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01. What sample volumes should be applied to average
packing columns?
Customer's detailed question: As
a guide, what ranges of sample volumes should
be used with the columns recommended for "small" and "average" sample
sizes, respectively?
MycoSep®112 AflaZon and MycoSep®113 Trich
columns are the same size and use extract amounts of
1-3ml, MycoSep®224 AflaZon and MycoSep®225
Trich columns use extract amounts of 3-5ml, MycoSep®226
AflaZon+ and MycoSep®227 Trich+ columns are the
same size and use extract amounts of 7-10ml. The difference
is the amount of packing material between the columns.
MycoSep®112 AflaZon, MycoSep®224 AflaZon and
MycoSep®226 AflaZon+ are the "white silica" columns,
MycoSep®113 Trich, MycoSep®225 Trich and MycoSep®227
Trich+ columns are the "dark alumina-charcoal" columns.
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02. What types of matrices are cleaned with double
packing columns?
Customer's detailed question:
Are the columns with "double packing volume
for additional purification" recommended
when using larger extract volumes or when
analyzing more complex sample matrices? If
they are for larger volumes, what is the
recommended volume range? If they are for
more complex matrices, what types of matrices
should be tested using these columns? How
do they compare with the columns for "small" or "average" sample
sizes?
MycoSep®112 AflaZon and MycoSep®113 Trich columns:
These columns are used for "easy" to clean up samples
(grains, corn extract, etc.) A small amount of 1 ml
of extract is passed through the column.
MycoSep®224 AflaZon and MycoSep®225 Trich columns:
These columns are used for grains and a maximum of
2ml extract is passed through the column.
MycoSep®226 AflaZon+ and MycoSep®227 Trich+ columns:
These columns are used for extra clean up; i.e. complex
matrices such as finished feeds, etc. A maximum of
4 ml of extract is passed through the column. However
when pushing less extract through the column, much
better clean up is possible.
These are the "MycoSep®" columns that function differently
than the "MultiSep®" columns. In the MycoSep® columns,
as the solvent moves through column, chromatographic
separation occurs. The toxin moves with a pure band
of solvent in front (Rf - relative front) due to toxin
solubility in the solvent and its lack of absorbability
in the matrix. Nearly all-interfering substances are
retained on the column.
Generally, it can be said that when less extract is
pushed through the columns, a higher degree of purification
can be attained. If you continue to elute (with higher
quantity of extract + solvent), other interferences
may be separated and removed.
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03. Which columns can be used for trichothecene clean up?
Customer's detailed question:
In the case of trichothecenes, which column
should be used for purification? What are
the specific advantages of using the specialized
columns for purified trichothecenes extracts?
"Dark" alumina-charcoal columns, MycoSep®225 Trich or MyoSep®227 Trich+ columns,
have better purification due to the added absorbent recovery for Trichothecenes.
Typically, they recover 85 - 90% of the toxin. When using "white" silica columns,
MycoSep®224 AflaZon or MycoSep®226 AflaZon+, you can obtain an 80-95% recovery (typically
good trichothecenes recovery through the white columns will lose some clean up ability).
The advantage of a clean up with MycoSep®224 AflaZon
or MycoSep®226 AflaZon+ is that other toxins can be
recovered from one extract in a single clean up step
as well: One can analyse Zearalenone, Zearalenols as
well as Aflatoxin B1, B2, G1 and G2 from the cleaned
extract by e.g. using highly sophisticated LC-MS/MS methodology.
The advantage of a clean up with MycoSep®225 Trich
or MycoSep®227 Trich+ is the higher purification efficiency
that is reached with this columns, this is especially
necessary if TLC or HPLC-UV is used for detection.
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04. Why use MycoSep®228 AflaPat instead of MycoSep®112, 224 AflaZon & 226 AflaZon+ for Patulin clean up?
Customer's detailed question: According to our information, the MycoSep®112, 224 AflaZon & 226
AflaZon+ columns are suitable for purification of Aflatoxins, Zearalenone, Patulin and a range of other
mycotoxins. For purification of Aflatoxins and Patulin, what are the specific advantages of using MycoSep®228
AflaPat column as compared with the MycoSep®226 AflaZon+ column?
The MycoSep®228 AflaPat column has an additional layer of packing material that is different
from the other columns. The packing material purifies matrices that other columns cannot purify.
However, the MycoSep®228 AflaPat columns cannot be used for Zearalenone analysis.
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05. Can the MycoSep®226 AflaZon+ & MycoSep®228 AflaPat columns be used for
purification of Aflatoxin M1
from milk?
Yes and No. The columns will purify for M1, but in order to get to acceptably low detection limits set by government
agencies too much solvent must be pushed thru, thus losing clean up ability. Because of this, IACs (immunoafinity columns)
are the preferred method for M1.
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06. What are the basic differences in the function of the MultiSep® and the MycoSep®
columns?
The main difference is the format: MultiSep® columns are columns in syringe format.
The extract runs through, the speed could be varied by applying vacuum or pressure.
The MycoSep® is a simple 10-second clean up format. The MycoSep® clean up column is pushed
into a test tube (containing the extract), forcing the extract to filter upwards through the
chemical packing in the column. The interferences adhere to the chemical packing in the column
and the purified extract passes through a membrane (frit) to the surface of the column.
Many of our columns are available in both formats with the same performance parameters.
Thus, the costumer can choose between two different formats according to the available
equipment in the lab.
Only one column is an exception of this rule: The Multisep®211 Fum column used for the
clean up of fumonisins works according to the classical SPE principle where 3 steps are
necessary for clean up: The columns are rinsed with a solvent to remove remaining interferences
bound by the packing. An elution step follows with the application of a solvent in
combination with another chemical to separate the bound mycotoxin from the absorbent and
allow it to pass downwards, out of the column into a separate collecting vessel.
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07. What is the basic procedure for using the
MultiSep®216 together with the MycoSep® 225 Trich or 227
Trich+ columns?
Essentially, the MultiSep®213 and MultiSep®216 columns are used when additional
clean up is necessary (complex matrixes - finished feed, etc.) In this case, we recommend
the use of a MycoSep® column followed by additional clean up of the purified extract in a
MultiSep® column. Similar to the MycoSep®227 Trich+ or MycoSep®225 Trich column, the
charcoal alumina packing in the MultiSep®213 or 216 column will bind the interferences.
Because of this absorption, minimal toxins retained must be rinsed off the columns using
extraction solvent. MultiSep®216 column require only 12ml of rinse while the MultiSep®213
column requires 25ml of rinse. The described procedure can be used for trichothecene clean up
only.
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08. For what types of sample matrices are the MultiSep® columns
generally suitable?
Same as MycoSep®; i.e. grains and feeds, but also some foods and silages.
Many MultiSep® columns are just another format of existing MycoSep®columns and have therefore the same performance characteristics and application fields as they have. Detailed information on the application fields can be looked up in our Romer® methods.
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09. When should I prefer MycoSep® for Trichothecene clean up?
Customer's detailed question: From an analytical point of view, under what circumstances would it be preferable to use a MycoSep® column for Trichothecenes clean up?
Whenever available MycoSep® clean up should be preferred: When the alternative would be liquid liquid partition the
lower consumption of hazardous solvents and the huge reduction of working time is a clear advantage.
Additionally, with MycoSep® clean up there are less sources for error.
When the alternative method would be Immunoaffinity DON column, the MycoSep® clean up should be preferred due to the
possibility of analysing other trichothecenes like Nivalenol, Fusarenon X, 3- and 15-Acetyldeoxynivalenol and the type A
trichothecenes after MycoSep® clean up as well, which is not possible after Immunoaffinity clean up. Analysing the other
fungal metabolites of this group gives a more complete picture of the contamination of the sample. The MycoSep®
clean up also has the clear advantage of less time consumption: About 30 seconds for the MycoSep® clean up and about 30
minutes for an Immunoaffinity clean up.
Only one reason is in favor of the Immunoaffinity column in this case: The Immunoaffinity column leads to a
concentration of the analyte and allows slightly better LODs - of course depending on the method used for detection.
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10. What it the shelf life of MycoSep® columns?
Customer's detailed question: You had previously advised us that the MycoSep® columns have an unlimited shelf life.
The "Directions for Use" indicate their performance is only guaranteed for 12 months after manufacture. Could you please
clarify this? We can understand you may wish to limit the warranty period for end-users,
but if the same applied to us as a distributor, it would obviously limit the amount of stock we could keep.
The columns may have an "indefinite" shelf life if properly stored in a desiccated, dark storage area. Moisture is harmful to their efficacy and, in most cases; conditions are not optimal for this type of claim. Therefore, we recommend a 12 month shelf life and most columns that we ship out should have at least 3/4 of the shelf life left.
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11. Is it possible to use Mycosep®225 Trich or Mycosep®227 Trich+
columns for the clean up of oil seeds for
DON analysis?
It is possible. It has to be taken care, that only few millilitres are
pushed through the Mycosep®227 Trich+ column. The Mycosep®227 Trich+ column
should be preferred as the additional packaging will help clean up the extract.
A matrix spike should be made additionally to gain information about the recovery
in this special matrix.
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12. Is LOD after clean up with Mycosep®226 AflaZon+
and Mycosep®228 AflaPat columns comparable with IAC clean up?
The limit of detection (LOD) does in most cases mainly depend on the instrumentation used.
If the FLD in use is sensitive enough very good LODs can be reached. E.g. for Aflatoxins
in spices after clean up with Mycosep®228 AflaPat columns LODs of 0.5 µg/kg were reported
by Akiyama. By using Mycosep columns LODs well beyond the regulated levels in the US or in
Europe can be achieved.
But of course due to the concentration step that is done by using Immunoaffinity
columns and the better clean up for some matrices even lower LODs can be reached.
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13. What is inside the columns?
The packing material differs from column type to column type. The all contain a
blend of polar and apolar adsorbents that are able to remove the common interferences
of food and feed extracts as proteins, carbohydrates, fatty acids and colours.
The mixture was designed in a way that a single toxin, a toxin group or multiple toxins
go through the column almost unaffected with a high recovery rate of usually more than 80%.
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14. How do the MycoSep® columns work?
The MycoSep® is a simple 10 to 30-second clean up format. The MycoSep® clean up
column is pushed into a test tube (containing the extract), forcing the extract to
filter upwards through the chemical packing in the column. The interferences adhere
to the chemical packing in the column and the purified extract passes through a membrane
(frit) to the surface of the column.
Nevertheless, it should be taken into consideration that the interferences and
the analyte is separated chromatographically. The consequence is that the interferences
are not bound to the column unlimited. If too much extract is pushed through the column
interferences might be again eluted leading to less resolved chromatograms. The optimum
volume for a special commodity might be slightly different from the volume we suggest for
most common commodities.
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15. How do the MultiSep® columns work?
All our MultiSep® columns except MultiSep® 211 Fum for Fumonisin clean up
work in a similar way as the MycoSep® columns. The extract is applied to the column
and drains through, the speed could be varied by applying vacuum or pressure, but gravity
usually does work sufficiently.
 MultiSep® columns
 MultiSep® 211 Fum columns
The interferences adhere to the chemical packing in the column and the purified extract passes
through a frit in the bottom of the column. A defined volume of the clean eluate can than be
transferred and further be processed.
Nevertheless, it should be taken into consideration that the interferences and
the analyte is separated chromatographically. The consequence is that the interferences
are not bound to the column unlimited. If too much extract is applied the column
interferences might be again eluted leading to less resolved chromatograms. The optimum
volume for a special commodity might be slightly different from the volume we suggest for
most common commodities.
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16. What is the difference between MycoSep® columns and the common
SPE format?
The main difference is that in common SPE one focuses on the immobilisation of the analyte.
With MycoSep® we focus on the immobilisation of interferences.
Thus, in common SPE one needs at least an additional washing step to remove interferences
from the column and then needs an elution step to elute the analyte molecules from the
column for detection.
Using the MycoSep® principles we bind the interferences and get a cleaned extract containing the analyte molecules within one step. No additional washing step or eluting step is necessary.
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17. Which extraction solvents can be used?
All columns are optimised for use with mixtures of acetonitrile and water. This is a
clear advantage as out of one extract many different mycotoxins can be analysed.
If other solvents were used for extraction the solvent should be removed, the residue should
be redissolved in acetonitrile/water.
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18. How many different columns are there?
Romer Labs® has a total of 23 columns:
9 Multisep® columns
9 Mycosep® columns
3 Star™ columns
2 Solsep® columns (Fluorometry)
Multisep® Columns (extract is filtered top down):
MultiSep® 211 Fum: Fumonisin (common SPE format)
MultiSep® 213 (double packing volume for additional purification): Trichothecene
MultiSep® 216 (single packing volume): Trichothecene
Multisep® 224 AflaZon (single packing): Aflatoxin B1, B2, G1, G2 and Zearalenone -
average sample sizes
Multisep® 226 AflaZon+ (double packing for extra purification): Aflatoxin B1, B2, G1, G2,
Zearalenone and Patulin - finished feed or processed food
Multisep® 228 AflaPat (extra clean up for complex sample matrices): Aflatoxin B1, B2, G1,
G2 and Patulin
MultiSep® 229 Ochra column: Ochratoxins
Multisep® 225 Trich (single packing clean up): Trichothecenes - average sample sizes
Multisep® 227 Trich+ (double packing clean up for extra purification): Trichothecenes -
finished feed or
processed food
MycoSep® columns (extract is filtered bottom up):
MycoSep® 112 AflaZon: for small samples using a small amount of extract
MycoSep® 224 AflaZon (single packing): Aflatoxin B1, B2, G1, G2 and Zearalenone -
average sample sizes
MycoSep® 226 AflaZon+ (double packing for extra purification): Aflatoxin B1, B2, G1,
G2, Zearalenone and Patulin - finished feed or processed food
MycoSep® 228 AflaPat (extra clean up for complex sample matrices): Aflatoxin B1, B2,
G1, G2 and Patulin
MycoSep® 229 Ochra (cereals, raisins, red wine, green coffee): Ochratoxins
MycoSep® 240 Mon: Moniliformin
MycoSep®113 Trich: Trichothecenes - for small samples using a small amount of extract
MycoSep® 225 Trich (single packing clean up): Trichothecenes - average sample sizes
MycoSep® 227 Trich+ (double packing clean up for extra purification): Trichothecenes -
finished feed or
processed food
Star™ columns (Immunaffinity columns):
AflaStar™ column: Aflatoxin B1, B2, G1, G2
ZearaStar™ column: Zearalenone
OchraStar™ column: Ochratoxins
Solsep® (2001 & 2002) columns
Solsep® 2001: Fluorimetric Aflatoxin testing of grain Fluorimetric Aflatoxin
Solsep® 2002: Fluorimetric Aflatoxin testing of cotton seed
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19. How is the column designation by mycotoxin and matrix?
Aflatoxin:
Grains: 224, 226 and 228 columns
Finished Feed (double packing for extra purification): 226 and 228 columns
Haylages/Silages, Spices, Botanicals: 228 columns
DON & Trichothecene A & B:
Grains: 225 (average sizes) column
Mixed Feeds: 225 + 216 (alumina-charcoal - single packing) columns
Additional purification: 227 (double packing) column
Additional purification: 227 column + 216 (alumina-charcoal) columns
Small samples and extract amounts: 113 column
Fumonisin:
Grains / Feeds: 211 column
Ochratoxin:
Grains / Green Coffee / Red wine / raisins: 229 column
Moniliformin:
Grains: 240 column
Patulin:
Juice, Juice Concentrate, Fruits, other matrices (double packing for extra
purification): 226, 228 columns
Zearalenone:
Additional purification: 224, 226 (double packing volume) columns
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