IntroductionMycotoxins are secondary metabolites produced by certaintypes of mould. These molecules are highly toxic to all animalorganisms, which have harmful effects even at very low doses(neurotoxic, nephrotoxic, hepatotoxic, enterotoxic, immunosuppressive, and teratogenicaction). The contamination of food (especially those of vegetable origin) by mycotoxigenicfungi can cause an accumulation of these metabolites in feed or food, jeopardizing the safetyand wholesomeness of use.The contamination can occur in the field, but also during the subsequent phases oftransportation, storage and/or processing, when the environmental conditions of temperatureand humidity able to develop fungal spores naturally present in the environment, resulting inthe production of mycotoxins.Liquid ChromatographyA P P L I C A T I O N N O T EDetermination ofmycotoxins in foodby LC and LC/MSMould Development ConditionsAspergillus spp. After collectionFieldPenicillium spp. After collectionFieldFusarium graminearum,F. culmorum, F. sportrichioidesFieldF. verticilioides, F. proliferatum FieldAuthorsMarco GoriRoberto TroianoPerkinElmerMonzaTab 1.
IntroductionGrapes are probably the first cultivatedfruit: its domestication by man began6000-8000 years ago across the areabetween the Caspian and Black Seas andover the centuries it spread all over theworlds temperate areas becoming oneof the most important fruit crops on themarket.More than 10,000 varieties of grapes arecultivated and used for different products.Grapes can be consumed as fresh (tablegrape) or dried (raisin) fruit, or they can be transformed in juice or wine, andalso used in vinegar or distilled to produce different kind of spirits (brandy,cognac).In the last decades, grapes have emerged as one of the fruits with the highestcontent in nutraceutical compounds, raising the interest of nutritionists andpharmaceutical companies for its important antioxidant and health-promotingsubstances.Aromatic compounds are one of the most important parameters in determiningthe quality of grape-derived products. This is true not only for wine, but alsofor unfermented grape juice and vinegar; moreover, aromatic compounds andprecursors contained in the grape berry play a key role affecting the quality ofits products.Gas Chromatography/Mass SpectrometryA P P L I C A T I O N N O T EAuthorsGiuseppe GenovaLife Science InstituteScuola SuperioreSant’Anna, Pisa ItalyGraziano MontanaroPerkinElmer ItalyQualitative Evaluation ofAroma-Active Compoundsin Grape and Grape-DerivedProducts by Means ofHeadspace SPME-GC/MSAnalysisThe oldest winemaking equipment, 4100 B.C.
2Internal standard selectionEven with qualitative analysis, the use of an InternalStandard (ISTD) can be useful to evaluate fiber efficiencyand compare results of different samples. For example, asthermal desorption and conditioning processes progressivelylead to fiber deterioration. As a matter of fact the recoveryyield drops down cycle-by-cycle, making it difficult tocompare the results of different analysis.By adding a constant amount of an internal standard to allof the samples it is possible to quantify the fiber extactionyelid sample-by-sample (evaluating the progressive reductionin the ISTD peak area) and establish a minimum thresholdto replace the fiber (e.g. when efficiency is less than 70%).Moreover, the amount of each analyte can be calculatedand expressed referring to the internal standard peak area(as % of ISTD).Important criteria to consider for selecting an internalstandard include:• it has to be volatile in the experimental conditionsconsidered and must bind with the fiber coating• its chromatographic peak should not overlap with otherpeaks to have a more accurate quantification• the same amount should be accurately added to allsamples to obtain confident results• the standard must be absent in all the samples andeasily detectable with GC/MS analysis. Deuteratedmolecules represent a good choice and for this analysis ap-Xylene-d10 was usedColumn selectionColumns must be appropriately selected according to theobjective of the analysis. When a quantitation determinationof a single molecule or a class of molecules is needed, it isimportant to choose a specific column with a high affinityfor your analytes. Diameter, phase thickness and columnlength must be selected in order to minimize the time ofanalysis and they must have the same timepeak to ensure the correct identification and quantitation ofthe analytes.Conversely, when the analytical purpose is to study acomplex and non-characterized sample like a food or abeverage, the column selection is subjected to differentconsiderations. When headspace is sampled it is importantto evaluate the relative analytes concentrations and obtainthe largest number of detectable peaks in order to have arepresentative “aromatic fingerprint.”This kind of data is very useful when the goal of the analysisis to compare similar products that undergo differentVarious classes of aromatic compounds contribute to theflavor profile of grapes and grape-based products withalcohols and esters providing the predominant contribution.Other classes include carbonyl compounds, terpens, organicsacids, and norisoprenoids.To investigate these patterns we choose to analyze redgrape berries, unfermented grape juice, wine and balsamicvinegar to characterize the main odorants and evaluate theirconcentration in these products using gas chromatographycoupled with mass spectrometry (GC/MS).Extraction of the aroma-active compounds was carried outusing the solid phase micro-extraction (SPME) approach,trapping the odorants contained in the headspace onto asorbent polymeric micro-cartridge that was subsequentlydesorbed into the GC injector.Preliminary trialsSolid phase micro-extraction is a powerful and versatiletechnique that allows a fast and solvent-free extractionand concentration of volatile compounds contained in theheadspace of the sample. It works by basically absorbingthe molecules onto a polymeric fiber constituted by differentsorbent materials according to the type of analytes tobe investigated.Working with the SPME technique requires a fine tuning ofmany variables that markedly affect the affect the extractionprocess and recovery yield. A brief overview on the mainparameters to be optimized, and the approach to befollowed in method optimization are given below.Fiber selectionMany different SPME coating materials and polymersare available on the market and their selection must bedone taking into account the analytical needs, the samplecomposition and the scientific literature.As aroma composition of grape and grape derived productsis very complex and heterogeneous, a multi-sorbent fiber wasselected in order to catch most of the volatiles containedin the headspace. The use of a triphasic fiber containingcarboxen, polydimethylsiloxane and divinylbenzene allowsa broad-spectrum interaction with many kind of analytes;linear and branched alcohols, esters, terpenes, carbonylcompounds and norisoprenoids are effectively trapped bythis fiber and thus can be subsequently desorbed into theGC injector.
3Extraction variablesSolid phase micro-extraction of the headspace is verysimilar to a classical static headspace analysis and a purgeand trap sampling. Like these two techniques, headspaceSPME is affected by many physical and chemical variablesthat markedly affect the headspace equilibration and theextraction process.First, it is important that prior to fiber exposition into theheadspace of the vial, the equilibrium between liquid andvapor phases must be reached for all the analytes. Pre-incubation temperature must be high enough to ensure afast headspace equilibration but low enough to prevent theformation of new compounds due to sample heating (e.g.Maillard products).A good compromise is to leave the samples at roomtemperature over night and then apply low temperature(40 °C) during pre-incubation time (20 minutes). In this wayis possible to achieve a more confident and reproducibleresult. Similarly the temperature applied during SPMEextraction, when the fiber is into the headspace, shouldbe chosen to avoid artifacts formation; usually 60-80 °C isconsidered a good compromise to have a good recoveryand speed-up the process, but it is possible to make alonger extraction at a lower temperature without alteringsample composition. It is necessary to point out that duringextraction, temperature has a dual effect: it promotes thepassage of less volatile analytes in the headspace, but at thesame time, disrupts the weak chemical interaction betweenthe fiber and the volatiles.Chromatograms reported in Figure 2 show how thecombination 40 °C for 30 min gives the best results whentreatments, for example, storage conditions or productiontechnologies. For this kind of analysis the choice of thecolumn must be done by selecting a stationary phasewith intermediate polarity to interact with many differentclasses of compounds; moreover, a column with a thickerphase ensures a massive retention and a better separationof analytes. Column length as well can enhance thechromatographic performance because many functionalgroups are available to interact with the analytes.For this analysis a PerkinElmer Elite-VMS 60 m x 0.32 mmx 1.8 μm (Part No. N9316655) was used to obtain thearomatic profile.Injection parametersInjection is another crucial step that needs to be studiedand optimized when using SPME: in this phase it is veryimportant to ensure a fast and massive desorption of theanalytes from the fiber. This could be obtained choosing anappropriate carrier flow and a desorption temperature highenough to release the volatiles into the column.A narrow non-packed liner (1 or 0.75 mm) must beinstalled in the injector to ensure a correct heating anda homogeneous carrier flow onto the fiber, improvingsignificantly the peak shape and separation. Injectiontemperature was set up at 250 °C, which is the maximumprogrammable temperature for the capillary column used,and a liner with an internal diameter of 1 mm was mountedin the injector.Carrier flow selection requires more consideration anddifferent preliminary trials to be optimized. A constant 1mL/min carrier flow was kept during the wholechromatographic run, but split flow in the injector wasclosed during the first minutes, to enhance the introductionof analytes in the column. Pressure pulse injection has beenused. Afterwards split was opened again and set up at 50mL/min until the end of the analysis.Different splitless times were investigated to evaluate theireffect on peak shape and signal intensity and thus choosethe best moment to re-open the split valve. In Figure 1 theeffect of three different splitless times (respectively 5, 2 and1 minutes) are summarized.It is possible to see how a longer splitless time (5 minutes)results in larger and smaller peaks compared with the onesobtained using 1 and 2 minutes. One minute is not enoughto ensure a sufficient introduction of the analytes in thecolumn. Two minutes of splitless time seems to be the bestchoice: peaks are well separated and MS identification ofthe compounds is easier and more confident.Figure 1. Chromatograms obtained with 5, 2 and 1 minute splitless injection mode.2
For example Carvone forms two mirror image forms orenantiomers: (–)-carvone smells like spearmint. Its mirrorimage, (+)-carvone, smells like caraway. Moreover, thesetwo compounds have a different olfactory threshold (OT, theminimum concentration needed to perceive the aroma) with(+) isomer having an OT value of 43 ppb whereas (-) isomerthreshold is equal to 600 ppb. In this case the two massspectra are very similar.These considerations are useful in defining our analyticalskills and in particular in choosing the detector type and itscharacteristics. The chromatographic separation of isomericmolecules is a prerequisite to be achieved optimizing all the GCparameters and choosing a suitable column in this challengingsituation.With a mass spectrometer, a mass spectrum can be obtainedfor each compound and searched against the NIST library. Iftwo isomers are listed in this library it is possible to have anidentification of their spectra, associated with a probabilityvalue that sometimes can reach more than 90% matching.In Figure 5 it is possible to understand how GC/MS analysisworks: in grape juice two geometrical isomers (geraniol andnerol) can be separated by a GC system giving two peaks inthe chromatogram; spectra can be extracted from each peakanalyzing the MS detector signals and the library searchgives a probable identification of the compounds.Matching values are very high (942 and 947 out of 1000 forgeraniol and nerol respectively), thus the identification can beconsidered trustworthy. As geraniol (cis) and nerol (trans) havedifferent odors and olfactory thresholds the discriminationand separate quantification of these two molecules is veryimportant in analyzing the grape product qualityExperimentalThe preliminary trial allows one to set up an optimizedprotocol for HS-SPME/GC/MS aroma profiling suitable notonly for grape juice analysis, but also for grape berries,balsamic vinegar and wine.compared to 60 °C for 20 min or 80 °C for 10 min.To increase volatile compounds concentration in headspaceand to speed up the equilibration process some salts can beadded to the sample. For example, adding sodium chloridein the vial better peaks are obtain in the GC/MS analysis(Figure 3). For this purpose 1.2 grams of high purity NaClwere added to each sample.Why a mass spectrometry detector?Olfactory is a very complex sense in humans and odorperception is a finely regulated process that allows ourbrain to work as one of the most advanced molecular labanalyzer. Thanks to a raveled system of receptors, glomeruliand neural networks our olfactive system can discernbetween two isomers of the same compound. Olfactoryreceptors act just as a molecular analyzer and have theability to detect the stereochemical features of the molecule:that’s why two enantiomers can smell completely differentto our nose (Figure 4).4Figure 4. Two optical isomers that have different odors.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).Figure 3. Chromatograms obtained without salts addition and adding 1,2 g ofNaCl to the sample.
The HS-SPME/GC/MS protocol chosen for the determinationof aroma active compounds in grape and grape-derivedproducts is summarized in the following paragraphs.Sample preparationSample preparation is very simple in order to minimize thevariability due to the analyst accuracy. All the samples wereprepared into a 22-mL clear glass crimped vial for headspaceanalysis (Part No. N6356471) with thin septa for SPME (PartNo. N6356564). NaCl was added to increase volatility ofsemi-volatile compounds, whereas a 50 ppb water solutionof deuterated p-xylene (IS) were added to monitor fiberextraction yield. To obtain a chromatogram with a goodpeak shape and resolution, weights and volumes wereadjusted 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 NaClUsing exactly the same protocol for grape and grape-derivedproducts it is possible to compare the results obtained withdifferent samples, having the same retention times and asimilar detector response for each detected compound. Theuse of CombiPAL®autosampler in all the SPME steps allowsone to achieve reproducible and confident results.5Figure 5. cis/trans isomers with different odors recognized tanks to MSspectra library searching.Instrumentation.Gas Chromatograph: Clarus 680 GC/MS with PSS injectorInjector: Programmable S/S injector (PSS)Detector: Clarus MSColumn: Elite VMS 60 x 0.32 x 1.8 (Part No. N9316655)Autosampler: CTC CombiPAL XT equipped for automatedSPMEAnalytical Method.Flow: 1 mL/minCarrier: HeliumSplit: 50 mL/min (2 min splitless)Injector: 250 °C, quartz liner 1 mm ID, Merlin SeptumMicrosealHeating Raps: 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 minMS conditionsTransfer line temperature: 220 °CSource temperature: 220 °CMass range: 28 - 250 amuScan time: 0.25 secInter scan delay: 0.025 secSolvent delay: 4.0 minSPME conditionsFiber type: Supelco®triphasic fiber PDMS-DVB-Carboxen1 cmEquilibration: 40 °C x 20 min (stirring at 500 rpm)HS Extraction: 40 °C x 20 minDesorption: 10 min in the GC injectorFiber conditioning: 10 min at 260 °CResultsThe results presented here demonstrate the effectiveness ofthis analytical technique to distinguish between both grapevarities and the products derived from them. The describedSPME approach shows good affinity and recovery rates
There was minimum sample preparation using theheadspace SPME technique, and it was possible to isolateand identify many compounds in each of these samples.Grape berries show a simpler profile and only 22 compoundswere identifiable, with prevalence of short chain alcoholsand C-6 aldehydes responsible of the green leaf-like aroma.Grape juice proved to be the most complex matrix with45 compounds clearly identifiable through mass spectra:terpenoids, alcohols and carbonyl are the most representedclasses.Wine showed a complex aromatic pattern with 37 significantpeaks; the first large peak corresponds to the ethanol andsaturates the MS detector due to its very high concentrationin this sample; esters and terpenes contribute to the fruity,floral notes.Overall 32 peaks were detected with prevalence of alcohols,short chain carboxylic acids and esters.Conclusions and perspectivesIn this paper we present an excellent analytical approachfor the fast and simple analysis of aromatic compounds invarious types of complex food matrices using HS-SPME/GC/MS. The method combines the ease of sample preparationof HS-SPME with the comprehensive identification qualitiesof GC/MS to allow for a comprehensive approach for use inthe food industry. We present the analysis of various grape-based products including raw fruit (fresh and dried), grapejuice, and grape-based wine and vinegar, and describe theunique chemical profile of each. For example, the similaritiesand differences of juice from Sangiovese and Aleaticogrape varieties is clearly demonstrated using this techniqueand allows for products based on each to be equallydifferentiated. Although the focus of this paper was grape-based products, this analytical approach is transferable tomany other fruit- and vegetable-based products.toward the main odorant classes of grape products andeffective product development and product QA/QC can beacheived using such a technique.Juices showed to have the most complex aromatic profileand it was possible to identify more than 40 compounds.Alcohols and aldehyde were the most represented classes,followed by terpenoids benzoic derivatives and C-13norisoprenoids. These latter are derived from carotenoidsand proved to be determinant in giving grape itscharacteristic flowery aroma (Figure 6).This analysis allows us to highlight the variety of differencesin the aromatic pattern between two different products.Figure 7 shows the comparison between two grape juicesobtained from two different Tuscan red grape varieties:• Sangiovese This is a red grape variety particularlyappreciated and exploited for its good phenoliccomposition and aging potential, but is known to be poorin terms of aromatic quality;• Aleatico This is a red Muscat-type variety with verycomplex, floral aroma mainly used in the production ofdessert sweet wines (passito).As expected the variety of differences is considerable:Sangiovese shows a chromatogram with fewer and smallerpeaks, where aldehydes and alcohols are predominantand terpenoids are present in traces. Conversely, Aleaticoshows a more complex profile and in the central part ofthe chromatogram many terpenoids are clearly separatedand 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 chromatogramsare showed in Figure 8.6Figure 6. Main aroma-active C-13 norisoprenoids commonly found in grape.Figure 7. Comparison of aromatic profiles of Sangiovese and Aleatico juices.
2The regulation sets the maximum contaminant limits (expressedin µg/kg), which vary depending on the quality of toxin andfood at issue.The table below shows the minimum allowed by law (Reg. EC1881/2006) for the presence of mycotoxins in different foodmatrices with the exception of foods consumed by infants andchildren.Methods of analysisHPLC with Conventional DetectorSome mycotoxins are naturally fluorescent (B2, G2, M1,...) orcan be easily made through such a chemical derivatization(Fumonisins) or by UV radiation. For these molecules is normallyused a fluorescence detector. In other cases, mycotoxins can bedetected by a common UV-VIS detector. Using a fluorimetricdetector has the indubitable advantage of high sensitivity but itis sometimes necessary to perform a pre- or post-columnderivatization process.A typical pre-column derivatization is that which is obtainedthrough the use of TFA while the most widespread systems ofpost-column derivatization are: Pickering, KobraCell or UVphoto-derivatization devices.In some cases, the addition of derivatization reagents is directlyperformed in the mobile phase, making it difficult to use agradient HPLC system when a final determination of moretoxins in the same chromatographic run is needed.As a result, in order to quantify mycotoxins within thedetection limits specified in Table 3, it is necessary an HPLCsystem equipped with UV-VIS and Fluorimetric Detectors aswell as derivatization system.Since these substances may be present not only in food butalso in animal feed, human exposure to contamination mayoccur not only through the direct consumption ofcontaminated food, but also consuming animal products, suchas milk, in case of presence of mycotoxins in the animal feedused.Mycotoxigenic mould belong to Penicillium, Aspergillus andFusarium species and the type of toxins synthesized variesdepending on the fungal genus and species considered.The high toxicity of mycotoxins, even in very lowconcentrations (in the order of ppb), makes them essential tobe controlled in those foods where it is most likely thedevelopment of toxigenic mould (cereals, nuts, milk, andcoffee). The classes of mycotoxins that frequently are found infoods are aflatoxins, ochratoxins, fumonisins, trichothecenesand zearalenone.RegulationsMycotoxin control in foods is regulated at Community level byReg. (EC) No. 1881/2006 (as amended) concerning thepresence of contaminants in food.Toxin Lowest conc. LC DetectrorAflatoxins(Aspergillusspp.)B1 2 ppb for B1 FL + deriv.B2 4 ppb for(B1+B2+G1+G2)FLG1 FL + deriv.G2 FLM1 0,05 ppb FLAspergillusand Penicil-lium spp.Ocratoxin A 2 ppb FLPatuline 10 ppb UVVISFusariumspp.Deoxynivalenol 200 ppb UVVISZearalenone 75 ppb FL/UVVISB1+B2 Fumonisins 800 ppb FL + deriv.T2+HT2 Toxin FL + deriv.Tab 3. (*) The limit for T2 and HT2 Toxins is not shown in the table asit has not been introduced yet a limit by LawTab 2.
3Instrumental Configuration:UHPLC Flexar FX-10Flexar AutosamplerFL Flexar DetectorChromera CDSKobra Cell™: 100 µAAnalytical Column: Brownlee Pinnacle DB C18 HPLC Column, 1.9 μm, 50 mm × 2.1 mm i.d.Mobile phase: Water / Acn / MeOh 75-10-15 + 119mg KBr + 350µl HNO3 4MFlow: 0,7 ml/min; 6000 psiFluorescence Detector: Excitation 362 nm Emission 435 nm Emission Bandwidth: WideInjection Volume: 2 µlTotal Run Time: 3 min.HPLC with MS DetectorIn order to deal with a complete analysis of mycotoxinsreported in Table 3 by means of an HPLC system equipped witha conventional detector, it is necessary an instrumentalconfiguration consisting of several detectors. Similarly, it isoccasionally necessary to operate with different methodsdepending on the toxin to be determined in a specific matrix.The use of a universal detector such as the MS detectors allowdeveloping a single analytical method without resorting to anysystem of derivatization. The MS detector identifies moleculesexploiting the ions generated by them when subjected to aprocess of ionization.This results, generally, in the determination of their molecularions or adducts dependent on their chemical nature and thecomposition of the mobile phase.Each toxin is analyzed in the most appropriate ionizationmethod: ESI + o ESI -Sample PreparationIn the case of analysis of mycotoxins in foodstuffs, samplingplays a crucial role and is the subject of a Special Regulation(Reg (EC 401/2006). In the process of sampling, it is necessaryto give particular attention due to the imperfect homogeneityof the sample.Once collected, the sample is subjected to the analyticalprocess that meets the criteria set by Reg. (EC) 401/2006 andReg. (EC) 882/2004 Annex III sets out the criteria for evaluatingthe method used.Assuming that the sampling is done properly, it is necessary toproceed with the preparation of the sample. In the case ofmycotoxins, it can be usually followed two ways:- Use of immunoaffinity columns (IAC)- Use of SPE columnsImmunoaffinity Columns: IACs are based on the use of mono-or polyclonal antibodies suitably immobilized on a solid phase.These are very selective and allow a fast extraction ofmycotoxins from usually complex food matrices. Normally thesample, prior to being loaded on the immunoaffinity column isFlexar FX 10
4Chromatographic and MS methodsThe chromatographic method provides a unique analyticalrun able to separate and determine all mycotoxins indicatedin Table 3 operating both in positive and negative ionization(with the exception of the patulin because of its poorionizability can not be quantified within the limits indicatedby law using this method, which requires a dedicatedsample preparation).homogenized, extracted (e.g. using methanol/water) and thendiluted. Before eluting analytes with a suitable solvent(methanol), the column is washed with suitable buffers. Themethanol phase can be generally further concentrated toimprove its detection limits. IAC columns can be used onlyonce.SPE (Solid Phase Extraction): SPE columns represent analternative to those of immunoaffinity. These are generallycheaper but at the same time less specific and selective. Theiruse provides an activation phase with methanol before loadingthe sample. Subsequent washing and elution release toxinsadsorbed.Analytical MethodInstrumentation Used:UHPLC Flexar FX-15 PerkinElmerFlexar AutosamplerDegassing SystemFlexar Peltier Column OvenSQ 300 MS with ESIChromera CDS Flexar FX 15Toxin IonizationModeCapillaryExit (V)Ion Dwell Time Tr (min)B1 ESI + 120 313.0 (M+H)+ 100 6.43B2 ESI + 120 315.0 (M+H)+ 100 6.05G1 ESI + 120 329.1 (M+H)+ 100 5.62G2 ESI + 120 331.0 (M+H)+ 100 5.28M1 ESI + 120 329.1 (M+H)+ 100 4.92Ochratoxin A ESI- -90 402.2 (M-H)- 100 11.91Deoxynivalenol ESI- -60 341.0 (M+HCOO)- 100 1.51Zearalenone ESI- -90 317.2 (M-H)- 100 11.74FB1+FB2 ESI + 120 FB1= 722.4FB2= 706.3(M+H)+(M+H)+20020011.2214.73T2+HT2 Toxins ESI + 120 T2= 447.0HT2=489.0(M+Na)+(M+Na)+1001008.256.80UHPLC MS DetectorAnalytic Column: HRes DB AQ C18 (1.9 um,100 mm, 2.1mm id – p/n N9303919)Cylinder Lens: - 4000VMobile phase End Plate.-5000VA. Ultrapure water for LCMS + 0.1% v/v HCOOH Capillary entrance: -6000 VB. Acetonitrile + 0.1% v/v HCOOH End Plate Temp.: HighLinear Gradient Drying gas Temp.: 350°CStep 0: Equilibration time 4’ – 90%A – 10%B Drying gas Temp.: 12 L/minStep 1: Run Time 12’ – 38%A – 62%B Nebulizer gas pressure: 80 psiStep 2: Run Time 4’ – 38%A – 62%BFlow: 0.65 mL/minColumn temperature: 50°CInjection volume: 5 uL Tab 4.Tab 5.
5The V value of the “capillary exit” has been optimized foreach chemical species analyzed in order to allow betterqualitative/quantitative analysis using the “RAMP” function.This feature allow finding the optimal value of "capillaryexit" verifying as the signal intensity varies with the variationof applied potential.Standard SolutionsStandard 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. Toxin Stan-dard 1(ppb)Stan-dard 2(ppb)Stan-dard 3(ppb)Stan-dard 4(ppb)Limit (ppb)B1 // 0.5 5.0 50.0 2B2 0.05 0.5 5.0 50.04 for(B1+B2+G1+G2)G1 0.05 0.5 5.0 50.0G2 0.05 0.5 5.0 50.0M1 // 0.5 5.0 50.0 0.05Ochra-toxin A1.0 10.0 100.0 1000.0 2Deoxyni-valenol1.0 10.0 100.0 1000.0 200Zearale-none1.0 10.0 100.0 1000.0 75FB1 andFB25.0 50.0 500.0 5000 800T2+HT2Toxins5.0 50.0 500.0 5000The range of the calibration curves was evaluated according toTable 3 considering the sample preparation.Calibration curves and the results have been obtained without anypre-concentration performed during the sample preparation. It istherefore possible to reduce approximately ten times the sensitivitylimits of the method using sample pre-concentration and possiblyincreasing the volume of injection.Calibration CurvesHere below some calibration curves are showed by way ofexample:Tab 6.
6MS Chromatograms and SpectraThis chromatogram shows chromatographic peaks of eachmycotoxin analyzed. The response is different due to thedifferent chemical nature; in the case of Aflatoxins(chromatogram in the bottom), the scale has been expanded tobetter highlight the chromatographic signal.Sample AnalysisCertified samples were analyzed as follow:- Peanuts for Aflatoxins- Corn Flour for DON (Biopure TM)The samples of peanuts were analyzed with two differentmethods of preparation: through specific immunoaffinitycolumns (Romer Labs Diagnostic GmbH) and using SPE columns(Supra-Clean C18 - 500 mg/3 ml p/n N9306438). For corn flourand milk were used IACs.Procedures specified by the manufacturer have been used withregards to IACs, while SPE columns have been performed fol-lowing the extraction and purification protocol:- Place 5 g of sample in a 100 mL beker and add a 100 mLsolution containing water/methanol 3:2 and 2 g of NaCl.- After 45’ stirring, leave the sample to precipitate. Centrifugatefor 15’.- Pass 10 mL of the supernatant on SPE column previously acti-vated 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.Peanuts:The sample was found to contain only the B1 Aflatoxin.The analysis conducted on the sample of Peanuts showed nosignificant difference in terms of results even when conductedwith the two extraction techniques indicated.As for the specific case, the results also show that by vary-ing the type of the washing solution do not have a significantchange in the measured value.