Freestyle Thermelute

Aflatoxin & Ochratoxin Analysis
A Completely Automated Solution with the

Freestyle, ThermELUTE, and SMART
Immunoaffinity Cleanup Columns
Visit us at www.LCTech.de!
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.
What LCTech does
supplies innovative products

and methods for the
preparation and analysis of
food, feed and environmental
samples.
Introduction
The goal: totally automated system
for Aflatoxins (B1, B2, G1, G2) and/or Ochratoxin A
From raw extract to chromatogram
=> in 15 minutes
=> without manual steps
HPLC
Introduction
The Components
quaternary or
isocratic pump
sampler
column
oven
detector
HPLC
UVETM
UV derivatiser
(only for Aflatoxins)
FREESTYLETM with ThermELUTE

and SPE modules
Raw extract
Chromatogram
Instrumentation
Sample preparation
and
Analysis
Any HPLC
FREESTYLE Basic
Pump(s)
SPE module
Autosampler
ThermELUTE module
Column oven
Racks and vials
Derivatizer (e. g. UVETM)
AflaCLEAN and/or OtaCLEAN
Fluorescence detector
SMART Immunoaffinity Cleanup

Columns
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
Analysis Instrumentation
Any HPLC possible
tested with DIONEX Ultimate 3000
Pump
Isocratic mode
Autosampler
Only needed for standard injection
Column switching
valve
If Aflatoxins and Ochratoxin A should be analyzed

a 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
ThermELUTE chromatograms
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 excitation
460 nm emission
Derivatizer
LCTech UVE
The goal:
Completely automated system
for Aflatoxin and/or Ochratoxin A analysis
Step 1:
Replace 1 mL or 3 mL immunoaffinity columns
with SMART Immunoaffinity columns
SMART Columns
SMART columns - advantages

What is so smart about SMART columnns?
Original size:
25 mm
They are completely comparable

to 1 mL or 3 mL immunoaffinity
columns
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
SMART Columns

They meet AOAC requirements
Loading capacity
100 ng B1
Loading capacity
100 ng Ochratoxin A
Recovery > 80 %
Recovery > 80 %
SMART Columns

Reduced Cleanup time
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
SMART Columns

They are applicable for all regulated matrices
All kinds of nuts:
All kinds of coffee
Hazelnut, walnut, brazil nut,

peanut, etc.
Red wine
pistachios, almonds, raisins, figs
Raisins
All kinds of cereals, corn, rice
Liquorice
All common spices
etc.
etc.
Beer
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
Completely automated solution

SPE Gripper
SMART
columns
ThermELUTE
module
Rack for SMART columns

Any HPLC

ThermELUTE , standard version (p/n 13572)
Heating element
Injection valve
Width:110 mm
Rack (p/n 13497)

for 60 SMART columns
Rack (p/n 13497) in tray (p/n 11915)

for SMART columns
ThermELUTE with Standards
Cooled rack for standards
Extraction and direct

injection system
Injection port
Injection valve
SMART Column
Gripper
Rack for up to 60 SMART columns
ThermELUTE module
SMART Column Gripper
How the system

operates:
SMART Column
gripper is taken up
by SPE tool
How the system

operates:
gripper takes up the

SMART column
Unused columns
Used columns
How the system

operates:
The SMART column

is now tightly
connected
to the system and
can be manipulated
by positive pressure
control
How the system

operates:
After handling steps

(loading, washing,
drying) column is
transfered into heating
element
Heating element (hidden)
How the system

operates:
Column is transfered
into heating element
The antigen-antibody
bond is broken by the
heat and the toxins are
eluted with water. No
organic solvent is
required.
Heating element (hidden)
SMART Columns
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.
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 L
into 700 L sample loop
Injection port
Injection valve
Sample loop
Advantages
Elution followed by whole sample large volume injection
No loss of analytes. No storage between cleanup and

analysis
Large volume injection in water / all analytes are injected

straight 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
Advantages
Elution followed by whole sample large
volume injection
Suitable for very strict detection limits
for baby food
Maximum level according Detection limit with
to EU Regulation
FREESTYLE +
1881/2006 *
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
Advantages
Elution followed by whole sample large volume injection
Single-use AflaCLEAN and OtaCLEAN SMART Cleanup

columns
- 100% initial performance of SMART columns for all samples
- No cross contamination
= Perfect analytical conditions

Increased sample throughput by parallelization of
sample preparation and analysis
FREESTYLE sample preparation time: 15 min* / sample
Sample preparation
with FREESTYLETM
Analysis
with HPLC
Automated
sample injection
1st sample
2nd sample
3rd sample
4rth etc
1st sample
2nd sample
3rd etc
HPLC analysis time: 15 min* / sample

* Ochratoxin A; Aflatoxin even faster
Completely automated solution - advantages
Throughput at its best

15 min / sample
Monday
Thuesday
4 samples per hour

96 samples per day
Monday Sunday
Day and night
Completely automated solution - advantages
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.
Main advantages
Completely automated
No cross contamination
Higher sensitivity (15 20x)
Faster analysis
Meets AOAC requirements
Shown in the chromatograms on the following slides
Ginger
Higher sensitivity compared to manual cleanup processing

Excellent chromatograms (less sample needed)
Ginger (10 ppb) with
ThermELUTE, 2.8 mL sample
400
ingwer blind 2,8ml halbe einwaage Emission

ingwer 10ppb 2,8ml halbe einwaage Emission
EM:460 nm
mV
Ginger (10 ppb), manual

processing, 14 mL sample
1 - 130206-FW - SOFTW ARE 5.2 U SYSTEMW ASCH Emissi
ingwer halbe einwaag
Emissi
60,0
mV
EM:460 nm
50,0
300
40,0
200
30,0
20,0
100
10,0
1
0,0 2
2
1
-50
0,0
min
2,0
4,0
6,0
8,0
10,0
12,0
15,0
10,0
0,0
min
2,0
4,0
6,0
8,0
10,0
12,0
15,0
Spices: Paprika

Paprika (10 ppb) with
300
paprika blind 2,8ml halbe einwaage Emission

paprika 10ppb 2,8ml halbe einwaage Emission
EM:460 nm
mV
Paprika (10 ppb), manual

1 - 130206-FW - SOFTW ARE 5.2 U SYSTEMW ASCH
Emissi
paprika
halbe
einwaag
Emissi
160
mV
EM:460 nm
250
125
200
100
150
75
100
50
50
25
0 21
2
1
-50
0,0
min
2,0
4,0
6,0
8,0
10,0
12,0
15,0
-20
0,0
min
2,0
4,0
6,0
8,0
10,0
12,0
15,0
Spices: Nutmeg

Nutmeg (10 ppb) with
300
muskatnuss blind 2,8ml halbe einwaageEmission

muskatnuss 10ppb 2,8ml halbe einwaage
Emission
EM:460 nm
mV
Nutmeg (10 ppb), manual

200
muskatnuss halbe einwaa

muskatnuss halbe einwaa
mV
Emissi
Emissi
EM:460 nm
250
150
200
150
100
100
50
50
0 21
1
2
-50
0,0
min
2,0
4,0
6,0
8,0
10,0
12,0
15,0
-20
0,0
min
2,0
4,0
6,0
8,0
10,0
12,0
15,0
Spices: Coriander

Coriander (10 ppb) with
300
koriander blind 2,8ml halbe einwaageEmission

koriander 10ppb 2,8ml halbe einwaageEmission
EM:460 nm
mV
Coriander (10 ppb), manual

110
1 - 130206-FW - SOFTW ARE 5.2 U SYSTEMW ASCH

Emissi
koriander halbe einwaag
Emissi
mV
EM:460 nm
250
80
200
150
60
100
40
50
20
1
2
1
2
-50
0,0
min
2,0
4,0
6,0
8,0
10,0
12,0
15,0
-10
0,0
min
2,0
4,0
6,0
8,0
10,0
12,0
15,0
Nuts: Hazelnut
Even low levels are detected. Not detected with manual

processing
Haselnuss 10ml blind
Emission
2 - 130205-FW-THERMELUTE AFLACLEAN PISTAZIE_HASELNUSS_ROSINE_ERDNUSS #50
300
EM:460 nm
mV
250
Hazelnut, not spiked
200
Black:
ThermELUTE detects even
lowest level
150
100
Red:
Not detected with manual
processing
50
0 2
1
-50
0,0
min
2,0
4,0
6,0
8,0
10,0
12,0
15,0
Nuts: Peanut
Even low levels are detected. Not detected with manual

processing
300
Erdnuss 10ml blind

Erdnuss blind manuell 3ml
Emission
Emission
EM:460 nm
mV
250
Peanut, not spiked
200
Black:
ThermELUTE detects even
lowest level
150
100
Red:
Not detected with manual
processing
50
0 1 2
-50
0,0
min
2,0
4,0
6,0
8,0
10,0
12,0
15,0
Less sample needed, thus faster processing
700
Probe 4 10ppb 2,5ml

Emission
Probe 5 10ppb 1ml
Emission
10ppb Probe mais manuell 3ml Sule Emission
EM:460 nm
mV
Cereals: Corn
Corn, 10 ppb
Black:
ThermELUTE, 2.5 mL
500
Red:
ThermELUTE, 1 mL
375
250
Orange:
Manual processing, 3 mL
125
2
1 3
-100
0,0
min
2,0
4,0
6,0
8,0
10,0
12,0
15,0
700
Haselnuss 2,5ml 10ppb

Haselnuss 1ml 10ppb
Haselnuss 10ppb manuell 3ml
mV
Nuts: Hazelnut
Emission
Emission
Emission
EM:460 nm
Hazelnut, 10 ppb
Black:
ThermELUTE, 2.5 mL
500
375
Red:
ThermELUTE, 1 mL
250
Orange:
125
1 3
2
-100
0,0
min
2,0
4,0
6,0
8,0
10,0
12,0
15,0
700
Rosinen 2,5ml 10ppb

Rosinen 1ml 10ppb
Rosinen 10ppb manuell 3ml
mV
Raisins
Emission
Emission
Emission
EM:460 nm
Raisins 10 ppb
Black:
ThermELUTE, 2.5 mL
500
375
Red:
ThermELUTE, 1 mL
250
Orange:
125
2 3
1
-100
0,0
min
2,0
4,0
6,0
8,0
10,0
12,0
15,0
700
Pistazien 2,5ml 10ppb

Pistazien 1ml 10ppb
Pistazien 10ppb manuell 3ml
mV
Nuts: Pistachio
Emission
Emission
Emission
EM:460 nm
Pistachio, 10 ppb
Black:
ThermELUTE, 2.5 mL
500
375
Red:
ThermELUTE, 1 mL
250
Orange:
125
1
2
-100
0,0
min
2,0
4,0
6,0
8,0
10,0
12,0
15,0
800
Erdnuss 2,5ml 10ppb

ERdnuss 1ml 10ppb
Erdnuss 10ppb manuell 3ml
mV
Nuts: Peanut
Emission
Emission
Emission
EM:460 nm
Peanut, 10 ppb
Black:
ThermELUTE, 2.5 mL
600
Red:
ThermELUTE, 1 mL
400
Orange:
200
2 3
1
-100
0,0
min
2,0
4,0
6,0
8,0
10,0
12,0
15,0
Methods - Aflatoxin
Nuts: Peanut
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 gram

matrix 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
Chromatogram of Peanuts

(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 using
AflaCLEAN SMART and
ThermELUTE.

matrix equivalents.
2.5 mL sample loading (3 mL/min)
Ready to inject within 7.5 min.
Methods - Aflatoxin
Nuts: Hazelnut
Chromatogram of Hazelnuts

(G2/G1/B2/B1 (1ng G2; 4ng G1;
procedure, 10 mL processed using
AflaCLEAN SMART and
ThermELUTE.

matrix equivalents.
10 mL sample loading (3mL/min)
Ready to inject within 10 min.
Methods - Aflatoxin
Nuts: Hazelnut
Chromatogram of Hazelnuts

(G2/G1/B2/B1) (1ng G2; 4ng G1;
procedure, 2.5 mL processed using
AflaCLEAN SMART and
ThermELUTE.

matrix equivalents.
2.5 mL sample loading (3 mL/min)
Ready to inject within 7.5 min.
Ochratoxin
Coffee
Chromatogram of Instant Coffee
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).
Methods - Ochratoxin
Coffee
Chromatogram of Roasted Coffee
10 mL roasted coffee sample after

extraction using methylene chloride
partitioning, spiked at the regulated
level of 5 ppb.
The chromatogram represents 0.05

Wine
Chromatogram of White Wine
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.
Beer
Chromatogram of Beer
10 mL Beer sample extracted

according to LCTech OtaCLEAN
SMART extraction and clean-up
procedure loaded onto OtaCLEAN
SMART column, spiked with 1 ppb
The chromatogram represents 1.42

Completely automated solution - further features
SPE Module
GPC Module
EVAporation
Module
ThermELUTE
Module
www.freestyle-robotic.com
www.chromspec.com
sales@chromspec.com
david.mazawa@pickeringlabs.com
Visit us at www.LCTech.de!

Freestyle Thermelute

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Aflatoxin & Ochratoxin Analysis

A Completely Automated Solution with the

Pickering Labs primarily makes HPLC Post-column

LC Tech has been the European distributor for Pickering Labs

Recently Pickering Labs has been distributing LC Tech

What LCTech does

supplies innovative products

FREESTYLETM with ThermELUTE

Racks and vials

Derivatizer (e. g. UVETM)

AflaCLEAN and/or OtaCLEAN

SMART Immunoaffinity Cleanup

Sample preparation with large volume injection

Automated and unattended handling

heating = breaking the antigen-antibody bond

Only needed for standard injection

If Aflatoxins and Ochratoxin A should be analyzed

For two columns (Afla & Ochra)

Standard guard and analytical columns

UV derivatizer, photochemical derivatization

WPS 3000 TSL

LCTech HPLC column (LCTech P/N 10522)

SMART columns - advantages

They are completely comparable

SMART columns - advantages

SMART columns - advantages

Cleanup process down

Cleanup process down

Incredible time reduction

SMART columns - advantages

All kinds of nuts:

All kinds of coffee

Hazelnut, walnut, brazil nut,

pistachios, almonds, raisins, figs

All kinds of cereals, corn, rice

All common spices

Completely automated solution

Rack for SMART columns

Completely automated solution

Completely automated solution

Rack (p/n 13497)

Rack (p/n 13497) in tray (p/n 11915)

ThermELUTE with Standards

Cooled rack for standards

Extraction and direct

Rack for up to 60 SMART columns

SMART Column Gripper

How the system

How the system

gripper takes up the

How the system

The SMART column

How the system

After handling steps

Heating element (hidden)

How the system

SMART columns - advantages

Normally you elute with organic

How the system

No loss of analytes. No storage between cleanup and

Large volume injection in water / all analytes are injected

Less sample required to stay within FLD range.

Aflatoxin B1: 5 ppt

Ochratoxin A: 500 ppt