www.LCTech-online.com As of: November 2019, Version: 1.3

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Fully Automated Mycotoxin Analysis

in the ppq Range FREESTYLE ThermELUTE™

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The robotic system FREESTYLE ThermELUTE™ processes your samples fully automated from the raw extract to the ready chromatogram with highest sensitivity, e.g. for Aflatoxin M1 even in the range of ppq. The system analyses up to 500 samples per week in combination with any HPLC system using the unique technology of thermal denaturation. After washing, the immunoaffinity column is heated, which causes the bond between toxin and antibody to break up. The toxins, in form of a large-volume, aqueous eluate, are directly eluted into the HPLC sample loop via partial filling and analysed by the HPLC. In the FREESTYLE ThermELUTE™ LCTech’s SMART columns are used. These immunoaffinity columns have a size of only 3 cm but their performance is comparable to the standard 1 mL and 3 mL immunoaffinity columns. The SMART columns require significant less sample through miniaturisation of the overall process, less solvent and less time and thus save costs. In the following we show some chromatograms processed using the protocol described below:

Extraction and dilution are corresponding to the protocol of the SMART columns (incl. dilution of the sample with PBS-puffer before loading onto the column)

Processing via FREESTYLE ThermELUTE™

Loading volume: 20 mL (milk / milk powder; extracted) with a flow rate of 1 mL/min. For the example shown below 10 mL milk were loaded onto the column.

Washing volume: 4 mL with a flow rate of 1 mL / min. Heating time: 8 min with 98 °C

The following chromatograms were obtained with a Dionex 3400 RS detector. Please note that the achieved results always depend on the HPLC and detector used, respectively.

Unique Automation FREESTYLE ThermELUTE™

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0.0 2.0 4.0 6.0 8.0 10.0 12.0-10,000

20,000

40,000

60,000

80,000

100,000 counts

min

M1

Chromatograms Aflatoxin M1 cleaned-up with AflaCLEAN M1 SMART

AflaCLEAN M1 SMART loaded with AflaCLEAN M1 SMART loaded with 100 pg/20 mL = represents 10 ppt 10 pg/20 mL = represents 1 ppt

AflaCLEAN M1 SMART loaded with AflaCLEAN M1 SMART loaded with 8.8 pg/20 mL = represents 0.9 ppt 7.5 pg/20 mL = represents 0.8 ppt

0.0 2.0 4.0 6.0 8.0 10.0 12.0-10,000

20,000

40,000

60,000

80,000

100,000counts

min

M1

0.0 2.0 4.0 6.0 8.0 10.0 12.0 -20,000

0

20,000

40,000

60,000

80,000

100,000 counts

min

M1

0.0 2.0 4.0 6.0 8.0 10.0 12.0 -70,000

500,000

1,000,000

1,500,000

2,000,000counts

min

M1

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AflaCLEAN M1 SMART loaded with 5 pg/20 mL = represents 0.5 ppt = 500 ppq Aflatoxins B/G cleaned-up with AflaCLEAN SMART

AflaCLEAN SMART loaded with AflaCLEAN SMART loaded with 0.05 ng/10 mL total toxin = 0.005ng/10mL total toxin = 17.9 ppt G2/B2 (referred to matrix); 1.8 ppt G2/B2 (referred to matrix); 71.4 ppt B1/G1 7.1 ppt B1/G1

0.0 2.0 4.0 6.0 8.0 10.0 12.0-10,000

20,000

40,000

60,000

80,000

100,000 counts

min

M1

0.0 2.0 4.0 6.0 8.0 10.0 12.0-10,000

50,000

100,000

150,000

200,000

250,000

300,000 counts

min

G2

G1

B2

B1

0.0 1.3 2.5 3.8 5.0 6.3 7.5 8.8 10.0 12.0-7,000

0

5,000

10,000

15,000

23,000 counts

min

G1

B2

B1

G2

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-10,000

40,000 counts M1

-10,000

40,000 counts M1

0.0 1.3 2.5 3.8 5.0 6.3 7.5 8.8 10.0 12.0 -10,000

40,000 counts

min

M1

AflaCLEAN SMART loaded with 0.0025 ng/10 mL total toxin = 0.9 ppt G2/B2 (referred to matrix), 3.6 ppt B1/G1

Reproducibility To show the reproducibility of the results we consecutively processed three independent samples.

Aflatoxin M1 with AflaCLEAN M1 SMART Aflatoxin B/G with AflaCLEAN SMART

Comparison of three chromatograms from three different samples. These comparisons show the excellent reproducibility even in this very low measurement range.

0.0 1.3 2.5 3.8 5.0 6.3 7.5 8.8 10.0 12.0-5,000

0

10,000

20,000

30,000

35,000 counts

min

G2

G1

B2

B1

-10,000

40,000 counts G1

B2 B1

-10,000

40,000 counts G1

B2 B1

0.0 1.3 2.5 3.8 5.0 6.3 7.5 8.8 10.0 12.0-10,000

40,000 counts

min

G1

B2 B1

G2

G2

G2

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Sensitivity In order to prove the sensitivity we cleaned-up a blind sample and a spiked sample and compared these two samples. Aflatoxin M1 with AflaCLEAN M1 SMART

Comparison of non spiked (blind sample), black and spiked sample with 500 ppq (red). very high sensitivity of M1.

-10,000

20,000

40,000 counts

0.0 1.3 2.5 3.8 5.0 6.3 7.5 8.8 10.0 12.0

-10,000

20,000

40,000 counts

min

M1

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Contact

LCTech GmbH Daimlerstraße 4 84419 Obertaufkirchen Germany Tel.: +49 8082 2717-0 Fax: +49 8082 2717-100 E-Mail: info@LCTech.de www.LCTech.de www.LCTech-online.com