Food Chemistry 146 (2014) 242–249

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Analytical Methods

Simultaneous determination of aflatoxin M1, ochratoxin A, zearalenone and a-zearalenol in milk by UHPLC–MS/MS L.C. Huang a,b,d,1, N. Zheng a,d,1, B.Q. Zheng c, F. Wen a,d, J.B. Cheng a,b, R.W. Han a,d, X.M. Xu a,d, S.L. Li a,d, J.Q. Wang a,d,e,⇑ a Ministry of Agriculture Laboratory of Quality & Safety Risk Assessment for Dairy Products (Beijing), Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, PR China b College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, PR China c Tangshan Livestock and Aquatic Products Quality Monitoring Center, Tangshan 06300, PR China d Ministry of Agriculture – Milk and Dairy Product Inspection Center (Beijing), Beijing 100193, PR China e State Key Laboratory of Animal Nutrition, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, PR China

a r t i c l e

i n f o

Article history: Received 21 January 2013 Received in revised form 24 August 2013 Accepted 8 September 2013 Available online 18 September 2013 Keywords: Milk Mycotoxin Matrix effect Matrix-matched calibration curve UHPLC–MS/MS

a b s t r a c t In this study, a sensitive and rapid method has been developed for the simultaneous determination of aflatoxin M1, ochratoxin A, zearalenone and a-zearalenol in milk by ultra high performance liquid chromatography combined with electrospray ionisation triple quadrupole tandem mass spectrometry (UHPLC–ESI–MS/MS). The milk samples were purified using Oasis HLB cartridge. The matrix effects were evaluated by determining the signal suppression–enhancement (SSE) and corrected by external matrix-matched calibration. The limits of quantity (LOQ) of the mycotoxins were in the range of 0.003–0.015 lg kg1. The high correlation coefficients (R2 P 0.996) were obtained in the range of 0.01–1.00 lg kg1 of the mycotoxins, along with good recovery (87.0–109%), repeatability (3.4–9.9%) and intra-laboratory reproducibility (4.0–9.9%) at the concentrations of 0.025, 0.1 and 0.5 lg kg1. The detected rates of the mycotoxins were from 16.7% to 96.7% in raw milk, liquid milk and milk powder samples collected from the dairy farms and supermarkets in Beijing. The method proposed is suitable for the simultaneous determination of aflatoxin M1, ochratoxin A, zearalenone, and a-zearalenol, and could be performed for analysing the mycotoxins in milk. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Mycotoxin contamination, especially in milk, has evoked global concern on feed and food safety due to the toxic effects of mycotoxins in animals and human (Blount, 1961; De Iongh, Vles, & Van Pelt, 1964; Jolly et al., 2007; Pattono, Gallo, & Civera, 2011; Strosnider et al., 2006; Williams et al., 2004). Mycotoxins, a series of secondary metabolites produced by moulds, mainly come from feed contaminated either in the field or during drying and storage. To ensure the quality and safety of animal-derived products, the levels of aflatoxin B1 (AFB1), ochratoxin A (OTA), and zearalenone (ZON) in feed have been limited to 10, 100, and 500 lg kg1, respectively, in China (GB13078-2001; GB13078.2-2006) 20 lg kg1 of total aflatoxins in USA (Ren et al., 2007), and 5 lg kg1 of AFB1 in EU (EC, 2006, 2007). These mycotoxins can ⇑ Corresponding author at: State Key Laboratory of Animal Nutrition, Ruminant Nutrition Laboratory, Institute of Animal Science, Chinese Academy of Agricultural Sciences, No. 2, Yuan Ming Yuan West Road, Haidian District, Beijing 100193, PR China. Tel.: +86 10 62815859; fax: +86 10 62897587. E-mail address: [email protected] (J.Q. Wang). 1 These two authors contributed equally. 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.09.047

be metabolized or transferred to raw milk in the forms of aflatoxin M1 (AFM1), OTA, ZON, and a-zearalenol (a-ZOL), which were stable throughout processing of dairy products and could exist in liquid milk and milk powder (Bullerman & Bianchini, 2007). The toxic effects of mycotoxins include mutagenic, teratogenic, carcinogenic, and immunosuppressive effects. The International Agency for Research on Cancer (IARC) has defined AFM1 and OTA as potential carcinogens, and they are included in Group 2B (IARC, 1993a, 1993b). Although ZON is considered non carcinogenic, it causes other adverse effects; in particular, it may affect reproduction in mammals because of its estrogenic effects. The estrogenic activity of a-ZOL is 3–4 times higher than that of the parent compound ZON (Minervini, Giannoccaro, Cavallini, & Visconti, 2005). To safeguard public health, the maximum residual levels (MRLs) of mycotoxins have been established by different national and international organizations (EC, 2006, 2007). The MRLs of AFM1 in milk are set at 0.5 lg kg1 in China and USA, and 0.05 lg kg1 in the European Union (EU). More restrictive levels are set for baby food in the EU, being 0.025 lg kg1 for AFM1, 0.5 lg kg1 for OTA, and 20 lg kg1 for ZON, respectively. To enforce the restrictive

L.C. Huang et al. / Food Chemistry 146 (2014) 242–249

regulation on mycotoxins in milk, the rapid, sensitive, and precise detection methodologies are in demand. The enzyme-linked immune sorbent assay (ELISA) has been widely used for mycotoxin detection (Kav, Col, & Kaan Tekinsen, 2011; Rastogi, Dwivedi, Khanna, & Das, 2004; Turner, Subrahmanyam, & Piletsky, 2009). However, ELISA suffers from the disadvantages of occasionally pseudo-positive and inaccurate results, which limit its further application (Beltrán et al., 2011; Ren et al., 2007; Sforza, Dall’Asta, & Marchelli, 2005). Therefore, some confirmatory and quantification methods have been developed based on gas chromatography (GC) (Valle-Algarra et al., 2005), high-performance liquid chromatography (HPLC) (Liu, Zhu, Cheng, & Senyuva, 2012; Solfrizzo, Panzarini, & Visconti, 2008), and LC or GC combined with mass spectrometry (MS) (Nielsen & Thrane, 2001; Njumbe Ediage, Diana Di Mavungu, Monbaliu, Van Peteghem, & De Saeger, 2011; Oueslati, Romero-González, Lasram, Frenich, & Vidal, 2012). The determination of 18 mycotoxins in bovine milk by LC–MS/MS has been reported (EC, 2002). The method requires two types of chromatographic columns for mycotoxin separation according to the electrospray ionisation modes, which makes the procedure relatively complex and tedious. Recently ultra-performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) has been used for mycotoxin analysis owing to its high selection and sensitivity (Xia et al., 2009). UHPLC–MS/MS has also been applied for the simultaneous determination of multi-mycotoxins in food and feed (Beltrán, Ibáñez, Sancho, & Hernández, 2009; Beltrán et al., 2011; Liu et al., 2012; Ren et al., 2007; Sørensen & Elbæk, 2005), blood plasma (Mathias, Siegrid, Patrick, & Siska, 2012), and urine samples (Desalegn et al., 2011). With great concern on the mycotoxin contamination of milk, the UHPLC–MS/MS method for multi-mycotoxins detection in milk is currently under development. Beltrán et al. (2011) used UHPLC– MS/MS for determination of aflatoxins, AFM1, and OTA in baby food and milk. However, this method could not detect OTA at its trace level of 0.025 lg kg1 in raw milk and milk powder, due to difficultly in purification. In this study, we aim to establish a sensitive and rapid UHPLC–MS/MS method for the simultaneous analysis of AFM1, OTA, ZON, and a-ZOL in raw milk, liquid milk, and milk powder to survey the actual contaminant situations in milk in China. In the purification steps, solid phase extraction (SPE) conditions were optimised by a full factorial experiment and a multilevel factorial experiment design. The matrix effects were evaluated by signal suppression–enhancement (SSE) and compensated by matrixmatched standards calibration. The method has been validated in three different matrices (raw milk, liquid milk, and milk powder) with good recovery (87.0–109%), repeatability (3.4–9.9%) and intra-laboratory reproducibility (4.0–9.9%). 2. Experimental 2.1. Reagents and chemicals The standards of AFM1, OTA, ZON, and a-ZOL were supplied by Sigma–Aldrich (BioReliance, USA). All standards were stored at 20 °C under dark. HPLC-grade methanol (MeOH), acetonitrile (ACN), and aqueous ammonia were obtained from Merck (USA), Fisher Scientific (USA), and Sigma (USA), respectively. Phosphate buffer solutions (PBS) of pH 2 and 10 were purchased from Hanna (USA). Milli-Q quality water (Millipore, USA) was used during all the analyses. The standard stock solutions (50 mg L1) of AFM1, OTA, ZON, and a-ZOL were prepared in MeOH. The combined standard solutions (500 lg L1) were prepared by transferring 1 mL of each stock solution of 50 mg L1 to a volumetric flask, followed by further dilution with MeOH up to a final volume of 100 mL. They were further diluted with MeOH/water (50/50, v/v) to the final

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concentrations of 5 and 1 lg L1 as the working solutions. The stock and combined standard solutions were stored at 20 °C, whereas the working solutions were stored at 4 °C before use. The non-contaminated milk was spiked with the combined standard solutions for mycotoxin standard calibration. The final concentrations of the mycotoxins were set at 0.01, 0.025, 0.05, 0.1, 0.5, and 1 lg kg1, respectively, which fell into the analytical range of 0.04–4 lg kg1. 2.2. Instrumentation The UHPLC system consisted of an Acquity UHPLC (UHPLC™, Waters, USA). The LC separations were performed on an UHPLC BEH C18 column (1.7 lm, 50 mm  2.1 mm, Waters, USA) using solvent A (MeOH) and solvent B (0.1% (v/v) aqueous ammonia) at a flow rate of 0.4 mL min1. A gradient programme was used as follow: 90% B (initial), 90–10% B (2.0 min), 10–90% B (0.1 min). A subsequent re-equilibration time (1.9 min) was allowed before next injection. In addition, the column and sample temperature were maintained at 40 and 25 °C, respectively. The LC column effluent was interfaced to a TQS™ Micromass Quattro Ultima triple-quadrupole MS equipped with an electrospray ion source (Micromass, Manchester, UK) and MassLynx V 4.1 software. Nitrogen was used as the desolvation gas and cone gas, and argon as the collision gas. 2.3. Sample collection and preparation Fifty samples were collected from dairy farms and supermarkets in Beijing, China in April 2012. Among them, 30 samples of raw milk were collected from 30 different dairy farms, 12 samples of liquid milk and 8 samples of milk powder from the supermarkets. All the samples were stored at 20 °C before analysis. Milk powder was reconstituted by following manufacturer instruction. Each homogenised milk sample was accurately weighed (2.0 g, precision: 0.1 mg) into 15 mL centrifuge tubes. Then, 8 mL of ACN was added to extract the mycotoxins from the sample and simultaneously precipitate proteins. The entire mixture was vortexed for 2 min using Vortex-Genie 2 (Scientific Industries, USA) and then put in an ultrasonic bath (KQ-500 DE, China) for 30 min. Finally, the extracts were centrifuged at 12,100g for 10 min at 4 °C, and the supernatant was collected. The supernatant was concentrated to 2 mL by evaporation at 50 °C under a nitrogen stream. The concentrate was mixed with 4 mL of water, and the pH was adjusted to 5.0 ± 0.2 with PBS. The solution was applied to an Oasis HLB cartridge (60 mg, 3 cm3, Waters, USA) at a flow rate of 0.5 mL min1. The cartridge was previously conditioned with 2 mL of MeOH and 2 mL of water. After washing with 2 mL of water, the mycotoxins were eluted with 4 mL of MeOH, and the eluate was evaporated to bare dryness at 50 °C. Another aliquot of supernatant was directly loaded into Mycosep 226 (Romer Labs, USA), and the eluent was dried by evaporation at 50 °C under a nitrogen stream. Each residue was re-dissolved in 500 lL of aqueous MeOH solution (50:50, v/v). After the solution was filtered through a polytetrafluoroethylene (PTFE) filter, 5 lL of the final extract solution was injected into the UHPLC–ESI–MS/MS system. The recovery R (%) was calculated according to the following equation, where the concentration of the standard solution was equal to the concentration spiked in the sample (Maragou, Rosenberg, Thomaidis, & Koupparis, 2008):

Rð%Þ ¼

signal from spiked sample  signal from unspiked sample signal from standard solution  100%

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L.C. Huang et al. / Food Chemistry 146 (2014) 242–249

Table 1 MS/MS parameters, SSE and variance of raw milk, liquid milk and milk powder. Precursor ion (m/z)

Production1 (m/z)

AFM1

329.0

OTA

402.0

ZON

316.8

a-ZOL

318.8

273.0⁄ 259.0 357.9⁄ 166.8 174.8⁄ 130.9 275.0⁄ 160.0

Compound

CE1 (eV)

CV1 (V)

TR1 (min)

Ionisation mode

5

33

1.48

21

20

21 21

Variance2

SSE (%) Raw milk

Liquid milk

Milk powder

Raw milk

Liquid milk

Milk powder

ESI+

65

66

68

307375.0a

313271.0a,b

322342.0b

0.99

ESI

177

175

173

7349.0

7244.5

7032.7

20

1.45

ESI

74

53

77

15053.3a

10323.0b

15779.0a

20

1.50

ESI

81

57

63

6321.0a

4221.7b

5053.4b

The milk matrixes with the same superscript letter (a, b, or c) per compound do not statistically differ in their slope at 95% or higher confidence level. 1 CE, collision energy; CV, cone voltage; TR, retention time; asterisk (⁄) indicates quantitative ion. 2 Variance, the difference among the slopes of the curves of each mycotoxin in the three different milk matrices.

Fig. 1. Selected ion chromatograms of MRM in 0.025 lg kg1 standard solution prepared in milk matrix.

2.4. Method validation The method developed was validated for detection of the mycotoxins in raw milk, liquid milk, and milk powder according to the guide of Commission Decision 2002/657 (EC, 2002). Method capa-

bility was evaluated by the following parameters: the linearity was evaluated by matrix-matched calibration curves in the range of 0.01–1 lg kg1; the LOD and LOQ were estimated by MassLynx V 4.1 instrumental software, from the chromatograms of the samples spiked with the mycotoxins at 0.025 lg kg1, at a signal-to-noise

L.C. Huang et al. / Food Chemistry 146 (2014) 242–249

245

Fig. 2. UHPLC–MS/MS chromatograms for AFM1, OTA, ZON, and a-ZOL corresponding to (a) milk spiked to 0.025 lg kg1 after direct injection of the raw diluted extract, (b) polymeric Oasis HLB SPE cartridge, and (c) Mycosep 226 cartridge.

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L.C. Huang et al. / Food Chemistry 146 (2014) 242–249

Fig. 3. (a) Standardised Pareto chart; (b) estimated response surface for AFM1. Water volume: 2 mL, SPE flow rate: 0.5 mL min1.

ratio (S/N)P3 and P10, respectively; The precision, repeatability (RSDr) and intra-laboratory reproducibility (RSDR) were determined on 6 replicated pooled samples of each milk matrix spiked with the mycotoxins at 0.025, 0.1 and 0.5 lg kg1, but RSDR was determined by different operators on different days.

3. Results and discussion 3.1. Optimisation of MS/MS conditions The ionisation mode and precursor ions were selected according to the highest relative intensity under positive- and negativemode electrospray ionisation (ESI+ and ESI) by infusing standard solutions via a syringe pump (Table 1). ESI mode was selected in this study for OTA analysis due to its higher sensitivity and stability in negative mode, although it could also be determined in ESI+ mode. ZON and a-ZOL were analysed in ESI mode, whereas AFM1 was analysed in ESI+ mode due to its higher ionisation efficiency. Positive/negative ion modes were switched automatically by positive/negative polarity switching in a single chromatographic run. For each compound, the collision energies and cone voltages were optimised in the multiple reaction monitoring (MRM) mode to find precursor ions and the two most intensive product ions for quantitation and qualitation, respectively (Table 1). ZON and a-ZOL were not separated completely in the chromatograghic separation system because of their similar structures and precursor ions. However, considering the fact that the characteristic product ions for quantitation and qualitation under MRM mode were 316.8 > 174.8, 316.8 > 130.8 and 318.8 > 275.0, 318.8 > 160.0 for

ZON and a-ZOL, respectively, ZON and a-ZOL could be successfully distinguished with no interference (Fig. 1). 3.2. Extraction of mycotoxins The effects of sample pH and extraction solvent on the extraction efficiency were evaluated. The sample pH was adjusted to 2–5 by acetic acid. No significant difference was observed in the recoveries of the four mycotoxins from acetic acid-treated samples. Two solvents of ACN and MeOH were used as extraction solvents. The recoveries with ACN were 92.8%, 92.3%, 94.5% and 90.4% for AFM1, OTA, ZON and a-ZOL, respectively, which were higher than these for MeOH (82.3%, 64.3%, 78.5% and 61.5% for AFM1, OTA, ZON and a-ZOL, respectively). The results were in accordance with those reported (Beltrán et al., 2009; Wang, Zhou, Liu, Yang, & Guo, 2011). 3.3. Optimisation of purification conditions Two cartridges of Oasis HLB and Mycosep 226 were selected to purify the mycotoxins from milk samples. The results showed that direct injection of the extract without purification treatments caused signal suppression because of the matrix effect (Fig. 2a); very weak signals were obtained for OTA and a-ZOL, making it impossible to accurate quantify OTA and a-ZOL after Mycosep 226 cartridge purification (Fig. 2c). The purifying efficiency was successfully improved and many impurities were removed when the supernatant was purified with Oasis HLB cartridges (Fig. 2b). Therefore, Oasis HLB cartridge was found to be more suitable than Mycosep 226 cartridge.

8.4 7.8 7.0 9.5 9.6 7.9 9.2 8.9 8.3 7.9 7.1 7.7

3.4. Matrix effect Matrix effect, caused by the co elution of matrix components, can affect the ionisation efficiency of the analytes (Gilar, Bouvier, & Compton, 2001; King, Bonfiglio, Fernandez-Metzler, Miller-Stein, & Olah, 2000; Taylor, 2005). SSE was applied to evaluate matrix effect on the signals of the mycotoxins from raw milk, liquid milk and milk powder. SSE for each analyte in each matrix was calculated and defined as a percentage of the matrix-matched calibration slope divided by the slope of the standard calibration in solvent (Rubert, Soler, & Mañes, 2011; Spanjer, Rensen, & Scholten, 2008; Sulyok, Krska, & Schuhmacher, 2007):

R ± SD = mean recovery ± standard deviation. Number of replicated: 6. a

Milk powder

Liquid milk

AFM1 OTA ZON a-ZOL AFM1 OTA ZON a-ZOL AFM1 OTA ZON a-ZOL Raw milk

y = 308295x + 115.1 y = 7198.3x + 38.7 y = 15094x  125.9 y = 6330.1x  10.5 y = 313267x  518.2 y = 7096.8x  98.8 y = 10940x + 23.6 y = 4466.4x + 38.2 y = 322342x + 1206.3 y = 7032.7x + 82.0 y = 15779x + 84.8 y = 4947.7x + 65.3

0.999 0.998 0.999 0.999 0.999 0.999 0.999 0.998 0.996 0.999 0.999 0.999 b

RSDr

8.3 7.4 6.0 9.8 9.6 7.7 9.2 8.3 8.2 7.8 6.6 7.6 97.7 ± 8.1 95.9 ± 7.1 95.2 ± 5.7 99.9 ± 9.8 97.2 ± 9.3 93.9 ± 7.2 96.5 ± 8.9 87.0 ± 7.2 101.9 ± 8.4 101.2 ± 7.9 101.2 ± 6.7 100.7 ± 7.7 8.0 8.5 6.2 8.4 9.0 6.7 8.3 7.8 8.5 9.6 6.0 8.9 7.9 8.3 6.2 7.9 8.4 6.3 8.0 6.7 8.0 9.1 6.8 8.5 100.3 ± 7.9 98.4 ± 8.2 94.8 ± 5.9 97.4 ± 7.7 98.3 ± 8.3 97.6 ± 6.1 95.4 ± 7.6 90.7 ± 6.1 94.5 ± 7.6 95.6 ± 8.7 96.4 ± 6.6 92.9 ± 7.9 9.5 9.6 4.0 8.1 7.8 9.5 9.6 9.2 9.7 9.9 8.8 9.5 9.5 9.5 3.4 8.1 6.7 9.4 9.5 9.9 9.4 9.4 8.5 9.5 0.003 0.012 0.003 0.009 0.006 0.009 0.006 0.015 0.003 0.009 0.003 0.012 0.001 0.004 0.001 0.003 0.002 0.003 0.002 0.005 0.001 0.003 0.001 0.004

99.5 ± 9.5 99.7 ± 9.5 99.5 ± 3.4 98.8 ± 8.0 109.0 ± 7.3 99.9 ± 9.4 99.4 ± 9.4 88.8 ± 8.8 90.8 ± 8.5 93.3 ± 8.8 91.3 ± 7.8 89.9 ± 8.5

R ± SDa RSDr RSDr R ± SD

a

0.025 lg kg1 (%)b LOQ (lg kg1) LOD (lg kg1) Coefficient (R2) Calibration curve (lg kg1) Compound

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The SPE pH of crude extraction solution (5–8.5), SPE flow rate (0.5–1.5 mL min1), water volume (2–6 mL), and eluting solution (0–100% ACN–MeOH solution) were tested at two indicated levels for SPE optimisation using Oasis HLB cartridge. A full factorial experimental design 24 + 1 centre point (Maragou et al., 2008) were conducted for spiked samples of AFM1, OTA, a-ZOL, and ZON at a concentration of 0.025 lg kg1 to determine optimum condition. The data was processed using the SAS 8.0 software. In all four cases, the critical influencing factors were found to be SPE pH of crude extraction solution and eluting solution, and each was presented in a standardised Pareto chart for AFM1 (Fig. 3a). The length of each bar representing the estimated effect divided by its standard error is proportional to the standardised effect. A first-order model was fitted to the data, and the corresponding response surface is shown in Fig. 3b. When SPE pH of crude extraction solution was increased from 5.0 to 8.5, the signal suppression was increased with declined peak area. The results indicated that the best signal was obtained at pH 5.0 of crude extraction solution with 100% MeOH as eluting solution. A multilevel factorial design with SPE flow rates at three levels (0.5, 1.0, and 1.5 mL min1) and water volumes for washing at three levels (2, 4, and 6 mL) was realised with the samples spiked by 0.025 lg kg1 of each AFM1, OTA, ZON, and a-ZOL. The signal was better when the flow rate and water volume were at 1.5 mL min1 and water volume at 2 mL, respectively. Therefore, the optimal SPE conditions with Oasis HLB cartridge were the crude extraction solution at pH 5.0, and eluting solution of 100% MeOH, flow rate at 1.5 mL min1, and water volume of 2 mL for washing.

SSEð%Þ ¼

Milk

Table 2 Calibration curves, relative repeatability standard deviation (RSDr) and intra-laboratory reproducibility standard deviation (RSDR).

RSDR

R ± SD

a

0.1 lg kg1 (%)b

RSDR

0.5 lg kg1 (%)b

RSDR

L.C. Huang et al. / Food Chemistry 146 (2014) 242–249

matrix-matched calibration slope  100% slope of standard calibration

SSE > 100%, signal enhancement; SSE = 100%, no absolute matrix effect; SSE < 100%, signal suppression. The results indicated that notable signal suppression occurred for AFM1, ZON, and a-ZOL in milk, whereas notable signal enhancement occurred for OTA (Table 1). Similar matrix effects of three milk matrices have been observed for AFM1 and OTA, whereas quite different effects have been observed for ZON and a-ZOL. Therefore, matrix-matched standards calibration curves are necessary to ensure the accuracy of the analytical results (Beltrán et al., 2009; EC, 2002; Wang et al., 2011). The difference among the slopes of the curves of each mycotoxin in the three different milk matrices was analysed by software SAS 8.0 (Table 1). If the slopes of the curves of each mycotoxin in the three milk matrices did not have statistically different slopes at 95% or higher confidence level, one calibration curve could be used for raw milk, liquid milk and milk powder. OTA produced curves with statistically the same slope for all the three matrices, whereas AFM1, ZON, and a-ZOL showed significantly different slopes. To avoid any over- or under-estimation of the mycotoxin residues, matrix-matched standards calibration curves of each mycotoxin were obtained in raw milk, liquid milk, and milk powder (Table 2).

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L.C. Huang et al. / Food Chemistry 146 (2014) 242–249 Table 3 Occurrence of mycotoxin residues in milk in Beijing. Milk (N1)

Mycotoxin

Mean ± SD (ng kg1)

Detectable rate (%)

Maximum (ng kg1)

Raw milk (30)

AFM1 OTA ZON a-ZOL AFM1 OTA ZON a-ZOL AFM1 OTA ZON a-ZOL

80.4 ± 87.7 56.7 ± 23.1 14.9 ± 6.0 24.3 ± 16.1 32.3 ± 16.5 26.8 ± 14.9 20.5 ± 11.1 36.7 ± 7.9 16.0 ± 8.2 27.0 ± 16.4 11.6 ± 1.1 43.1 ± 18.5

80.0 96.7 23.3 93.3 33.3 91.7 16.7 41.7 25.0 62.5 25.0 37.5

237.4 84.1 45.8 73.5 46.0 57.9 28.3 45.1 21.8 49.4 12.4 64.3

Liquid milk (12)

Milk powder (8)

1

N, Number of samples analysed.

3.5. Method capability

References

Good linearity, with correlations coefficients (R2) higher than 0.996, was obtained for all mycotoxins in the range of 0.01–1 lg kg1 (Table 2). The LODs and LOQs of selected mycotoxins in milk were in the range of 0.001–0.005 and 0.003–0.015 lg kg1 (Table 2), respectively, which were sensitive enough to meet the requirement of the EU regulations for the corresponding maximum levels of mycotoxins in milk. The LOQs were lower than reported results (EC, 2002; Xia et al., 2009) and comparable to the results developed by using immunoaffinity clean-up pretreatment (Beltrán et al., 2011). Good recoveries (87.0–109.0%), RSDr (3.4–9.9%) and intra-laboratory RSDR (4.0–9.9%) were obtained (Table 2).

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In this study, a sensitive and rapid method has been developed for the simultaneous determination of aflatoxin M1, ochratoxin A, zearalenone and α-zea...
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