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 HPLC From raw extract to chromatogram => in 15 minutes => without manual steps
Introduction The Components quaternary or isocratic pump sampler column oven detector HPLC UVE TM UV derivatiser (only for Aflatoxins) FREESTYLE TM with ThermELUTE and SPE modules Raw extract Chromatogram
Instrumentation Sample preparation and Analysis Any HPLC FREESTYLE Basic SPE module ThermELUTE module Racks and vials AflaCLEAN and/or OtaCLEAN SMART Immunoaffinity Cleanup Columns Pump(s) Autosampler Column oven Derivatizer (e. g. UVE TM ) Fluorescence detector
Sample preparation with large volume injection FREESTYLE Basic ThermELUTE module 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 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 Autosampler Column switching valve Column Oven Column(s) Derivatizer Fluorescence detector Software Isocratic mode Only needed for standard injection If Aflatoxins and Ochratoxin A should be analyzed a switching valve is recommended For two columns (Afla & Ochra) Standard guard and analytical columns UV derivatizer, photochemical derivatization Standard 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 Derivatizer 365 nm excitation 460 nm emission 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 - advantages SMART Columns 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 - advantages SMART Columns What is so smart about SMART columnns? They meet AOAC requirements Loading capacity 100 ng B1 Loading capacity 100 ng Ochratoxin A Recovery > 80 % Recovery > 80 %
SMART columns - advantages SMART Columns What is so smart about SMART columnns? 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 - advantages SMART Columns What is so smart about SMART columnns? They are applicable for all regulated matrices All kinds of nuts: Hazelnut, walnut, brazil nut, peanut, etc. pistachios, almonds, raisins, figs All kinds of cereals, corn, rice All common spices etc. All kinds of coffee Red wine Beer Raisins Liquorice etc.
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 Any HPLC Rack for SMART columns
Completely automated solution ThermELUTE, standard version (p/n 13572) Heating element Injection valve Width:110 mm
Completely automated solution 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 Used columns Unused 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 - advantages 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 µl into 700 µl 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 to EU Regulation 1881/2006 * Aflatoxin B1: 100 ppt Ochratoxin A: 500 ppt Detection limit with FREESTYLE + ThermELUTE Aflatoxin B1: 5 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
Completely automated solution Increased sample throughput by parallelization of sample preparation and analysis FREESTYLE sample preparation time: 15 min* / sample Sample preparation with FREESTYLE TM 1st sample 2nd sample 3rd sample 4rth etc Analysis with HPLC 1st sample 2nd sample 3rd etc Automated sample injection HPLC analysis time: 15 min* / sample * Ochratoxin A; Aflatoxin even faster
Completely automated solution - advantages Throughput at its best 15 min / sample 4 samples per hour 96 samples per day Monday Thuesday 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.
ThermELUTE chromatograms 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 Higher sensitivity compared to manual cleanup processing Excellent chromatograms (less sample needed) Ginger Ginger (10 ppb) with ThermELUTE, 2.8 ml sample Ginger (10 ppb), manual processing, 14 ml sample 400 mv ingwer blind 2,8ml halbe einwaage Emission ingwer 10ppb 2,8ml halbe einwaage Emission EM:460 nm 1-130206-FW- SOFTWARE 5.2 U SYSTEMWASCH Emissi ingwer halbe einwaag Emissi 60,0 mv EM:460 nm 300 50,0 40,0 200 30,0 100 20,0 10,0 21 1 0,0 2-50 min 0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0 10,0 min 0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0
ThermELUTE chromatograms Higher sensitivity compared to manual cleanup processing Excellent chromatograms (less sample needed) Spices: Paprika Paprika (10 ppb) with ThermELUTE, 2.8 ml sample Paprika (10 ppb), manual processing, 14 ml sample 300 mv paprika blind 2,8ml halbe einwaage Emission paprika 10ppb 2,8ml halbe einwaage Emission EM:460 nm 1-130206-FW- SOFTWARE 5.2 U SYSTEMWASCH Emissi 160 paprika halbe einwaag Emissi mv EM:460 nm 250 200 150 100 50 125 100 75 50 25 0 21 21-50 min 0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0-20 min 0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0
ThermELUTE chromatograms Higher sensitivity compared to manual cleanup processing Excellent chromatograms (less sample needed) Spices: Nutmeg Nutmeg (10 ppb) with ThermELUTE, 2.8 ml sample Nutmeg (10 ppb), manual processing, 14 ml sample 300 mv muskatnuss blind 2,8ml halbe einwaageemission muskatnuss 10ppb 2,8ml halbe einwaageemission EM:460 nm 200 mv muskatnuss halbe einwaa muskatnuss halbe einwaa Emissi Emissi EM:460 nm 250 200 150 150 100 100 50 50 0 21 21-50 min 0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0-20 min 0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0
ThermELUTE chromatograms Higher sensitivity compared to manual cleanup processing Excellent chromatograms (less sample needed) Spices: Coriander Coriander (10 ppb) with ThermELUTE, 2.8 ml sample Coriander (10 ppb), manual processing, 14 ml sample 300 mv koriander blind 2,8ml halbe einwaageemission koriander 10ppb 2,8ml halbe einwaageemission EM:460 nm 1-130206-FW- SOFTWARE 5.2 U SYSTEMWASCH Emissi koriander halbe einwaag Emissi 110 mv EM:460 nm 250 200 80 150 60 100 40 50 20 0 21 21-50 min 0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0-10 min 0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0
ThermELUTE chromatograms 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 mv EM:460 nm 250 200 150 100 50 Hazelnut, not spiked Black: ThermELUTE detects even lowest level Red: Not detected with manual processing 0 2 1-50 min 0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0
ThermELUTE chromatograms Nuts: Peanut Even low levels are detected. Not detected with manual processing 300 mv Erdnuss 10ml blind Erdnuss blind manuell 3ml Emission Emission EM:460 nm 250 200 150 100 50 Peanut, not spiked Black: ThermELUTE detects even lowest level Red: Not detected with manual processing 0 1 2-50 min 0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0
ThermELUTE chromatograms Less sample needed, thus faster processing Cereals: Corn 700 mv 500 375 250 125 Probe 4 10ppb 2,5ml Emission Probe 5 10ppb 1ml Emission 10ppb Probe mais manuell 3ml Säule Emission EM:460 nm Corn, 10 ppb Black: ThermELUTE, 2.5 ml Red: ThermELUTE, 1 ml Orange: Manual processing, 3 ml 21 3-100 min 0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0
ThermELUTE chromatograms Less sample needed, thus faster processing Nuts: Hazelnut 700 mv Haselnuss 2,5ml 10ppb Haselnuss 1ml 10ppb Haselnuss 10ppb manuell 3ml Emission Emission Emission EM:460 nm Hazelnut, 10 ppb 500 Black: ThermELUTE, 2.5 ml 375 250 125 Red: ThermELUTE, 1 ml Orange: Manual processing, 3 ml 21 3-100 min 0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0
ThermELUTE chromatograms Less sample needed, thus faster processing Raisins 700 mv Rosinen 2,5ml 10ppb Rosinen 1ml 10ppb Rosinen 10ppb manuell 3ml Emission Emission Emission EM:460 nm Raisins 10 ppb 500 Black: ThermELUTE, 2.5 ml 375 250 125 Red: ThermELUTE, 1 ml Orange: Manual processing, 3 ml 21 3-100 min 0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0
ThermELUTE chromatograms Less sample needed, thus faster processing Nuts: Pistachio 700 mv Pistazien 2,5ml 10ppb Pistazien 1ml 10ppb Pistazien 10ppb manuell 3ml Emission Emission Emission EM:460 nm Pistachio, 10 ppb 500 Black: ThermELUTE, 2.5 ml 375 250 125 Red: ThermELUTE, 1 ml Orange: Manual processing, 3 ml 21 3-100 min 0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0
ThermELUTE chromatograms Less sample needed, thus faster processing Nuts: Peanut 800 mv Erdnuss 2,5ml 10ppb ERdnuss 1ml 10ppb Erdnuss 10ppb manuell 3ml Emission Emission Emission EM:460 nm Peanut, 10 ppb 600 Black: ThermELUTE, 2.5 ml 400 Red: ThermELUTE, 1 ml 200 Orange: Manual processing, 3 ml 21 3-100 min 0,0 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 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 using AflaCLEAN SMART and ThermELUTE. Chromatogram represents 0.07 gram matrix 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 Hazelnuts Nuts: Hazelnut 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 Chromatogram of Hazelnuts Nuts: Hazelnut 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 using AflaCLEAN SMART and ThermELUTE. Chromatogram represents 0.07 gram matrix 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.
Ochratoxin Chromatogram of Instant Coffee 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 Chromatogram of Roasted Coffee 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 gram matrix equivalents.
Methods - Ochratoxin Chromatogram of White Wine 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.
Methods - Ochratoxin Chromatogram of Beer 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 gram matrix equivalents.
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!