aflatoxin & ochratoxin analysis a completely automated solution … freestyle... · 2013. 7....
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Visit us at www.LCTech.de!
Aflatoxin & Ochratoxin Analysis
A Completely Automated Solution with the Freestyle, ThermELUTE, and SMART
Immunoaffinity Cleanup Columns
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Background
• Pickering Labs primarily makes HPLC Post-column instruments and reagents for a variety of post-column applications.
• LC Tech has been the European distributor for Pickering Labs for about 20 years.
• Recently Pickering Labs has been distributing LC Tech products in North America along with help from Chromatographic Specialties in Canada.
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What LCTech does
supplies innovative products and methods for the preparation and analysis of food, feed and environmental samples.
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Introduction
The goal: totally automated system for Aflatoxins (B1, B2, G1, G2) and/or Ochratoxin A
HPLC
From raw extract to chromatogram
=> in 15 minutes
=> without manual steps
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Introduction
The Components
UVETMUV derivatiser(only for Aflatoxins)
ChromatogramFREESTYLETM with ThermELUTE and SPE modules
Raw extract
quaternary or isocratic pump
sampler
columnoven
detector
HPLC
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Instrumentation
Sample preparation Analysisand
FREESTYLE Basic
SPE module
ThermELUTE module
Racks and vials
AflaCLEAN and/or OtaCLEAN
SMART Immunoaffinity Cleanup
Columns
Any HPLC Pump(s)
Autosampler
Column oven
Derivatizer (e. g. UVETM)
Fluorescence detector
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Sample preparation with large volume injection
FREESTYLE Basic Automated and unattended handling of the immunoaffinity column:- loading of a precise volume - washing- drying in case of AflaCLEAN SMART- column transfer into the ThermELUTE module
ThermELUTE module heating = breaking the antigen-antibody bond- elution of the analytes with water and - large volume injection into HPLC sample loop
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Analysis Instrumentation
Any HPLC possibletested with DIONEX Ultimate 3000
Pump Isocratic mode
Autosampler Only needed for standard injection
Column switching valve
If Aflatoxins and Ochratoxin A should be analyzeda switching valve is recommended
Column Oven For two columns (Afla & Ochra)
Column(s) Standard guard and analytical columns
Derivatizer UV derivatizer, photochemical derivatization
Fluorescence detector Standard
Software Standard
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Dionex Ultimate 3000 details
Pump LPG 3400a
Sampler WPS 3000 TSL
Mobile Phase Methanol/Water/Acetonitrile, 30/60/15
Flow rate 1.2 mL/min
Column Oven 36 °C
Column LCTech HPLC column (LCTech P/N 10522)
Flourescence detector RF 2000, FLD
Fluorescence wavelength 365 nm excitation460 nm emission
Derivatizer LCTech UVE
ThermELUTE – chromatograms
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The goal: Completely automated systemfor Aflatoxin and/or Ochratoxin A analysis
Step 1:
Replace 1 mL or 3 mL immunoaffinity columns with SMART Immunoaffinity columns
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What is so smart about SMART columnns?
SMART columns - advantages
Original size: 25 mm
They are completely comparableto 1 mL or 3 mL immunoaffinitycolumns
But,-less time is needed-less sample is needed-less solvent is needed-Less waste
And -toxin concentration is equivalent to 1 mL or 3 mL format
SMA
RT
Col
umns
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What is so smart about SMART columnns?
They meet AOAC requirements
SMART columns - advantages
Loading capacity100 ng B1
Loading capacity100 ng Ochratoxin A
Recovery > 80 % Recovery > 80 %
SMA
RT
Col
umns
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What is so smart about SMART columnns?
Reduced Cleanup time
SMART columns - advantages
Cleanup process down to less than 9 minutes
Cleanup process down to less than 13 minutes
Incredible time reduction
- less sample volume = less time for sample loading
- high flow rate
SMA
RT
Col
umns
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What is so smart about SMART columnns?
They are applicable for all regulated matrices
SMART columns - advantages
All kinds of nuts:Hazelnut, walnut, brazil nut, peanut, etc. pistachios, almonds, raisins, figsAll kinds of cereals, corn, riceAll common spicesetc.
All kinds of coffeeRed wineBeerRaisinsLiquoriceetc.
SMA
RT
Col
umns
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The goal: Completely automated system for Aflatoxin and/or Ochratoxin A analysis
Step 1:
Replace 1 mL or 3 mL immunoaffinity columns by SMART Immunoaffinity columns
Step 2:
High end Completely automated solution with FREESTYLE and ThermELUTE module
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Completely automated solution
ThermELUTE module
Rack for SMART columnsAny HPLC
SMART columns
SPE Gripper
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Completely automated solution
Width:110 mm
ThermELUTE , standard version (p/n 13572)
Heating element
Injection valve
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Rack (p/n 13497) for 60 SMART columns
Rack (p/n 13497) in tray (p/n 11915) for SMART columns
Completely automated solution
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ThermELUTE with Standards
Injection valve
Injection port
Extraction and directinjection system
Cooled rack for standards
SMART Column Gripper
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ThermELUTE module
Rack for up to 60 SMART columns
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SMART Column Gripper
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How the system operates:
SMART Column gripper is taken up by SPE tool
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How the system operates:
gripper takes up the SMART column
Used columns
Unused columns
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How the system operates:
The SMART column is now tightly connected to the system and can be manipulated by positive pressure control
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How the system operates:
After handling steps (loading, washing, drying…) column is transfered into heating element
Heating element (hidden)
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How the system operates:
Column is transfered into heating element
Heating element (hidden)
The antigen-antibody bond is broken by the heat and the toxins are eluted with water. No organic solvent is required.
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SMART columns - advantages
Original size: 25 mm
Normally you elute with organic solvent; either methanol or acetonitrile. This destroys the antigen-antibody bond and elutes the toxins. The disadvantage is that you cannot inject high volumes of methanol onto the Reverse Phase column. You usually have to evaporate your cleaned sample and re-suspend in a small volume prior to injection onto the Reverse Phase column.
When you are eluting with water you are injecting all the toxin that is bound to the SMART column (~700ul) directly onto the Reverse Phase column.
SMA
RT
Col
umns
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How the system operates:
Column is now in the heating element
The toxins are eluted into the injection valve after thermal antigen-antibody bond cleavage.
Sample was eluted as a partial loop fill with 400 µL into 700 µL sample loop
Sample loop
Injection valve
Injection port
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Elution followed by whole sample large volume injection
Advantages
No loss of analytes. No storage between cleanup and analysis
Large volume injection in water / all analytes are injectedstraight from the column
- No evaporation
- No adsorption by the glass wall of storage vials
- No loss of recovery = highest sentivity
Less sample required to stay within FLD range.- Less matrix on the immunoaffinity column
- Faster sample clean-up
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Elution followed by whole sample large volume injection
Advantages
Suitable for very strict detection limits for baby food
Maximum level accordingto EU Regulation 1881/2006 *
Detection limit with FREESTYLE + ThermELUTE
Aflatoxin B1: 100 ppt Aflatoxin B1: 5 ppt
Ochratoxin A: 500 ppt Ochratoxin A: 30 ppt
* COMMISSION REGULATION (EC) No 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs
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Elution followed by whole sample large volume injection
Advantages
Single-use AflaCLEAN and OtaCLEAN SMART Cleanup columns
- 100% initial performance of SMART columns for all samples
- No cross contamination
= Perfect analytical conditions
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Increased sample throughput by parallelization of sample preparation and analysis
Completely automated solution
FREESTYLE sample preparation time: 15 min* / sample
1st sample
1st sample
Sample preparation with FREESTYLETM
Analysis with HPLC
2nd sample
2nd sample 3rd sample
HPLC analysis time: 15 min* / sample
4rth … etc
3rd … etc
* Ochratoxin A; Aflatoxin even faster
Automated sample injection
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Throughput at its best
Completely automated solution - advantages
15 min / sample
4 samples per hour
96 samples per day
Monday Thuesday
Monday – Sunday
Day and night
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Loading capacity:
90 samples (1 SMART column / sample & samples presented in 16 mL vials)
Quantity of samples can be increased by reduction of sample volume and by choosing smaller vials.
Completely automated solution - advantages
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Main advantages
Completely automated
No cross contamination
Higher sensitivity (15 – 20x)
Faster analysis
Meets AOAC requirements
Shown in the chromatograms on the following slides
ThermELUTE – chromatograms
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Higher sensitivity compared to manual cleanup processing
Excellent chromatograms (less sample needed)
ThermELUTE – chromatograms
Ginger (10 ppb) with ThermELUTE, 2.8 mL sample
Ginger (10 ppb), manual processing, 14 mL sample
Ginger
0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,010,0
0,0
10,0
20,0
30,0
40,0
50,0
60,0
1 - 130206-FW- SOFTWARE 5.2 U SYSTEMWASCH Emissiingwer halbe einwaag Emissi
mV
min
2
1
EM:460 nm
0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0-50
100
200
300
400
ingwer blind 2,8ml halbe einwaage Emissioningwer 10ppb 2,8ml halbe einwaage Emission
mV
min
21
EM:460 nm
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Higher sensitivity compared to manual cleanup processing
Excellent chromatograms (less sample needed)
ThermELUTE – chromatograms
Paprika (10 ppb) with ThermELUTE, 2.8 mL sample
Paprika (10 ppb), manual processing, 14 mL sample
Spices: Paprika
0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0-20
25
50
75
100
125
160
1 - 130206-FW- SOFTWARE 5.2 U SYSTEMWASCH Emissipaprika halbe einwaag Emissi
mV
min
21
EM:460 nm
0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0-50
0
50
100
150
200
250
300
paprika blind 2,8ml halbe einwaage Emissionpaprika 10ppb 2,8ml halbe einwaage Emission
mV
min
21
EM:460 nm
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Higher sensitivity compared to manual cleanup processing
Excellent chromatograms (less sample needed)
ThermELUTE – chromatograms
Nutmeg (10 ppb) with ThermELUTE, 2.8 mL sample
Nutmeg (10 ppb), manual processing, 14 mL sample
Spices: Nutmeg
0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0-20
50
100
150
200
muskatnuss halbe einwaa Emissimuskatnuss halbe einwaa Emissi
mV
min
21
EM:460 nm
0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0-50
0
50
100
150
200
250
300
muskatnuss blind 2,8ml halbe einwaageEmissionmuskatnuss 10ppb 2,8ml halbe einwaageEmission
mV
min
21
EM:460 nm
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Higher sensitivity compared to manual cleanup processing
Excellent chromatograms (less sample needed)
ThermELUTE – chromatograms
Coriander (10 ppb) with ThermELUTE, 2.8 mL sample
Coriander (10 ppb), manual processing, 14 mL sample
Spices: Coriander
0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0-10
20
40
60
80
110
1 - 130206-FW- SOFTWARE 5.2 U SYSTEMWASCH Emissikoriander halbe einwaag Emissi
mV
min
21
EM:460 nm
0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0-50
0
50
100
150
200
250
300
koriander blind 2,8ml halbe einwaageEmissionkoriander 10ppb 2,8ml halbe einwaageEmission
mV
min
21
EM:460 nm
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Even low levels are detected. Not detected with manual processing
ThermELUTE – chromatograms
Hazelnut, not spiked
Black:ThermELUTE detects even lowest level
Red:Not detected with manual processing
Nuts: Hazelnut
0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0-50
0
50
100
150
200
250
300
Haselnuss 10ml blind Emission2 - 130205-FW-THERMELUTE AFLACLEAN PISTAZIE_HASELNUSS_ROSINE_ERDNUSS #50mV
min
21
EM:460 nm
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Even low levels are detected. Not detected with manual processing
ThermELUTE – chromatograms
Peanut, not spiked
Black:ThermELUTE detects even lowest level
Red:Not detected with manual processing
Nuts: Peanut
0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0-50
0
50
100
150
200
250
300
Erdnuss 10ml blind EmissionErdnuss blind manuell 3ml Emission
mV
min
21
EM:460 nm
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Less sample needed, thus faster processing
ThermELUTE – chromatograms
Corn, 10 ppb
Black:ThermELUTE, 2.5 mL
Red:ThermELUTE, 1 mL
Orange:Manual processing, 3 mL
Cereals: Corn
0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0-100
125
250
375
500
700
Probe 4 10ppb 2,5ml EmissionProbe 5 10ppb 1ml Emission
10ppb Probe mais manuell 3ml Säule EmissionmV
min
321
EM:460 nm
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Less sample needed, thus faster processing
ThermELUTE – chromatograms
Hazelnut, 10 ppb
Black:ThermELUTE, 2.5 mL
Red:ThermELUTE, 1 mL
Orange:Manual processing, 3 mL
Nuts: Hazelnut
0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0-100
125
250
375
500
700
Haselnuss 2,5ml 10ppb EmissionHaselnuss 1ml 10ppb Emission
Haselnuss 10ppb manuell 3ml EmissionmV
min
321
EM:460 nm
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Less sample needed, thus faster processing
ThermELUTE – chromatograms
Raisins 10 ppb
Black:ThermELUTE, 2.5 mL
Red:ThermELUTE, 1 mL
Orange:Manual processing, 3 mL
Raisins
0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0-100
125
250
375
500
700
Rosinen 2,5ml 10ppb EmissionRosinen 1ml 10ppb Emission
Rosinen 10ppb manuell 3ml EmissionmV
min
321
EM:460 nm
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Less sample needed, thus faster processing
ThermELUTE – chromatograms
Pistachio, 10 ppb
Black:ThermELUTE, 2.5 mL
Red:ThermELUTE, 1 mL
Orange:Manual processing, 3 mL
Nuts: Pistachio
0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0-100
125
250
375
500
700
Pistazien 2,5ml 10ppb EmissionPistazien 1ml 10ppb Emission
Pistazien 10ppb manuell 3ml EmissionmV
min
321
EM:460 nm
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Less sample needed, thus faster processing
ThermELUTE – chromatograms
Peanut, 10 ppb
Black:ThermELUTE, 2.5 mL
Red:ThermELUTE, 1 mL
Orange:Manual processing, 3 mL
Nuts: Peanut
0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0-100
200
400
600
800
Erdnuss 2,5ml 10ppb EmissionERdnuss 1ml 10ppb Emission
Erdnuss 10ppb manuell 3ml EmissionmV
min
321
EM:460 nm
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Chromatogram of Peanuts Spiked with 10ppb total aflatoxin (G2/G1/B2/B1) (1ng G2; 4ng G1; 1ng B2; 4 ng B1) extracted according to LCTech extraction and clean-up procedure, 10 mL processed using AflaCLEAN SMART and ThermELUTE.
Chromatogram represents 0.28 grammatrix equivalents.
Clean-up time consists of
10 mL sample loading (3mL/min)
2 mL washing (3mL/min)
6 min ThermELUTE treatment
Ready to inject within 10 min.
Methods - Aflatoxin
Nuts: Peanut
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Chromatogram of Peanuts Spiked with 10ppb total aflatoxin (G2/G1/B2/B1) (1ng G2; 4ng G1; 1ng B2; 4 ng B1) extracted according to LCTech extraction and clean-up procedure, 2.5 mL processed usingAflaCLEAN SMART and ThermELUTE.
Chromatogram represents 0.07 grammatrix equivalents.
Clean-up time consists of
2.5 mL sample loading (3 mL/min)
2 mL washing (3mL/min)
6 min ThermELUTE treatment
Ready to inject within 7.5 min.
Methods - Aflatoxin
Nuts: Peanut
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Spiked with 10ppb total aflatoxin (G2/G1/B2/B1 (1ng G2; 4ng G1; 1ng B2; 4 ng B1) extracted according to LCTech extraction and clean-up procedure, 10 mL processed usingAflaCLEAN SMART and ThermELUTE.
Chromatogram represents 0.28 grammatrix equivalents.
Clean-up time consists of
10 mL sample loading (3mL/min)
2 mL washing (3mL/min)
6 min ThermELUTE treatment
Ready to inject within 10 min.
Methods - Aflatoxin
Chromatogram of HazelnutsNuts: Hazelnut
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Spiked with 10ppb total aflatoxin (G2/G1/B2/B1) (1ng G2; 4ng G1; 1ng B2; 4 ng B1) extracted according to LCTech extraction and clean-up procedure, 2.5 mL processed usingAflaCLEAN SMART andThermELUTE.
Chromatogram represents 0.07 grammatrix equivalents.
Clean-up time consists of
2.5 mL sample loading (3 mL/min)
2 mL washing (3mL/min)
6 min ThermELUTE treatment
Ready to inject within 7.5 min.
Methods - Aflatoxin
Chromatogram of HazelnutsNuts: Hazelnut
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10 mL instant coffee sample after extraction using methylene chloride partitioning, spiked at the regulated level of 10 ppb.
The chromatogram represents 0.01��� gram matrix equivalents.
By using PICKERING post-column derivatization sensitivity could be increased by a factor of approx. 4 and chromatography (injection peak/matrix interference could be dramatically reduced).
Ochratoxin
Chromatogram of Instant CoffeeCoffee
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10 mL roasted coffee sample after extraction using methylene chloride partitioning, spiked at the regulated level of 5 ppb.
The chromatogram represents 0.05 gram matrix equivalents.
Methods - Ochratoxin
Chromatogram of Roasted CoffeeCoffee
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10 mL White Wine sample extracted according to LCTech OtaCLEAN SMART extraction and clean-up procedure loaded onto OtaCLEAN SMART column, spiked at the regulated level of 2 ppb.
The chromatogram represents 1 gram matrix equivalents.
Methods - Ochratoxin
Chromatogram of White WineWine
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10 mL Beer sample extracted according to LCTech OtaCLEANSMART extraction and clean-up procedure loaded onto OtaCLEANSMART column, spiked with 1 ppb
The chromatogram represents 1.42 gram matrix equivalents.
Methods - Ochratoxin
Chromatogram of BeerBeer
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SPE Module
EVAporation Module
GPC Module
ThermELUTE Module
Completely automated solution - further features
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Visit us at www.LCTech.de!
www.freestyle-robotic.comwww.chromspec.com
sales@chromspec.comdavid.mazawa@pickeringlabs.com
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