrile•2mL Water
Loading•6mL of loading solution
Washing of interferents (W1)•3mL NaHCO3 1% in water
Drying 30 secondsWashing of interferents (W2)
•1mL DiethyletherElution (E)
•4mL Ethyl AcetateThe elution fraction was then evaporated anddissolved in water -0,1% HCOOH before HPLCanalysis.
MatrixC°
µg/kg
Mean
µg/kgR% %RSDr
Babyfood
(n=3)150 136.5 91 0.4
AFFINIMIP® SPE Deoxynivalenol
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
Deoxynivalenol (DON)
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.
Cleanup with a 6mL/100mg AFFINIMIP® SPEDeoxynivalenol cartridge.
Equilibration•2mL Acetonitrile•2mL Water
Loading•6mL of loading solution
Washing of interferents (W1)•3mL NaHCO3 1% in water
Drying 30 secondsWashing of interferents (W2)
•1mL DiethyletherElution (E)
•4mL Ethyl AcetateThe elution fraction was then evaporated anddissolved in water-0.1% formic acid beforeHPLC analysis.
AFFINIMIP® SPE Deoxynivalenol
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
0 5 10 15 20 25 30
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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 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.
Cleanup with a 6mL/200mg AFFINIMIP® SPEDeoxynivalenol cartridge.
Equilibration•2mL Acetonitrile•2mL Water
Loading•2mL of loading solution
Washing of interferents (W1)•3mL NaHCO3 1% in water
Drying 30 secondsWashing of interferents (W2)
•1mL DiethyletherElution (E)
•4mL Ethyl AcetateThe elution fraction was then evaporated anddissolved in water -0,1% HCOOH before HPLCanalysis.
AFFINIMIP® SPE Deoxynivalenol
Deoxynivalenol (DON)
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
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
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
AFFINIMIP® SPE FumoZON
PROTOCOL OF CLEANUPSample preparation
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.
Equilibration•2mL Acetonitrile•2mL Water
Loading•6mL of loading solution
Washing of interferents•6mL 60/40 Water/ACN
Elution (E)•2mL Methanol – 2% Acetic Acid
The elution fraction was then evaporated anddissolved in water before HPLC analysis.
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
5 10 15 20 25 30
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Inte
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6.19
26.95
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3.52
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3.52
PROTOCOL OF CLEANUPSample preparation
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.
Equilibration•2mL Acetonitrile•2mL Water
Loading•6mL of loading solution
Washing of interferents•6mL 60/40 Water/ACN
Elution (E)•2mL Methanol – 2% Acetic Acid
The elution fraction was then evaporated anddissolved in water before HPLC analysis.
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
AFFINIMIP® SPE FumoZON
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
Catalog number: PI-FS104-02 for 25 cartridges, 3mLPI-FS104-03 for 50 cartridges, 3mL
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
Catalog number: PI-FS104-02 for 25 cartridges, 3mLPI-FS104-03 for 50 cartridges, 3mL
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
PROTOCOL OF CLEANUPSample preparation
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.
Time (min) % water % ACN
0 65 35
2 65 35
12 50 50
20 50 50
20.5 65 35
35 65 35
Minutes
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
mVo
lts
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0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
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100
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400
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600
700
800
900
1000
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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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100
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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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100
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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
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
mVo
lts
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Bisphenol A
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
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 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
Equilibration•3mL Methanol -2% Acetic Acid•3mL Acetonitrile•3mL Water
LoadingUp to 40mL 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
39
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.
Time (min) % water % ACN
0 65 35
2 65 35
12 50 50
20 50 50
20.5 65 35
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
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 13,5 14,0
mV
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-100
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1200
1300
1400 Fluo
2-MD1.dat
Fluo
2-md2.dat
Fluo
4-md1.dat
Fluo
4-md2.dat
Fluo
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
AFFINIMIP® SPE Bisphenol A
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
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.
AFFINIMIP® SPE Bisphenol A
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 CANNED FOOD (Liquid form)
PROTOCOL OF CLEANUPSample preparation
Cleanup with a 3mL or 6mL/100mgAFFINIMIP® SPE Bisphenol A cartridge
Equilibration•3mL Methanol -2% Acetic Acid•3mL Acetonitrile•3mL Water
Loading10mL liquid from canned food after filterpaper filtration
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
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
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.
Time (min) % water % ACN
0 65 35
2 65 35
12 50 50
20 50 50
20.5 65 35
35 65 35
Minutes
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
mV
olts
-80
-60
-40
-20
0
20
40
60
80
100
120
140
160
180
200
220
Minutes
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
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Minutes
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-40
-20
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Minutes
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
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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).
C° (µg/L) Mean (µg/L) Recoveries % % RSDR
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
-100
-50
0
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
-100
-50
0
50
100
150
200
250
300
350
400
450
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).
C° (µg/L) Mean (µg/L) Recoveries % % RSDR
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
AFFINIMIP® SPE Bisphenol A
Minutes
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
mVo
lts
-50
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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
Cleanup with a 3mL or 6mL/100mgAFFINIMIP® SPE Bisphenol A cartridge
Equilibration•3mL Methanol -2% Acetic Acid•3mL Acetonitrile•3mL Water
Loading20mL loading solution
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
42
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.
Time (min) % water % ACN
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).
Regulations for Bisphenol A: Europe (directive 2011/8/EU) : Specific migration limit in food from packaging of 0.6mg/kg
Minutes
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
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-50
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Fluo
MD1.dat
Fluo
md2.dat
Fluo
mnd.dat
Bisphenol A
Recovery yield : 97-99%
ZOOM
Bisphenol A
AFFINIMIP® SPE Bisphenol A
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 BEER
PROTOCOL OF CLEANUPSample preparationThe beer is degassed by sonication for 1 hour.
Cleanup with a 3mL or 6mL/100mgAFFINIMIP® SPE Bisphenol A cartridge
Equilibration•3mL Methanol -2% Acetic Acid•3mL Acetonitrile•3mL Water
Loading10mL of degassed beer
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
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
43
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.
Time (min) % water % ACN
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:
- under repeatability conditions (n=3, % RSDr)
- under reproducibility conditions ( % RSDR).
Regulations for Bisphenol A: Europe (directive 2011/8/EU) : Specific migration limit in food from packaging of 0.6mg/kg
AFFINIMIP® SPE Bisphenol A
DETERMINATION OF BISPHENOL A IN RED/WHITE WINES
PROTOCOL OF CLEANUP
Cleanup with a 3mL or 6mL/100mgAFFINIMIP® SPE Bisphenol A cartridge
Equilibration•3mL Methanol -2% Acetic Acid•3mL Acetonitrile•3mL Water
LoadingUp to 10mL of wine
Washing of interferents•9mL Water•6mL Water/Acetonitrile (60/40)
Drying 1 minuteElution (E)
3mL MethanolThe elution fraction was then evaporated anddissolved in the mobile phase before HPLCanalysis.
RESULTS
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
44
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.
Time (min) % water % ACN
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
AFFINIMIP® SPE Bisphenol A
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
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MND.dat
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md1.dat
Fluo
md2.dat
Fluo
md3.dat
Bisphenol A
Regulations for Bisphenol A: Europe (directive 2011/8/EU) : Specific migration limit in food from packaging of 0.6mg/kg
DETERMINATION OF BISPHENOL A IN COLA DRINKS
PROTOCOL OF CLEAUNP
Cleanup with a 3mL or 6mL/100mgAFFINIMIP® SPE Bisphenol A cartridge
Equilibration•3mL Methanol -2% Acetic Acid•3mL Acetonitrile•3mL Water
Loading6mL of Cola drinks after 30min degassingwith ultrasounds
Washing of interferents•9mL Water•6mL Water/Acetonitrile (60/40)
Drying 3 minuteElution (E)
3mL MethanolThe elution fraction was then evaporated anddissolved in the mobile phase before HPLCanalysis.
RESULTS
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
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
AFFINIMIP® SPE Bisphenol A
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
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.
Time (min) % water % ACN
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
Cleanup with a 3mL or 6mL/100mgAFFINIMIP® SPE Bisphenol A cartridge
Equilibration•3mL Methanol -2% Formic Acid•3mL Acetonitrile•3mL Water
Loading9mL of Milk
Washing of interferents•9mL Water•6mL Water/Acetonitrile (60/40)
Drying 3 minuteElution (E)
3mL Methanol (E1)3mL Acetonitrile (E2)
The elution fractions E1 and E2 were gathered,evaporated and dissolved in the mobile phasebefore HPLC analysis.
RESULTS
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
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).
AFFINIMIP® SPE Bisphenol A
Matrice
Spiked at
10ng/mL
Mean
concentration
(µg/kg)
Recoveries
%
BPA 10.85 108.5
BADGE 7.5 75
Bisphenol A BADGE
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.
Time (min) % water % ACN
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
AFFINIMIP® SPE Bisphenol A
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
Equilibration•3mL Methanol -2% Acetic Acid•3mL Acetonitrile•3mL Water
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
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
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
Equilibration•2mL Acetonitrile•2mL Water
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)
2mL MethanolThe elution fraction was then evaporated anddissolved in the mobile phase before HPLCanalysis.
RESULTS
Catalog number:1mL-50mg sorbentPI-FS110-02A for 25 cartridgesPI-FS110-03A for 50 cartridges
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.
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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:.
- under repeatability conditions (n=3, % RSDr)
C° (µg/kg) Mean (µg/kg) Recoveries % % RSDr
16.0 15.4 96.1 3.3
- under reproducibility conditions ( % RSDR).
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
Equilibration•2mL Acetonitrile•2mL Water
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))
Drying 1 minWashing of interferents (W2)
0.25mL Diethyl etherElution (E)
2mL MethanolThe elution fraction was then evaporated anddissolved in the mobile phase before HPLCanalysis.
RESULTS
Catalog number:1mL-50mg sorbentPI-FS110-02A for 25 cartridgesPI-FS110-03A for 50 cartridges
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
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 321 (ESI-)Injection volume: 20µL.
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
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NL:1.32E5
Channel A UV UrineD1-E
NL:1.32E5
Channel A UV urined2-e
NL:1.32E5
Channel A UV UrineND-E
AFFINIMIP® SPE Chloramphenicol
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
Equilibration•2mL Acetonitrile•2mL Water
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))
Drying 1 minWashing of interferents (W2)
0.25mL Diethyl etherElution (E)
2mL MethanolThe elution fraction was then evaporated anddissolved in the mobile phase before HPLCanalysis.
RESULTS
Catalog number:1mL-50mg sorbentPI-FS110-02A for 25 cartridgesPI-FS110-03A for 50 cartridges
51
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 321 (ESI-)Injection volume: 20µL.
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
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AFFINIMIP® SPE Chloramphenicol
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
Equilibration•1mL Acetonitrile•2mL Water
Loading5mL of diluted urine
Washing of interferents (W1)•3mL Water•3mL Water/Acetonitrile (60/40)
Drying 30 secondsElution (E)
1.5mL Methanol – 2% Formic acidThe elution fraction was then evaporated anddissolved in the mobile phase before HPLCanalysis.
RESULTS
Catalog number:3mL-50mg cartridgePI-DG102-02 for 25 cartridgesPI-DG102-03 for 50 cartridges
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.
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MDA
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MDMA
MDEA
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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
Equilibration•1mL Acetonitrile•2mL Water
Loading2.5mL of diluted serum
Washing of interferents (W1)•3mL Water•3mL Water/Acetonitrile (60/40)
Drying 30 secondsElution (E)
1.5mL Methanol – 2% Formic acidThe elution fraction was then evaporated anddissolved in the mobile phase before HPLCanalysis.
RESULTS
Catalog number:3mL-50mg cartridgePI-DG102-02 for 25 cartridgesPI-DG102-03 for 50 cartridges
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
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Methamphetamine
MDA
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MDEA
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Methamphetamine
MDA
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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).
Cleanup with a 1mL/10mg AFFINIMIP® SPE
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
The elution fraction was then evaporated and
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)
Drying 10 secondsWashing of interferents (W2)
•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
Catalog number:3mL-100mg sorbentPI-FS103-02 for 25 cartridgesPI-FS103-03 for 50 cartridges
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
PROTOCOL OF CLEANUPSample preparation
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
Equilibration•3mL Acetonitrile•3mL Water
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.
Catalog number:3mL-100mg sorbentPI-FS103-02 for 25 cartridgesPI-FS103-03 for 50 cartridges
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
Cleanup with a 3mL/100mg AFFINIMIP® SPEPhenolics cartridge
Equilibration•3mL Acetonitrile•3mL Water
Loading•Up to 2mL of red or white wine
Washing of interferents•3mL Water / Acetonitrile (80/20 v/v)
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).
Catalog number:3mL-100mg sorbentPI-FS103-02 for 25 cartridgesPI-FS103-03 for 50 cartridges
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
AFFINIMIP® SPE Phenolics
DETERMINATION OF CARNOSIC ACID IN MEAT
PROTOCOL OF CLEANUPSample preparation
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.
Cleanup with a 3mL/100mg AFFINIMIP® SPEPhenolics cartridge
Equilibration•3mL Acetonitrile•3mL Water
Loading•Up to 80mL of loading solution
Washing of interferents•3mL Water / Acetonitrile (60/40 v/v)
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
Catalog number:3mL-100mg sorbentPI-FS103-02 for 25 cartridgesPI-FS103-03 for 50 cartridges
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 Phenolics
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
AFFINIMIP® SPE 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
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