General Procedure of Testing

Transcription

1 Reference Testing

2 General Procedure of Testing Sampling Grinding Extraction Purification Analysis

3 Sampling Errors 100% Error 88% Sampling Error 10% Subsampling Error 2% Analysis Error LOT Sample Subsample Analysis (Whitaker & Dicken,1974)

4 Sampling the DistributionProblem Even distribution, e.g. Proteins Uneven distribution, e.g. Mycotoxins

5 General Procedure of Testing Sampling Grinding Extraction Purification Analysis

6 Clean-up Products Signal of extract w/o purification: %F FLD1 A, Ex=335, Em=440 (L:\BACKUP\HPLC_1~1\DEZ03\DATA\031229\ D) mi Signal of extract with purification: LU FLD1 A, Ex=360, Em=440), Aflatoxin total standard, 1ppb *FLD1 A, Ex=360, Em=440, Aflatoxin total standard, 100ppb min

7 Romer Labs offers: Clean-up Products 1-step purification 30 seconds for purification 2-step purification 2 minutes for purification 3-step purification 20 minutes for purification

8 Clean-up Products 1-step purification: columns are available in two formats columns are packed with mixtures of adsorbents interferences stick to the packing material mycotoxins go through un-effected MycoSep MultiSep

9 Work flow: The MycoSep format 1. push crude extract through column 30 seconds 2. use purified extract for analysis Push Crude extract Purified extract, contains mycotoxins Impurities, retained on the column

10 available columns: The MycoSep format NEW! MycoSep 112 AflaZon (Aflatoxins, Zearalenone) MycoSep 224 AflaZon (Aflatoxins, Zearalenone) MycoSep 226 AflaZon+ (Aflatoxins, Zearalenone) MycoSep 228 AflaPat (Aflatoxins, Patulin) MycoSep 113 Trich (A and B Trichothecenes) MycoSep 225 Trich (A and B Trichothecenes) MycoSep 227 Trich+ (A and B Trichothecenes) MycoSep 230 Niv (Nivalenol) MycoSep 229 Ochra (Ochratoxin A and B) MycoSep 150 Ergot (Ergot Alkaloide) NEW! MycoSep 231 Fum (Fumonisins) MycoSep 240 Mon (Moniliformin)

11 Work flow: The MultiSep format 1. let crude extract drain through column 2. use purified extract for analysis Crude extract Impurities, retained on the column Purified extract, contains mycotoxins

12 available columns: The MultiSep format MultiSep 224 AflaZon (Aflatoxins, Zearalenone) MultiSep 226 AflaZon+ (Aflatoxins, Zearalenone) MultiSep 228 AflaPat (Aflatoxins, Patulin) MultiSep 225 Trich (A and B Trichothecenes) MultiSep 227 Trich+ (A and B Trichothecenes) MultiSep 213 (additional purification for Trich.) MultiSep 216 (additional purification for Trich.) MultiSep 229 Ochra (Ochratoxin A and B)

13 Clean-up Products 2-step purification: columns are in a spin format columns are packed with mixtures of adsorbents interferences stick to the packing material mycotoxins go through un-effected designed for multi-mycotoxin analysis MycoSpin

14 Work flow: The MycoSep TM format 1. apply crude extract to column 2. Vortex for 1 minute 3. Spin down into a new tube 4. Use purified extract for analysis Crude extract Impurities, retained in the column Purified extract, contains mycotoxins

15 The MycoSep TM format Available: MycoSpin 400 Multitoxin column for LC-MS/MS purification for multiple toxins incl. Aflatoxins (B1, B2, G1, G2) Ochratoxin A Deoxynivalenol T-2 Toxin HT-2 Toxin Zearalenone

16 3-step purification Clean-up Products two different types of columns are available: column contains antibodies that specifically binds the toxin of interest interferences are washed from the column mycotoxins are eluted from the column Star Line Immunaffinity columns

17 Work flow: The StarLine TM format 1. apply crude extract to column 2. specific antibodies bind mycotoxin 3. wash interferences from column 4. elute mycotoxins Crude extract Analyte, retained on the column Impurities are washed from the column Analyte elutes from the column

18 The StarLine TM format Sample addition Rinsing Elution Toxin Impurity

19 The StarLine TM available columns: 3ml format AflaStar TM R column Aflatoxin B1, B2, G1 and G2 AflaStar TM M1 R column Aflatoxin M1 ZearaStar TM column Zearlenone, Zearalenol (α+β) OchraStar TM column Ochratoxin A and B FumoniStar TM column Fumonisin B1, B2 and B3 DonStar TM R column Deoxynivalenol

20 Clean-up Products Critical steps MycoSep /MultiSep Chromatographic process optimize volume! Interferences may elute, when too much extract is pushed through The dirtier the extract, the less extract may be pushed through If to few extract is pushed through recovery might be bad Application briefs give optimum for standard commodities like corn for unusual commodities customer must test.

21 Clean-up Products Critical steps MycoSep /MultiSep ph sensitive watch ph! Some mycotoxins only pass the column with good recovery at a certain ph. E.g. Zearalenone Be sure to use fresh acid if recommended and not to forget it. Flow rate Try to use the recommended flow rates (approx. 8ml/min) The chromatographic process needs a certain constant flow rate to perform optimal

22 Clean-up Products Critical steps StarLine TM Storage Do never freeze the columns it destroys them. Do not expose them to hot temperature. Do not store them at room temperature for more than a few days Store them at 5-6 C. Applied solution composition Do only apply solvent diluted with buffer as recommended in the box inserts Pure solvent destroys the antibody

23 Clean-up Products Critical steps StarLine TM Applied solution volume Try to keep the total applied volume low It reduces time It enhances the performance Too much applied solution can wash out some analyte Applied solution ph and temperature Be sure the buffer is o.k. and buffers the system The antibodies are sensitive against ph Be sure all solvents and the columns are adjusted to this temperature. Antibodies show the best performance at room temperature

24 Clean-up Products Critical steps StarLine TM Flow rates The flow rate when the sample is applied is critical The antibody needs time to react with the toxin The washing step can be quite fast The elution takes time The solvent has to be able to reach all the pores.

25 Clean-up Products Critical steps StarLine TM Pressure Positive pressure or negative pressure (vacuum) can be applied to get the liquid through the column Make sure to work with slight pressures huge differences in pressure deteriorate the performance Antibodies perform best in an environment similar to their natural: physiological salt concentration, temperature like in the body