Sample clean-up using immunoaffinity columns prior to detection with HPLC or LC-MS/MS
R-Biopharm Group Sample clean-up using IAC prior to HPLC or LC-MS/MS September 2011 Sample clean-up using immunoaffinity columns prior to detection with HPLC or LC-MS/MS R-Biopharm AG An der neuen Bergstraße 17 64297 Darmstadt, Germany [email protected] www.r-biopharm.com tel: 49 (0) 61 51 - 81 02-0 Fax: 49 (0) 61 51 – 81 02-20 e-mail: [email protected] website: www.r-biopharm.com Speaker: Claire Milligan Date: 29th September 2011 R-Biopharm Rhône Ltd Block 10 Todd Campus West of Scotland Science Park Acre Road, GLASGOW G20 0XA tel: +44 (0) 141 945 2924 fax: +44 (0) 141 945 2925 e-mail: [email protected] website: www.r-biopharm.com Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity September 2011 Principle of immunoaffinity columns columns •Affinity chromatography is a method of separating mixtures based on a Introduction to mycotoxins Analysis of mycotoxins highly specific interaction between an antigen and an antibody. •Immunoaffinity columns can be used prior to injection on an HPLC or LS-MS/MS system to selectively isolate and concentrate an antigen from Analysis of vitamins a sample. Analysis of antibiotics •R-Biopharm currently offers immunoaffinity columns for the detection of mycotoxins, vitamins and antibiotics in food and feed samples. Conclusions Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity September 2011 Principle of immunoaffinity columns columns •An immunoaffinity column contains a gel suspension of antibody specific Introduction to mycotoxins Analysis of mycotoxins to the antigen of interest. •Following extraction of the antigen the sample extract is diluted and passed through the immunoaffinity column. Analysis of vitamins Analysis of antibiotics •Any antigen which is present in the sample is retained by the antibody within the gel suspension. •The column is washed to remove unbound material and the antigen is Conclusions then released following elution with solvent. •The eluate is collected prior to analysis by HPLC or LC-MS/MS. Sample clean-up using IAC prior to HPLC or LC-MS/MS September 2011 Principle of immunoaffinity columns SAMPLE The sample extract is passed through the column. Antigen Other material WASHING The antibody isolates and concentrates the antigen and retains it in the column. ELUTION Passage of solvent through the column denatures the antibody and releases the antigen. Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity September 2011 Principle of immunoaffinity columns columns •The result is improved clean-up and concentration of the antigen from Introduction to mycotoxins Analysis of mycotoxins food and feed samples giving a much cleaner chromatogram and therefore providing more accurate and sensitive detection. 1,200 Analysis of vitamins Analysis of antibiotics 071106 #5 [modified by Administrator] mV Fluorescence 1,200 1,000 1,000 800 800 600 600 071106 #9 [modified by Administrator] mV Fluorescence 2 - HT-2 - 19.098 2 - HT-2 - 19.042 400 400 Conclusions 1 - T-2 - 10.034 200 200 0 0 -200 0.0 min 5.0 10.0 15.0 20.0 25.0 30.0 35.0 Chromatogram obtained without IAC clean-up for T-2 & HT-2 1 - T-2 - 10.073 -200 0.0 min 5.0 10.0 15.0 20.0 25.0 30.0 35.0 Chromatogram obtained without IAC clean-up for T-2 & HT-2 Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity September 2011 Introduction to mycotoxins columns • Introduction to mycotoxins risk of chronic exposure to aflatoxins through contaminated food and medicinal plants. Analysis of mycotoxins Analysis of vitamins More than 5 billion people in developing countries worldwide are at • The presence of mycotoxins in food is unavoidable. • Mycotoxins impair human health and cause economic losses in livestock due to the loss or reduction of livestock or in treating Analysis of antibiotics animals or humans suffering from the effects of mycotoxins. Conclusions • In 1988 the International Agency for Research on Cancer (IARC) placed aflatoxin B1 on the list of human carcinogens. Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity September 2011 Introduction to mycotoxins columns • FAO estimates that 25% of the world food crops are affected by mycotoxins each year. Introduction to mycotoxins • Crop loss due to aflatoxins contamination costs US producers more Analysis of mycotoxins than $100 million per year on average including $26 million for Analysis of vitamins Analysis of antibiotics Conclusions peanuts alone. Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity September 2011 Introduction to mycotoxins columns • To ensure that food entering or being produced within a country contains little or no mycotoxins, there is the need to set official Introduction to mycotoxins legislative levels for mycotoxins. Analysis of mycotoxins • Analysis of vitamins For mycotoxins there are already EU limits for total aflatoxins, aflatoxins B1, aflatoxin M1, ochratoxin A, zearalenone, DON, fumonisin and patulin. EU legislative levels for T-2 & HT-2 are Analysis of antibiotics pending. Conclusions Sample clean-up using IAC prior to HPLC or LC-MS/MS Introduction to mycotoxins •Over 70 countries world wide currently have legislation in place for mycotoxins. September 2011 Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity September 2011 Introduction to mycotoxins columns • To reduce the chance of mycotoxins from entering the food chain it is important to test not only raw commodities but also processed food Introduction to mycotoxins and feed. Analysis of mycotoxins • Analysis of vitamins To achieve accurate results it is necessary to ensure correct sampling, extraction and to use high quality, validated test kits for screening and for quantitative analysis. Analysis of antibiotics Conclusions Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity Analysis of mycotoxins columns Option of results required: Introduction to mycotoxins Analysis of mycotoxins Quantitative analysis (result given in form X ppb) with HPLC and fluorescence detection Analysis of vitamins Analysis of antibiotics Semi quantitative analysis (result given in form >X <Y ppb) visual reading of fluorescence, TLC, fluorometry Conclusions Qualitative analysis (result given in form < or > X ppb ) visual reading of cards September 2011 Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity Analysis of mycotoxins columns Option of results required: Introduction to mycotoxins Analysis of mycotoxins Quantitative analysis (result given in form X ppb) with HPLC and fluorescence detection Analysis of vitamins Analysis of antibiotics Semi quantitative analysis (result given in form >X <Y ppb) visual reading of fluorescence, TLC, fluorometry Conclusions Qualitative analysis (result given in form < or > X ppb ) visual reading of cards September 2011 Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity September 2011 Analysis of mycotoxins columns Immunoaffinity columns suit laboratories with highly trained personnel Introduction to mycotoxins Analysis of mycotoxins and well equipped facilities that need to meet the following requirements • Analysis of vitamins Analysis of antibiotics Conclusions Quantitative kits which comply with EU and international legislative limits. • Accurate results. • Ability to analyse different food commodities. • Suitable for use with a range of methods in order to give optimum results. Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity September 2011 Analysis of mycotoxins columns AFLAPREP®, EASI-EXTRACT ® AFLATOXIN, AFLAPREP ® M Introduction to mycotoxins OCHRAPREP® Analysis of mycotoxins AFLAOCHRA PREP® Analysis of vitamins EASI-EXTRACT ® ZEARALENONE Analysis of antibiotics DONPREP ® Conclusions FUMONIPREP ® EASI-EXTRACT ® T-2 & HT-2 DZT MS-PREP ® Sample clean-up using IAC prior to HPLC or LC-MS/MS September 2011 Analysis of mycotoxins AFLAPREP® EASI-EXTRACT® AFLATOXIN P07, P07/500 (50 or 500 columns) RP70, RP70N, RP70/500 (10 or 50 or 500 columns) 1ml column 3ml column Antibody Monoclonal Antibody (B1, B2, G1, G2) Monoclonal Antibody (B1, B2, G1, G2) Extraction Methanol / acetonitrile / chloroform Methanol / acetonitrile PBS PBS 1ml of 100% methanol 1.5ml of 100% methanol Evaporation No No Derivatisation Yes Yes Fluorescence HPLC Fluorescence HPLC Product code Format Wash solution Elution Detection Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity Analysis of mycotoxins columns AFLAPREP® M Introduction to mycotoxins Product code Analysis of mycotoxins Format P04 (25 columns) 1ml column Antibody Monoclonal Antibody (M1) Extraction N/A Wash solution PBS Analysis of vitamins Analysis of antibiotics Conclusions Elution 1.25ml of 40% methanol: 60% acetonitrile Evaporation No Derivatisation No Detection Fluorescence HPLC September 2011 Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity Analysis of mycotoxins columns OCHRAPREP® Introduction to mycotoxins Product code Analysis of mycotoxins Format P14, P14B, P14/500 (10 or 50 or 500 columns) 3ml column Antibody Monoclonal Antibody (OTA) Extraction Methanol / sodium bicarbonate Analysis of vitamins Analysis of antibiotics Wash solution Conclusions Elution PBS 1ml of 98% methanol : 2% acetic acid Evaporation No Derivatisation No Detection Fluorescence HPLC September 2011 Sample clean-up using IAC prior to HPLC or LC-MS/MS September 2011 Analysis of mycotoxins DONPREP® EASI-EXTRACT® ZEARALENONE FUMONIPREP® P50, P50B, P50/500 (10, 50 or 500 columns) RP90, RP90N, RP90/500 (10, 50 or 500 columns) P31, P31/500 (25 or 250 columns) 3ml column 3ml column 3ml column Antibody Monoclonal antibody (DON) Monoclonal antibody (ZON) Monoclonal antibody (FUM B1, B2, B3) Extraction Water Acetonitrile Acetonitrile : methanol : water H2O PBS PBS 1.5ml of 100% methanol 1.5ml of 100% acetonitrile 1.5ml of 100% methanol Evaporation Yes No No Derivatisation No No Yes UV HPLC Fluorescence HPLC Fluorescence HPLC Product code Format Wash solution Elution Detection Sample clean-up using IAC prior to HPLC or LC-MS/MS September 2011 Analysis of mycotoxins EASI-EXTRACT® T-2 & HT-2 AFLAOCHRA PREP® DZT MS-PREP® P43, P43B (10 or 50 columns) P89, P89B (10 or 50 columns) P73, P73B (10 or 50 columns) 3ml column 1ml column 1ml column Antibody Monoclonal antibodies (T-2, HT-2) Monoclonal Antibodies (B1, B2, G1, G2, OTA) Monoclonal antibodies (DON, ZON, T-2, HT-2) Extraction Methanol Methanol Methanol H2O PBS H2O 1.5ml of 100% methanol 1ml of 100% methanol 1ml of 100% methanol Evaporation Yes No No Derivatisation Yes Yes No Fluorescence HPLC Fluorescence HPLC LC-MS/MS Product code Format Wash solution Elution Detection Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity September 2011 Analysis of mycotoxins columns • Introduction to mycotoxins Various methods have been developed for the analysis of aflatoxins in a wide range of food and feed samples. • Cereals • Nuts • Spices Analysis of antibiotics • Cocoa Conclusions • Rice • Coconut • Animal feed • and many more…….. Analysis of mycotoxins Analysis of vitamins Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity September 2011 Analysis of mycotoxins columns • Introduction to mycotoxins Analysis of mycotoxins The extraction method and solvent varies depending on the commodity of interest. • For example, for extracting aflatoxin from peanuts a methanol extraction is more efficient while an acetonitrile extraction for cocoa is Analysis of vitamins Analysis of antibiotics preferred. • The antibody contained in the gel of an immunoaffinity column will have a particular tolerance to the level of solvent applied. If too much Conclusions solvent is applied to the immunoaffinity column this can affect recoveries. Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity September 2011 Analysis of mycotoxins columns • Introduction to mycotoxins Analysis of mycotoxins Analysis of vitamins Analysis of antibiotics Conclusions Solvent tolerance information for AFLAPREP® and EASI-EXTRACT® AFLATOXIN – Solvent Solvent Tolerance Methanol 30 % Acetonitrile 2.5 % Sample clean-up using IAC prior to HPLC or LC-MS/MS September 2011 Analysis of mycotoxins •Recovery information for EASI-EXTRACT® AFLATOXIN – Commodity Extraction Solvent Spike Level AFT B1 Recovery AFT B2 Recovery AFT G1 Recovery AFT G2 Recovery TOTAL AFT Recovery Peanuts 60% MeOH 10ppb 90 % 93 % 90 % 78 % 90 % Maize 80% MeOH 4ppb 98 % 88 % 87 % 91 % 92 % Paprika 80% MeOH 10ppb 94 % 102 % 93 % 102 % 98 % Figs 80% MeOH 10ppb 81 % 80 % 79 % 75 % 79 % Animal feed 60 % A CN 50ppb - - - - 102 % Sample clean-up using IAC prior to HPLC or LC-MS/MS September 2011 Analysis of mycotoxins •Example chromatogram for the analysis of paprika using EASI-EXTRACT® AFLATOXIN – Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity September 2011 Analysis of mycotoxins columns • Introduction to mycotoxins Analysis of mycotoxins Aflatoxins B2 and G2 fluoresce brightly under UV light and can easily be detected by HPLC with a fluorescence detector. • Aflatoxins B1 and G1 do not fluoresce naturally to such a high degree and must be derivatised using iodine or bromine. Analysis of vitamins Analysis of antibiotics • During derivatisation the chemical structures of aflatoxin B1 and G1 is changed to a more fluorescent form, increasing the fluorescent signal in each case for detection by HPLC. Conclusions Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity September 2011 Analysis of mycotoxins columns • Introduction to mycotoxins Analysis of mycotoxins Analysis of vitamins Derivatisation of aflatoxin B1 and G1 can be carried out using various derivatising procedures and agents in order to enhance their fluorescence – • Trifluoracetic acid – derivatisation occurs prior to HPLC column. • Iodine – derivatisation occurs after the HPLC column but before the detector. Analysis of antibiotics • Pyridium bromide perbromide – derivatisation occurs after the Conclusions HPLC column but before the detector. • KOBRA® CELL • PHRED / UVE System. Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity September 2011 Analysis of mycotoxins columns • Derivatisation by Trifluoracetic acid (TFA) Introduction to mycotoxins Injection valve Analysis of mycotoxins Analysis of vitamins Analysis of antibiotics HPLC column Mobile phase HPLC pump Eluate derivatised with TFA then Conclusions injected onto the HPLC Fluorescence detector Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity September 2011 Analysis of mycotoxins columns • Derivatisation by Trifluoracetic acid (TFA) Introduction to mycotoxins Analysis of mycotoxins Advantages Disadvantages Pre-column derivatisaion method only requires one HPLC pump Highly manipulative and numerous steps means that this method requires experience to obtain good derivatisation Traditional deivaisation method for aflatoxins TFA is a harmful, corrosive chemical therefore regular handling should be discouraged Analysis of vitamins Analysis of antibiotics Conclusions The incorporation of an evaporation step leads to aflatoxin losses Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity September 2011 Analysis of mycotoxins columns • Derivatisation by Iodine Introduction to mycotoxins HPLC columnn Analysis of mycotoxins Reaction coil Injector Analysis of vitamins Analysis of antibiotics Pump Mobile Phase Water bath At 60oC Clean-Up Conclusions Sample Saturated iodine solution Detector Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity September 2011 Analysis of mycotoxins columns • Derivatisation by Iodine Introduction to mycotoxins Analysis of mycotoxins Advantages Disadvantages Official and widely used method Requires a second HPLC pump and water bath or column jacket which increases the capital cost Reliable and accurate results The saturated iodine solution needs to be freshly prepared daily because it is unstable Requires no additional sample preparation following elution from immunoaffinity column Maintenance and cleaning is important in order to avoid iodine crystallising in-line and blocking flow Analysis of vitamins Analysis of antibiotics Conclusions Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity September 2011 Analysis of mycotoxins columns • Derivatisation by Pyridium bromide perbromide (PBPB) Introduction to mycotoxins HPLC columnn Analysis of mycotoxins Reaction coil Injector Analysis of vitamins Analysis of antibiotics Pump Mobile Phase Clean-Up Detector Conclusions Sample 50mg/L PBPB solution Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity September 2011 Analysis of mycotoxins columns • Derivatisation by Pyridium bromide perbromide (PBPB) Introduction to mycotoxins Advantages Analysis of mycotoxins Publications demonstrating reliable Requires a second HPLC pump and accurate results which increases the capital cost Analysis of vitamins Requires no additional sample preparation following elution from immunoaffinity column The PBPB solution can be difficult to dissolve Short reaction time (only 30cm reaction coil required) at ambient temperature Maintenance and cleaning is important in order to avoid PBPB crystallising in-line and blocking flow No water bath is required PBPB is hazardous to handle Peak area is larger than with iodine derivatisation Mostly used in the UK, not an internally used method Analysis of antibiotics Conclusions Disadvantages Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity columns Introduction to mycotoxins Analysis of mycotoxins •KOBRA® CELL = KOk BRomine Apparatus •The KOBRA® CELL is a unique system Analysis of mycotoxins Analysis of vitamins which offers a popular alternative derivatisation method with testing for aflatoxins in conjunction with HPLC. Analysis of antibiotics Conclusions September 2011 Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity columns Introduction to mycotoxins September 2011 Analysis of mycotoxins •The KOBRA® CELL is an electrochemical cell consisting of a platinum working electrode and a stainless steel auxiliary electrode separated from another by a membrane. These layers are sandwiched between a Analysis of mycotoxins Analysis of vitamins rigid plastic housing. •The CELL is fitted between the HPLC column and the detector that generateds the derivatisation agent, bromine, on-line from potassium Analysis of antibiotics Conclusions bromide and nitric acid present in the mobile phase. •The derivatisation results in a significant increase in the fluorescent signals of the modified forms of aflatoxin B1 and G1. Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity columns Introduction to mycotoxins Analysis of mycotoxins Analysis of vitamins Analysis of antibiotics Conclusions Analysis of mycotoxins •KOBRA® CELL structure. September 2011 Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity columns Introduction to mycotoxins Analysis of mycotoxins Analysis of vitamins Analysis of antibiotics Conclusions Analysis of mycotoxins •KOBRA® CELL structure. September 2011 Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity columns September 2011 Analysis of mycotoxins •Derivatisation by KOBRA® CELL Introduction to mycotoxins HPLC columnn Analysis of mycotoxins Injector Analysis of vitamins Analysis of antibiotics KOBRA® CELL Pump Mobile Phase Clean-Up Conclusions Sample The length and diameter of tubing between CELL and the detector is critical to allow derivatisation to occur Detector Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity columns Introduction to mycotoxins Analysis of mycotoxins Analysis of vitamins Analysis of antibiotics Conclusions Analysis of mycotoxins •KOBRA® CELL fitting guide September 2011 Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity columns Introduction to mycotoxins September 2011 Analysis of mycotoxins •Chromatograms of aflatoxin standards obtained with and without the KOBRA® CELL. Without KOBRA® CELL Analysis of mycotoxins Analysis of vitamins Analysis of antibiotics Conclusions With KOBRA® CELL Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity columns Introduction to mycotoxins September 2011 Analysis of mycotoxins •Advantages of a KOBRA® CELL compared to traditional methods – • No daily preparation of derivatising reagents. • No second pump required. Analysis of mycotoxins Analysis of vitamins • No water bath or column heater required. • Comparable detection limit to other derivatisation methods. • Low maintenance electrochemical cell. Analysis of antibiotics Conclusions • Easy to install. • Sharp peaks, no risk of peak broadening normally associated with the introduction of a derivatisation agent. • No odour, non-hazardous derivatising agent. Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity columns Introduction to mycotoxins Analysis of mycotoxins •There are direct competitor products to the KOBRA® CELL available on the market – • Cobra Zelle or Coring Cell. Analysis of mycotoxins Analysis of vitamins Analysis of antibiotics Conclusions September 2011 • CK by BK or Le Cell Kerbaje. • Romer Cell or Romer Derivatisation Unit. Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity columns Analysis of mycotoxins • All are based on the KOBRA® CELL and are made by Coring systems. Introduction to mycotoxins • Analysis of mycotoxins Analysis of vitamins Vicam originally sold a Vicam Cell however Coring now only deal with Romer and Libios. • The word ‘Cobra’ with ‘Cell’ can’t be used as a name in any form as it trades on the KOBRA® CELL registration mark. Analysis of antibiotics Conclusions September 2011 Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity columns Introduction to mycotoxins Analysis of mycotoxins •There are some competitor photochemical derivatisation units available on the market – • PHRED by Aura Industries. Analysis of mycotoxins Analysis of vitamins Analysis of antibiotics Conclusions September 2011 • UVE by LC Tech. Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity columns Introduction to mycotoxins Analysis of mycotoxins •PHRED – PHotochemical Reactor for Enhanced Detection of aflatoxins. •Consists of a lamp holder, a low pressure mercury lamp at 254nm and PTFE reaction coils. Analysis of mycotoxins Analysis of vitamins Analysis of antibiotics Conclusions September 2011 Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity columns Introduction to mycotoxins September 2011 Analysis of mycotoxins •The PTFE reaction coils are 5 – 20cm in length. •Delay coils 30m for peak separation. •The aflatoxin B1 is converted to B2a by hydroxylation of the double Analysis of mycotoxins Analysis of vitamins bond (water is added to the aflatoxin molecule and excited by the UV light). •The run time is only 9 minutes ex/e = 365 / 415nm. Analysis of antibiotics Conclusions •AFT / OTA / ZEA multi-mycotoxin method run time is 21 minutes ex/e = 315 / 415nm. •The end user prices is approximately 1500 US $. •Some published references, albeit quite old however highly credible. Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity columns Introduction to mycotoxins Analysis of mycotoxins Analysis of vitamins Analysis of antibiotics Conclusions September 2011 Analysis of mycotoxins •The UVE derivatisation module is similar to PHRED. •It consists of a low pressure mercury UV lamp = 9W at 254nm. Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity columns Introduction to mycotoxins September 2011 Analysis of mycotoxins •Reactor loop is fluorocarbon, longer life than PTFE / Teflon. •Lamp and reactor loop cooled by fan which is meant to extend the shelf life. Analysis of mycotoxins Analysis of vitamins •Leak proof due to drain tube. •CE certification. •The end user prices is approximately 1800 €. Analysis of antibiotics Conclusions •The quartz coil will need replaced after about 9 months (medium use) due to loss of signal. This costs about 700 – 900 €. This is not covered by warranty. Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity columns Introduction to mycotoxins September 2011 Analysis of mycotoxins •The disadvantages of the photochemical derivatisation units compared to the KOBRA® CELL – • UV wavelength shifts and UV light diminishes over time = loss of Analysis of mycotoxins Analysis of vitamins effectiveness. And reduction in derivatisation • PTFE becomes blocked due to age & UV damage. • Complex knitting of reaction coils creates blank zones where UV Analysis of antibiotics Conclusions light cannot reach. • Column heater/jacket is required. • Leaking & damage may be a problem. • Breakdown time is significant due to frequent leaking. Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity columns Introduction to mycotoxins September 2011 Analysis of mycotoxins •The advantages of the KOBRA® CELL compared to the photochemical derivatisation units – • KOBRA® CELL is the only device listed by CEN reference Analysis of mycotoxins Analysis of vitamins method for import control. • KOBRA® CELL is an officially recognized AOAC method. • KOBRA® CELL is tested under ISO 9001 Quality Manufacturing Analysis of antibiotics Conclusions Systems. • KOBRA® CELL is recommended by several competitors. Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity September 2011 Analysis of mycotoxins columns • Introduction to mycotoxins Analysis of mycotoxins In recent years we have seen a need for analysis of multiple mycotoxins and different mycotoxin combinations. • There is also more pressure being placed on analysts to test more therefore there is a need for faster and less labour intensive Analysis of vitamins Analysis of antibiotics methods. • Multi-mycotoxin immunoaffinity columns, which can be used in conjunction with LC-MS/MS offers the solution to these problems. Conclusions • LC-MS/MS is one of the most advanced techniques for mycotoxin analysis and many analysts and official labs are moving towards this technique. Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity September 2011 Analysis of mycotoxins columns • Introduction to mycotoxins Analysis of mycotoxins LC-MS/MS is a hyphenated technique, combining the separation power of HPLC, with the detection power of mass spectrometry. • Even with a very sophisticated MS instrument, HPLC is required to remove the interferences from a sample that would impact on Analysis of vitamins Analysis of antibiotics ionisation. • In all cases there is the need for an interface (ion source) between the HPLC and MS. The ion source will eliminate the solvent from the Conclusions HPLC eluant and generate gas phase ions, which are then transferred to the optics of the mass spectrometer. Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity September 2011 Analysis of mycotoxins columns Introduction to mycotoxins • LC-MS/MS is particularly suitable for analysis of multi parameters. • One extraction method can be used for the analysis of different mycotoxins enabling the simultaneous detection of more than one Analysis of mycotoxins mycotoxin. Analysis of vitamins • However, extraction and clean-up techniques are often necessary prior to analysis in order to ensure well separated peaks without Analysis of antibiotics interference from matrix components. Conclusions • Immunoaffinity columns can also help to remove interfering components from a range of products which can otherwise result in quenching of the ions. Sample clean-up using IAC prior to HPLC or LC-MS/MS September 2011 Analysis of mycotoxins Example ion chromatogram for the analysis of Dried Distillers Grain with and without cleanup with DZT MS-PREP® Sample Name: "DDG IAC cleanup" Sample ID: "" File: "FL DDG 261110 Cleanup vs no cleanup.wiff" Peak Name: "DON 295.3 / 264.9" Mass(es): "295.3/264.9 amu" Comment: "" Annotation: "" Sample Index: 20 Sample Type: Unknown Concentration: N/A Calculated Conc: 0.04674 ng Acq. Date: 26/11/2010 1500 Acq. Time: 17:04:58 Modified: No Proc. Algorithm: Analyst Classic Bunching Factor: 2 Noise Threshold: 3.61 cps Area Threshold: 18.07 cps ,Num. Smooths: 4 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 sec Expected RT: 2.64 min Use Relative RT: No DON 295.3 / 264.9 DZT clean-up 1450 1350 1300 1250 1200 Int. Type: Base To Base Retention Time: 2.621 min Area: 7643. counts Height: 1551. cps Start Time: 2.52 min End Time: 2.80 min Sample Name: "DDG no cleanup" Sample ID: "" File: "FL DDG 261110 Cleanup vs no cleanup.wiff" Peak Name: "DON 295.3 / 264.9" Mass(es): "295.3/264.9 amu" Comment: "" Annotation: "" Sample Index: 24 Sample Type: Unknown Concentration: N/A 1500 Calculated Conc: 0.05932 ng Acq. Date: 26/11/2010 1450 Acq. Time: 18:02:37 2.62 1400 1150 1100 1050 DON 295.3 / 264.9 No clean-up Modified: Yes Proc. Algorithm: Analyst Classic Bunching Factor: 5 Noise Threshold: 3.61 cps Area Threshold: 18.07 cps ,Num. Smooths: 3 Sep. Width: 0.20 Sep. Height: 0.50 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 6.00 RT Window: 30.0 sec Expected RT: 2.64 min Use Relative RT: No 2.64 1400 1350 1300 1250 1200 Int. Type: Base To Base Retention Time: 2.636 min Area: 9700. counts Height: 1256. cps Start Time: 2.50 min End Time: 2.86 min 1150 1100 1050 1000 950 1000 900 950 850 In ten sity , c ps 900 In te nsi ty , c ps 850 800 800 750 700 750 650 700 600 650 2.32 550 600 3.01 500 550 450 500 400 450 350 3.23 400 300 350 250 300 200 250 150 200 100 150 50 100 0 50 0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 Time, min 2.2 2.4 Sample Name: "DDG IAC cleanup" Sample ID: "" File: "FL DDG 261110 Cleanup vs no cleanup.wiff" Peak Name: "DON 295.3 / 138.2" Mass(es): "295.3/138.2 amu" Comment: "" Annotation: "" Sample Index: 20 Sample Type: Unknown Concentration: N/A 530 Calculated Conc: 0.04782 ng 520 Acq. Date: 26/11/2010 Acq. Time: 17:04:58 510 Modified: No Proc. Algorithm: Analyst Classic Bunching Factor: 3 Noise Threshold: 1.99 cps Area Threshold: 9.95 cps ,Num. Smooths: 5 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 sec Expected RT: 2.62 min Use Relative RT: No 2.6 2.8 3.0 3.2 3.4 3.6 2.62 DON 295.1 / 138.2 DZT clean-up 490 480 470 460 450 440 430 420 410 Int. Type: Base To Base Retention Time: 2.615 min Area: 2747. counts Height: 531.4 cps Start Time: 2.52 min End Time: 2.77 min 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 Time, min 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 3.8 500 400 390 380 370 DON 295.3 / 138.2 No clean-up Sample Name: "DDG no cleanup" Sample ID: "" File: "FL DDG 261110 Cleanup vs no cleanup.wiff" Peak Name: "DON 295.3 / 138.2" Mass(es): "295.3/138.2 amu" Comment: "" Annotation: "" Sample Index: 24 Sample Type: Unknown Concentration: N/A Calculated Conc: 0.07697 ng Acq. Date: 26/11/2010 600 Acq. Time: 18:02:37 2.64 580 Modified: Yes Proc. Algorithm: Analyst Classic Bunching Factor: 4 Noise Threshold: 1.99 cps Area Threshold: 9.95 cps ,Num. Smooths: 5 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 sec Expected RT: 2.62 min Use Relative RT: No 560 540 520 500 480 Int. Type: Base To Base Retention Time: 2.638 min Area: 4422. counts Height: 541.5 cps Start Time: 2.53 min End Time: 2.92 min 460 440 360 420 350 340 400 330 320 380 310 360 300 290 340 280 270 In ten sity , c ps In ten sity , c ps • 260 250 240 320 300 280 230 220 260 210 200 240 190 220 180 2.77 170 200 160 150 180 140 130 160 120 140 110 2.95 100 3.01 120 90 80 2.42 100 70 3.60 60 3.15 2.16 0.97 60 40 30 2.07 40 3.26 3.43 2.33 1.97 20 1.53 10 0 3.73 80 50 3.91 1.86 20 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 Time, min 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 Time, min 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 Sample clean-up using IAC prior to HPLC or LC-MS/MS September 2011 Analysis of mycotoxins Example ion chromatogram for the analysis of Dried Distillers Grain with and without cleanup with DZT MS-PREP® - Sample Name: "DDG IAC cleanup" Sample ID: "" File: "FL DDG 261110 Cleanup vs no cleanup.wiff" Peak Name: "T2 484.2 / 305.1" Mass(es): "484.2/305.1 amu" Comment: "" Annotation: "" Sample Index: 22 Sample Type: Unknown 380 Concentration: N/A Calculated Conc: 0.001343 ng Acq. Date: 26/11/2010 370 Acq. Time: 17:33:48 Modified: Yes Proc. Algorithm: Analyst Classic Bunching Factor: 2 Noise Threshold: 7.86 cps Area Threshold: 39.31 cps ,Num. Smooths: 3 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 sec Expected RT: 5.53 min Use Relative RT: No 360 Int. Type: Base To Base Retention Time: 5.517 min Area: 1717. counts Height: 378.3 cps Start Time: 5.43 min End Time: 5.67 min 290 5.52 T-2 484.2 / 305.1 DZT clean-up 350 340 330 320 310 300 280 270 260 250 240 230 T-2 484.2 / 305.1 No clean-up Sample Name: "DDG no cleanup" Sample ID: "" File: "FL DDG 261110 Cleanup vs no cleanup.wiff" Peak Name: "T2 484.2 / 305.1" Mass(es): "484.2/305.1 amu" Comment: "" Annotation: "" Sample Index: 24 Sample Type: Unknown Concentration: N/A Calculated Conc: 0.0007464 ng 230 Acq. Date: 26/11/2010 Acq. Time: 18:02:37 225 Modified: No Proc. Algorithm: Analyst Classic Bunching Factor: 1 Noise Threshold: 7.86 cps Area Threshold: 39.31 cps ,Num. Smooths: 3 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 sec Expected RT: 5.53 min Use Relative RT: No 5.52 220 215 210 205 200 195 190 185 180 Int. Type: Base To Base Retention Time: 5.520 min Area: 954.5 counts Height: 204.8 cps Start Time: 5.42 min End Time: 5.60 min 175 170 4.10 165 160 155 150 145 140 135 220 130 125 Intensity, cps Intensity, cps 210 200 190 180 120 115 4.14 110 105 170 100 160 95 150 90 85 140 80 130 75 120 70 110 65 4.37 60 100 4.26 55 5.73 90 4.21 50 80 45 70 40 60 35 4.80 5.27 5.01 5.32 5.22 5.15 5.66 5.10 5.06 4.85 4.52 20 30 15 5.74 20 10 5.39 4.15 10 0 4.90 4.71 4.58 4.48 25 40 5.40 4.65 4.43 30 50 4.1 4.44 4.2 4.3 4.4 4.80 4.5 4.6 4.7 4.8 5.15 4.9 5.0 5.1 5 5.19 5.2 0 5.3 5.4 5.5 5.6 5.7 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5.8 5.0 5.1 5.2 5.3 5.4 5.5 5.6 5.7 Time, min Time, min Sample Name: "DDG IAC cleanup" Sample ID: "" File: "FL DDG 261110 Cleanup vs no cleanup.wiff" Peak Name: "T2 484.2 / 245.2" Mass(es): "484.2/245.2 amu" Comment: "" Annotation: "" Sample Index: 22 Sample Type: Unknown Concentration: N/A 220 Calculated Conc: 0.001468 ng Acq. Date: 26/11/2010 215 Acq. Time: 17:33:48 5.52 T-2 484.2 / 245.2 DZT clean-up 210 Modified: Yes Proc. Algorithm: Analyst Classic Bunching Factor: 3 Noise Threshold: 9.57 cps Area Threshold: 47.87 cps ,Num. Smooths: 6 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 sec Expected RT: 5.52 min Use Relative RT: No 205 200 195 190 185 180 175 Int. Type: Base To Base Retention Time: 5.519 min Area: 1165. counts Height: 222.0 cps Start Time: 5.40 min End Time: 5.69 min 170 165 160 155 150 145 140 135 130 T-2 484.2 / 245.2 No clean-up Sample Name: "DDG no cleanup" Sample ID: "" File: "FL DDG 261110 Cleanup vs no cleanup.wiff" Peak Name: "T2 484.2 / 245.2" Mass(es): "484.2/245.2 amu" Comment: "" Annotation: "" Sample Index: 24 Sample Type: Unknown Concentration: N/A Calculated Conc: 0.0006378 ng 110 Acq. Date: 26/11/2010 108 Acq. Time: 18:02:37 5.51 106 Modified: Yes Proc. Algorithm: Analyst Classic Bunching Factor: 2 Noise Threshold: 9.57 cps Area Threshold: 47.87 cps ,Num. Smooths: 6 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 sec Expected RT: 5.52 min Use Relative RT: No 104 102 100 98 96 94 92 90 88 86 Int. Type: Base To Base Retention Time: 5.508 min Area: 506.2 counts Height: 94.72 cps Start Time: 5.41 min End Time: 5.64 min 84 82 80 78 76 74 72 70 125 68 120 66 64 115 62 110 60 Intensity, cps Intensity, cps • 105 100 95 90 58 56 54 52 50 85 48 80 46 44 75 42 70 40 65 38 60 36 34 55 32 50 4.74 30 4.68 28 45 5.28 26 40 5.21 24 35 22 30 18 5.05 20 5.38 5.11 20 25 4.61 16 14 15 12 10 10 8 5 0 6 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5.0 Time, min 5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 4 2 0 4.6 4.7 4.8 4.9 5.0 5.1 5.2 Time, min 5.3 5.4 5.5 5.6 5.7 5.8 5.8 Sample clean-up using IAC prior to HPLC or LC-MS/MS September 2011 Analysis of mycotoxins Example ion chromatogram for the analysis of Dried Distillers Grain with and without cleanup with DZT MS-PREP® Sample Name: "DDG IAC cleanup" Sample ID: "" Fil e: "FL DDG 261110 Cle anup vs no cle anup.wiff" Peak Name: "HT2 442.1 / 263.1" Mass(es): "442.1/263.1 amu" Comment: "" Annotation: "" Sample Index: 20 Sample Type: Unknown Concentration: N/A Calculated Conc: 0.001873 ng 110 Acq. Date: 26/11/2010 108 Acq. Time: 17:04:58 Sample Name: "DDG no cle anup" Sample ID: "" File: "FL DDG 261110 Cleanup vs no cleanup.wiff" Peak Name: "HT2 442.1 / 263.1" Mass(es): "442.1/263.1 amu" Comment: "" Annotation: "" Sam ple Inde x: 24 Sam ple Type : Un know n Con cent rati on: N/A Cal cula ted Conc : 0. 0011 20 ng 102 Acq . Da te: 26 /11/ 2010 100 Acq . Ti me: 18 :02: 37 4.88 HT-2 442.1 / 263.1 DZT clean-up 106 Modified: Yes Proc. Algorithm: Analyst Classic Bunching Factor: 3 Noise Threshold: 7.92 cps Area Threshold: 39.61 cps ,Num. Smooths: 8 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 sec Expected RT: 4.91 min Use Relative RT: No 104 102 100 98 96 94 92 90 88 86 Int. Type: Base To Base Retention Time: 4.881 min Area: 591.9 counts Height: 110.6 cps Start Time: 4.76 min End Time: 5.04 min 84 HT-2 442.1 / 263.1 No clean-up Mod ifie d: RT Wind ow: Exp ecte d RT : Use Rel ativ e RT : Yes 30.0 4.9 1 No 94 92 Int . Ty pe: M anua l Ret enti on T ime: 4.89 9 min Are a: 354 .0 cou nts Hei ght: 7 2.65 cp s Sta rt T ime: 4.8 3 min End Tim e: 4.9 9 min 90 4.75 88 86 5.07 84 82 80 78 76 74 72 80 70 78 68 76 4.20 66 74 72 64 70 62 68 60 66 58 64 4.05 56 Inte n sity , c ps 62 60 Inten s ity , c p s 4.90 98 96 sec min 82 58 56 54 52 48 52 46 50 44 48 42 46 4.62 50 54 4.11 40 44 38 42 4.45 36 40 38 34 36 32 34 30 32 28 30 4.38 4.32 5.02 4.52 5.57 26 28 24 26 22 24 20 22 5.45 18 20 18 16 16 14 14 12 12 10 10 6 5.34 5.26 4 4.30 5.12 5.79 4 5.22 5.72 2 5.80 0 0 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5.0 5.1 5.2 5.3 5.4 5.5 5.6 5.7 4.1 5.8 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5.0 5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 Time, min Time, min Sample Name: "DDG IAC cleanup" Sample ID: "" File: "FL DDG 261110 Cleanup vs no cleanup.wiff" Peak Name: "HT2 442.1 / 215.0" Mass(es): "442.1/215.0 amu" Comment: "" Annotation: "" Sample Index: 20 Sample Type: Unknown Concentration: N/A 120 Calculated Conc: 0.001956 nG Acq. Date: 26/11/2010 Acq. Time: 17:04:58 4.88 HT-2 442.1 / 215.0 DZT clean-up 115 110 105 100 95 Int. Type: Base To Base Retention Time: 4.878 min Area: 634.7 counts Height: 119.7 cps Start Time: 4.74 min End Time: 5.04 min 5.68 6 5.51 4.70 4.43 2 Modified: Yes Proc. Algorithm: Analyst Classic Bunching Factor: 2 Noise Threshold: 5.98 cps Area Threshold: 29.88 cps ,Num. Smooths: 7 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 sec Expected RT: 4.90 min Use Relative RT: No 5.62 5.50 8 8 90 85 HT-2 442.1 / 215.0 No clean-up Sample Name: "DDG no cleanup" Sample ID: "" File: "FL DDG 261110 Cleanup vs no cleanup.wiff" Peak Name: "HT 2 442.1 / 215.0" Mass(es): "442.1/215.0 amu" Comment: "" Annotation: "" Sam ple Inde x: 24 Sam ple Type : Un know n Con cent rati on: N/A 82 Cal cula ted Conc : 0. 0011 14 nG Acq . Da te: 26 /11/ 2010 Acq . Ti me: 18 :02: 37 80 Mod ifie d: Yes Pro c. A lgor ithm : An alys t Cl assi c Bun chin g Fa ctor : 2 Noi se T hres hold : 5.9 8 cps Are a Th resh old: 2 9.88 c ps ,Nu m. S moot hs: 7 Sep . Wi dth: 0.2 0 Sep . He ight : 0.0 1 Exp . Pe ak R atio : 5.0 0 Exp . Ad j. R atio : 4.0 0 Exp . Va l. R atio : 3.0 0 RT Wind ow: 30.0 sec Exp ecte d RT : 4.9 0 min Use Rel ativ e RT : No 4.92 78 76 74 72 70 68 66 4.13 64 Int . Ty pe: Ba se T o Ba se Ret enti on T ime: 4.91 6 min Are a: 361 .6 cou nts Hei ght: 6 4.24 cp s Sta rt T ime: 4.8 1 min End Tim e: 5.0 2 min 62 60 58 56 80 54 75 52 50 4.58 70 48 4.63 46 Intensity, cps 65 Intensity, cps • 60 55 4.18 44 4.52 42 40 5.48 38 36 50 4.05 34 45 32 4.26 30 40 28 4.69 26 35 25 5.07 4.79 4.34 24 30 5.13 22 5.82 4.45 20 4.11 18 5.18 20 5.28 16 4.69 5.35 14 15 12 5.66 10 10 4.23 4.56 5.71 5.45 5.29 5.08 5.63 8 5 6 4 0 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5.0 5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 2 Time, min 0 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5.0 Time, min 5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 Sample clean-up using IAC prior to HPLC or LC-MS/MS September 2011 Analysis of mycotoxins Example ion chromatogram for the analysis of Dried Distillers Grain with and without cleanup with DZT MS-PREP® Sample Name: "DDG IAC cleanup" Sample ID: "" File: "FL DDG 261110 Cleanup vs no cleanup.wiff" Peak Name: "ZON 317.3 / 131.1" Mass(es): "317.3/131.1 amu" Comment: "" Annotation: "" Sample Index: 21 Sample Type: Unknown Concentration: N/A 1250 Calculated Conc: 0.006518 ng Acq. Date: 26/11/2010 Acq. Time: 17:19:23 1200 Modified: Yes Proc. Algorithm: Analyst Classic Bunching Factor: 3 1150 Noise Threshold: 5.19 cps Area Threshold: 25.95 cps ,Num. Smooths: 6 1100 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 1050 Exp. Val. Ratio: 3.00 RT Window: 30.0 sec Expected RT: 6.12 min 1000 Use Relative RT: No Int. Type: Base To Base Retention Time: 6.091 min Area: 6188. counts Height: 1253. cps Start Time: 5.93 min End Time: 6.29 min 6.09 ZON 317.1 / 131.1 DZT clean-up 950 900 850 800 750 ZON 317.1 / 131.1 No clean-up Sample Name: "DDG no cleanup" Sample ID: "" File: "FL DDG 261110 Cleanup vs no cleanup.wiff" Peak Name: "ZON 317.3 / 131.1" Mass(es): "317.3/131.1 amu" Comment: "" Annotation: "" Sample Index: 24 Sample Type: Unknown Concentration: N/A Calculated Conc: 0.008176 ng 1550 Acq. Date: 26/11/2010 Acq. Time: 18:02:37 1500 Modified: No Proc. Algorithm: Analyst Classic 1450 Bunching Factor: 3 Noise Threshold: 5.19 cps Area Threshold: 25.95 cps 1400 ,Num. Smooths: 4 Sep. Width: 0.20 Sep. Height: 0.01 1350 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 1300 RT Window: 30.0 sec Expected RT: 6.12 min 1250 Use Relative RT: No Int. Type: Base To Base Retention Time: 6.096 min Area: 7762. counts Height: 1571. cps Start Time: 5.98 min End Time: 6.31 min 1150 1100 1050 1000 950 900 650 850 600 Intensity, cps Inten sity, cps 6.10 1200 700 550 800 750 500 700 450 650 400 600 350 550 500 300 450 250 400 200 350 150 300 100 250 200 50 0 150 5.95 6.00 6.05 6.10 6.15 6.20 6.25 6.30 6.35 6.40 6.45 6.50 Time, min 6.55 6.60 6.65 6.70 6.75 6.80 6.85 6.90 6.95 7.00 7.05 7.10 100 50 0 Sample Name: "DDG IAC cleanup" Sample ID: "" File: "FL DDG 261110 Cleanup vs no cleanup.wiff" Peak Name: "ZON 317.3 / 175.2" Mass(es): "317.3/175.2 amu" Comment: "" Annotation: "" Sample Index: 21 Sample Type: Unknown Concentration: N/A Calculated Conc: 0.006706 ng 1080 Acq. Date: 26/11/2010 Acq. Time: 17:19:23 1060 6.08 ZON 317.1 / 175.2 DZT clean-up 1040 Modified: Yes Proc. Algorithm: Analyst Classic Bunching Factor: 3 Noise Threshold: 6.21 cps Area Threshold: 31.05 cps ,Num. Smooths: 4 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 sec Expected RT: 6.12 min Use Relative RT: No 1020 1000 980 960 940 920 900 880 860 Int. Type: Base To Base Retention Time: 6.084 min Area: 5444. counts Height: 1099. cps Start Time: 5.97 min End Time: 6.26 min 840 820 800 780 760 740 720 700 680 660 640 620 600 580 560 ZON 317.1 / 175.2 No clean-up 540 520 500 480 5.95 6.00 6.05 Sample Name: "DDG no cleanup" Sample ID: "" File: "FL DDG 261110 Cleanup vs no cleanup.wiff" Peak Name: "ZON 317.3 / 175.2" Mass(es): "317.3/175.2 amu" Comment: "" Annotation: "" Sample Index: 24 Sample Type: Unknown Concentration: N/A Calculated Conc: 0.008130 ng 1400 Acq. Date: 26/11/2010 Acq. Time: 18:02:37 6.10 6.15 6.20 6.25 6.30 6.35 6.40 6.45 6.50 Time, min 6.55 6.60 6.65 6.15 6.20 6.25 6.30 6.35 6.40 6.45 6.50 Time, min 6.55 6.60 6.65 6.70 6.75 6.80 6.85 6.90 6.95 7.00 7.05 7.10 6.10 1350 Modified: Yes Proc. Algorithm: Analyst Classic Bunching Factor: 1 Noise Threshold: 6.21 cps Area Threshold: 31.05 cps ,Num. Smooths: 5 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 sec Expected RT: 6.12 min Use Relative RT: No 1300 1250 1200 1150 1100 Int. Type: Base To Base Retention Time: 6.097 min Area: 6601. counts Height: 1398. cps Start Time: 5.97 min End Time: 6.24 min 1050 1000 950 900 850 800 Intensity, cps Intensity, cps • 750 700 650 460 440 600 420 400 550 380 500 360 340 450 320 300 400 280 260 350 240 220 300 200 180 250 160 200 140 120 150 100 80 100 60 40 6.69 50 20 0 5.95 6.00 6.05 6.10 6.15 6.20 6.25 6.30 6.35 6.40 6.45 6.50 Time, min 6.55 6.60 6.65 6.70 6.75 6.80 6.85 6.90 6.95 7.00 7.05 7.10 0 5.95 6.00 6.05 6.10 6.70 6.75 6.80 6.85 6.90 6.95 7.00 7.05 7.10 Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity September 2011 Analysis of mycotoxins columns • Introduction to mycotoxins Analysis of mycotoxins Direct injection of complex matrices causes ion suppression therefore there is the requirement for some form of clean-up. • Although solid phase clean-up columns are often used there is still some ion suppression, this is lowered when using immunoaffinity Analysis of vitamins Analysis of antibiotics columns. • There is a need to use matrix matched standards for direct injection or with solid phase clean-up however there is reduced requirement Conclusions for matrix matched standards when using immunoaffinity columns. Sample clean-up using IAC prior to HPLC or LC-MS/MS September 2011 Analysis of mycotoxins •Recovery information for DZT MS-PREP® – Commodity Extraction Solvent Spike Level Maize Wheat 70% MeOH 800ppb DON, 80ppb ZEA, 100ppb T-2 & HT-2 Oats DON Recovery ZEA Recovery T-2 Recovery HT-2 Recovery 92 % 94% 109% 106% 98% 74% 103% 103% 82% 84% 99% 97% Animal feed 80% ACN 5ppm DON, 1ppm ZEA, T-2 & HT-2 87% 101% 104% 104% Baby food 80% MeOH 200ppb DON, T-2 & HT-2, 20ppb ZEA 91% 89% 106% 104% Beer N/A 20ppb DON, 2ppb ZEA, T-2 & HT-2 89% 89% 99% 99% Sample clean-up using IAC prior to HPLC or LC-MS/MS September 2011 Analysis of mycotoxins •Example ion chromatogram for the analysis of a cereal sample using DZT MS-PREP® – Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity September 2011 Analysis of vitamins columns • Introduction to mycotoxins Analysis of mycotoxins Analysis of vitamins Analysis of antibiotics Conclusions Vitamins cannot be synthesized in sufficient quantities and therefore must be obtained from the diet. • Thirteen vitamins are currently universally recognized. Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity September 2011 Analysis of vitamins columns • Introduction to mycotoxins Analysis of mycotoxins The analysis of vitamins is carried out to check that foods have been fortified with the correct amount of vitamin. • It should however, be noted that foods are generally over fortified with vitamin to account for losses due to degradation over time. Analysis of vitamins • Analysis of vitamins can often be problematic due to the very small levels of vitamin present and the complexity of the sample so method Analysis of antibiotics sensitivity and sample preparation are particularly important. Conclusions • Enzymes are used to cleave the protein and peptides from the food and release the vitamin. • Immunoaffinity columns can then be used to selectively isolate and concentrate the vitamin from a sample. Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity Analysis of vitamins columns EASI-EXTRACT ® VITAMIN B12 Introduction to mycotoxins EASI-EXTRACT ® VITAMIN B12 (LGE) Analysis of mycotoxins EASI-EXTRACT ® FOLIC ACID Analysis of vitamins EASI-EXTRACT ® BIOTIN Analysis of antibiotics • Conclusions These immunoaffinity columns were produced since the detection of the above vitamins can be particularly problematic by HPLC or LC-MS/MS. September 2011 Sample clean-up using IAC prior to HPLC or LC-MS/MS September 2011 Analysis of vitamins EASI-EXTRACT® VITAMIN B12 EASI-EXTRACT® VITAMIN B12 (LGE) EASI-EXTRACT® FOLIC ACID EASI-EXTRACT® BIOTIN P80 , P80B (10 or 50 columns) P88, P88B (10 or 50 columns) P81, P81B (10 or 50 columns) P82, P82B (10 or 50 columns) Format 3ml column 10ml column 3ml column 3ml column Antibody Monoclonal Monoclonal Monoclonal Monoclonal 50 mM sodium acetate 50 mM sodium acetate 0.1M Phosphate buffer 0.1M Phosphate buffer Pepsin, α-amylase, 1% KCN Pepsin, α-amylase, 1% KCN Pancreatin, 10% sodium ascorbate Pancreatin, 10% sodium ascorbate Water Water Water PBS 100% methanol 100% methanol Water containing 0.2% TFA : Acetonitrile (70 : 30 v/v) Water containing 0.2% TFA : Acetonitrile (70 : 30 v/v) Yes Yes No Yes UV HPLC UV HPLC UV HPLC UV HPLC Product code Extraction buffer Enzymes Wash buffer Elution Evaporation Detection Sample clean-up using IAC prior to HPLC or LC-MS/MS September 2011 Analysis of vitamins •Recovery information for EASI-EXTRACT® VITAMIN 12 – Commodity Vitamin B12 Claim Vitamin B12 Concentration Recovered Vitamin B12 Tablets 2000 µg / 0.849 g 1990 µg / 0.849 g Energy Drink 0.1 µg / 100ml 0.107 µg / 100ml Milk Based Infant Feed 1.25µg / 100 g 1.47 µg / 100 g Protein Shake 5.26 µg / 100g 1.17 µg / 100 g Yoghurt 3.5µg / 100 g 5.42 µg / 100 g Animal Feed 2 µg / 100g 3.08 µg / 100 g Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity September 2011 Analysis of vitamins columns • Introduction to mycotoxins In response to a series of customer requests, RBR have developed a protocol for Vitamin B12 determination in cocoa powder. Analysis of mycotoxins • 10.0 MARCH 8TH VIT B12 STD CURVE PLUS COCOA #8 [modif ied by Administrator] UV_VIS_2 mAU W VL:361 nm Cocoa powder is a 8.0 Analysis of vitamins particularly difficult 6.0 Analysis of antibiotics matrix to analyse. 1 - Vitamin B12 - 12.800 4.0 Conclusions 2.0 Chromatogram obtained without sufficient clean-up -1.0 0.0 min 5.0 10.0 15.0 20.0 25.0 30.0 35.0 Sample clean-up using IAC prior to HPLC or LC-MS/MS September 2011 Analysis of vitamins 1. Weigh 5g of sample into an amber glass bottle & add 50ml 50mM sodium acetate buffer (pH4). Place on magnetic stirrer & begin stirring. 2. Add 2g pepsin, 0.5g alpha-amylase & 2ml 1% KCN solution & stir for 10 min. 3. Place sample in shaking water bath at 37oC for 30 min, then transfer to a 100oC shaking water bath for 30 min. Cool sample to room temperature. 4. Transfer sample to a 100ml volumetric flask & fill to mark with 50mM sodium acetate buffer and centrifuge the sample at 4000rpm for 15 minutes. 5. Apply 30ml of sample to EASI-EXTRACT® VITAMIN B12. 6. Wash with 20ml of 2% Tween 20 followed by a 10ml water wash. 7. Elute column with 3ml MeOH & evaporate to dryness at 60-70oC. 8. Reconstitute samples in 300µl 0.025% TFA & inject 100 µl onto HPLC. Sample clean-up using IAC prior to HPLC or LC-MS/MS September 2011 Analysis of vitamins •Example chromatogram for analysis of cocoa powder using EASI-EXTRACT® VITAMIN B12 – 5.00 B12 300307 [C OC OA AN ALY SIS] #1 [m odif ied by Adm inis t rator]U V_VI S_2 m AU W VL:361 nm 1 - B12 - 13.416 2.00 0.00 -2. 00 0.0 m in 10.0 20.0 30.0 35. 0 Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity September 2011 Analysis of antibiotics columns • Introduction to mycotoxins Analysis of mycotoxins In some countries antibiotics are used to promote animal growth and to treat sick animals caused by poor hygiene conditions on farms. • Antibiotic residue levels in edible tissues indicate drug misuse, are contrary to regulations and may have health and economic Analysis of vitamins Analysis of antibiotics implications. • Due to the toxicity of chloramphenicol and resistance to this antibiotic, it is no longer used as a first line agent. Conclusions • In most countries the drug is banned for use in food producing animals. Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity September 2011 Analysis of antibiotics columns • Introduction to mycotoxins Analysis of mycotoxins Analysis of vitamins Analysis of antibiotics Conclusions Surveillance and testing of antibiotics has increased therefore there is a need for a rapid, easy to perform and inexpensive test. • To ensure compliance appropriate analytical methods are required. • Analysis of antibiotics can often be problematic due to the very small levels present so methods which improve sensitivity and limits of detection are particularly important. Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity September 2011 Analysis of antibiotics columns • Introduction to mycotoxins Analysis of mycotoxins With certain matrices there can sometimes be problems with false positives results in ELISAs due to matrix effects. • Complex matrices can also contain pigments making it difficult to detect low levels of chloramphenicol by HPLC or LC-MS/MS. Analysis of vitamins Analysis of antibiotics Conclusions • Therefore, clean-up with immunoaffinity columns prior to HPLC or LS-MS/MS help to eliminate these issues and to improve detection. Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity September 2011 Analysis of antibiotics columns EASI-EXTRACT® CHLORAMPHENICOL Introduction to mycotoxins Product code Analysis of mycotoxins Format 3ml column Antibody Monoclonal Analysis of vitamins Extraction buffer Analysis of antibiotics Wash buffer Elution Conclusions Evaporation Detection P300 , P300B (10 or 50 columns) McIlvaine Buffer Water 100% methanol Yes – for HPLC analysis only UV HPLC or LC-MS/MS Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity September 2011 Analysis of antibiotics columns • Weigh 5g of sample into a centrifuge tube. • Add 5ml of McIlvanie buffer (pH 2.5). • Vortex for 20 seconds or mix for 30 minutes on an orbital shaker. • Double filter the extract through Whatman No. 113. • Pass 2ml of extract through the column. Analysis of antibiotics • Wash the column with 10ml of water. Conclusions • Elute the antibiotic from the column using 100% methanol Introduction to mycotoxins Analysis of mycotoxins Analysis of vitamins (2ml if analysing by HPLC or 1ml if analysing by LC-MS/MS). • For HPLC analysis, evaporate and reconstitute in mobile phase. Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity September 2011 Analysis of antibiotics columns • Introduction to mycotoxins Recovery information for EASI-EXTRACT® CHLORAMPHENCIOL – Commodity Spike Level HPLC Recovery LC-MS/MS Recovery 48 ppb 104 % - 0.48 ppb - 96 % 48 ppb 106 % - 0.51 ppb - 100 % Bee Pollen 0.51 ppb - 83 % Royal Jelly 0.54 ppb - 85 % Prawn 0.15 ppb - 85 % Crab 0.3 ppb - 95 % Analysis of mycotoxins Chinese Honey Analysis of vitamins Analysis of antibiotics Conclusions Brazilian Honey Sample clean-up using IAC prior to HPLC or LC-MS/MS Analysis of antibiotics •Example chromatogram for HPLC analysis of honey using EASI-EXTRACT® CHLORAMPHENCIOL– September 2011 Sample clean-up using IAC prior to HPLC or LC-MS/MS September 2011 Analysis of antibiotics •Example chromatogram for LC-MS/MS analysis of prawn using EASI-EXTRACT® CHLORAMPHENCIOL– TIC of -MRM (4 pairs): from Sample 17 (Sample017) of 190511 Prawn.wiff (Turbo Spray) Max. 1333.3 cps. 4.35 1333 1300 1200 1100 1000 900 Intensity, cps 800 700 600 500 400 300 200 100 0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Time, min 4.0 4.5 5.0 5.5 6.0 6.5 Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity September 2011 Analysis of antibiotics columns • Introduction to mycotoxins Analysis of mycotoxins Analysis of vitamins Analysis of antibiotics Conclusions RBR took part in a recent FAPAS round (02169 for prawns) with the EASI-EXTRACT® CHLORAMPHENICOL columns. • RBR were lab number 67 and we obtained a Z-score of 0. • We reported a value of 1.89ppb, the assigned value of the sample was also 1.89ppb. Sample clean-up using IAC prior to HPLC or LC-MS/MS Analysis of antibiotics •Overview of Z-scores for FAPAS round 02169 - September 2011 Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity September 2011 Analysis of antibiotics columns • Introduction to mycotoxins set at a low wavelength of 280 nm. Due to the low wavelength there can be a lot of background noise on the chromatogram. Analysis of mycotoxins Analysis of vitamins Analysis of antibiotics For HPLC analysis chloramphenicol is detected using UV detector • The LOD of the honey HPLC method is 20 ng/ml. • The LOQ of the honey HPLC method is 60 ng/ml. • The use of immunoaffinity columns in conjunction with LC-MS/MS offers lower detection limits compared to HPLC analysis. Conclusions • The LOD of the LC-MS/MS method is 0.2 ng/ml. • The LOQ of the LC-MS/MS method is 0.6 ng/ml. Sample clean-up using IAC prior to HPLC or LC-MS/MS Principle of immunoaffinity September 2011 Conclusions columns • Introduction to mycotoxins Analysis of mycotoxins accompanied by Certificate of Conformance. • Columns can be stored at room temperature. • Technical support and training is available from an experienced Analysis of vitamins Analysis of antibiotics Conclusions Columns are manufactured to ISO 9001 Quality Standards and are team. • RBR also provide a range of application notes to aid analysis. Sample clean-up using IAC prior to HPLC or LC-MS/MS September 2011
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