Determination of mycotoxins in cereals by liquid chromatography tandem mass spectrometry

Food Chemistry 130 (2012) 1055–1060

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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

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 LC–MS/MS Validation Cereal

a b s t r a c t A liquid chromatography tandem mass spectrometry (LC–MS/MS) method is described for simultaneous determination of aflatoxins (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 quantification (LOQ) ranged 0.01–20 and 0.02–40 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 aflatoxins, 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. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Mycotoxins are toxic secondary metabolites produced by filamentous 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 significant 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. LC–MS 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. ESI–MS/MS in the positive mode has been used for the analysis of mycophenolic acid, penicillic acid and roquefortine C (together with five aflatoxins and ochratoxin A) in molded cheese (Kokkonen, Jestoi, & Rizzo, 2005). MS method has been created to facilitate detection and identification in fungal cultures (Nielsen & Smedsgaard, 2003). Different ⇑ Corresponding author. Tel.: +60 389468393; fax: +60 389423552. E-mail addresses: [email protected], [email protected] (S. Jinap). 0308-8146/$ - see front matter Ó 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 LC–MS/MS methods for the quantification 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 LC–MS/MS method for the simultaneous determination of aflatoxins, 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 LC–MS/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

Precursor ion (m/z)

Product ions (m/z)

Collision energy (V)

AFB1

313.04[M+H]+ 313.04[M+H]+

241 285

31 27

AFB2

315.06[M+H]+ 315.06[M+H]+

259 287

AFG1

329.05[M+H]+ 329.05[M+H]+

AFG2

Tube lens

Q1 PW

Q3 PW

94 94

0.4 0.4

0.7 0.7

17 80

114 114

0.4 0.4

0.7 0.7

243 283

27 5

131 131

0.4 0.4

0.7 0.7

331.07[M+H]+ 331.07[M+H]+

245 275

22 21

170 170

0.4 0.4

0.7 0.7

ZEA

319.0[M+H]+ 319.0[M+H]+

185 187

21 21

170 170

0.4 0.4

0.7 0.7

OTA

404.00[M+H]+ 404.00[M+H]+

221 239

21 21

170 170

0.4 0.4

0.7 0.7

DON

295.00[M H] 295.00[M H]

247 265

25 25

150 150

0.4 0.4

0.7 0.7

FB1

722.05[M+H]+ 722.05[M+H]+

334 352

35 35

180 180

0.4 0.4

0.7 0.7

FB2

706.04[M+H]+ 706.04[M+H]+

318 336

32 35

191 191

0.4 0.4

0.7 0.7

T2

489.00[M+Na]+ 489.00[M+Na]+

245 387

30 25

170 170

0.4 0.4

0.7 0.7

HT2

447.00[M+Na]+ 447.00[M+Na]+

285 345

30 27

170 170

0.4 0.4

0.7 0.7

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

obtained from Sigma–Aldrich (St. Louis, MO, USA). T2 and HT2-toxin were from Biopure (Tulln, Austria). De-ionised distilled water was obtained from a Milli-Q purification 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 (Sigma–Aldrich, St. Louis, MO, USA) in distilled water. Certified reference material for DON (maize flour containing 474 ± 30 ng/g DON), ZEA (maize flour containing 60 ± 9 ng/g ZEA), fumonisins (maize flour containing 2406 ± 612 ng/g FB1 and 630 ± 116 ng/g FB2) and OTA (wheat flour containing 2.7 ± 1 ng/g OTA), were supplied by the Sigma Aldrich (St. Louis, MO, USA). Fluted filter paper and GF/A glass microfiber filter 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 aflatoxins (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 flask, 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 May–June 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 final 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 filter, and injected into the LC–MS/MS (Spanjer, Rensen, & Scholten, 2008; Sulyok et al., 2006; Sulyok et al., 2007. To investigate the matrix effect, five concentrations of standards were added to blank matrix after extraction and injected three times each. 2.4. LC–MS/MS apparatus and conditions LC analysis was performed using Finnegan TSQ quantum ultra mass (Thermo Scientific, 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 Scientific, 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. Quantification 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 (0–1%), different flow rates (0.2–0.3 mL/min), injection volumes (10–30 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 15.74

100 50 2.05 3.76 4.91 6.75

0 100

12.56 14.25 16.19 18.13 15.65

21.33 23.37

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

50 Relative Abundance

9.55

NL: 1.61E6 TIC F: + c ESI SRM ms2 313.040 [240.995-241.005, 284.995-285.005] MS std100ppb

0 100 50 0 100 50 0 100 50 0 100 50 0 0

5

10

15

20

25

Time (min) Fig. 1. LC–MS/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, 0–20% and final, 75–95% 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 aflatoxins B1, B2, G1, G2, OTA, ZEA, DON, FB1,

FB2,T2-toxin, and HT2-toxin were made in the concentration ranges of 0.01–100 ng/mL for (AFB1, AFB2, AFG1, AFG2), 0.2– 2000 ng/mL for ZEA, 0.02–200 ng/mL for OTA, 0.4–4000 ng/mL for DON, 0.4–4000 ng/mL for (FB1, FB2), 0.4–4000 ng/mL for (T2-toxin, and HT2-toxin). The solutions were filtered through a 0.45 lm Whatman (Milford, MA, USA) membrane filter. Linear regression was used to plot the peak area ratio (y) of each mycotoxin against its concentration. The evaluation of each point was repeated five 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 LC–MS/MS method for simultaneous determination of 11 mycotoxins. Mycotoxins

LOD in standard solution (ng/g)

LOQ in standard solution (ng/g)

LOD in corn (ng/g)

LOQ in corn (ng/g)

LOD in rice (ng/g)

LOQ in rice (ng/g)

Linear range (ng/g)

R2

Regression equation

AFB1 AFB2 AFG1 AFG2 OTA ZEA DON FB1 FB2 T2 HT2

0.05 0.25 0.05 0.5 0.01 0.1 5 10 10 2 2

0.1 0.5 0.1 1 0.02 0.2 10 20 20 4 4

0.3 0.5 0.1 0.8 0.1 0.5 10 20 20 5 5

0.6 1 0.2 1.5 0.2 1 20 40 40 10 10

0.25 0.45 0.06 0.6 0.06 0.4 8 20 20 4 4

0.5 0.8 0.1 1 0.15 0.8 15 40 40 8 8

0.01–100 0.01–100 0.01–100 0.01–100 0.02–200 0.2–2000 0.4–4000 0.4–4000 0.4–4000 0.4–4000 0.4–4000

0.9995 0.9984 0.9984 0.9952 0.9969 0.9955 0.9509 0.9939 0.9934 0.9866 0.9846

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

Table 3 Recovery of LC–MS/MS method for simultaneous determination of 11 mycotoxins. Mycotoxins

AFB1 AFB2 AFG1 AFG2 OTA ZEA DON FB1 FB2 T2 HT2

Spiked level (ng/g)

2.5 0.75 2.5 0.75 6.25 62.5 125 62.5 62.5 125 125

Rice

Oat

Maize

Wheat

Barley

Rec. (%)

RSD (%)

Rec. (%)

RSD (%)

Rec. (%)

RSD (%)

Rec. (%)

RSD (%)

Rec. (%)

RSD (%)

108.1 92.5 83.6 86.9 88.4 89.6 88.4 89.3 89.2 97.4 96.9

6.8 9.1 9.0 6.9 12.1 13.2 11.1 11.9 12.1 10.4 10.2

105.1 89.9 87.5 76.8 86.8 87.9 89.3 88.1 87.1 96.8 95.1

7.4 9.7 9.8 9.2 11.9 11.8 11.9 12.5 12.3 11.7 12.1

106.1 89.9 85.8 84.1 86.1 88.0 87.2 87.5 85.9 95.9 97.3

7.1 9.2 8.4 8.3 11.8 12.1 11.9 14.4 11.9 11.7 11.8

106.4 88.5 84.4 82.9 88.2 87.9 85.9 87.9 86.4 96.3 95.3

6.9 8.1 9.9 8.4 12.7 10.9 9.9 12.3 12.7 12.2 11.9

103.6 87.8 83.9 82.9 86.6 87.3 87.6 89.6 87.8 95.9 97.6

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, aflatoxin B1; AFB2, aflatoxin B2; AFG1, aflatoxin G1; AFG2, aflatoxins 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 five 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. LC–MS/MS method development Different proportions of methanol:acetic acid (0.1%) or acetonitrile:acetic acid (0.1%) (5:95–95:5 v/v) was used for elution of mycotoxins in LC–MS/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 flow rate of 0.25 mL/min with a gradient elution programme. The gradient elution was started at 5% methanol (0–8 min) with a linear increase to 90% (8–22 min). Then 90% methanol of the isocratic period (8–22 min) was decreased to 5% methanol in 3 min (22–25 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 certified reference materials. Maycotoxin

Contamination level (ng/g)

Measured values (ng/g)

DON ZEA FB1 FB2 OTA

474 ± 30 60 ± 9 2406 ± 612 630 ± 116 2.7 ± 1

456 ± 36 57 ± 7 2355 ± 278 543 ± 56 2.3 ± 0.2

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

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F. Soleimany et al. / Food Chemistry 130 (2012) 1055–1060 Table 5 Mycotoxin contamination in cereal samples in Malaysian market. Mycotoxins

Number of naturally contaminated samples (range of contamination (ng/g))

N

Rice 50

Wheat 20

Barley 10

Oat 10

Maize meal 10

100

Aflatoxins

28 (0.15–4.54)

15 (0.2–3.2)

5 (0.26–2.59)

4 (0.12–1.94)

8(0.15–1.8)

60 (0.12–4.54)

OTA ZEA DON FB1 FB2 T2 HT2

21 (0.2–4.34) 12 (1.5–51.1) 13 (12.5–81.2) 7 (41.3–53.2) 3 (40.1–61.5) 7 (10.5–95.2) 7 (8.1–26.1)

7 (0.15–2.11) 6 (1.42–12.74) 10 (22.8–112.5) 3 (42.5–69.1) 3 (42.0–75.3) 3 (11.2–53.1) 3 (9.3–18.7)

3 3 5 3 3 1 1

3 0 3 3 1 1 1

6 4 5 3 3 4 4

40 (0.1–5.76) 25 (0.95–81.1) 36(20.5–109) 19 (41.3–209.3) 13 (40.1–113.5) 16 (10.5–50.7) 16 (8.1–81.8)

Occurrence

40

15

7

Type of cereal

Total

(0.18–2.84) (0.95–20.26) (27.9–72.5) (45.5–97.7) (43.1–72.5) (12.7–55.9) (10.1–30.7)

(0.1–0.2) (ND) (22.7–100.2) (49.5–177.3) (57.3) (39.5) (25.2)

7

8

(0.1–5.76) (1–13.47) (35–109) (48.2–209.3) (58.7–113.5) (31.7–50.7) (29.5–81.8)

77

N, number of samples analysed; ND, not detected; aflatoxins (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 final 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 LC–MS/MS parameters part. Fig. 1 shows LC–MS/ 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).

3.2. LC–MS/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 0.01–20 ng/g and 0.02–40 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).

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), (52.6–89.2%), Sulyok et al. (2006), (75– 108%), Spanjer et al. (2008), (46–115%), Monbaliu et al. (2009), (76–105%) 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 confirmed the trueness of measurements. The measured values are shown in Table 4. 3.3. Application of the LC–MS/MS method to commercial cereal samples The LC–MS/MS method was applied in the quantification of aflatoxins (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 LC–MS/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 aflatoxins 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 aflatoxins, 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 0.01–20 ng/g and 0.02–40 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

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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 aflatoxins. 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 financial support through the Research University Grant Scheme (RUGS), project number 02-01-07-0024RU. References Bennett, J. W., & Klich, M. (2003). Mycotxins. Clinical Microbiology Reviews, 16, 497–516. Berthiller, F., Schuhmacher, R., Buttinger, G., & Krska, R. (2005). Rapid simultaneous determination of major type A and B-trichothecenes as well as zearalenone in maize by high performance liquid chromatography–tandem mass spectrometry. Journal of Chromatography A, 1062, 209–216. Biselli, S., & Hummert, C. (2005). 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. Food Additives and Contaminants, 22, 752–760. Cavaliere, C., Foglia, P., Pastorini, E., Samperi, R., & Lagana, A. (2005). Development of a multiresidue method for analysis of major Fusarium mycotoxins in corn meal using liquid chromatography/tandem mass spectrometry. Rapid Communications in Mass Spectrometry, 19, 2085–2093. Delmulle, B., Saeger, S. D., Adams, A., Kimpe, N. D., & Peteghem, C. V. (2006). Development of a liquid chromatography/tandem mass spectrometry method for the simultaneous determination of 16 mycotoxins on cellulose filters and in fungal cultures. Rapid Communications in Mass Spectrometry, 20, 771–776. EC (European Communities) No. 1881/2006, European commission Regulation (EC) No. 1881/2006 of 19 December, 2006. Setting maximum levels for certain contaminants in foodstuffs. Official Journal of the European union, L364/5, 15–17. EC (European Communities) No. 401/2006, European commission Regulation (EC) No. 401/2006 of 23 February, 2006. Laying down the methods of sampling and analysis for the official control of the levels of mycotoxins in foodstuffs. Official Journal of the European union, L70/12, 20–23. EC (European Communities) No. 657/2002, of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results. Official Journal of the European union, L221/8, 1– 29.

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