Food Chemistry 130 (2012) 10551060

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Food Chemistry
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Analytical Methods

Determination of mycotoxins in cereals by liquid chromatography tandem mass spectrometry

F. Soleimany a, S. Jinap a,, F. Abas b
a b

Center of Excellence for Food Safety Research (CEFSR), Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia Department of Food Science, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia

a r t i c l e

i n f o

a b s t r a c t
A liquid chromatography tandem mass spectrometry (LCMS/MS) method is described for simultaneous determination of aatoxins (AFB1, AFB2, AFG1 and AFG2), ochratoxin A (OTA), zearalenone (ZEA), deoxynivalenol (DON), fumonisins (FB1 and FB2), T2 and HT2-toxin in cereals. One-step extraction using solvent mixtures of acetonitrile:water:acetic acid (79:20:1) without any clean-up was employed for extraction of these mycotoxins from cereals. The mean recoveries of mycotoxins in spiked cereals ranged from 76.8% to 108.4%. Limits of detection (LOD) and quantication (LOQ) ranged 0.0120 and 0.0240 ng/g, respectively. The developed method has been applied for the determination of mycotoxins in 100 cereal samples collected from Malaysian markets. A total of 77 cereal samples (77%) contaminated with at least one of these mycotoxins. Occurrence of mycotoxins in commercial cereal samples were 70%, 40%, 25%, 36%, 19%, 13%, 16, and 16% for aatoxins, OTA, ZEA, DON, FB1, FB2, T2 and HT2-toxin, respectively. The results demonstrated that the procedure was suitable for the determination of mycotoxins in cereals and could be implemented for the routine analysis. 2011 Elsevier Ltd. All rights reserved.

Article history: Received 24 February 2011 Received in revised form 14 June 2011 Accepted 31 July 2011 Available online 6 August 2011 Keywords: Simultaneous determination Mycotoxin LCMS/MS Validation Cereal

1. Introduction Mycotoxins are toxic secondary metabolites produced by lamentous fungi that are growing on agricultural commodities and present in a lot of feed and foodstuffs. Commodities and products frequently contaminated with mycotoxins include corn, wheat, barley, rice, oats, nuts, milk, cheese, peanuts and cottonseed (Bennett & Klich, 2003). Several simultaneous determinations of mycotoxin methods have been reported, offering a signicant advantage over the conventional single mycotoxin determination method, using thin-layer chromatography (TLC), gas chromatography (GC), and liquid chromatography (LC) (Rahmani, Jinap, & Soleimany, 2009). The complex composition of food and feed matrices as well as the wide range of physical and chemical properties of mycotoxins requires selective and sensitive detection techniques, such as mass spectrometry, especially for multi-toxin methods. LCMS is one of the most advanced techniques available for the detection of mycotoxins. Multi-mycotoxin methods are also highly desired and therefore strongly attracting interest. ESIMS/MS in the positive mode has been used for the analysis of mycophenolic acid, penicillic acid and roquefortine C (together with ve aatoxins and ochratoxin A) in molded cheese (Kokkonen, Jestoi, & Rizzo, 2005). MS method has been created to facilitate detection and identication in fungal cultures (Nielsen & Smedsgaard, 2003). Different
Corresponding author. Tel.: +60 389468393; fax: +60 389423552.
E-mail addresses: jinap@food.upm.edu.my, sjinap@gmail.com (S. Jinap). 0308-8146/$ - see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.07.131

proportions of methanol or acetonitrile have been used for coextraction of mycotoxins (Berthiller, Schuhmacher, Buttinger, & Krska, 2005; Biselli & Hummert, 2005; Klotzel, Lauber, & Humpf, 2006). Triple quadrupole LCMS/MS methods for the quantication of trichothecenes and ZEA in cereals were presented in 2005, after extraction with acetonitrile/water and using ESI (Biselli & Hummert, 2005) or APCI (Berthiller et al., 2005) interfaces. Cavaliere, Foglia, Pastorini, Samperi, and Lagana (2005) presented their method for the determination of eight trichothecenes, three fumonisins, ZEA and alpha-zearalenol in corn samples were using ESI triple quadrupole MS in both polarity modes. A positive ion mode ESI triple quadrupole MS method for the simultaneous determination of 16 mycotoxins was developed by Delmulle, Saeger, Adams, Kimpe, and Peteghem (2006). The aim of the present study is to develop a LCMS/MS method for the simultaneous determination of aatoxins, OTA, ZEA, DON, FB1, FB2, T2 and HT2-toxin. This method would be suitable for the detection of these mycotoxins in the cereal samples, especially, when many of samples needed to be analysed in a short time. The LCMS/MS method has also been successfully applied to 100 cereal samples which collected from Malaysian markets. 2. Experimental 2.1. Materials and reagents The analytical standards of mycotoxins AFs (AFB1, AFB2, AFG1, and AFG2), OTA, ZEA, DON, and fumonisins (FB1, FB2) were

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Table 1 Precursor and product ions and Ms/MS conditions of analytes. Analyte AFB1 AFB2 AFG1 AFG2 ZEA OTA DON FB1 FB2 T2 HT2 Precursor ion (m/z) 313.04[M+H]+ 313.04[M+H]+ 315.06[M+H]+ 315.06[M+H]+ 329.05[M+H]+ 329.05[M+H]+ 331.07[M+H]+ 331.07[M+H]+ 319.0[M+H]+ 319.0[M+H]+ 404.00[M+H]+ 404.00[M+H]+ 295.00[MH] 295.00[MH] 722.05[M+H]+ 722.05[M+H]+ 706.04[M+H]+ 706.04[M+H]+ 489.00[M+Na]+ 489.00[M+Na]+ 447.00[M+Na]+ 447.00[M+Na]+ Product ions (m/z) 241 285 259 287 243 283 245 275 185 187 221 239 247 265 334 352 318 336 245 387 285 345 Collision energy (V) 31 27 17 80 27 5 22 21 21 21 21 21 25 25 35 35 32 35 30 25 30 27 Tube lens 94 94 114 114 131 131 170 170 170 170 170 170 150 150 180 180 191 191 170 170 170 170 Q1 PW 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 Q3 PW 0.7 0.7 0.7 0.7 0.7 0.7 0.7 0.7 0.7 0.7 0.7 0.7 0.7 0.7 0.7 0.7 0.7 0.7 0.7 0.7 0.7 0.7

AFB1, aatoxin B1; AFB2, aatoxin B2; AFG1, aatoxin G1; AFG2, aatoxins G2; OTA, ochratoxin A; ZEA, zearalenone; DON, deoxynivalenol, fumonisins (FB1 and FB2); T2, T2toxin; HT2, HT2-toxin.

obtained from SigmaAldrich (St. Louis, MO, USA). T2 and HT2-toxin were from Biopure (Tulln, Austria). De-ionised distilled water was obtained from a Milli-Q purication system (Bedford, MA, USA). HPLC grade methanol and acetic acid were from Merck (Darmstadt, Germany). Phosphate buffered solution (PBS) was prepared by dissolving PBS tablets (SigmaAldrich, St. Louis, MO, USA) in distilled water. Certied reference material for DON (maize our containing 474 30 ng/g DON), ZEA (maize our containing 60 9 ng/g ZEA), fumonisins (maize our containing 2406 612 ng/g FB1 and 630 116 ng/g FB2) and OTA (wheat our containing 2.7 1 ng/g OTA), were supplied by the Sigma Aldrich (St. Louis, MO, USA). Fluted lter paper and GF/A glass microber lter paper were from Whatman (Maidstone, UK). 2.2. Stock and working standard preparation A volume of 50 lL of OTA (50,000 ng/mL) was dissolved in methanol to produce a 2500 ng/mL stock solution. One millilitre of this individual stock solution of OTA was mixed with 1 mL of aatoxins (AFB1, AFB2, AFG1, AFG2), mixed standard solution (B1and G1 1000 ng/mL and B2, G2 300 ng/mL), 500 lL of ZEA standard solution (50,000 ng/mL), 250 lL of DON standard solution (200,000 ng/mL), 494 lL of FB1 standard solution (50,600 ng/mL), 501 lL of FB2 standard solution (49,900 ng/mL), 495 lL of T2-toxin standard solution (100,900 ng/mL), and 487 lL of HT2-toxin standard solution (102,700 ng/mL) in a 10 mL volumetric ask, and later topped up with methanol to achieve a stock solution of AFB1, AFB2, AFG1, AFG2, OTA, ZEA, FB1, FB2, T2-toxin, HT2-toxin, and DON at the concentration of 100, 30, 100, 30, 250, 2500, 2500, 2500, 5000, 5000 and 5000 ng/mL, respectively. This stock solution was then used to prepare the working standard solution. All stock and working standard solutions were stored in amber vials at 20 C. 2.3. Samples and sample preparation A total of 100 cereal samples including rice (N = 50), wheat (N = 20), oat (N = 10), barley (N = 10) and Maize meal (N = 10) were

collected from Malaysian General Markets in Kuala Lumpur during MayJune 2010. The number of samples was according to cereal consumption in Malaysia. The samples were kept in dark cool room 4 C until analysis. The ground cereal sample (10 g) was homogenised with 40 mL of the organic extraction solvent mixture (acetonitrile:water:acetic acid, 79:20:1) (Sulyok, Berthiller, Krska, & Schuhmacher, 2006) by shaking for 60 min on an orbital shaker (model 711 VDRI, Asal, Milan, Italy). The supernatants were then centrifuged at 3000 rpm for 10 min (Allegra X-22R centrifuge, Beckman Coulter, Palo Alto, CA, USA) as described by Sulyok, Krska, and Schuhmacher (2007). Then, 0.5 mL of the nal extract of the one-step extraction was diluted with the same amount of acetonitrile:water:acetic acid (20:79:1) passed through a 0.22 lm lter, and injected into the LCMS/MS (Spanjer, Rensen, & Scholten, 2008; Sulyok et al., 2006; Sulyok et al., 2007. To investigate the matrix effect, ve concentrations of standards were added to blank matrix after extraction and injected three times each. 2.4. LCMS/MS apparatus and conditions LC analysis was performed using Finnegan TSQ quantum ultra mass (Thermo Scientic, CA, USA) system consisting of a binary pump, a degasser, a column oven, and an auto sampler. Analysis was done on a column, 150 mm, 4.6, 3 lm particle size C18 columns (Thermo Scientic, CA, USA). The column temperature was kept at 30 C. The capillary voltage was 3 kV, and nitrogen was used as the spray gas. Source temperature and desolvation temperature were set at 120 and 400 C, respectively. Mycotoxins were analysed in selected reaction monitoring (SRM) channels. Quantication was carried out by matrix-matched standard calibration. To obtain best chromatogram with lowest noise signal, different proportions of mobile phase consisted of methanol or acetonitrile and acetic acid (01%), different ow rates (0.20.3 mL/min), injection volumes (1030 lL) and ionisation methods including electrospray ionisation (ESI) and atmospheric pressure chemical ionisation (APCI) in positive and negative ion modes were studied.

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RT: 0.00 - 25.01 100 50 0 100 50 Relative Abundance 0 100 50 0 100 50 0 100 50 0 100 50 0 0 2.05 3.76 4.91 6.75 9.55 12.56 14.25 16.19 18.13 15.65 21.33 23.37 15.74 NL: 1.61E6 TIC F: + c ESI SRM ms2 313.040 [240.995-241.005, 284.995-285.005] MS std100ppb

NL: 1.13E5 TIC F: + c ESI SRM ms2 315.060 [258.995-259.005, 286.995-287.005] 0.33 1.75 21.19 14.95 18.18 24.74 MS std100ppb 5.98 6.82 9.53 10.90 NL: 5.75E5 15.35 TIC F: + c ESI SRM ms2 329.050 [242.995-243.005, 282.995-283.005] MS std100ppb 18.18 2.38 3.25 7.15 9.34 13.93 14.83 21.59 23.01 NL: 8.08E4 15.23 TIC F: + c ESI SRM ms2 331.070 [244.995-245.005, 274.995-275.005] 18.19 20.10 0.44 9.76 12.84 24.75 MS std100ppb 3.55 5.36 6.35 NL: 6.39E5 18.16 TIC F: + c ESI SRM ms2 319.000 [184.995-185.005, 186.995-187.005] MS std100ppb 2.30 3.12 6.08 7.39 8.79 13.34 14.48 16.99 18.75 22.11 23.90 NL: 1.40E4 15.20 TIC F: + c ESI SRM ms2 404.000 [220.995-221.005, 238.995-239.005] 13.94 13.44 15.93 18.36 20.25 23.75 MS std100ppb 6.58 7.50 0.14 2.85 10.66 5 10 Time (min) 15 20 25

Fig. 1. LCMS/MS chromatogram of mycotoxins at 2.5 AFB1, 0.75 AFB2, 2.5 AFG1, 0.75 AFG2, 6.25 OTA, 62.5 ZEA, 62.5 FB1, 62.5 FB2, 125 T2-toxin, 125 HT2-toxin, and 125 ng/g for DON.

Different mobile phases consisted of different proportions of methanol or acetonitrile were used (initial, 020% and nal, 7595% of methanol or acetonitrile percentages of the gradient mobile phase). 2.5. Method validation The method was validated according to the European Commission regulation for performance of analytical methods (EC No. 657/ 2002) and by analysis of replicate spiked samples of cereals according to the recommendations of Gilbert and Anklam (2002). Linear regression analysis was conducted for the mixture of AFs, OTA, ZEA, OTA, DON, FB1, FB2, T2-toxin, and HT2-toxin. The eight-point calibration curves for aatoxins B1, B2, G1, G2, OTA, ZEA, DON, FB1,

FB2,T2-toxin, and HT2-toxin were made in the concentration ranges of 0.01100 ng/mL for (AFB1, AFB2, AFG1, AFG2), 0.2 2000 ng/mL for ZEA, 0.02200 ng/mL for OTA, 0.44000 ng/mL for DON, 0.44000 ng/mL for (FB1, FB2), 0.44000 ng/mL for (T2-toxin, and HT2-toxin). The solutions were ltered through a 0.45 lm Whatman (Milford, MA, USA) membrane lter. Linear regression was used to plot the peak area ratio (y) of each mycotoxin against its concentration. The evaluation of each point was repeated ve times. Recovery experiments were carried out in triplicate by spiking ground cereals at following levels: 2.5, 0.75, 2.5, 0.75, 6.25, 62.5, 62.5, 62.5, 125, 125, and 125 ng/g for AFB1, AFB2, AFG1, AFG2, OTA, ZEA, FB1, FB2, T2-toxin, HT2-toxin, and DON, respectively. Spiked samples were left overnight at room temperature for solvent evaporation. For the evaluation of matrix effect, blank

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Table 2 Linearity and sensitivity of LCMS/MS method for simultaneous determination of 11 mycotoxins. Mycotoxins AFB1 AFB2 AFG1 AFG2 OTA ZEA DON FB1 FB2 T2 HT2 LOD in standard solution (ng/g) 0.05 0.25 0.05 0.5 0.01 0.1 5 10 10 2 2 LOQ in standard solution (ng/g) 0.1 0.5 0.1 1 0.02 0.2 10 20 20 4 4 LOD in corn (ng/g) 0.3 0.5 0.1 0.8 0.1 0.5 10 20 20 5 5 LOQ in corn (ng/g) 0.6 1 0.2 1.5 0.2 1 20 40 40 10 10 LOD in rice (ng/g) 0.25 0.45 0.06 0.6 0.06 0.4 8 20 20 4 4 LOQ in rice (ng/g) 0.5 0.8 0.1 1 0.15 0.8 15 40 40 8 8 Linear range (ng/g) 0.01100 0.01100 0.01100 0.01100 0.02200 0.22000 0.44000 0.44000 0.44000 0.44000 0.44000 R2 0.9995 0.9984 0.9984 0.9952 0.9969 0.9955 0.9509 0.9939 0.9934 0.9866 0.9846 Regression equation Y = 199859X Y = 16736.1X Y = 126930X Y = 16531X Y = 186849 Y = 11006.5X Y = 0.119999X Y = 321797X Y = 335929X Y = 204492X Y = 36419.6X

LOD, limit of detection; LOQ, limit of quantication; AFB1, aatoxin B1; AFB2, aatoxin B2; AFG1, aatoxin G1; AFG2, aatoxins G2; OTA, ochratoxin A; ZEA, zearalenone; DON, deoxynivalenol, fumonisins (FB1 and FB2); T2, T2-toxin; HT2, HT2-toxin.

Table 3 Recovery of LCMS/MS method for simultaneous determination of 11 mycotoxins. Mycotoxins Spiked level (ng/g) Rice Rec. (%) AFB1 AFB2 AFG1 AFG2 OTA ZEA DON FB1 FB2 T2 HT2 2.5 0.75 2.5 0.75 6.25 62.5 125 62.5 62.5 125 125 108.1 92.5 83.6 86.9 88.4 89.6 88.4 89.3 89.2 97.4 96.9 RSD (%) 6.8 9.1 9.0 6.9 12.1 13.2 11.1 11.9 12.1 10.4 10.2 Oat Rec. (%) 105.1 89.9 87.5 76.8 86.8 87.9 89.3 88.1 87.1 96.8 95.1 RSD (%) 7.4 9.7 9.8 9.2 11.9 11.8 11.9 12.5 12.3 11.7 12.1 Maize Rec. (%) 106.1 89.9 85.8 84.1 86.1 88.0 87.2 87.5 85.9 95.9 97.3 RSD (%) 7.1 9.2 8.4 8.3 11.8 12.1 11.9 14.4 11.9 11.7 11.8 Wheat Rec. (%) 106.4 88.5 84.4 82.9 88.2 87.9 85.9 87.9 86.4 96.3 95.3 RSD (%) 6.9 8.1 9.9 8.4 12.7 10.9 9.9 12.3 12.7 12.2 11.9 Barley Rec. (%) 103.6 87.8 83.9 82.9 86.6 87.3 87.6 89.6 87.8 95.9 97.6 RSD (%) 7.8 8.3 9.4 8.9 12.8 14.2 12.6 12.1 10.4 11.7 12.3

Rec., recovery (%); RSD, relative standard deviation (%); AFB1, aatoxin B1; AFB2, aatoxin B2; AFG1, aatoxin G1; AFG2, aatoxins G2; OTA, ochratoxin A; ZEA, zearalenone; DON, deoxynivalenol, fumonisins (FB1 and FB2); T2, T2-toxin; HT2, HT2-toxin.

samples from each type of cereal (rice, wheat, oat, maize and barley) were extracted, and the sample extracts were spiked with ve concentrations of each mycotoxin ranging from 0.1 to 50 ng/g AFs, 0.2 to 100 ng/g OTA, 2 to 1000 ng/g ZEA, 4 to 2000 ng/g DON, FB1, FB2, T2-toxin, and HT2-toxin.

3. Results and discussion 3.1. LCMS/MS method development Different proportions of methanol:acetic acid (0.1%) or acetonitrile:acetic acid (0.1%) (5:9595:5 v/v) was used for elution of mycotoxins in LCMS/MS. However, there are different outputs that could be considered as chromatographic responses to be optimised; peak area and signal/noise were considered in this study. Mycotoxins have a wide range of polarity strengths; therefore a mobile phase with a variable degree of hydrophobicity over the time course of each analysis was required. The chromatography was thus started with a mobile phase having a low organic solvent content for the elution of the more polar mycotoxins, and the organic solvent content was gradually increased to elute the less polar mycotoxins within a reasonable time frame. The best chromatograms of all mycotoxins with lowest noise obtained using mobile phase consisted of methanol and acetic acid (0.1%) at a ow rate of 0.25 mL/min with a gradient elution programme. The gradient elution was started at 5% methanol (08 min) with a linear increase to 90% (822 min). Then 90% methanol of the isocratic period (822 min) was decreased to 5% methanol in 3 min (2225 min).

Among different studied injection volumes, 20 lL injection provided lowest noise signal in chromatograms. ESI ionisation was selected due to its better chromatograms for all mycotoxins in comparison with APCI. Application of two positive and negative ion modes with ESI showed that except for DON, other mycotoxins exhibited better fragmentation patterns in positive mode. Consequently, the optimised multiple reaction monitoring (MRM) parameters were obtained in positive and negative ion mode as described in Table 1. The ressults were in agreement with previous reports (Delmulle et al., 2006; Lattanzio, Solfrizzo, Powers, & visconti, 2007; Monbaliu et al., 2009; Ren et al., 2007; Spanjer et al., 2008; Sulyok et al., 2006; Sulyok et al., 2007; Tanaka, Takino, Sugita-Konishi, & Tanaka, 2006; Ventura et al., 2006). Conversely, there are some reports on using ESI () for some mycotoxins (Ren et al., 2007) in the presence of ammonium acetate in the mobile phase. In addition, there are other reports where 5 mM ammonium acetate was used in the mobile phase (Delmulle et al., 2006; Sulyok et al., 2006), but in this study using of same mobile phase shown

Table 4 Mycotoxin measurement using certied reference materials. Maycotoxin DON ZEA FB1 FB2 OTA Contamination level (ng/g) 474 30 60 9 2406 612 630 116 2.7 1 Measured values (ng/g) 456 36 57 7 2355 278 543 56 2.3 0.2

OTA, ochratoxin A; ZEA, zearalenone; DON, deoxynivalenol, fumonisins (FB1 and FB2).

F. Soleimany et al. / Food Chemistry 130 (2012) 10551060 Table 5 Mycotoxin contamination in cereal samples in Malaysian market. Mycotoxins Number of naturally contaminated samples (range of contamination (ng/g)) Type of cereal N Aatoxins OTA ZEA DON FB1 FB2 T2 HT2 Occurrence Rice 50 28 (0.154.54) 21 (0.24.34) 12 (1.551.1) 13 (12.581.2) 7 (41.353.2) 3 (40.161.5) 7 (10.595.2) 7 (8.126.1) 40 Wheat 20 15 (0.23.2) 7 (0.152.11) 6 (1.4212.74) 10 (22.8112.5) 3 (42.569.1) 3 (42.075.3) 3 (11.253.1) 3 (9.318.7) 15 Barley 10 5 (0.262.59) 3 3 5 3 3 1 1 7 (0.182.84) (0.9520.26) (27.972.5) (45.597.7) (43.172.5) (12.755.9) (10.130.7) Oat 10 4 (0.121.94) 3 0 3 3 1 1 1 7 (0.10.2) (ND) (22.7100.2) (49.5177.3) (57.3) (39.5) (25.2) Maize meal 10 8(0.151.8) 6 4 5 3 3 4 4 8 (0.15.76) (113.47) (35109) (48.2209.3) (58.7113.5) (31.750.7) (29.581.8) Total 100

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60 (0.124.54) 40 (0.15.76) 25 (0.9581.1) 36(20.5109) 19 (41.3209.3) 13 (40.1113.5) 16 (10.550.7) 16 (8.181.8) 77

N, number of samples analysed; ND, not detected; aatoxins (AFB1, AFB2, AFG1 and AFG2); OTA, ochratoxin A; ZEA, zearalenone; DON, deoxynivalenol, fumonisins (FB1 and FB2); T2, T2-toxin; HT2, HT2-toxin.

high noises in chromatograms. Although organic acids are the most common mobile phase additive for LC separations that employ MS detection, it may be necessary under certain circumstances to use more neutral conditions, either because the analytes are sensitive to acids or they do not exhibit optimal resolution at low pH. When acids are not suitable, volatile salts, like ammonium formate or ammonium acetate, may be the additives of choice. However, compared to organic acids their use is much more complex. One issue is the limited solubility of the salts in organic solvents; another issue is the changing pH value during a gradient. On the other hand, the mildly acidic pH provided by the salts permits both positive and negative ion mode detection. Consequently, the nal optimised mobile phase contained only methanol, water and acetic acid (0.1%). Different types of tuning solutions were tested to select the most abundant m/z value. Furthermore, different collision energies were used to determine the most abundant product ions. The best peak shapes with lowest noise was achieved in best LC conditions as described in LCMS/MS parameters part. Fig. 1 shows LCMS/ MS chromatogram of these mycotoxins in selective reaction mode (SRM). All mycotoxins were detected in almost 18 min, consequently it provided fast determination of multi-mycotoxins. However, it was not possible to avoid the co-elution of some mycotoxins. For example AFG2 was eluted after 15.30 min and AFG1 was after 15.42 min. The co-elution of some mycotoxin compounds was accepted because the related compounds show different transitions (Delmulle et al., 2006; Sulyok et al., 2006).

Recovery values (Table 3) ranged from 76.8% to 108.4% for all mycotoxins. The recovery results was better than those reported by Delmulle et al. (2006), (52.689.2%), Sulyok et al. (2006), (75 108%), Spanjer et al. (2008), (46115%), Monbaliu et al. (2009), (76105%) for relevant mycotoxins. Relative standard deviation of recoveries (RSD%) for this procedure was lower than 12.7% for all mycotoxins. These recovery and RSD results were within satisfactory levels as recommended by the European Commission regulation (EC 401/2006). Three measurements of mycotoxins using CRMs conrmed the trueness of measurements. The measured values are shown in Table 4. 3.3. Application of the LCMS/MS method to commercial cereal samples The LCMS/MS method was applied in the quantication of aatoxins (AFB1, AFB2, AFG1 and AFG2), OTA, ZEA, DON, fumonisins (FB1 and FB2), T2 and HT2-toxin in 100 cereal samples collected from Malaysian markets. All collected cereal samples were analysed according to newly developed LCMS/MS multi-mycotoxin method. Of these samples, 40 out of 50 rice samples (75%), 15 out of 20 wheat samples (75%), seven out of 10 oat samples (70%), seven out of 10 barley samples (70%) and 8 out of 10 maize meal samples (80%), and 77 out of 100 total samples (77%) were contaminated with at least one mycotoxin. A total of 4% of the samples (4 out of 100) exceeded the proposed regulatory level of 4 and 5 ng/g for the total AFs and OTA, respectively. Two maize meal samples were contaminated, with 5.28, 5.76 ng/g of OTA, and two rice samples were contaminated with aatoxins at levels of 4.32 and 4.54 ng/g (Table 5). 4. Conclusion A triple quadrupole tandem mass spectrometer method using ESI+ and ESI in SRM mode was developed and validated for simultaneous determination of aatoxins, OTA, ZEA, DON, fumonisins, T2 and HT2-toxins. All of the 11 mycotoxins studied were well separated in 18 min by C18 short column (150 mm, 4.6, 3 lm) using gradient elution with aqueous solution of acetic acid (0.1%) and methanol as mobile phase. The validation data including linearity, sensitivity and recovery showed that this method is acceptable to be used for mycotoxin determination for cereals. The LOD and LOQ ranged at 0.0120 ng/g and 0.0240 ng/g, respectively. The application of method on commercial cereal samples collected from Malaysian markets showed that 77% of cereals were contaminated with at least one of these 11 mycotoxins. A total of 4

3.2. LCMS/MS method validation Method performance was evaluated for each mycotoxin at contamination levels, and they were compared with the relevant established European Commission regulations (EC 1881/2006). Linearity and sensitivity results are reported in Table 2. The method exhibited good linearity over the relevant working range, and R2 was between 0.950 for DON and 0.999 for AFB1. There was significant difference among the LODs in the standard solution and in matrices. LODs of mycotoxins standard solutions were far lower than LODs in matrixes. The LODs and LOQs of standards and matrixes ranged between 0.0120 ng/g and 0.0240 ng/g, respectively, which are acceptable because they were far below the European regulations for correspondent maximum levels of mycotoxins in foods. The LODs were lower than those reported by Sulyok et al. (2006) and comparable to those reported by other authors who use clean-up such as Ventura et al. (2006); Lattanzio et al. (2007) for AFB1, AFG1, OTA, ZEA, and Spanjer et al. (2008).

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F. Soleimany et al. / Food Chemistry 130 (2012) 10551060 Gilbert, J., & Anklam, E. (2002). Validation of analytical methods for determining mycotoxins in foodstuff. Trends in Analytical Chemistry, 21, 468486. Klotzel, M., Lauber, U., & Humpf, H. U. (2006). A new solid phase extraction clean-up method for the determination of 12 type A and B trichothecenes in cereals and cereal-based food by LCMS/MS. Molecular Nutrition & Food Research, 50, 261269. Kokkonen, M., Jestoi, M., & Rizzo, A. (2005). Determination of selected mycotoxins in mould cheeses with liquid chromatography coupled to tandem with mass spectrometry. Food Additives and Contaminants, 22, 449456. Lattanzio, V. M. T., Solfrizzo, M., Powers, S., & visconti, A. (2007). Simultaneous determination of aatoxins, ochratoxin A and Fusarium toxins in maize by liquid chromatography/ tandem mass spectrometry after multitoxin immunoafnity clean up. Rapid Communications in Mass Spectrometry, 21, 32533261. Monbaliu, S., Van Poucke, C., Van Peteghem, C., Van Poucke, K., Heungens, K., & De Saeger, S. (2009). Development of a multi-mycotoxin liquid chromatography/ tandem mass spectrometry method for sweet pepper analysis. Rapid Communications in Mass Spectrometry, 23, 311. Nielsen, K. F., & Smedsgaard, J. (2003). Fungal metabolite screening: Database of 474 mycotoxins and fungal metabolites for dereplication by standardised liquid chromatography UV mass spectrometry methodology. Journal of Chromatography A, 1002, 111136. Rahmani, A., Jinap, S., & Soleimany, F. (2009). Qualitative and quantitative analysis of mycotoxins. Comprehensive Review in Food Science and Food Safety, 8, 202251. Ren, Y., Zhang, Y., Shao, S., Cai, Z., Feng, L., Pan, H., et al. (2007). Simultaneous determination of multi-component mycotoxin contaminants in foods and feeds by ultra-performance liquid chromatography tandem mass spectrometry. Journal of Chromatography A, 1143, 4864. Spanjer, M. C., Rensen, P. M., & Scholten, J. M. (2008). LC/MS/MS multimethod for mycotoxins after single extraction, with validation data for peanut, pistachio, wheat, maize, cornakes, raisins and gs. Food Additives and Contaminants, 4, 472489. Sulyok, M., Berthiller, F., Krska, R., & Schuhmacher, R. (2006). Development and validation of a liquid chromatography/tandem mass spectrometric method for the determination of 39 mycotoxins in wheat and maize. Rapid Communications in Mass Spectrometry, 20, 26492659. Sulyok, M., Krska, R., & Schuhmacher, R. (2007). Application of a liquid chromatographytandem mass spectrometric method to multi-mycotoxin determination in raw cereals and evaluation of matrix effects. Food Additives and Contaminants, 24, 11841195. Tanaka, H., Takino, M., Sugita-Konishi, Y., & Tanaka, T. (2006). Development of a liquid chromatography/time-of-ight mass spectrometric method for the simultaneous determination of trichothecenes, zearalenone and aatoxins in foodstuffs. Rapid Communications in Mass Spectrometry, 20, 14221428. Ventura, M., Guillen, D., Anaya, I., Broto-Puig, F., liberia, J. L. L., Agut, M., et al. (2006). Ultra performance liquid chromatography/tandem mass spectrometry for the simultaneous analysis of aatoxins B1, G1, B2, G2 and ochratoxin A in beer. Rapid Communications in Mass Spectrometry, 20, 31993204.

out of 100 samples (4%) (2 rice and 2 maize meal samples) exceeded the proposed European regulatory level of 4 and 5 ng/g for the total AFs and OTA, respectively. However; the contamination levels were lower than 5.76 ng/g for OTA and 4.54 ng/g for total aatoxins. The results also indicated that the method could be used for trace analysis of multi-component mycotoxin contaminants in cereals. Acknowledgement The authors acknowledge Universiti Putra Malaysia for the nancial support through the Research University Grant Scheme (RUGS), project number 02-01-07-0024RU. References
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