A P P L I C AT I O N N O T E
mycotoxins in food
by LC and LC/MS
Mycotoxins are secondary metabolites produced by certain
types of mould. These molecules are highly toxic to all animal
organisms, which have harmful effects even at very low doses
(neurotoxic, nephrotoxic, hepatotoxic, enterotoxic, immunosuppressive, and teratogenic
action). The contamination of food (especially those of vegetable origin) by mycotoxigenic
fungi can cause an accumulation of these metabolites in feed or food, jeopardizing the safety
and wholesomeness of use.
The contamination can occur in the field, but also during the subsequent phases of
transportation, storage and/or processing, when the environmental conditions of temperature
and humidity able to develop fungal spores naturally present in the environment, resulting in
the production of mycotoxins.
F. culmorum, F. sportrichioides
F. verticilioides, F. proliferatum
A P P L I C AT I O N N O T E
Life Science Institute
Sant’Anna, Pisa Italy
The oldest winemaking equipment, 4100 B.C.
Qualitative Evaluation of
in Grape and Grape-Derived
Products by Means of
Grapes are probably the first cultivated
fruit: its domestication by man began
6000-8000 years ago across the area
between the Caspian and Black Seas and
over the centuries it spread all over the
world's temperate areas becoming one
of the most important fruit crops on the
More than 10,000 varieties of grapes are
cultivated and used for different products.
Grapes can be consumed as fresh (table
grape) or dried (raisin) fruit, or they can be transformed in juice or wine, and
also used in vinegar or distilled to produce different kind of spirits (brandy,
In the last decades, grapes have emerged as one of the fruits with the highest
content in nutraceutical compounds, raising the interest of nutritionists and
pharmaceutical companies for its important antioxidant and health-promoting
Aromatic compounds are one of the most important parameters in determining
the quality of grape-derived products. This is true not only for wine, but also
for unfermented grape juice and vinegar; moreover, aromatic compounds and
precursors contained in the grape berry play a key role affecting the quality of
Various classes of aromatic compounds contribute to the
flavor profile of grapes and grape-based products with
alcohols and esters providing the predominant contribution.
Other classes include carbonyl compounds, terpens, organics
acids, and norisoprenoids.
To investigate these patterns we choose to analyze red
grape berries, unfermented grape juice, wine and balsamic
vinegar to characterize the main odorants and evaluate their
concentration in these products using gas chromatography
coupled with mass spectrometry (GC/MS).
Extraction of the aroma-active compounds was carried out
using the solid phase micro-extraction (SPME) approach,
trapping the odorants contained in the headspace onto a
sorbent polymeric micro-cartridge that was subsequently
desorbed into the GC injector.
Solid phase micro-extraction is a powerful and versatile
technique that allows a fast and solvent-free extraction
and concentration of volatile compounds contained in the
headspace of the sample. It works by basically absorbing
the molecules onto a polymeric fiber constituted by different
sorbent materials according to the type of analytes to
Working with the SPME technique requires a fine tuning of
many variables that markedly affect the affect the extraction
process and recovery yield. A brief overview on the main
parameters to be optimized, and the approach to be
followed in method optimization are given below.
Many different SPME coating materials and polymers
are available on the market and their selection must be
done taking into account the analytical needs, the sample
composition and the scientific literature.
As aroma composition of grape and grape derived products
is very complex and heterogeneous, a multi-sorbent fiber was
selected in order to catch most of the volatiles contained
in the headspace. The use of a triphasic fiber containing
carboxen, polydimethylsiloxane and divinylbenzene allows
a broad-spectrum interaction with many kind of analytes;
linear and branched alcohols, esters, terpenes, carbonyl
compounds and norisoprenoids are effectively trapped by
this fiber and thus can be subsequently desorbed into the
Internal standard selection
Even with qualitative analysis, the use of an Internal
Standard (ISTD) can be useful to evaluate fiber efficiency
and compare results of different samples. For example, as
thermal desorption and conditioning processes progressively
lead to fiber deterioration. As a matter of fact the recovery
yield drops down cycle-by-cycle, making it difficult to
compare the results of different analysis.
By adding a constant amount of an internal standard to all
of the samples it is possible to quantify the fiber extaction
yelid sample-by-sample (evaluating the progressive reduction
in the ISTD peak area) and establish a minimum threshold
to replace the fiber (e.g. when efficiency is less than 70%).
Moreover, the amount of each analyte can be calculated
and expressed referring to the internal standard peak area
(as % of ISTD).
Important criteria to consider for selecting an internal
• it has to be volatile in the experimental conditions
considered and must bind with the fiber coating
• its chromatographic peak should not overlap with other
peaks to have a more accurate quantification
• the same amount should be accurately added to all
samples to obtain confident results
• the standard must be absent in all the samples and
easily detectable with GC/MS analysis. Deuterated
molecules represent a good choice and for this analysis a
p-Xylene-d10 was used
Columns must be appropriately selected according to the
objective of the analysis. When a quantitation determination
of a single molecule or a class of molecules is needed, it is
important to choose a specific column with a high affinity
for your analytes. Diameter, phase thickness and column
length must be selected in order to minimize the time of
analysis and they must have the same time
peak to ensure the correct identification and quantitation of
Conversely, when the analytical purpose is to study a
complex and non-characterized sample like a food or a
beverage, the column selection is subjected to different
considerations. When headspace is sampled it is important
to evaluate the relative analytes concentrations and obtain
the largest number of detectable peaks in order to have a
representative “aromatic fingerprint.”
This kind of data is very useful when the goal of the analysis
is to compare similar products that undergo different
treatments, for example, storage conditions or production
technologies. For this kind of analysis the choice of the
column must be done by selecting a stationary phase
with intermediate polarity to interact with many different
classes of compounds; moreover, a column with a thicker
phase ensures a massive retention and a better separation
of analytes. Column length as well can enhance the
chromatographic performance because many functional
groups are available to interact with the analytes.
For this analysis a PerkinElmer Elite-VMS 60 m x 0.32 mm
x 1.8 μm (Part No. N9316655) was used to obtain the
Injection is another crucial step that needs to be studied
and optimized when using SPME: in this phase it is very
important to ensure a fast and massive desorption of the
analytes from the fiber. This could be obtained choosing an
appropriate carrier flow and a desorption temperature high
enough to release the volatiles into the column.
A narrow non-packed liner (1 or 0.75 mm) must be
installed in the injector to ensure a correct heating and
a homogeneous carrier flow onto the fiber, improving
significantly the peak shape and separation. Injection
temperature was set up at 250 °C, which is the maximum
programmable temperature for the capillary column used,
and a liner with an internal diameter of 1 mm was mounted
in the injector.
Carrier flow selection requires more consideration and
different preliminary trials to be optimized. A constant 1
mL/min carrier flow was kept during the whole
chromatographic run, but split flow in the injector was
closed during the first minutes, to enhance the introduction
of analytes in the column. Pressure pulse injection has been
used. Afterwards split was opened again and set up at 50
mL/min until the end of the analysis.
Different splitless times were investigated to evaluate their
effect on peak shape and signal intensity and thus choose
the best moment to re-open the split valve. In Figure 1 the
effect of three different splitless times (respectively 5, 2 and
1 minutes) are summarized.
It is possible to see how a longer splitless time (5 minutes)
results in larger and smaller peaks compared with the ones
obtained using 1 and 2 minutes. One minute is not enough
to ensure a sufficient introduction of the analytes in the
column. Two minutes of splitless time seems to be the best
choice: peaks are well separated and MS identification of
the compounds is easier and more confident.
Figure 1. Chromatograms obtained with 5, 2 and 1 minute splitless injection mode.
Solid phase micro-extraction of the headspace is very
similar to a classical static headspace analysis and a purge
and trap sampling. Like these two techniques, headspace
SPME is affected by many physical and chemical variables
that markedly affect the headspace equilibration and the
First, it is important that prior to fiber exposition into the
headspace of the vial, the equilibrium between liquid and
vapor phases must be reached for all the analytes. Preincubation temperature must be high enough to ensure a
fast headspace equilibration but low enough to prevent the
formation of new compounds due to sample heating (e.g.
A good compromise is to leave the samples at room
temperature over night and then apply low temperature
(40 °C) during pre-incubation time (20 minutes). In this way
is possible to achieve a more confident and reproducible
result. Similarly the temperature applied during SPME
extraction, when the fiber is into the headspace, should
be chosen to avoid artifacts formation; usually 60-80 °C is
considered a good compromise to have a good recovery
and speed-up the process, but it is possible to make a
longer extraction at a lower temperature without altering
sample composition. It is necessary to point out that during
extraction, temperature has a dual effect: it promotes the
passage of less volatile analytes in the headspace, but at the
same time, disrupts the weak chemical interaction between
the fiber and the volatiles.
Chromatograms reported in Figure 2 show how the
combination 40 °C for 30 min gives the best results when
compared to 60 °C for 20 min or 80 °C for 10 min.
To increase volatile compounds concentration in headspace
and to speed up the equilibration process some salts can be
added to the sample. For example, adding sodium chloride
in the vial better peaks are obtain in the GC/MS analysis
(Figure 3). For this purpose 1.2 grams of high purity NaCl
were added to each sample.
Why a mass spectrometry detector?
Olfactory is a very complex sense in humans and odor
perception is a finely regulated process that allows our
brain to work as one of the most advanced molecular lab
analyzer. Thanks to a raveled system of receptors, glomeruli
and neural networks our olfactive system can discern
between two isomers of the same compound. Olfactory
receptors act just as a molecular analyzer and have the
ability to detect the stereochemical features of the molecule:
that’s why two enantiomers can smell completely different
to our nose (Figure 4).
For example Carvone forms two mirror image forms or
enantiomers: (–)-carvone smells like spearmint. Its mirror
image, (+)-carvone, smells like caraway. Moreover, these
two compounds have a different olfactory threshold (OT, the
minimum concentration needed to perceive the aroma) with
(+) isomer having an OT value of 43 ppb whereas (-) isomer
threshold is equal to 600 ppb. In this case the two mass
spectra are very similar.
These considerations are useful in defining our analytical
skills and in particular in choosing the detector type and its
characteristics. The chromatographic separation of isomeric
molecules is a prerequisite to be achieved optimizing all the GC
parameters and choosing a suitable column in this challenging
Figure 4. Two optical isomers that have different odors.
With a mass spectrometer, a mass spectrum can be obtained
for each compound and searched against the NIST library. If
two isomers are listed in this library it is possible to have an
identification of their spectra, associated with a probability
value that sometimes can reach more than 90% matching.
Figure 2. Different combination of time and temperature of extraction
(80 °C x 10 min; 60 °C x 20 min; 40 °C x 30 min).
In Figure 5 it is possible to understand how GC/MS analysis
works: in grape juice two geometrical isomers (geraniol and
nerol) can be separated by a GC system giving two peaks in
the chromatogram; spectra can be extracted from each peak
analyzing the MS detector signals and the library search
gives a probable identification of the compounds.
Matching values are very high (942 and 947 out of 1000 for
geraniol and nerol respectively), thus the identification can be
considered trustworthy. As geraniol (cis) and nerol (trans) have
different odors and olfactory thresholds the discrimination
and separate quantification of these two molecules is very
important in analyzing the grape product quality
Figure 3. Chromatograms obtained without salts addition and adding 1,2 g of
NaCl to the sample.
The preliminary trial allows one to set up an optimized
protocol for HS-SPME/GC/MS aroma profiling suitable not
only for grape juice analysis, but also for grape berries,
balsamic vinegar and wine.
Using exactly the same protocol for grape and grape-derived
products it is possible to compare the results obtained with
different samples, having the same retention times and a
similar detector response for each detected compound. The
use of CombiPAL® autosampler in all the SPME steps allows
one to achieve reproducible and confident results.
Gas Chromatograph: Clarus 680 GC/MS with PSS injector
Injector: Programmable S/S injector (PSS)
Detector: Clarus MS
Column: Elite VMS 60 x 0.32 x 1.8 (Part No. N9316655)
Autosampler: CTC CombiPAL XT equipped for automated
Flow: 1 mL/min
Split: 50 mL/min (2 min splitless)
Injector: 250 °C, quartz liner 1 mm ID, Merlin Septum
Step 1: 40 °C x 5 min
Step 2: 10 °C/min --> 120 °C (Hold x 5 min)
Step 3: 2 °C/min --> 180 °C (Hold x 2 min)
Step 4: 10 °C/min --> 230 °C (Hold x 5 min)
Total GC run time: 60 min
Figure 5. cis/trans isomers with different odors recognized tanks to MS
spectra library searching.
The HS-SPME/GC/MS protocol chosen for the determination
of aroma active compounds in grape and grape-derived
products is summarized in the following paragraphs.
Transfer line temperature: 220 °C
Source temperature: 220 °C
Mass range: 28 - 250 amu
Scan time: 0.25 sec
Inter scan delay: 0.025 sec
Sample preparation is very simple in order to minimize the
variability due to the analyst accuracy. All the samples were
prepared into a 22-mL clear glass crimped vial for headspace
analysis (Part No. N6356471) with thin septa for SPME (Part
No. N6356564). NaCl was added to increase volatility of
semi-volatile compounds, whereas a 50 ppb water solution
of deuterated p-xylene (IS) were added to monitor fiber
extraction yield. To obtain a chromatogram with a good
peak shape and resolution, weights and volumes were
adjusted for each sample type as follows:
• grape: 3 frozen cut berries + 2 mL H2O + 1.2 g NaCl +
100 µL IS
• juice: 3.9 mL + 100 µL IS + 1.2 g NaCl
• wine: 3.9 mL + 100 µL IS + 1.2 g NaCl
• vinegar: 500 µL + 100 µL IS + 1.2 g NaCl
Solvent delay: 4.0 min
Fiber type: Supelco® triphasic fiber PDMS-DVB-Carboxen
Equilibration: 40 °C x 20 min (stirring at 500 rpm)
HS Extraction: 40 °C x 20 min
Desorption: 10 min in the GC injector
Fiber conditioning: 10 min at 260 °C
The results presented here demonstrate the effectiveness of
this analytical technique to distinguish between both grape
varities and the products derived from them. The described
SPME approach shows good affinity and recovery rates
toward the main odorant classes of grape products and
effective product development and product QA/QC can be
acheived using such a technique.
There was minimum sample preparation using the
headspace SPME technique, and it was possible to isolate
and identify many compounds in each of these samples.
Juices showed to have the most complex aromatic profile
and it was possible to identify more than 40 compounds.
Alcohols and aldehyde were the most represented classes,
followed by terpenoids benzoic derivatives and C-13
norisoprenoids. These latter are derived from carotenoids
and proved to be determinant in giving grape its
characteristic flowery aroma (Figure 6).
Grape berries show a simpler profile and only 22 compounds
were identifiable, with prevalence of short chain alcohols
and C-6 aldehydes responsible of the green leaf-like aroma.
This analysis allows us to highlight the variety of differences
in the aromatic pattern between two different products.
Figure 7 shows the comparison between two grape juices
obtained from two different Tuscan red grape varieties:
• Sangiovese This is a red grape variety particularly
appreciated and exploited for its good phenolic
composition and aging potential, but is known to be poor
in terms of aromatic quality;
Grape juice proved to be the most complex matrix with
45 compounds clearly identifiable through mass spectra:
terpenoids, alcohols and carbonyl are the most represented
Wine showed a complex aromatic pattern with 37 significant
peaks; the first large peak corresponds to the ethanol and
saturates the MS detector due to its very high concentration
in this sample; esters and terpenes contribute to the fruity,
Overall 32 peaks were detected with prevalence of alcohols,
short chain carboxylic acids and esters.
• Aleatico This is a red Muscat-type variety with very
complex, floral aroma mainly used in the production of
dessert sweet wines (passito).
As expected the variety of differences is considerable:
Sangiovese shows a chromatogram with fewer and smaller
peaks, where aldehydes and alcohols are predominant
and terpenoids are present in traces. Conversely, Aleatico
shows a more complex profile and in the central part of
the chromatogram many terpenoids are clearly separated
and identifiable (eucalyptol, terpinolene, limonene, linalool,
terpineol and nerol are the most abundant).
This protocol of analysis has also been applied to red wines,
vinegar and grape berry and the resulting chromatograms
are showed in Figure 8.
Figure 6. Main aroma-active C-13 norisoprenoids commonly found in grape.
Figure 7. Comparison of aromatic profiles of Sangiovese and Aleatico juices.
Conclusions and perspectives
In this paper we present an excellent analytical approach
for the fast and simple analysis of aromatic compounds in
various types of complex food matrices using HS-SPME/GC/
MS. The method combines the ease of sample preparation
of HS-SPME with the comprehensive identification qualities
of GC/MS to allow for a comprehensive approach for use in
the food industry. We present the analysis of various grapebased products including raw fruit (fresh and dried), grape
juice, and grape-based wine and vinegar, and describe the
unique chemical profile of each. For example, the similarities
and differences of juice from Sangiovese and Aleatico
grape varieties is clearly demonstrated using this technique
and allows for products based on each to be equally
differentiated. Although the focus of this paper was grapebased products, this analytical approach is transferable to
many other fruit- and vegetable-based products.
Since these substances may be present not only in food but
also in animal feed, human exposure to contamination may
occur not only through the direct consumption of
contaminated food, but also consuming animal products, such
as milk, in case of presence of mycotoxins in the animal feed
Mycotoxigenic mould belong to Penicillium, Aspergillus and
Fusarium species and the type of toxins synthesized varies
depending on the fungal genus and species considered.
The high toxicity of mycotoxins, even in very low
concentrations (in the order of ppb), makes them essential to
be controlled in those foods where it is most likely the
development of toxigenic mould (cereals, nuts, milk, and
coffee). The classes of mycotoxins that frequently are found in
foods are aflatoxins, ochratoxins, fumonisins, trichothecenes
The regulation sets the maximum contaminant limits (expressed
in µg/kg), which vary depending on the quality of toxin and
food at issue.
The table below shows the minimum allowed by law (Reg. EC
1881/2006) for the presence of mycotoxins in different food
matrices with the exception of foods consumed by infants and
Methods of analysis
HPLC with Conventional Detector
Some mycotoxins are naturally fluorescent (B2, G2, M1,...) or
can be easily made through such a chemical derivatization
2 ppb for B1
FL + deriv.
4 ppb for
FL + deriv.
FL + deriv.
FL + deriv.
Tab 3. (*) The limit for T2 and HT2 Toxins is not shown in the table as
it has not been introduced yet a limit by Law
(Fumonisins) or by UV radiation. For these molecules is normally
used a fluorescence detector. In other cases, mycotoxins can be
detected by a common UV-VIS detector. Using a fluorimetric
detector has the indubitable advantage of high sensitivity but it
is sometimes necessary to perform a pre- or post-column
A typical pre-column derivatization is that which is obtained
through the use of TFA while the most widespread systems of
post-column derivatization are: Pickering, KobraCell or UV
Mycotoxin control in foods is regulated at Community level by
Reg. (EC) No. 1881/2006 (as amended) concerning the
presence of contaminants in food.
In some cases, the addition of derivatization reagents is directly
performed in the mobile phase, making it difficult to use a
gradient HPLC system when a final determination of more
toxins in the same chromatographic run is needed.
As a result, in order to quantify mycotoxins within the
detection limits specified in Table 3, it is necessary an HPLC
system equipped with UV-VIS and Fluorimetric Detectors as
well as derivatization system.
0,7 ml/min; 6000 psi
Excitation 362 nm
Emission 435 nm
Emission Bandwidth: Wide
Total Run Time:
HPLC with MS Detector
In order to deal with a complete analysis of mycotoxins
reported in Table 3 by means of an HPLC system equipped with
a conventional detector, it is necessary an instrumental
configuration consisting of several detectors. Similarly, it is
occasionally necessary to operate with different methods
depending on the toxin to be determined in a specific matrix.
The use of a universal detector such as the MS detectors allow
developing a single analytical method without resorting to any
system of derivatization. The MS detector identifies molecules
exploiting the ions generated by them when subjected to a
process of ionization.
This results, generally, in the determination of their molecular
ions or adducts dependent on their chemical nature and the
composition of the mobile phase.
UHPLC Flexar FX-10
FL Flexar Detector
Each toxin is analyzed in the most appropriate ionization
method: ESI + o ESI -
In the case of analysis of mycotoxins in foodstuffs, sampling
plays a crucial role and is the subject of a Special Regulation
(Reg (EC 401/2006). In the process of sampling, it is necessary
to give particular attention due to the imperfect homogeneity
of the sample.
Once collected, the sample is subjected to the analytical
process that meets the criteria set by Reg. (EC) 401/2006 and
Reg. (EC) 882/2004 Annex III sets out the criteria for evaluating
the method used.
Assuming that the sampling is done properly, it is necessary to
proceed with the preparation of the sample. In the case of
mycotoxins, it can be usually followed two ways:
Flexar FX 10
Brownlee Pinnacle DB C18 HPLC
Column, 1.9 μm, 50 mm × 2.1
Water / Acn / MeOh 75-10-15 +
119mg KBr + 350µl HNO3 4M
Use of immunoaffinity columns (IAC)
Use of SPE columns
Immunoaffinity Columns: IACs are based on the use of monoor polyclonal antibodies suitably immobilized on a solid phase.
These are very selective and allow a fast extraction of
mycotoxins from usually complex food matrices. Normally the
sample, prior to being loaded on the immunoaffinity column is
homogenized, extracted (e.g. using methanol/water) and then
diluted. Before eluting analytes with a suitable solvent
(methanol), the column is washed with suitable buffers. The
methanol phase can be generally further concentrated to
improve its detection limits. IAC columns can be used only
SPE (Solid Phase Extraction): SPE columns represent an
alternative to those of immunoaffinity. These are generally
cheaper but at the same time less specific and selective. Their
use provides an activation phase with methanol before loading
the sample. Subsequent washing and elution release toxins
Flexar FX 15
Chromatographic and MS methods
The chromatographic method provides a unique analytical
run able to separate and determine all mycotoxins indicated
in Table 3 operating both in positive and negative ionization
(with the exception of the patulin because of its poor
ionizability can not be quantified within the limits indicated
by law using this method, which requires a dedicated
UHPLC Flexar FX-15 PerkinElmer
Flexar Peltier Column Oven
SQ 300 MS with ESI
Analytic Column: HRes DB AQ C18 (1.9 um,100 mm, 2.1
mm id – p/n N9303919)
Cylinder Lens: - 4000V
A. Ultrapure water for LCMS + 0.1% v/v HCOOH
Capillary entrance: -6000 V
B. Acetonitrile + 0.1% v/v HCOOH
End Plate Temp.: High
Drying gas Temp.: 350°C
Step 0: Equilibration time 4’
– 90%A – 10%B
Drying gas Temp.: 12 L/min
Step 1: Run Time 12’
– 38%A – 62%B
Nebulizer gas pressure: 80 psi
Step 2: Run Time 4’
– 38%A – 62%B
Flow: 0.65 mL/min
Column temperature: 50°C
Injection volume: 5 uL
The V value of the “capillary exit” has been optimized for
each chemical species analyzed in order to allow better
qualitative/quantitative analysis using the “RAMP” function.
This feature allow finding the optimal value of "capillary
exit" verifying as the signal intensity varies with the variation
of applied potential.
Standard mixtures in contraction varying from 1 to 1000 ppb
(1, 10, 100 and 1000 ppb) for each analyte were prepared,
starting from a certificate standard (Biopure TM) in a Water/
Acetonitrile (1:1) + 0.1% Formic Acid mixture.
The range of the calibration curves was evaluated according to
Table 3 considering the sample preparation.
Calibration curves and the results have been obtained without any
pre-concentration performed during the sample preparation. It is
therefore possible to reduce approximately ten times the sensitivity
limits of the method using sample pre-concentration and possibly
increasing the volume of injection.
Here below some calibration curves are showed by way of
MS Chromatograms and Spectra
This chromatogram shows chromatographic peaks of each
mycotoxin analyzed. The response is different due to the
different chemical nature; in the case of Aflatoxins
(chromatogram in the bottom), the scale has been expanded to
better highlight the chromatographic signal.
Certified samples were analyzed as follow:
- Peanuts for Aflatoxins
- Corn Flour for DON (Biopure TM)
The samples of peanuts were analyzed with two different
methods of preparation: through specific immunoaffinity
columns (Romer Labs Diagnostic GmbH) and using SPE columns
(Supra-Clean C18 - 500 mg/3 ml p/n N9306438). For corn flour
and milk were used IACs.
Procedures specified by the manufacturer have been used with
regards to IACs, while SPE columns have been performed following the extraction and purification protocol:
- Place 5 g of sample in a 100 mL beker and add a 100 mL
solution containing water/methanol 3:2 and 2 g of NaCl.
- After 45’ stirring, leave the sample to precipitate. Centrifugate
- Pass 10 mL of the supernatant on SPE column previously activated with passages of methanol (2 x 3 mL) and distilled water
(2 x 3 mL) using a suitable vacuum manifold.
- Leave to dry for several minutes under vacuum
- Wash with 3 mL of a water/methanol mixture 9:1.
- Leave to dry for several minutes under vacuum.
- Elute the SPE column in 3 mL of pure methanol.
- Leave to dry the solution thus collected in a nitrogen stream;
- Re-suspend with 0.5 mL of a water/acetonitrile solution 1:1.
The sample is now ready for analysis.
The sample was found to contain only the B1 Aflatoxin.
The analysis conducted on the sample of Peanuts showed no
significant difference in terms of results even when conducted
with the two extraction techniques indicated.
As for the specific case, the results also show that by varying the type of the washing solution do not have a significant
change in the measured value.