Z Zelinková

O Novotný

J Schůrek

J Velíšek

J Hajšlová

M Doležal

Occurrence of 3-MCPD fatty acid esters in human breast milk

Keywords: 3-MCPD, Nutrition, Process contaminants -3-MCPD Food Types: Baby food, Milk Brest milk, 3-chloropropane-1, 2-diol, 3-MCPD, bound 3-MCPD, 3-MCPD fatty acid esters, organochlorine contaminants, organohalogens

A series of twelve breast milk samples were analysed by GC/MS operated in SIM mode for 3chloropropane-1,2-diol (3-MCPD). Whilst none of the samples contained 3-MCPD above the limit of detection of 3 µg/kg milk, all of them contained high amounts of 3-MCPD esterified with higher fatty acids. The levels of 3-MCPD released by hydrolysis of these esters (bound 3-MCPD) ranged from the limit of detection (300 µg/kg, expressed on fat basis) to 2195 µg/kg; with a mean level of bound 3-MCPD of 1014 µg/kg, which corresponded to 35.5 µg/kg milk. The presence of bound 3-MCPD was confirmed using GC×GC-TOF-MS analysis for four randomly selected breast milk samples. Six breast milks collected from one of the nursing mothers 14-76 days after childbirth contained bound 3-MCPD within the range of 328-2078 µg/kg fat (mean 930 µg/kg fat). The calculated bound 3-MCPD content of these samples was within the range of 6 µg/kg milk and 19 µg/kg milk (mean 12 µg/kg milk). The major types of 3-MCPD esters were the symmetric diesters with lauric, palmitic, and oleic acids and asymmetric diesters with palmitic acid/oleic acid among which 3-chloro-1,2propanediol 1,2-dioleate prevailed.

Introduction

3-Chloropropane-1,2-diol (known as 3-MCPD) is a representative of the so-called food borne or food processing contaminants. 3-MCPD was identified in acid-hydrolysed vegetable protein (acid-HVP) in 1981 [START_REF] Davídek | New chlorine-containing compounds in protein hydrolysates[END_REF] where it originates as a reaction product of phospholipids, acylglycerols and glycerol with hydrochloric acid. More recently, it has been shown that 3-MCPD occurs as a racemic mixture of its enantiomers, (R)-3-MCPD and (S)-3-MCPD [START_REF] Velíšek | Optical isomers of chloropropanediols: mechanisms of their formation and decomposition in protein hydrolysates[END_REF]. In view of its toxicity, the European Commission's Scientific Committee on Food (SCF) has proposed a provisional total daily intake (TDI) level of 2 µg/kg body weight/day for the amount of 3-MCPD that can be consumed daily over a lifetime 2 without appreciable harm to consumers health [START_REF] Scf | 1,2-diol (3-MCPD) updating the SCF opinion of[END_REF]. The TDI was adopted on 8 March 2001 and applies from 5 April 2002. Similarly, the Joint FAO/WHO Expert Group on Food Additives (JECFA) set a provisional maximum tolerable daily intake (PMTDI) of 2 µg/kg body weight/day in 2001 (JECFA 2001). A regulatory limit of 20 µg/kg, based on a 40% dry matter content, has been adopted for 3-MCPD in liquid acid-HVP and soy sauce and came into force in the European Union in 2002 (EC 2001).

Several recent studies have demonstrated that 3-MCPD is not only the contaminant typical for acid-HVP, soy sauces and related products but it also occurs in a wide range of retail outlet and home-made foods as well as in various food ingredients formulated without addition of acid-HVP [START_REF] Crews | Effect of domestic cooking on the levels of 3monochloropropane-1,2-diol in food[END_REF], 2002, Breitling-Utzmann et al. 2003, Hamlet et al. 2002, 2004a, 2004b, Divinová et al. 2004[START_REF] Doležal | Occurrence of 3chloropropane-1,2-diol and its esters in coffee[END_REF].

In raw acid-HVP, 3-MCPD occurs mainly as a free compound and, to a minor extent, esterified with higher fatty acids [START_REF] Velíšek | New chlorinecontaining organic compounds in protein hydrolysates[END_REF]. Rather surprising was a finding of fatty acid esters of 3-MCPD in the neutral fraction of goat's milk lipids [START_REF] Cerbulis | Occurrence of diesters of 3-chloro-1,2-propanediol in the neutral lipid fraction of goat´s milk[END_REF]; at that time, their occurrence was attributed to the use of chlorine-based sanitizers.

Recent findings document that 3-MCPD esters occur in a wide variety of both unprocessed and processed foods and in various food ingredients [START_REF] Svejkovská | Esters of 3chloropropane-1,2-diol in foodstuffs[END_REF], Hamlet & Sadd 2004[START_REF] Doležal | Occurrence of 3chloropropane-1,2-diol and its esters in coffee[END_REF], Svejkovská et al. 2006[START_REF] Zelinková | Fatty acids esters of 3chloropropane-1,2-diol in edible oils[END_REF][START_REF] Divinová | Free and bound 3-chloropropane-1,2-diol in coffee surrogates and malts[END_REF]. Fatty acid esters of 3-MCPD thus represent a new class of food contaminants as 3-MCPD can be easily released from these compounds by a lipase-catalysed hydrolysis reaction (Hamlet and Sadd 2004).

Generally, the amount of 3-MCPD in any food or food ingredient released from 3-MCPD esters by hydrolysis (bound 3-MCPD) largely exceeds that of free 3-MCPD. The study of [START_REF] Svejkovská | Esters of 3chloropropane-1,2-diol in foodstuffs[END_REF] reports on the presence of bound 3-MCPD in 20 selected retail food products. High levels (in the range of 280-2420 µg/kg) of bound 3-MCPD were found in salty crackers, pickled herrings, doughnuts, crisp bread, dark malt and French fries. Compared to the free form, they were 5 to 157 times higher. Analysis of white bread (Hamlet and Sadd 2004) showed that the highest levels of bound 3-MCPD were found in the crust (547 µg/kg) and toast (160 µg/kg) while in the crumb they were much lower amount (26 µg/kg) and 3 exceeded the free 3-MCPD levels 6 to 52 times. The level of bound 3-MCPD in roasted coffee was relatively low and varied between 6 µg/kg (soluble coffee) and 390 µg/kg (decaffeinated coffee) and exceeded the free 3-MCPD level 8 to 33 times [START_REF] Doležal | Occurrence of 3chloropropane-1,2-diol and its esters in coffee[END_REF]. Coffee surrogates contained bound 3-MCPD in the range of 145-1184 µg/kg, the highest level was found in roasted barley [START_REF] Divinová | Free and bound 3-chloropropane-1,2-diol in coffee surrogates and malts[END_REF]. In this case the bound 3-MCPD levels were higher 32 to 81 times. In malts [START_REF] Divinová | Free and bound 3-chloropropane-1,2-diol in coffee surrogates and malts[END_REF]), the bound 3-MCPD levels ranged from 4 to 650 µg/kg and the highest amount was found in roasted malts (463-650 µg/kg). The bound 3-MCPD levels exceeded the free 3-MCPD levels 0.4 to 36 times.

Recently, it has been shown [START_REF] Zelinková | Fatty acids esters of 3chloropropane-1,2-diol in edible oils[END_REF]) that edible oils, notably the refined edible oils including refined olive oils, contain relatively high levels of bound 3-MCPD. Amounts ranging from < 300 µg/kg to 2462 µg/kg have been found. Analysis of coffee creamers, cream aerosols and bouillon cubes, produced using refined vegetable oils, revealed that these products did not contain free 3-MCPD but their bound 3-MCPD levels ranged from 110 to 730 µg/kg product [START_REF] Karšulínová | Analysis of the lipid fraction of coffee creamers, cream aerosols and bouillon cubes[END_REF]).

Considering the above facts, the esterified 3-MCPD extends the list of toxic chlorinated chemicals to which humans can be exposed through diet. The bound 3-MCPD content in food lipids is comparable or even higher than levels of chlorinated persistent organic pollutants (POPs) found for instance in some fish, which is in most cases their major dietary source. It should be noted that POPs represented e.g. by dioxins, organochlorine pesticides and/or polychlorinated biphenyls (PCBs), are of great concern since they bioaccumulate in adipose tissues of biota throughout the food chain, at top of which are humans, and create a lasting toxic body burden. During lactation, transfer of POPs into human brest milk occurs, thus breastfeeding provides a significant source of exposure to POPs early in infant life, the effects of which are unknown, and is the subject of a growing body of research. However, despite the possibility of harm from environmental contaminants in breast milk, experimental evidence suggests that, barring certain health issues, human breast milk is the best source of nourishment for human infants. In any case, measures aimed at reduction of toxic chemicals, regardless of how they are formed during processing or are due to environmental pollution, have to be adopted to minimise the risk of toxic effects.

While many data illustrating bioaccumulation of various halogenated environmental pollutants in human milk are available (e.g. [START_REF] Heifetz | Mother's milk or mother's poison? Pesticides in breast milk[END_REF][START_REF] Golding | Unnatural constituents of breast milk -medication, lifestyle, pollutants, viruses[END_REF] [START_REF] Kalantzi | Different levels of polybrominated diphenyl ethers (PBDEs) and chlorinated compounds in breast milk from two UK regions[END_REF]), the preliminary information on the occurrence of persistent chlorine-containing processing contaminants, such as 3-MCPD esters, has been mentioned only recently [START_REF] Velíšek | Historical perspective on 3-MCPD in foods and recent research. 3-MPCD Stakeholders[END_REF]. Based on the assumption of similarity in bioaccumulation potential in human fat tissue and possible transfer of lipophilic 3-MCPD esters into human milk, we attempted to analyse them in the isolated breast milk fat.

Materials and methods

Chemicals

3-Chloropropane-1,2-diol (> 98%, 3-MCPD) was purchased from E. Merck (Darmstadt, Germany), 3-MCPD-d 5 (99.4%) from Dr. Ehrenstorfer (Augsburg, Germany), phenylboronic acid (PBA, ≥ 97%) from Fluka Chemie (Buchs, Switzerland). 1,2-Diacyl-3-chloro-1,2propanediols, symmetric and asymmetric (mixed) diesters of 3-MCPD with lauric, myristic, palmitic, stearic, and oleic acids, respectively, were synthesised according to [START_REF] Kraft | Massenspektrometrische Strukturuntersuchung stellungsisomerer Fettsäureester der Halogenpropandiole (Desoxyglyceride)[END_REF] and purified on a silica gel column using light petroleum ether/diethyl ether mixtures (see below). Isotopically labelled 3-MCPD-d5 1,2-dipalmitate, i.e. was synthesised employing 3-MCPD-d 5 and purified using the same procedures. The GC purity of symmetric 3-MCPD diesters was in the range of 94.4% and 99.4%. The asymmetric 3-MCPD diesters were mixtures with the corresponding symmetric 3-MCPD diesters and their content was in the range of 44.4% and 49.3%. All other reagents and solvents were of analytical purity.

Samples

Human breast milk samples were collected from healthy native Czech mothers living in the Prague and East Bohemia regions. Three samples were collected within 1-2 weeks, 7 samples within 1-2 months, 1 sample after 4 months and 1 sample after 11 months after childbirth.

Ages of the mothers were within the range of 18-36 years (mean 28.1, median 28.5). One of the mothers (29 years old) provided us with a series of 6 samples collected 14, 49, 70, 71, 74 and 76 days after childbirth. The breast milk samples were collected in cleaned glass containers and stored at -20 o C until analysis, then thawed and used for isolation of milk fat.

Isolation of milk fat

Milk (about 20 ml) was transferred to a 100 ml separatory funnel by a volumetric cylinder, weighed and 2 ml of saturated solution of potassium oxalate, 20 ml of ethanol (96%, v/v) and 40 ml of a mixture of hexane-diethyl ether (1:1, v/v) were added. The mixture was shaken for 15 minutes and the aqueous layer was transferred to a second separatory funnel. To this funnel, 10 ml of ethanol and 20 ml of hexane-diethyl ether mixture were added and the content was shaked for additional 5 minutes. The aqueous layer was discarded and the upper layer combined with the upper organic layer from the first separatory funnel. The extract was then twice partitioned with 5 ml of water, the aqueous layer was again discarded, the upper layer was dried over anhydrous sodium sulphate and evaporated to dryness at 40 o C using a rotary vacuum evaporator [START_REF] Schenzler | European standardization of methods for pesticide residue analysis in foods -current status[END_REF]. The residue was redissolved in hexane (2 ml) and a 0.5 ml aliquot was dried at 80 o C to determine the lipid content gravimetrically.

Analysis of 3-MCPD diesters

The isolated milk fat (about 200 mg) dissolved in hexane (1 ml) containing 3-MCPD-d5 1,2dipalmitate (7.26 µg) was placed onto the top of a silica gel column 330 x 20 mm (silica gel 60, 70-230 mesh, Merck, Darmstadt, Germany). The flask was washed out with 2 ml of a mixture of light petroleum ether (b.p. 40-65 o C) with diethyl ether (95:5, v/v) and the column was eluted at a flow rate of 4 ml/min using 500 ml of the solvent. The eluent containing 3-MCPD diesters was evaporated using a rotary evaporator and the residue was dissolved in tetrahydrofurane (200 µl). An aliquot of this solution (1 µl) was analysed by GC/MS.

Determination of free and bound 3-MCPD

Free and bound 3-MCPD were analysed following the method described earlier [START_REF] Zelinková | Fatty acids esters of 3chloropropane-1,2-diol in edible oils[END_REF]. Briefly, the milk fat (5 g) with added 3-MCPD-d 5 (internal standard) was extracted with a hexane:acetone (1:1, v/v) mixture and obtained extract was derivatised with phenylboronic acid and used for the determination of free 3-MCPD by GC/MS. For the determination of bound 3-MCPD by GC/MS, the milk fat (100 mg) was dissolved in tetrahydrofurane, treated with 1.8 ml sulphuric acid solution (98%, 1.8 ml in 100 ml methanol), neutralised using saturated NaHCO 3 solution, spiked with 3-MCPD-d 5 and derivatised with phenylboronic acid. Three parallel examinations of each milk sample were made.

Orthogonal gas chromatography (GC x GC) coupled with high-speed time-of-flight mass spectrometry (TOF-MS) was used for confirmation of results obtained by the above method.

Four randomly selected samples were used for this purpose.

Instrumentation and operating conditions

Statistical methods

Statistical evaluation of the obtained results was done employing the computer program SPSS

for Windows, Release 11.0.0, Standard Version (SPSS Inc., Chicago, IL, USA).

Results and discussion

The advantages of breast-feeding both for infant and mother have been widely acknowledged: it offers superior nutrition, protection against infection, enhancement of the immune system, a contraceptive effect while lactating, economic benefits, and emotional support. At the same time, mother's milk is a good pollution indicator. The pharmacokinetics of POPs transfer from mother to infant via breastfeeding is a complex process that is strongly influenced by a particular chemical characteristic of all POPs, their distinct affinity for fat. When a woman begins lactating, her fat stores are mobilized to efficiently excrete lipids and correspondingly also POPs are excreted to breast milk. She effectively transfers her own body burden of pollutants to her newborn. The lipid content in breast milk may have concentrations of POPs ten times higher than lipids of ordinary food [START_REF] Heifetz | Mother's milk or mother's poison? Pesticides in breast milk[END_REF][START_REF] Bauchner | Environmental contaminants and human milk[END_REF]. Considering high amounts of lipophilic 3-MCPD esters in various foodstuffs, we hypothesized on potential transfer of these chemicals into breast milk in a similar way as known for POPs. It should be noted that, until now, no information on the excretion of chlorinated food contaminants such as 3-MCPD esters into human milk has been available.

The preliminary experiments employing conventional GC/MS procedure (unit resolution, single quadrupole mass analyser operated in a selected ion monitoring mode) for analysis of derivatised extract obtained from milk fat hydrolysate indicated the presence of 3-MCPD, nevertheless, due to a high chemical noise in such a complex matrix and relatively low selectivity of selected ions, unbiased proof of target analyte presence in human milk fat was needed. For this purpose, a novel approach represented by comprehensive -orthogonal gas chromatography (GCxGC) coupled with high speed time of flight mass spectrometry (TOF-MS) was employed for analysis of randomly selected 4 breast milk samples. This challenging technique offers both high resolution power on the GC side and full mass spectral information, even at low analyte level, enabling identification. In Figure 1, there is shown an example of the chromatographic record obtained by GCxGC-TOF-MS analysis of sample no.

1; the presence of bound 3-MCPD in breast milk fat (analyte released from the respective esters by hydrolysis) was unambiguously demonstrated. The content of bound 3-MCPD determined by this technique in samples no. 1, 2, 6 and 11 was 1147, 771, 618, and 705 µg/kg fat, respectively. These results were in good agreement with data obtained by routine GC/MS method that was used for analysis of the whole set of samples (Table I). As shown in Table I, free 3-MCPD was not detected in any of milk samples, while the bound 3-MCPD was present in all of them. Its levels ranged from the LOD (100 µg/kg fat) up to 2195 µg/kg fat, the mean value was 1014 µg/kg fat. Considering a large variability of fat content in mother's milk (1.7% to 7.2%, mean 3.8%), then the calculated bound 3-MCPD content of the breast milks lays within the range of <11 µg/kg and 76 µg/kg (mean 35.5 µg/kg milk).

To visualise the relationships between the bound 3-MCPD in individual breast milk samples and the other individual parameters (variables), partial correlation analysis was done using the data presented in Table I. The only meaningful positive correlation (r = 0.735) was obtained between the level of bound 3-MCPD content in breast milk and its fat content (Pearson correlation was significant at the 0.01 level, 2 tailed). The fat content or mother's age as well as the date of milk sample collection after childbirth did not correlate with any other variable. In the final phase of this study, the individual naturally occurring 3-MCPD esters were investigated in samples no. 1, 2, 6, and 11 employing the GC/MS method for examination of the diester fraction isolated from human milk fat. According to our previous results, the 3-MCPD diesters prevail over the 3-MCPD monoesters (Zelinková et al. 2007). The major types of 3-MCPD diesters in breast milk no. 1 were symmetric diesters with lauric, palmitic, and oleic acids and asymmetric diesters with palmitic acid/oleic acid (Table II). The asymmetric diesters bearing 2 different acyl groups (i.e. oleoyl/palmitoyl) can be seen in 2 GC/MS records corresponding to m/z 331 and m/z 357, which are formed by the fission of [M-RCOOH] + involving either palmitic or oleic acid. As it can be seen, the major 3-MCPD diester of this breast milk sample was 1,2-dioleoyl-3-chloro-1,2-propanediol (3117 µg/kg fat), which corresponds to the average fatty acid composition of human milk lipids (western diet) where the weight % and range of oleic acid is 31.0% and 22.6-38.7%, respectively [START_REF] Donangelo | Human milk: composition and nutritional value[END_REF]. It is highly probable that several other minor 3-MCPD diesters and monoesters occur in the analysed sample as well. The bound 3-MCPD content calculated using the determined levels of the individual 3-MCPD diesters was 1668 µg/kg fat, which is in a good agreement (134%) with the value given in Table I (1263 µg/kg fat). Analogous results were obtained analyzing breast milk samples no. 2 (505 µg/kg fat, 69%), no. 6 (603 µg/kg fat, 90%), and no. 11 (851 µg/kg fat, 113%), respectively. Figure 2 is an example of chromatograms obtained analysing the individual diesters in the breast milk sample no. 1.

Table III summarises the results obtained by analysing the second series of samples collected from one of the nursing mothers 14, 49, 70, 71, 74 and 76 days after childbirth (sample no. 13-18). As shown here, their fat content was relatively low and ranged from 0.92% to 1.93% (mean 1.48%). All these samples contained bound 3-MCPD within the range of 328 µg/kg fat and 2078 µg/kg fat (mean 930 µg/kg fat). The calculated bound 3-MCPD content of these samples was within the range of 6 µg/kg milk and 19 µg/kg milk (mean 12 µg/kg milk) but no meaningful correlation between any variables was found.

Question arises about the origin of 3-MCPD esters in human breast milk. It has been already

shown that various foodstuffs such as crackers, donuts (doughnuts), cookies, French fries (chips), baked goods, snack foods, fried foods, many other processed foods, and, especially, the refined vegetable oils may contain elevated levels of 3-MCPD esters [START_REF] Svejkovská | Esters of 3chloropropane-1,2-diol in foodstuffs[END_REF], Hamlet and Sadd 2004[START_REF] Doležal | Occurrence of 3chloropropane-1,2-diol and its esters in coffee[END_REF][START_REF] Zelinková | Fatty acids esters of 3chloropropane-1,2-diol in edible oils[END_REF][START_REF] Divinová | Free and bound 3-chloropropane-1,2-diol in coffee surrogates and malts[END_REF]. It is, therefore, highly probable that these and some other dietary items become the Currently, there is no information available on how are these esters metabolised, to which extent are they hydrolysed or biosynthesised in the body, to which extent they deposit in tissues and how do they influence the properties and functions of tissues (if they really do it) is not known.

Regarding dietary intake the following scenario could be considered. The baby is breastfed for up to 4 months with only his/her mother's milk with the average content of bound 3-MCPD of 35.5 µg/kg (Table I) and 3-MCPD esters of the milk are totally hydrolysed in his/her body by lipases so that 3-MCPD is released in its free form. An average daily intake of mother's milk by the baby is about 750 ml (approximate density of 1 g/ml) with a range of 570-900 ml per day (Figure 3). Under these conditions the amount of 3-MCPD taken by the baby per day is 26.625 µg. Considering tolerable daily intake (TDI) of 2 µg/kg body weight/day (JECFA 2001) and the ideal weight score of a newborn baby 3.25 kg (on average boys are 0.3 kg heavier and girls 0.3 kg lighter than the ideal weight score), then his/her daily intake of 3-MCPD is 4.1 times higher than that corresponding to TDI. The TDI level of 6.5 µg/day is exceeded after the consumption of 183 ml of such breast milk. The situation significantly improves with the age of the baby (the body weight increases) as at the age of 4 months he/she takes daily only 2.1 times higher amount of 3-MCPD than the amount corresponding to TDI (Figure 3). MCPD esters are principally processing contaminants. It has been shown, for example, that appropriate manufacturing controls the levels of MCPD esters in edible oils but strategies to reduce these compounds in other food products have not yet been fully explored. Identifying primary routes of 3-MCPD esters exposure, their mitigation, metabolism and/or biosynthetic pathways and biological effects are subjects for further research.

  analyses were generally carried out on an Agilent Technologies 6890N gas chromatograph (Agilent Technologies, Palo Alto, CA, USA) equipped with a Series 5975 quadrupole mass selective detector Agilent 5973 MSD (70 eV) and data processing system (MSD Productivity ChemStation, Revision D.02.00 SP1).4-Chloromethyl-2-phenyl-1,3,2-dioxaborolane, the product obtained by derivatization of 3-MCPD with phenylboronic acid, was analysed using a GC capillary column Equity-1 (30 m x 0.25 mm x 1 µm; Supelco, Bellefonte, PA, USA). The injector was held at 250 o C (splittles), the column temperature was programmed from 80 o C (1 min) to 300 o C (37 min) at a rate of 10 o C/min. Helium at a flow rate of 0.8 ml/min was used as the carrier gas, 1 µl sample was injected. For quantification purposes, single ion monitoring was used to monitor ions at m/z 147 (3-MCPD) and at m/z 150 (3-MCPD-d 5 ). Ions at m/z 91and 196 (3-MCPD) and at m/z 93 and 201 (3-MCPD-d 5 ) were used as qualifiers. The performance characteristics were as follows: the limit of detection (LOD) was 100 µg/kg milk fat, RSD (3 injections, 0.3282 µg 3-MCPD/ml) = 5.9%, linearity (0.005-8.575 µg 3-MCPD/ml), r 2 = 0.9998.The GC×GC/TOF-MS system, used for examination of 4 selected samples, consisted of a HP 6890 (Agilent Technologies, Palo Alto, CA, USA) gas chromatograph with split-splittles injector. The detector was Pegasus III, high speed time-of-flight mass spectrometer (LECO, St. Joseph, MI, USA) with 10 ml/min pumping capacity and operated in electron ionisation mode (EI). Inside the GC oven, a dual-stage jet modulator and the secondary oven were mounted. Resistively heated air was used as a medium for hot jets, while cold jets were supplied by gaseous nitrogen, secondary cooled by liquid nitrogen. The instrumental set-up of GC consisted of a primary column DB5ms (30 m × 0.25 mm × 0.25 µm; Agilent Technologies, Palo Alto, CA, USA) and a secondary column BPX50 (2.2 m × 0.1 mm × 0.1 µm; SGE Ringwood, Australia). The oven temperature program was as follows: 80°C for 1 min, 10°C/min to 250°C, 5 min at 250°C, secondary oven was held 5°C above the main oven; helium flow rate was 1.0 ml/min; injection mode: splittles for 1.0 min; injection temperature: 270°C; modulation time: 5 s (hot pulse 1.5 s); modulation temperature offset: 15°C. The instrumental of the mass spectrometer was as follows: solvent delay 350 s; acquisition rate 125 Hz; mass range 45-500 amu; ion source temperature 220°C; transfer line temperature 270°C; detector voltage -1700V. The performance characteristics were as follows: LOD = 3 MCPD/kg milk fat, RSD (5 injections, 0.0515 µg 3-MCPD/ml) = 6 .5%, linearity (0.01-0.515 µg 3-MCPD/ml), r 2 = 0.9942. GC/MS analysis of 3-MCPD diesters was performed on a capillary column DB-1HT (15 m x 0.25 mm i.d., film thickness 0.1 µm, Agilent Technologies, Palo Alto, CA, USA). The injector was held at 300 o C (pulsed splittles), the column temperature was programmed from 170 o C (1 min) to 305 o C at a rate of 5 o C/min and then to 400°C (10 min) at a rate of 40 o C/min. Helium at a flow rate of 0.7 ml/min was used as the carrier gas, 1 µl sample was injected. Ionization was performed by electron impact at 70 eV and temperature of 250°C. For quantification purposes, single ion monitoring was used to monitor the ions [M-RCOOH] + at m/z 275 (3-MCPD 1,2-dilaurate), m/z 303 (3-MCPD 1,2-dimyristate), m/z 331 (3-MCPD 1,2dipalmitate), m/z 336 (3-MCPD-d5 1,2-dipalmitate), m/z 331 (mixed palmitate and oleate of 3-MCPD), m/z 357 (3-MCPD 1,2-dioleate), and m/z 359 (3-MCPD 1,2-distearate). The ions [M-RCOOH+2] + and [RCO] + were monitored for confirmation.

  3-MCPD esters occurring in breast milk.

Figure 1 .

 1 Figure 1. Detection and identification of 3-MCPD released from breast milk fat (sample no. 1) by CGxGC-TOF-MS. A=contour plot of particular part of chromatogram with target analyte, B=mass spectrum of 3-MCPD after derivatization using 4-chloromethyl-2-phenyl-1,3,2dioxaborolane, C=3D chromatogram showing target analyte (zoom).

Figure 2 .

 2 Figure 2. GC/MS analysis of 3-MCPD fatty acid esters in breast milk sample no. 1.

Figure 3 .

 3 Figure 3. Ideal weight score, TDI and 3-MCPD/TDI during the first four months after childbirth. ♦ = ideal weight score in kg (calculated from: http://www.medindia.net/patients/calculators/ ideal_weight_result.asp), ▪ = TDI in µg/day, •

Table I .

 I Characteristics of breast milk samples, their content of fat and bound 3-MCPD.

	Sample Age Collected* Fat content Bound 3-MCPD RSD Bound 3-MCPD
	no. [years] [months]	[%]	[µg/kg fat]	[%] [µg/kg breast milk]
	1	18	1	5.00	1263	8.8	63
	2	24	4	3.40	729	3.9	25
	3	26	1	2.40	471	4.1	11
	4	27	0.25	4.27	1789	1.0	76
	5	28	11	1.70	916	3.5	16
	6	28	1	3.90	671	6.0	26
	7	29	0.25	2.02	2195	7.2	44
	8 9 10 11 12	29 29 30 33 36 F 0.5 1 2 2 2 o r	2.93 3.75 6.60 7.20 2.80	1952 <300 <300 753 833	3.5 --5.3 13.5	57 <11 <20 54 23
	* After childbirth.	P
			e
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						O n l
							y

Table II .

 II Levels of individual 3-MCPD diesters in breast milk sample no. 1.

	Fatty acids in 3-MCPD diesters	Diesters content	Bound 3-MCPD
		[µg/kg fat]	[µg/kg fat]
	Lauric/Lauric	662	154
	Myristic/Myristic	nd	nd
	Palmitic/Palmitic	2816	530
	Palmitic/Oleic	2580	465
	Oleic/Oleic Stearic/Stearic Total nd=not detected. F o r	3117 nd 9175	539 nd 1688
	P
	e
	e r
		R
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		v i e
		w
			O n l
			y

Table III .

 III The dynamics of bound 3-MCPD during breast feeding (39 years old mother).

	Sample Collected* Fat content Bound 3-MCPD RSD Bound 3-MCPD
	no.	[days]	[%]	[µg/kg fat]	[%] [µg/kg breast milk]
	13	14	1.67	612	4.7	10
	14	49	1.28	1450	2.1	19
	15	70	1.76	328	3.0	6
	16	71	0.92	2078	21.4	19
	17	74	1.31	617	9.4	8
	18	76	1.93	493	5.2	10
	* After childbirth.

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manuscriptcentral.com/tfac Email: fac@tandf.co.

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants

Acknowledgement

This work was substantially part of the Research Project MSM6046137305 supported by the Ministry of Education and Youth of the Czech Republic. Development of analytical methods aimed at confirmation of bound 3-MCPD was supported by the project no. 2B06168 -National program of research.


Veronica Maria

Teresa Lattanzio

Michele Solfrizzo

V M T Lattanzio

A Visconti

Determination of trichothecenes in cereals and derived products by liquid chromatography tandem mass spectrometry

Keywords: trichothecenes, Mycotoxins -trichothecenes Food Types: Cereals, Processed foods LC-MS/MS, nivalenol, deoxynivalenol, T-2 toxin, HT-2 toxin, cereals, cereal products, mycotoxins

published or not. The documents may come

Determination of trichothecenes in cereals and derived

products by liquid chromatography tandem mass spectrometry

different Fusarium species that may develop in field mainly in wheat, maize, barley and oats [START_REF] Bottalico | Fusarium diseases of cereals: species complex and related mycotoxin profiles in Europe[END_REF][START_REF] Who | Safety Evaluation of Certain Mycotoxins in Food. Prepared by the Fifty-sixth Meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA). WHO Food Additives Series 47/FAO Food and Nutrition Paper[END_REF]. Trichothecenes are responsible for a wide range of disorders in animals including feed refusal, weight loss and vomiting and have been found to inhibit protein, DNA and RNA synthesis, and to have immunosuppressive and cytotoxic effects [START_REF] Visconti | Cytotoxic and immunotoxic effects of Fusarium mycotoxins using a rapid colorimetric bioassay[END_REF][START_REF] Prelusky | Toxicology of mycotoxins[END_REF][START_REF] Visconti | Problems associated with Fusarium mycotoxins in cereals[END_REF], [START_REF] Who | Safety Evaluation of Certain Mycotoxins in Food. Prepared by the Fifty-sixth Meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA). WHO Food Additives Series 47/FAO Food and Nutrition Paper[END_REF]. The European Scientific Committee on Food (SCF) evaluated in a set of opinions the Fusarium toxins, including DON, NIV, T-2 and HT-2 [START_REF] Scf | Opinion of the Scientific Committee on Food on Fusarium toxins. Parts 6: Group evaluation of T-2 toxin, HT-2 toxin, nivalenol and deoxynivalenol, adopted on 26[END_REF] and established a fully tolerable daily intake (TDI) for DON at 1 µg/kg body weight/day, a temporary TDI (t-TDI) for NIV at 0.7µg/kg body weight/day, and a combined t-TDI for T-2 and HT-2 at 0.06 µg/kg body weight/day. Health risks associated with human exposure to Fusarium toxins are worldwide recognized and depend on the extend to which they are assumed in a diversified diet. The major source of dietary intake of trichothecenes are products made from cereals, in particular wheat and maize. Processing of cereal crops into foods and commercial or home preparation of cereal based foods may decrease the level of mycotoxins but does not completely eliminate them [START_REF] Visconti | Reduction of deoxynivalenol during durum wheat processing and spaghetti cooking[END_REF]). Therefore, the presence of trichothecenes in the human diet, and especially in the diet of vulnerable populations such as infants, is of great concern. However, limited information are available on the distribution of these mycotoxins in cereal based foods [START_REF] Lombaert | Mycotoxins in infant cereal foods from the Canadian retail market[END_REF][START_REF] Jestoi | Levels of mycotoxins and sample cytotoxicity of selected organic and conventional grain-based products purchased from Finnish and Italian markets[END_REF], especially in infant foods, also due to the lack of reliable methods for trichothecene determination at low levels (µg/kg).

A recent data collection on the occurrence of Fusarium toxins in food in the European Union (EU), performed within an ad hoc SCOOP project [START_REF] Scoop | SCOOP TASK 3.2.10: Collection of occurrence data of Fusarium toxins in food and assessment of dietary intake by the population of EU Member States[END_REF], showed that the dietary intakes of Fusarium toxins for the entire population and adults are often lesser than the TDIs for the respective toxin. Nevertheless for risk groups like infants and young children, the dietary intake of DON was found close to the TDI, and T-2 and HT-2 intakes were found to be higher than the t-TDI. As underlined in the same SCOOP report, intake data were strongly influenced 4 by the limit of detection (LOD) of the analytical method used since negative samples (80% for T-2 and 86% for HT-2) were assumed to contain toxins at level of LOD/2. Therefore an overestimation of the intake was reported since LODs were up to 100 µg/kg.

Sensitive, accurate and reliable methods of analysis for these toxins in cereals and cereal-based foods are needed to better estimate human exposure and to assess compliance of food with European Union regulations, which are already in place for DON and programmed for T-2 and HT-2. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is becoming the technique of choice for the simultaneous determination of type-A and type-B trichothecenes [START_REF] Berger | Quantitative determination and structure elucidation of type-A and B-trichothecenes by HPLC/Ion trap multiple mass spectrometry[END_REF][START_REF] Berthiller | Rapid simultaneous determination of major type A-and B-trichothenenes as well as zearalenone in maize by high performance liquid chromatography-tandem mass spectrometry[END_REF][START_REF] Biselli | Development of a multicomponent method for Fusarium toxins using LC-MS/MS and its application during a survey for the content of T-2 toxin and deoxynivalenol in various feed and food samples[END_REF][START_REF] Cavaliere | Development of a multiresidue method for analysis of major Fusarium mycotoxins in corn meal using liquid chromatography/tandem mass spectrometry[END_REF].

Besides the high sensitivity, tandem mass spectrometry provides also the highest degree of certainty in analyte identification and may be employed in accordance with recent European Union guidelines to obtain data with relevant unambiguity (European Commission 2002).

Although the high selectivity of LC-MS/MS instruments should allow to reduce and simplify sample preparation, the presence of co-extracted matrix compounds can affect the analyte ionization, leading to signal suppression or enhancement that affect the accuracy of the measurement.

Methods using MycoSep ® #227 multifunctional clean-up column (containing charcoal, celite and alumina) are simple and used in several laboratories for simultaneous analysis of type-A and type-B trichothecenes, although low NIV recoveries have been reported (Langseth & Rundberget 1998[START_REF] Berthiller | Rapid simultaneous determination of major type A-and B-trichothenenes as well as zearalenone in maize by high performance liquid chromatography-tandem mass spectrometry[END_REF][START_REF] Biselli | Development of a multicomponent method for Fusarium toxins using LC-MS/MS and its application during a survey for the content of T-2 toxin and deoxynivalenol in various feed and food samples[END_REF].

In The method was used to analyze 57 samples of durum ground wheat obtained from wheat produced in Apulia region (South Italy) and 9 cereal-based food samples collected from retail market.

Materials and Methods

Chemicals and Reagents

Acetonitrile and methanol (both HPLC grade) were purchased from Mallinckrodt Baker (Milan, Italy). Ultrapure water was produced by a Millipore Milli-Q system (Millipore, Bedford, MA, USA). Ammonium acetate (for mass spectrometry), NIV, DON, T-2, and HT-2, were purchased from Sigma-Aldrich (Milan, Italy). Oasis ® HLB columns were purchased from Waters (Milan, Italy), MycoSep ® #227 columns from Romer Labs ® Diagnostic GmbH (Herzogenburg, Austria).

Filter papers (Whathman no. 4) were obtained from Whathman International Ltd., Maidstone, UK. HPLC syringe filters (0.45 µm) were from Alltech (Deerfield, IL, USA).

Sample preparation and clean up with Oasis ® HLB columns

Ground samples (10 g) were extracted with 50 ml acetonitrile/water (84:16, v/v) -20 ng toxin injected (corresponding to 10 -1000 µg/kg when referred to the initial sample).

For LC-MS/MS infusion experiments two solutions of each trichothecene, at concentration of 1 µg/ml, were prepared in methanol/water (40/60, v/v) with or without ammonium acetate (5mM).

Recovery experiments

For recovery experiments from the Oasis ® HLB columns, dried blank crude sample extracts (equivalent to 1 g matrix) of infant semolina were spiked with 100 µg/kg of each toxin, dissolved in 1 ml methanol/water (10/90, v/v) and passed throughout different Oasis ® HLB columns. Then each column was eluted with 1 ml of a methanol/water mixtures having different methanol content (10, 20, 30, 40, 45, 50, 55, 60, 70, 100%). Twenty microliters of each eluate were analyzed by LC-MS/MS.

Recovery experiments for the whole method were carried out in quadruplicate by spiking cerealbased food samples with each toxin at levels of 10, 100, 500, and 1000 µg/kg, and raw cereals at 100 µg/kg. Spiked samples were left overnight to allow solvent evaporation prior to extraction.

After breaking lumps, samples were extracted and the filtered extracts were purified by Oasis ® HLB or Mycosep ® #227 as described in Sample preparation sections.

In all experiments, the amounts of recovered toxins were calculated by using matrix-matched calibration. The t-test was applied to statistically quantify the slope difference between standard and matrixmatched calibration curves using a significance alpha level of 0.05 (degrees of freedom = 8, critical t value = 2.3). t values were calculated by using the following formula

t (n-4) = (b 1 -b 2 )/es (b1-b2)
were:

N-4 are the degree of freedom; b 1 and b 2 are the slopes of the two calibration curves to be compared; es (b1-b2) is the standard error of the difference between the two slopes calculated as described by [START_REF] Soliani | Manuale di statistica per la ricerca e la professione[END_REF].

LC parameters

The analytical column was a Synergi Hydro ® (150 mm × 3 mm, 4 µm particles) (Phenomenex, USA), preceded by an Aqua C18 guard column (4 mm x 2 mm, 10 µm particles) (Phenomenex, USA). The flow rate of the mobile phase was 400 µl/min, while the injection volume was 20 µl.

The column effluent was directly transferred into the APCI interface, without splitting. Two consecutive isocratic mixtures of methanol/water containing 5 mM of ammonium acetate were used. In particular, 20% methanol was pumped for 9 min followed by 80% methanol for 8 min.

The column was re-equilibrated with 20% methanol for 5 min. Before LC-MS/MS analysis all samples were filtered through 0.45 µm membrane filter.

MS parameters

For LC-MS/MS analyses, the APCI interface was used with the following settings: temperature (TEM) 450 ºC and 380 ºC, for negative and positive ion mode, respectively. Curtain gas (CUR), nitrogen, 30 psi; nebulizer gas (GS1), air, 50 psi; auxiliary gas (GS2), air, 30 psi; corona discharge needle current (NC), -3 or +3 µA, respectively. The mass spectrometer operated in MRM (multiple reaction monitoring) mode, with a dwell time of 100 ms. TEM, 300 ºC, CUR, 20 psi, GS1, 15 psi, GS2, air, 30 psi, ionspray voltage + 4500 V or -4500V, respectively.

Results and discussion

LC-MS/MS optimization

Preliminary studies were conducted to select, for each analyzed trichothecene, the most suitable precursor ion, and to identify the specific transitions to be used in the MRM (multiple reaction monitoring) acquisition mode. Moreover, since in LC -MS detection the response is strongly dependent on the interface conditions [START_REF] Razzazi-Fazeli | Determination of nivalenol and deoxynivalenol in wheat using liquid chromatography -mass spectrometry with negative ion atmospheric pressure chemical ionization[END_REF][START_REF] Scf | Opinion of the Scientific Committee on Food on Fusarium toxins. Parts 6: Group evaluation of T-2 toxin, HT-2 toxin, nivalenol and deoxynivalenol, adopted on 26[END_REF][START_REF] Pallaroni | Optimization of atmospheric pressure chemical ionization interface parameters for the simultaneous determination of deoxynivalenol and zearalenone using HPLC/MS[END_REF], the interface parameters were optimized in order to obtain maximum sensitivity for all tested mycotoxins. For this purpose, trichothecene solutions were directly infused into the mass Based on these results we decided to use the APCI interface in the successive steps of method development.

MS/MS experiments were performed to find the most intense and specific transitions for each trichothecene. For selective detection three transitions were chosen for DON and NIV, and five transitions for T-2 and HT-2, that exhibited a more complex fragmentation pattern. Collision energy (CE) and collision cell exit potential (CXP) were optimized for each selected transition.

The selected transitions for the four toxins and the resulting MRM method are reported in Table 1.

A good separation of NIV, DON, T-2 and HT-2 was obtained by using a Synergi Hydro ® column and two isocratic compositions of methanol/water containing 5 mM ammonium acetate, i.e. 20/80 (v/v) for the separation of NIV and DON, and 80/20 (v/v) for the separation of HT-2

and T-2. Retention times were: 4.1 min for NIV, 7.8 min for DON, 14.8 min for HT-2, and 15.5 min for T-2. As reported in Table 1, the selected precursor ions required the ionization polarity to be switched from negative (for the detection of NIV and DON) to positive (for the detection of HT-2 and T-2). The time windows defined from the two isocratic steps corresponded to the two periods of polarity switching of the APCI interface.

By operating under isocratic conditions it was also possible to define two distinct optimal temperatures of the heated nebulizer in the LC-MS/MS run. It is well documented that the temperature of the nebulizer (TEM) could significantly affect the APCI performances in terms of ionization efficiency [START_REF] Razzazi-Fazeli | Determination of nivalenol and deoxynivalenol in wheat using liquid chromatography -mass spectrometry with negative ion atmospheric pressure chemical ionization[END_REF][START_REF] Scf | Opinion of the Scientific Committee on Food on Fusarium toxins. Parts 6: Group evaluation of T-2 toxin, HT-2 toxin, nivalenol and deoxynivalenol, adopted on 26[END_REF][START_REF] Pallaroni | Optimization of atmospheric pressure chemical ionization interface parameters for the simultaneous determination of deoxynivalenol and zearalenone using HPLC/MS[END_REF]. The optimal temperature is affected not only by the type of sample, but also by the flow rate and the mobile The reconstitution in 10% methanol of dried extracts of infant semolina, spiked at 100 µg/kg of each toxin, allowed complete dissolution of the four toxins. When passed through the Oasis ® HLB column the four toxins were quantitatively retained without any loss of loaded toxins.

Mixtures with different methanol/water ratio were then tested for the washing and elution steps in order to achieve quantitative recoveries for all trichothecenes together with an efficient removal of matrix interfering compounds. Quantitative elution of NIV and DON was obtained by passing 1 ml methanol/water 45/55 (v/v). After washing with 1 ml methanol/water 55/45, quantitative elution of HT-2 and T-2 was obtained by passing 1 ml methanol. A washing step before the elution of NIV and DON was not possible since mixtures of methanol/water with methanol percentages between 10 and 45% resulted in significant loss of NIV. On the other hand a washing step with 55% methanol was possible before the elution of HT-2 and T-2 that occurred with methanol percentage >60%.

Matrix effect study

Despite the high sensitivity and selectivity, the influence of matrix components on the analyte ionization could represent a limit to the accuracy of the LC-MS/MS methods. The co-elution of for the cereals and cereal-based foods considered in this study. Six points standard calibration curves and six points matrix-matched calibration curves were performed for each of the 4 toxins and the 8 matrices tested herein. The t-test was applied to statistically evaluate the slope differences between standard and matrix-matched calibration curves, giving a significance alpha level of 0.05, with 8 degrees of freedom. Under these conditions, the critical t value is 2.3. For t values higher than 2.3, the slope difference is considered statistically significant, i.e.

considerable matrix effects occurs.

The obtained t values, are reported in Table 5. This study revealed that the presence of co-eluting matrix molecules resulted in a general enhancement (20/32 cases) of APCI response of all analyzed mycotoxins, even though in some cases, such as semolina, no significant matrix effects were observed. As expected, the strongest matrix effects were observed for NIV, having this compound the shortest retention time. Moreover, the slope of the regression varied considerably from matrix to matrix also revealing changing influences of co-eluting matrix compounds on the analyte signal. It was evident, for example, that no general regression curve could be used for different raw cereals, but calibration for each individual cereal grain was necessary. On the basis of these findings we conclude that, due to the observed matrix effects, the matrix-matched calibration is necessary for reliable quantitative analyses.

Our results are in agreement with [START_REF] Biselli | Development of a multicomponent method for Fusarium toxins using LC-MS/MS and its application during a survey for the content of T-2 toxin and deoxynivalenol in various feed and food samples[END_REF] that recently performed a similar evaluation of matrix effect by comparison of slope differences between standard and matrix calibration for soybean and oats. The authors reported a strong signal suppression, up to 40%, in soybean extracts purified with Mycosep ® #227 and "nearly no matrix effects" for oats, ground wheat and rye extract, although statistical evaluation of slope differences was not performed.

Matrix effects in trichothecene determination by LC/MS are well known since the first reports, and different approaches have been proposed to overcome this problem. For example, Razzazi- The use of isotope labeled internal standard is another mode to correct matrix effects in LC-MS/MS analyses. However few isotope labeled standards are commercially available. In particular, they are available for DON and T-2 but not for NIV and HT-2 [START_REF] Häubl | Suitability of a fully 13 C isotope labeled internal standard for the determination of the mycotoxin deoxynivalenol by LC-MS/MS without clean up[END_REF][START_REF] Bretz | Stable isotope dilution of the Fusarium mycotoxins deoxynivalenol and 3-acetyldeoxynivalenol[END_REF]. To date the most frequently used approach is the matrix-matched calibration [START_REF] Laganà | Liquid chromatography/tandem mass spectrometry for the identification and determionation of trichothecenes in maize[END_REF][START_REF] Cavaliere | Development of a multiresidue method for analysis of major Fusarium mycotoxins in corn meal using liquid chromatography/tandem mass spectrometry[END_REF][START_REF] Dall'asta | Simultaneous detection of type A and type B trichothecenes by LC/ESI-MS by using NaCl as cationization agent[END_REF][START_REF] Razzazi-Fazeli | Simultaneous determination of major B-trichothecenes and de-epoxy-metabolite of deoxynivalenol in pig urine and maize using highperformance liquid chromatography-mass spectrometry[END_REF]). An alternative approach has been recently suggested to minimize or eliminate matrix effect by a drastic dilution of the sample extract combined with the use of high sensitive MS/MS instruments [START_REF] Sulyok | Development and validation of a liquid chromatography/tandem mass spectrometric method for the determination of 39 mycotoxins in wheat and maize[END_REF]). However, signal enhancement/suppression was still observed for wheat and maize confirming that matrix effect have to be always carefully evaluated.

Performances of the analytical method

Method validation was performed in three different cereals (wheat, barley, and maize) and five cereal based foods (infant semolina, infant biscuits, bacon biscuits, cocoa wafers and coconut snacks). Matrix-matched calibration curves for all analytes were linear over the working range of 10 µg/kg -1000 µg/kg, for all tested matrices, with r values between 0.9997 and 0.9999. Limits of detection for all investigated matrices are reported in Table 2. LOD values (S/N = 3) ranged from 2.5 to 4.0 µg/kg for NIV, from 2.8 to 5.3 µg/kg for DON, from 0.4 to 1.7 µg/kg for HT-2, and from 0.4 to 1.0 µg/kg for T-2. These results confirm that the use of the MRM detection mode allows not only a high degree of selectivity, but also high sensitivity. Similar LODs have been obtained by other authors [START_REF] Berthiller | Rapid simultaneous determination of major type A-and B-trichothenenes as well as zearalenone in maize by high performance liquid chromatography-tandem mass spectrometry[END_REF][START_REF] Biselli | Development of a multicomponent method for Fusarium toxins using LC-MS/MS and its application during a survey for the content of T-2 toxin and deoxynivalenol in various feed and food samples[END_REF], Lagana' et al. 2003) that developed MRM methods for trichothecene detection, while previous methods based on Single Ion Monitoring (SIM) generally achieved higher detection limits [START_REF] Dall'asta | Simultaneous detection of type A and type B trichothecenes by LC/ESI-MS by using NaCl as cationization agent[END_REF][START_REF] Razzazi-Fazeli | Determination of nivalenol and deoxynivalenol in wheat using liquid chromatography -mass spectrometry with negative ion atmospheric pressure chemical ionization[END_REF][START_REF] Scf | Opinion of the Scientific Committee on Food on Fusarium toxins. Parts 6: Group evaluation of T-2 toxin, HT-2 toxin, nivalenol and deoxynivalenol, adopted on 26[END_REF][START_REF] Scoop | SCOOP TASK 3.2.10: Collection of occurrence data of Fusarium toxins in food and assessment of dietary intake by the population of EU Member States[END_REF].

Results of recovery experiments from infant semolina, infant biscuits, bacon biscuits, cocoa wafers and coconut snacks are reported in 2002).

Performances of the present method were compared with those of a previously reported method using Mycosep ® #227 columns for clean up of the extract [START_REF] Berthiller | Rapid simultaneous determination of major type A-and B-trichothenenes as well as zearalenone in maize by high performance liquid chromatography-tandem mass spectrometry[END_REF]. For this purpose recovery experiments were carried out in both cereals and cereal-based foods spiked at 100 µg/kg of each toxin and the results are reported in Table 4. Good and comparable recovery values were obtained for DON, T-2 and HT-2 with the two methods, whereas significantly higher NIV recoveries were obtained for all tested matrices with the present method as compared with the method using the Mycosep columns. For cereal-based products the different recoveries of NIV were more pronounced between the two types of column as shown in Figure 2. In particular, for infant semolina, infant biscuits, cocoa wafers and coconut snacks mean NIV recoveries were 35% and 80% for Mycosep ® #227 and Oasis ® HLB columns, respectively. The low NIV recoveries (31-37%, see Table 4) obtained from cereal-based products (infant semolina, infant biscuits, cocoa wafers, coconut snacks) purified with Mycosep ® #227columns could be correlated to their sugar content considering that recoveries from raw cereals and bacon biscuits were higher and in the range of 56-66%. By using Mycosep ® 226 column clean up, higher recoveries for NIV (89%), but low recoveries for the less polar HT-2 and T-2 toxins (54% and 71%, respectively) have been reported in wheat [START_REF] Biselli | Development of a multicomponent method for Fusarium toxins using LC-MS/MS and its application during a survey for the content of T-2 toxin and deoxynivalenol in various feed and food samples[END_REF]. The use of Oasis ® HLB columns for clean up provided good recoveries for all toxins and matrices tested herein showing a wide range of applicability of the present method. Acceptable recoveries have been reported in corn and corn meal for the four toxins considered in this study after a time consuming clean up procedure by carbograph-4 cartridge applied to 1 l of diluted extract [START_REF] Laganà | Liquid chromatography/tandem mass spectrometry for the identification and determionation of trichothecenes in maize[END_REF][START_REF] Cavaliere | Development of a multiresidue method for analysis of major Fusarium mycotoxins in corn meal using liquid chromatography/tandem mass spectrometry[END_REF]. A widespread contamination of DON (8 positives out of 9 tested samples) was found in cerealbased food samples, and DON contamination levels were significantly higher as compared to T-2 and HT-2. Three samples were found contaminated with the four mycotoxins whereas the other samples contained different combinations of toxins. Interestingly, in most cereal-based food samples, the co-occurrence of two or more trichothecenes was found, confirming the need and the relevance of analytical methods for simultaneous monitoring of these trichothecenes in cereal-based foods. The percentage of positive samples for T-2 and HT-2 was higher than that reported for similar products in the SCOOP ( 2003) report. This finding can be attributed to the higher sensitivity of the method used herein. The levels of T-2 and HT-2 found in this study were lower than the mean levels reported in the SCOOP report supporting the hypothesis that the SCOOP data were overestimated due to the high LOD of the analytical methods reported therein.

Conclusions

F

  Determination of trichothecenes in cereals and derived products by liquid chromatography tandem mass spectrometry Introduction Trichothecenes causing most concerns for human health are T-2 toxin (T-2) and HT-2 toxin (HT-2), as representative of type-A trichothecenes, deoxynivalenol (DON) and nivalenol (NIV), as representative of type-B trichothecenes. These toxins occurr in cereal grains and are produced by

  this paper we describe a new sample preparation procedure for determination of NIV, DON, HT-2 and T-2 in cereals and cereal-based products by LC-MS/MS. The developed method, using Oasis ® HLB SPE columns to cleanup sample extracts, allows high recoveries for both polar (NIV, DON) and non polar (HT-2, T-2) toxins. A detailed investigation of matrix effects in different cereal and cereal-based foods has also been carried out.

For

  method development three raw cereals, namely wheat, maize and barley, and five cerealbased foods, namely infant semolina, infant biscuits, bacon flavored biscuits, wafers and coconut snacks containing wheat, barley, oats, rice and maize were used. All these samples were collected from the Italian retail market, between 2005 and 2006. Durum ground wheat samples were obtained from grains cultivated in Apulia region (South of Italy) in 2005 and 2006. Instrumentation LC-MS/MS analyses were performed on a Q TRAP ® MS/MS system, from Applied Biosystems (Foster City, CA, USA) equipped with either an APCI (atmospheric pressure chemical ionization) or an ESI (electrospray ionization) interface and a 1100 Series micro-LC system comprising a binary pump and a microautosampler from Agilent Technologies (Waldbronn, Germany).

  Matrix effects were evaluated by comparing standard and matrix-matched calibration curves.

  spectrometer and analyzed with two different interfacing systems, ESI and APCI. In agreement with previously published studies[START_REF] Berthiller | Rapid simultaneous determination of major type A-and B-trichothenenes as well as zearalenone in maize by high performance liquid chromatography-tandem mass spectrometry[END_REF][START_REF] Biselli | Development of a multicomponent method for Fusarium toxins using LC-MS/MS and its application during a survey for the content of T-2 toxin and deoxynivalenol in various feed and food samples[END_REF][START_REF] Razzazi-Fazeli | Simultaneous quantification of Atrichothecene mycotoxins in grains using liquid chromatography-atmospheric pressure chemical ionization mass spectrometry[END_REF], NIV and DON showed higher signal intensities in negative ionization mode, while T-2 and HT-2 toxins were more easily ionized in positive mode. Since the formation of sodiate adducts was observed using both ESI and APCI interfaces, 5 mM ammonium acetate was added to the solvent mixture, leading to mass spectra containing almost exclusively a single trichothecene-adduct ion species: the [M+CH 3 COO] -in the negative ionization, or the [M+NH 4 ] + adduct in the positive ionization. The following precursor ions were therefore chosen to develop the MRM acquisition method: [M+CH 3 COO] -for NIV (m/z 371.1) and DON (m/z 355.0), and [M+NH 4 ] + for T-2 (m/z 484.0) and HT-2 (m/z 442.0). the interface parameters, the best results in terms of signal intensities were observed with APCI interface, giving for the precursor ions of all studied trichothecenes signals about five fold more intense as compared to that obtained with the ESI interface in agreement with the results obtained by[START_REF] Berthiller | Rapid simultaneous determination of major type A-and B-trichothenenes as well as zearalenone in maize by high performance liquid chromatography-tandem mass spectrometry[END_REF]. Other authors found that for type-B trichothecenes ESI interface performed better than APCI[START_REF] Laganà | Liquid chromatography/tandem mass spectrometry for the identification and determionation of trichothecenes in maize[END_REF][START_REF] Biselli | Development of a multicomponent method for Fusarium toxins using LC-MS/MS and its application during a survey for the content of T-2 toxin and deoxynivalenol in various feed and food samples[END_REF]. However they selected [M-H] -as precursor ion and the possibility to work with acetate adducts was not investigated. No comparison between ESI and APCI for T-2 and HT-2 have been reported by[START_REF] Biselli | Development of a multicomponent method for Fusarium toxins using LC-MS/MS and its application during a survey for the content of T-2 toxin and deoxynivalenol in various feed and food samples[END_REF], working with sodiate adducts [M+Na] + for these toxins.

  Low temperature produces incomplete vaporization of the sample, whereas too high temperature could induce thermal degradation of analytes. Moreover, the probe temperature should be inversely correlated to the percentage of organic solvent in the mobile phase. Under our operating conditions the optimal temperature was 450 ºC for the detection of NIV and DON (20% methanol in the mobile phase), and 380 ºC for the detection of HT-2 and T-2 (80% methanol in the mobile phase).Development of the cleanup procedureOne of the aims of this work was to find a suitable SPE column allowing the simultaneous purification of type-A and -B trichothecenes, that vary considerably in polarity, from NIV (the most polar trichothecene) to T-2 toxin. For this reason we chose to test the performances of the Oasis ® HLB column, filled with a patented hydrophilic/lipophilic balanced polymer [poly(divinylbenzene-co-N-vinylpyrrolidone)], which retains both polar and non-polar compounds.

  results in the enhancement or suppression of the analyte chromatographic signal. A detailed investigation of possible matrix effects was thus performed

Fazeli

  et al. developed a double clean up procedure, using Mycosep #227 ® and 226 ® columns, to eliminate matrix effects in the determination of T-2 and HT-2 in oats (Razzazi-Fazeli et al. 2002).

  contaminated samplesThe total ion chromatogram (sum of all monitored MRM transitions) of a purified extract from a sample of bacon flavored biscuits naturally contaminated with NIV, near to its LOD, DON at 191 µg/kg, HT-2 at 8 µg/kg, and T-2 at 5 µg/kg is shown in Figure1. The developed method has been applied to the analysis of 57 samples of ground wheat produced by durum wheat cultivated in Apulia, a region of Southern Italy, and of 9 cereal-based food samples collected from the Italian retail market in2005-2006. Results are summarized in Tables 6 for ground wheat and Table7for cereal-based foods. Analyses of ground wheat samples revealed HT-2 to be the most frequently occurring trichothecene, with a percentage of positive samples of 44%, followed by DON and T-2 found in 18% and 11% of samples respectively. Nivalenol was found only in one out of 57 samples. The highest concentration values were 63.5, 17.3, 14.1, and 1.9 µg/kg for NIV, DON, HT-2 and T-2 respectively. In particular, DON contamination levels were quite below the maximum limit established by the European Commission (2005) in wheat flour, i.e. 750 µg/Kg. These data indicated the Apulia region as a low risk area for NIV, DON, HT-2 and T-2 contamination in agreement with[START_REF] Pascale | Occurrence of deoxynivalenol in cereals from experimental field in different Italian regions[END_REF] who reported that the northern Italian regions seem to be more susceptible to DON contamination than southern regions.

Figure 1 :

 1 Figure 1: Total ion chromatogram (sum of all monitored MRM transitions) of an extract of bacon flavored biscuits naturally contaminated with NIV, near to its LOD, 191 µg/kg DON, 8 µg/kg HT-2, and 5 µg/kg T-2. The mobile phase was methanol/water (20/80) containing 5 mM of ammonium acetate (0-9 min, APCI negative ion mode) followed by methanol/water (80/20) containing 5 mM of ammonium acetate (9-17 min, APCI positive ion mode) at flow rate of 400 µl/min.

Figure 2 :

 2 Figure 2: Recovery values of NIV from raw cereals and cereal-based foods, spiked at 100 µg/kg, after clean up with Mycosep ® 227 or Oasis ® HLB columns.

  For tuning experiments with the APCI interface, standard solutions of each mycotoxin (10 µg/ml) were infused, using a T-piece, at 10 µl/min into a flow of 400 µl/min of methanol/water (40/60 v/v), with or without ammonium acetate (5mM), delivered by the LC pump. The infusion was performed by using an Harvard infusion pump (model 11, South Natick, MA, USA). The ESI interface was solely used for tuning experiments performed by direct infusion at flow rate of 20 µl/min of the 1 µg/ml standard solutions of individual trichothecenes in methanol/water (40/60, v/v) with or without ammonium acetate (5mM). Interface parameters were as follows:

	F o
	r
	P
	e e r
	R e v i e w
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	9

Quantification of trichothecenes was performed by measuring peak areas in the TIC chromatogram (total ion chromatogram, sum of all monitored transitions) and by comparing them with the relevant calibration curve.

Table 3

 3

	. Recoveries for type-B trichothecenes for the

Table 1 :

 1 MS/MS parameters for detection of trichothecenes in the multiple reaction monitoring (MRM) method.

	Food Additives and Contaminants
	A sensitive, accurate and precise method for the determination of DON, NIV, T-2 and HT-2 was
	developed and applied to several cereals and cereal-based foods. The use of triple quadrupole
	analyzer in the MRM detection mode allowed unambiguous identification of the selected
	trichothecenes in complexes matrices at µg/kg levels. The method revealed its general
	applicability and robustness among a wide range of cereals and cereal-based foods, with
	detection limits generally lower than 5 µg/kg for DON and NIV and 1 µg/kg for T-2 and HT-2.
	The use of reversed phase SPE columns (Oasis ® HLB) provided good sample clean up and high
	recoveries for all studied trichothecenes although matrix-matched calibration was necessary for o accurate quantitative LC-MS/MS analyses. In particular, NIV recoveries resulted significantly
	r higher than those obtained when Mycosep ® #227 columns were used.
	P The performance of this method meet the CEN criteria at concentrations much lower than the e lowest concentrations reported in the Commission Regulation (EC) No 401/2006 (European e r Commission 2006) for DON, HT-2 and T-2.
	The co-occurrence of NIV, DON, HT-2 and T-2 was found in cereals or cereal-based products R produced or commercialized in Italy. This method can be used for multi-mycotoxin analysis of e food commodities for both control purposes and surveys aimed to accurately measure human v i w e exposure to this group of trichothecenes.
	O n
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	16

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Table 2 :

 2 Limits of detections (S/N = 3) of NIV, DON, HT-2 and T-2 in the tested cereals and

	cereal-based foods.
			LOD (µg/kg)
		NIV	DON	HT-2	T-2
	Infant Semolina	2.5	5.3	0.7	0.4
	Infant Biscuits	4.0	4.6	0.8	0.7
	Coconut Snacks	2.6	3.8	0.4	0.4
	Bacon Biscuits	3.4	3.0	1.7	1.0
	Cocoa Wafers	3.0	4.0	0.8	0.5
	Wheat	2.6	2.8	0.6	0.4
	Maize	2.7	5.1	0.8	0.4
	Barley	3.6	5.1	0.9	0.5

Table 3 :

 3 Recoveries of NIV, DON, HT-2 and T-2 from cereal-based foods, spiked at different toxin levels, after clean up with Oasis ® HLB column. Quantification was performed by using matrix-

	matched calibration curves.
		Spiking level		Recovery, % (RSD r , %)
		(µg/kg)
			NIV	DON	HT-2	T-2
	Infant Semolina 1000	79.2 (4.0)	91.6 (2.3)	105.5 (5.7)	105.7 (3.4)
		500	78.5 (3.0)	94.0 (2.9)	108.6 (2.1)	107.5 (2.8)
		100	75.0 (3.6)	83.2 (5.2)	100.9 (6.9)	99.8 (6.1)
		10	77.8 (3.8)	97.0 (1.0)	99.2 (3.0)	98.8 (4.1)
	Infant Biscuits	1000	76.2 (3.4)	85.0 (2.3)	104.5 (4.2)	90.0 (4.0)
		500	83.2 (3.1)	94.8 (3.8)	109.6 (1.0)	100.2 (0.9)
		100	79.4 (2.3)	87.2 (1.6)	109.3 (1.2)	108.4 (4.4)
		10	99.1 (1.3)	84.2 (1.9)	98.3 (1.1)	101.5 (3.9)
	Bacon Biscuits	1000	76.5 (4.4)	84.3 (2.3)	89.1 (4.9)	88.2 (6.3)
		500	83.8 (1.8)	98.0 (4.0)	93.0 (1.0)	100.7 (4.0)
		100	75.8 (1.1)	86.3 (4.1)	83.2 (4.4)	88.2 (5.1)
		10	77.7 (1.6)	80.4 (4.5)	85.3 (3.6)	98.6 (2.0)
	Cocoa Wafers	1000	79.7 (3.4)	94.7 (4.0)	99.5 (1.4)	98.2 (3.2)
		500	82.6 (2.4)	98.1 (5.7)	101.3 (5.6)	101.5 (3.1)
		100	72.3 (6.8)	80.2 (1.1)	89.6 (3.9)	85.2 (0.6)
		10	78.7 (3.4)	100.3 (4.6)	86.3 (5.6)	93.8 (3.8)
	Coconut Snacks 1000	75.0 (7.6)	97.9 (4.2)	80.6 (2.1)	101.1 (6.7)
		500	73.1 (2.1)	81.0 (1.0)	88.8 (1.5)	91.0 (2.2)
		100	73.4 (5.4)	94.3 (1.4)	73.2 (7.9)	91.6 (9.7)
		10	71.8 (5.9)	80.0 (4.2)	73.6 (4.1)	88.1 (2.3)
	RSD r , relative standard deviation (n = 4).

Table 4 :

 4 Comparison of recovery values of NIV, DON, HT-2, and T-2 from cereal-based foods and raw cereals, spiked at 100 µg/kg, obtained after clean up with Mycosep ® #227, and Oasis ® HLB columns. Quantification was performed by using matrix-matched calibration curves.

	Sample

Table 5 :

 5 t values (α = 0.05) relevant to the slope differences between standard (5 points) and matrix-assisted (5 points) calibration graphs in different cereal and cereal-based foods. Calibration range: [0.2 -20] ng injected. Degrees of freedom = 8; critical t value = 2.3.

	Sample	NIV	DON	HT-2	T-2
	Infant Semolina	0.52	-0.42	0.64	0.88
	Infant Biscuits	8.80	2.20	0.81	0.43
	Bacon Biscuits	5.57	4.66	4.69	6.45
	Cocoa Wafers	10.98	3.50	3.24	0.83
	Coconut Snacks	9.66	0.54	0.06	-0.06
	Wheat	15.72	2.97	4.19	5.36
	Barley	12.90	0.35	2.49	5.36
	Maize	2.93	8.65	4.29	4.29

Table 6 .

 6 NIV, DON, HT-2 and T-2 content in naturally contaminated samples of ground wheat produced from grain cultivated in Apulia region (South Italy) in2005 -2006.

		Food Additives and Contaminants	Page 26 of 30
	Toxin	Positives/total	% Positives	Mean of positives (µg/kg)	Range of contamination (µg/kg)
	NIV	1/57	1.7	63.5	-
	DON	10/57	17.5	7.7	3.3 -17.3
	HT-2	25/57	43.8	3.4	0.8 -14.1
	T-2	6/57	10.5	1.3	0.5 -1.9

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Table 7 .

 7 NIV, DON, HT-2 and T-2 content in naturally contaminated samples of cereal-based food samples collected from the Italian retail market.

	µg/kg

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Acknowledgements

This work was carried out with the financial support of the Project MIUR n. 201 -D.M. 1105 of 9.10.2002. Italian Minister of University and Research is acknowledged.