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Determination of aflatoxin M1 in ice cream samples using immunoaffinity columns and ultra-high performance liquid chromatography coupled to tandem mass spectrometry
Food Control 56 (2015) 34e40
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Determination of aflatoxin M1 in ice cream samples using immunoaffinity columns and ultra-high performance liquid chromatography coupled to tandem mass spectrometry , Gustavo Antonio Pen ~ uela Duvan Esteban Hoyos Ossa*, Diego Alfonso Hincapie GDCON Research Group, Faculty of Engineering, University Research Headquarters (SIU), University of Antioquia, Street 70 # 52 e 21, Medellin, Colombia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 December 2014 Received in revised form 12 February 2015 Accepted 10 March 2015 Available online 18 March 2015
In this work, a highly sensitive method was developed for the selective determination of aflatoxin M1 mycotoxin in ice cream samples using ultra high performance liquid chromatographyetandem mass spectrometry (UHPLC-MS/MS). The low detection values required, of mg/kg, made necessary sample concentration and cleaning using highly targeted immunoaffinity columns for this compound after extraction from matrix using a phosphate buffered saline solution (PBS). Method validation was carried out for ice cream matrix by means of recovery experiments using fortified samples near to the lower end of the calibration curve and a higher level. All recoveries were found in the range of 70e120 % with % RSD less than 25%. Quantitation was performed using external calibration with standards prepared in mobile phase. Validated method was used to determine the content of AFM1 in samples of ice cream bought in local stores. Compound identification was performed using the quantitation/qualification ratios for monitored transitions (Q/q1 and Q/q2). © 2015 Elsevier Ltd. All rights reserved.
Keywords: Aflatoxin M1 Ice cream Immunoaffinity columns UHPLC-MS/MS Chemical compounds studied in this article: Aflatoxin M1 (PubChem CID: 15558498)
1. Introduction Mycotoxins are toxic secondary metabolites produced by fungi. These types of substances have very different chemical structures, but all are generally chemical compounds of relatively low molecular mass. Mycotoxins are distributed in a wide range of food and feed, and constitute a risk for human and animal health, to induce potent and diverse biological effects (Peraica, Radic, Luci c, & Pavlovi c, 1999). Some of these substances exhibit carcinogenic, teratogenic, mutagenic and neurotoxic effects,etc; while others show antitumor capacity, cytotoxic and antimicrobial properties (Kumar, Basu, & Rajendran, 2008; Steyn, 1995). The effects on human and animal health, known as mycotoxicosis, depend among other factors on the toxicity of the mycotoxin, the degree of exposure, age, the amount consumed and nutrition status of the individual (Steyn, 1995). Human ingestion of mycotoxins is due to the consumption of foods prepared from contaminated sources with these substances and the consumption of animal source foods with metabolites residues of this kind of toxins (as aflatoxin M1 in
* Corresponding author. Tel.: þ57 4 2196689; fax: þ57 4 2196571. E-mail address: [email protected] (D.E. Hoyos Ossa). http://dx.doi.org/10.1016/j.foodcont.2015.03.011 0956-7135/© 2015 Elsevier Ltd. All rights reserved.
milk) (Steyn, 1995). These toxins are produced mostly by 5 fungi genera: Aspergillus, Fusarium, Alternaria, Penicillium and Claviceps (Steyn, 1995). Aflatoxins, ochratoxins, trichothecenes, zearalenone and fumonisins are the most important groups of mycotoxins often found in foodstuffs and agricultural commodities (Kumar et al., 2008; Zain, 2001). Due to the risk of contamination by mycotoxins, products like peanuts, cereals (barley, maize, rice, sorghum and oats), spices (like black pepper) and chili are considered of global significance over human health beings (Kumar et al., 2008). The Aflatoxins are produced mainly by Aspergillus flavus and Aspergillus parasiticus, A. flavus produces only B aflatoxins, and A. parasiticus produces B and G aflatoxins (Peraica et al., 1999). The oxidative metabolites of aflatoxin B1 and B2 are respectively the aflatoxin M1 and M2, which can be found in milk or milk products obtained from animals that have consumed contaminated feed (Peraica et al., 1999). By eating foods contaminated with aflatoxin B1, mammals excrete hydrolyzed aflatoxin M1 (AFM1) in the urine, faeces and milk. The AFM1 excreted in cow milk has toxic properties similar to that of aflatoxin B1, the most carcinogenic of aflatoxins (Meerdink & Abvt, 2002). Cow milk is one of the key ingredients for ice cream preparation, and is used for its contribution of fat and non-fat solids. In ice cream
D.E. Hoyos Ossa et al. / Food Control 56 (2015) 34e40
preparation, milk can be added both liquid and powder, and therefore the presence of AFM1 residues in milk is transferred to the finished product, since it has been demonstrated that thermal treatment used in dairy industry, like pasteurization and sterilization, cannot deactivate this type of mycotoxin (Aguilera-Luiz, Plaza~ os, Romero-Gonza lez, Vidal, & Frenich, 2011; Prandini et al., Bolan 2009). In order to protect public health, maximum levels of mycotoxins have been established in certain foods (European Commission, 2006), including AFM1 in milk and dairy products with a value of 0.050 mg/kg. There are several methodologies for the determination of mycotoxins in dairy products, such as ELISA (Enzyme-linked inmunosorbent assay), thin layer chromatography (TLC) and solid phase extraction using immunoaffinity columns and subsequent quantification by liquid chromatography, either high performance liquid chromatography (HPLC) or ultra-high performance liquid chromatography (UHPLC) (Shephard et al., 2013). For ice cream, different studies have been conducted in recent years in order to evaluate the presence of this type of mycotoxin in this kind of product, either using thin layer chromatography (Fallah, 2010), ELISA (Nilchian & Rahimi, 2012) or HPLC (Iqbal, Asi, & Jinap, 2013) but the combination of immunoaffinity column and UHPLC for the determination of AFM1 in ice cream has not been reported (Shephard et al., 2013). HPLC hyphenated to different detection methods has been, after ELISA, one of the most important techniques in the determination of mycotoxins in food matrices and within these, the fluorescence detection has been preferred (Shephard et al., 2013). In recent years, the introduction of UHPLC allowed to carry out this analysis in shorter run times and better chromatographic resolution than HPLC, and coupled to mass spectrometry has become a highly sensitive and selective technique to quantify and confirm the presence of such contaminants in food (Beltr an et al., 2011). The aim of this work was to develop a highly sensitive liquid chromatography method which could be able to quantify and confirm the presence of aflatoxin M1 in ice cream samples at the level required by European Legislation. Particular attention was placed on the sample preparation, in order to eliminate interferences and concentrate the analyte. Immunoaffinity columns Aflaprep M were selected due to their capacity to selectively clean the ice cream matrix and concentrate the mycotoxin. The optimized method using immunoaffinity columns and UHPLC-MS/MS technology was validated with satisfactory results and applied to the analysis of commercial ice cream samples.
2. Materials and methods 2.1. Chemicals , AFM1 standard of 2000 mg/L was bought to Micotox (Bogota Colombia). Working solutions of 100, 10 and 1 mg/L were prepared in LC-MS grade methanol obtained from Merck (Darmstadt, Germany) and stored at 20 C in amber vials until use. Immunoaffinity columns Aflaprep M were purchased from R-Biopharm (Glasgow, Scotland). Deionized water used for the preparation of
35
mobile phases and the buffer solution was supplied by a UV Synergy equipment obtained from Millipore (Bedford, USA). Monobasic anhydrous potassium phosphate was purchased from Sigma Aldrich (Steinheim, Germany). Sodium chloride, potassium chloride and dibasic potassium phosphate were purchased from Merck (Darmstadt, Germany) and were used for the preparation of phosphate buffered saline solution (PBS). Ammonium formate (purity > 99.995%) was used for mobile phase preparation and was supplied by Sigma Aldrich (Germany). The PBS buffer was prepared as follows: For 1 L of solution, 8 g of sodium chloride, 0.2 g of potassium chloride, 1.44 g of dibasic potassium phosphate and 0.24 g of monobasic anhydrous potassium phosphate were weighed and diluted with deionized water. 2.2. Apparatus For UHPLC e MS/MS analysis, an ACQUITY UPLC H-Class equipment (Waters, USA) was used. An ACQUITY UPLC BEH C18 column (2.1 mm (ID) 50 mm, 1.7 mm particle size) was used for the chromatographic separation. A Waters XEVO TQD mass spectrometer (Manchester, UK) equipped with an electrospray ionization source (ESI) was used for all experiments. Equipment control and data acquisition were performed with Masslynx v. 4.1 software (Waters, USA). In addition, a BOECO U-320 centrifuge (Hamburg, Germany) equipped with temperature control was used for the sample preparation. A New Brunswick Scientific Excella E2 shaker was used to perform the mechanical agitation of the samples. 2.3. Extraction and cleanup Ten grams of ice cream melted at room temperature were weighed into a 125 mL e Erlenmeyer flask, then 100 mL of PBS solution were added and the mixture was mechanically stirred for 30 min at 270 rpm. Then, the sample was evenly divided into two centrifuge tubes of 50 mL and centrifuged at 5000 rpm for 5 min at 5 C. Following this, the supernant fat was discarded and the process repeated 2 more times. For solid phase extraction using immunoaffinity columns, the free e fat sample was passed through an Aflaprep M column at a flow not greater than 5 mL/min. Once the sample has passed, the column was washed with about 20 mL of PBS solution. Finally, the toxin was eluted from column using 1.5 mL of a methanol: acetonitrile (40:60 v/v) solution and collected on a 25 mL e clear vial. The extract was concentrated to dryness using nitrogen and then diluted to 5.0 mL using a water: methanol (95:5 v/v) 5 mM in ammonium formate solution. This solution was filtered through 0.22 mm PVDF filters and about 1 mL of sample was deposited on a 2 mL amber vial and then was injected in the UHPLC-MS/MS equipment. 2.4. UHPLC conditions Waters ACQUITY UPLC H-Class equipment coupled to a XEVO TQD mass spectrometer was used for the analysis. The mobile phase consisted of water:methanol (95:5 v/v) 5 mM in ammonium formate (A) and methanol:water (95:5 v/v) 5 mM in ammonium
Table 1 Monitored transitions for AFM1. Compound
Formula
Transition
Parent ion (m/z)
Cone voltage (V)
Daughter ions (m/z)
Collision energy (V)
Ionization mode
Aflatoxin M1
C17H12O7
1 2 3
328.96 328.96 328.96
42 42 42
273.01 258.98 229.03
26 24 48
ESI þ ESI þ ESI þ
36 D.E. Hoyos Ossa et al. / Food Control 56 (2015) 34e40 Fig. 1. Comparison of AFM1 standards chromatograms obtained by HPLC-FLD and UHPLC-MS/MS. The chromatogram (a) represents a 0.100 mg/L AFM1 standard obtained by HPLC-FLD (AFM1 at 5.40 min) and the chromatogram (b) represents a 0.050 mg/L AFM1 standard obtained by UHPLC-MS/MS (AFM1 at 2.98 min).
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37
Table 2 ANOVA for response surface (quadratic model, Box e Behnken design). Factors
Sum of squares
Mean square
F Value
p- value
A: Source temperature B: Desolvation temperature C: Desolvation gas flow D: Cone gas flow AA AB AC AD BB BC BD CC CD DD Lack-of-fit Random error
56581.3 1.0286 107 43320.1 625177 16689.9 83521.0 289.0 11236.0 96584.4 15376.0 45796.0 29466.6 32580.3 66628.0 110398. 36658.8
56581.3 1.0286 107 43320.1 625177 16689.9 83521.0 289.0 11236.0 96584.4 15376.0 45796.0 29466.6 32580.3 66628.0 11039.8 9164.7
6.17 1122.35 4.73 68.22 1.82 9.11 0.03 1.23 10.54 1.68 5.00 3.22 3.55 7.27 1.20
0.0679 0.0000 0.0954 0.0012 0.2485 0.0392 0.8677 0.3303 0.0315 0.2649 0.0891 0.1474 0.1324 0.0543 0.4644
R2 ¼ 98.7327%. R2 (adjusted) ¼ 97.4654%. DurbineWatson statistic ¼ 2.24485 (p ¼ 0.6668).
formate (B). The run was carried out in gradient mode according to the following program: at 0 min 100% A, 0.5 min 100% A, 1 min 100% B, 4 min 100% B, 5 min 100% A, 8 min 100% A; for a total run time of 8.0 min. The mobile phase flow was set at 0.35 mL/min and the injection volume was 50 mL. Column temperature was set at 40 C. The capillary voltage of Waters Xevo TQD was set to 3.6 kV for ESI þ. The source temperature was set at 120 C and desolvation gas temperature (N2) at 600 C to a flow of 800 L/h and a cone gas flow of 29 L/h. Xevo TQD was used in selected reaction monitoring mode (SRM). The collision energy (CE) and the cone voltage (CV) were optimized for the monitored compound via AFM1 standard infusion. Table 1 shows the transitions monitored for AFM1 compound.
of ice cream at two different levels, 0.050 and 0.150 mg/kg. Specificity was assessed by comparing the signals obtained at the expected retention time for a reagent blank and fortified ice cream samples. From the chromatograms of the ice cream samples spiked at 0.050 mg/kg, the LOQ and LOD were estimated regarding a signal to noise ratio of 10 and 3 respectively. Q/q ratios were evaluated from reference standards in initial mobile phase and compared to those experimentally obtained from spiked samples.
3. Results and discussion 3.1. Optimization of analysis conditions for UHPLC-MS/MS
2.5. Validation In order to ensure the method quality for the determination of AFM1 in ice cream the following parameters were evaluated: linearity, accuracy, precision, specificity, limit of quantification (LOQ), limit of detection (LOD) and Q/q ratios of the SRM transitions monitored, which were used for quantification (Q/q1) and confirmation (Q/q2) of positive findings. Linearity was estimated by analysis of six calibration standards prepared in initial mobile phase in range from 0.050 to 1.0 mg/L. Accuracy and precision were estimated by apparent recovery experiments of the target analyte, spiking five contaminated samples
Exploratory tests showed better differentiation between the signals obtained for AFM1 compound and the baseline using UHPLC-MS/MS compared to the signal provided by HPLC-FLD, even in lower concentrations. Fig. 1 shows the chromatograms obtained in the exploratory tests for the compound AFM1 using HPLC-FLD for a 0.100 mg/L AFM1 standard (a) and UHPLC-MS/MS chromatogram (TIC mode) for a 0.050 mg/L AFM1 standard (b). According to the Commission Decision of August 12th of 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results, liquid chromatography with fluorescence or spectrometric detection can be used for the
Fig. 2. Peak area residuals vs. run number.
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Table 3 Optimized values for analyzed factors. Factor
Low
High
Optimum
Source temperature Desolvation temperature Desolvation gas flow Cone gas flow
120.0 400.0 800.0 20.0
150.0 600.0 1000.0 100.0
120.0 600.0 800.02 29.015
analysis of mycotoxins residues, but the use of chromatography without spectrometric detection are not suitable on their own as confirmatory methods, due the incapacity to provide information on the chemical structure of the analyte (The Commission of the European Communities, 2002). The unequivocal identification of the compound and the clear distinction between the analyte signal and the baseline were the key features to choose the technology UHPLC-MS/MS for this work. Also, the sample cleaning obtained just with immunoaffinity columns was not enough to observe a well e resolved AFM1 peak in HPLC analysis of ice cream samples using a fluorescence detector. The use of ultra-high performance liquid chromatography for methodology development provides superior chromatographic resolution, higher sensitivity and shorter run times as compared with conventional liquid chromatography (Swartz, 2005). The total run time obtained was just 8.0 min that includes 2.0 min for column stabilization.
Two mobile phase pairs were initially evaluated in order to proceed to a subsequent optimization. The pairs tested were: 1) H2O:MeOH 95:5 (v/v) 5 mM ammonium formate and MeOH:H2O 95:5 (v/v) 5 mM ammonium formate solution, 2) H2O:MeOH 95:5 (v/v) 5 mM ammonium acetate and MeOH:H2O 95:5 (v/v) 5 mM ammonium acetate solution. The results showed a better response for mobile phase pair 1 evaluated under the same type of gradient, flow of 0.35 mL/min and 50 mL of injection. The operating conditions of XEVO TQD mass spectrometer were optimized using a BoxBehnken experimental design (Ferreira et al., 2007), which was performed using the following factors: source temperature, desolvation temperature, desolvation gas flow and cone gas flow (Nitrogen). A total of 29 runs were carried out by injecting an AFM1 standard prepared in methanol at a concentration of 1.0 mg/L. Statistical analysis of each factor is shown in Table 2. Results showed that the second-order model used to fit data explained 98.73% of the variability. The adjusted R2 (97.46%) indicated that none model terms could be discarded. The DurbineWatson statistic (p ¼ 0.6668) shows no significant correlation between the residuals, lack of fit was not significant (p ¼ 0.4644) and the residuals showed no trend in their behavior by plotting them against the runs as shown in Fig. 2. All this confirmed the model validity to adjust the response. Finally, the desirability function of DerringerSuich was used to calculate the optimum analysis conditions regarding all factors, which are shown in Table 3 and were used for UHPLC analysis for each one of samples processed.
Fig. 3. UHPLC e MS/MS chromatograms for AFM1 in ice cream matrix: (a) Reagent blank, b) Non e spiked sample and c) Spiked sample with 0.150 mg/kg by addition of 150 mL of a 10 mg/L AFM1 standard solution.
D.E. Hoyos Ossa et al. / Food Control 56 (2015) 34e40
3.2. Sample treatment The sample preparation and therefore mycotoxin extraction from the food matrix was the most critical and the most time consuming step. Foods are complex sample matrices which make highly selective extraction methods necessary and because of the low concentrations at which it is common to find the compound AFM1, methods such as solid phase extraction using immunoaffinity columns are one of the options. Several studies have been conducted in recent years in order to assess the presence of this type of mycotoxin in this kind of product, either by using thin layer chromatography (Fallah, 2010), ELISA (Nilchian & Rahimi, 2012) or HPLC (Iqbal et al., 2013). The results showed presence of AFM1 in ice cream at 69.4, 29.0 and 43.0% of the ice cream samples analyzed respectively. The QuEChERS methodology and immunoaffinity columns were tested in order to assess both the AFM1 recovery from ice cream and cleaning of the obtained extract. Immunoaffinity columns, given the ability of the methodology to selectively clean the matrix and n et al., 2011), were selected as concentrate the compound (Beltra the most suitable technique for sample preparation. The results showed satisfactory percentages of recovery in the range of 72e102 % for spiked ice cream samples. Due to the high fat and solids contents in ice cream samples, solid phase extraction using immunoaffinity columns was difficult.
39
Centrifugation at 5 C for 5 min was used to precipitate the greater amount of solids as possible and to extract fat from the top of the 50 mL centrifuge tubes. This was done after the mechanical stirring.
3.3. Methodology validation The decision of the European Union Commission of 2002 concerning to the performance of analytical methods and the interpretation of results (The Commission of the European Communities, 2002) is applicable to analytical methodologies for the determination of certain residues in products of animal origin, such as ice cream, in whose preparation milk is used. The decision of the European Union Commission in 2002 aimed to fulfill Directive 96/23/EC of April 29th, 1996, within which mycotoxins as substances to be measured and monitored in animal products are included. This document was used as a basis for validation of the methodology developed. The developed method was validated prior to be used for the analysis of commercial ice cream samples. A correlation coefficient (R2) of 0.9979 for compound AFM1 using weighted linear calibration by external standard method was obtained in the range of 0.050e1.0 mg/L (Calibration standards of 0.050, 0.100, 0.200, 0.300, 0.600 and 1.0 mg/L were prepared). Individual residuals deviation was below 20% for each one of the calibration standards.
Fig. 4. Confirmation of samples with AFM1 using retention time and Q/q1 and Q/q2 ratios. Chromatogram (a) represents one of the spiked samples and chromatogram (b) one of the analyzed commercial samples.
40
D.E. Hoyos Ossa et al. / Food Control 56 (2015) 34e40
Method accuracy was estimated at 2 levels, one near to the lowest concentration level of the calibration curve and the other at an intermediate level. For this, 2 samples contaminated with AFM1 were analyzed and subsequently spiked (each one by fivefold) in order to calculate recovery percentages. This was done because the absence of an AFM1 mycotoxin e free matrix during the exploratory trials. Samples used for the recovery experiments were spiked to 0.050 and 0.150 mg/kg adding 50 and 150 mL of a 10 mg/L AFM1 standard solution respectively. Satisfactory recoveries were obtained in the range from 76 to 102 % with relative standard deviations (% RSD) of 18.85 for the low level (n ¼ 5) and 22.90 for the other level (n ¼ 5). These were satisfactory results regarding the low concentration working range and the complexity of the matrix evaluated. Recovery for each sample with a known added amount of AFM1 was calculated according to the Equation (A).
Recovery ð%Þ ¼ ððC2 C1 Þ=Cadd Þ*100
(A)
where: C2: Concentration calculated for spiked sample. C1: Concentration calculated for contaminated sample without spiking. Cadd: Amount of AFM1 added in terms of concentration. Using signal to noise ratio calculated from the chromatograms of the ice cream samples spiked at 0.050 mg/kg, the LOQ and the LOD were estimated. The LOD varied between 0.0004 and 0.0009 mg/kg and the LOQ had values between 0.001 and 0.003 mg/kg. The method was considered to be highly specific as no interferences were observed in reagent blank at AFM1 retention time. Fig. 3 shows a chromatogram for a reagent blank (a), a sample contaminated with AFM1 (b) and for this same sample spiked with AFM1 compound (c). Q/q ratios deviations below 20% were obtained for each one of the spiked and commercial samples analyzed in relation to the values measured for calibration solutions. Fig. 4 shows the Q/q ratios and the deviation obtained for a spiked (a) and a commercial sample (b) of ice cream analyzed with the developed method. 3.4. Application to commercial ice cream samples The developed and optimized method was applied to the analysis of commercial ice cream samples from different trademarks and flavors obtained from local stores. For 7 of the 15 samples analyzed, the presence of the mycotoxin was confirmed using AFM1 retention time and Q/q ratios obtained from calibration curve. For three of these positive samples the concentration measured was above the lowest calibration level (0.025 mg/kg in sample, equivalent to 0.050 mg/L in the calibration curve according to the sample treatment) and just one sample (0.058 mg/kg) exceeded the limit fixed by the European Union regulation (0.05 mg/ kg; LMR for AFM1 in raw milk, heat-treated milk and milk for the manufacture of milk-based products) (European Commission, 2006). 4. Conclusions An efficient and highly selective method for the determination of aflatoxin M1 in ice cream samples was developed, optimized and validated. The methodology was focused on ice cream products given that previous studies have shown residuality of this mycotoxin in milk used in their manufacture. Both the conventional liquid chromatography with fluorescence detection (HPLC-FLD)
and ultra-high performance liquid chromatography coupled to tandem e mass spectrometry (UHPLC-MS/MS) were tested. The first one showed poor resolution because of the interference of the matrix, while the second successfully enabled the development of a highly sensitive method, which in turn enabled compound presence confirmation by mean of selected reactions monitoring (SRM). Methodology was validated in ice cream matrix at 2 different levels and applied to the quantification and confirmation of AFM1 in different commercial samples.
Acknowledgment This project was conducted with funding from the Departan “COLmento Administrativo de Ciencia, Tecnología e Innovacio CIENCIAS” within the Call 566 e 2012: Young Researchers and Innovators Program 2012. Funding and infrastructure necessary to perform the experiments described in the project were supplied by the Grupo Diag stico y Control de la Contaminacio n (GDCON), which provided its no materials and equipment for this research work.
References ~ os, P., Romero-Gonza lez, R., Vidal, J. L. M., & Aguilera-Luiz, M. M., Plaza-Bolan Frenich, A. G. (2011). Comparison of the efficiency of different extraction methods for the simultaneous determination of mycotoxins and pesticides in milk samples by ultra high-performance liquid chromatography-tandem mass spectrometry. Analytical and Bioanalytical Chemistry, 399(8), 2863e2875. http:// dx.doi.org/10.1007/s00216-011-4670-7. Yus n, E., Ib ~ ez, M., Sancho, J. V., Corte s, M.A., ndez, F. (2011). Beltra an a, V., & Herna UHPLCeMS/MS highly sensitive determination of aflatoxins, the aflatoxin metabolite M1 and ochratoxin A in baby food and milk. Food Chemistry, 126(2), 737e744. http://dx.doi.org/10.1016/j.foodchem.2010.11.056. European Commission. (2006). Commission regulation (EC) N0 1881/2006. Official Journal of the European Communities, 1881, 1e26. Fallah, A. A. (2010). Aflatoxin M1 contamination in dairy products marketed in Iran during winter and summer. Food Control, 21(11), 1478e1481. http://dx.doi.org/ 10.1016/j.foodcont.2010.04.017. Ferreira, S. L. C., Bruns, R. E., Ferreira, H. S., Matos, G. D., David, J. M., Brand~ ao, G. C., et al. (2007). Box-Behnken design: an alternative for the optimization of analytical methods. Analytica Chimica Acta, 597(2), 179e186. http://dx.doi.org/ 10.1016/j.aca.2007.07.011. Iqbal, S. Z., Asi, M. R., & Jinap, S. (2013). Variation of aflatoxin M1 contamination in milk and milk products collected during winter and summer seasons. Food Control, 34(2), 714e718. http://dx.doi.org/10.1016/j.foodcont.2013.06.009. Kumar, V., Basu, M. S., & Rajendran, T. P. (2008). Mycotoxin research and mycoflora in some commercially important agricultural commodities. Crop Protection, 27, 891e905. http://dx.doi.org/10.1016/j.cropro.2007.12.011. Meerdink, G. L., & Abvt, D. (2002). Mycotoxins. Clinical Techniques in Equine Practice, 1(2), 89e93. Nilchian, Z., & Rahimi, E. (2012). Aflatoxin M1 in Yoghurts. Cheese and Ice-Cream in Shahrekord-Iran, 19(5), 621e624. http://dx.doi.org/10.5829/ idosi.wasj.2012.19.05.65172. Peraica, M., Radi c, B., Luci c, A., & Pavlovi c, M. (1999). Toxic effects of mycotoxins in humans. Bulletin of the World Health Organization, 77(9), 754e766. Prandini, A., Tansini, G., Sigolo, S., Filippi, L., Laporta, M., & Piva, G. (2009). On the occurrence of aflatoxin M1 in milk and dairy products. Food and Chemical Toxicology: An International Journal Published for the British Industrial Biological Research Association, 47(5), 984e991. http://dx.doi.org/10.1016/j.fct.2007.10.005. Shephard, G. S., Berthiller, F., Burdaspal, P. a., Crews, C., Jonker, M. a., Krska, R., et al. (2013). Developments in mycotoxin analysis: an update for 2011e2012. World Mycotoxin Journal, 6(1), 3e30. http://dx.doi.org/10.3920/WMJ2012.1492. Steyn, P. S. (1995). Mycotoxins, general view, chemistry and structure. Toxicology Letters, 82/83, 843e851. Swartz, M. E. (2005). UPLCTM: an introduction and review. Journal of Liquid Chromatography & Related Technologies, 28(7e8), 1253e1263. http://dx.doi.org/ 10.1081/JLC-200053046. The Commission of the European Communities. (2002). Commission decision 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 Communities, 8e36. Zain, M. E. (2001). Toxicity, metabolism, and impact of mycotoxins on humans and animals. Toxicology, 167, 101e134. http://dx.doi.org/10.1016/S0300-483X(01) 00471-1.
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Food Control journal homepage: www.elsevier.com/locate/foodcont
Determination of aflatoxin M1 in ice cream samples using immunoaffinity columns and ultra-high performance liquid chromatography coupled to tandem mass spectrometry , Gustavo Antonio Pen ~ uela Duvan Esteban Hoyos Ossa*, Diego Alfonso Hincapie GDCON Research Group, Faculty of Engineering, University Research Headquarters (SIU), University of Antioquia, Street 70 # 52 e 21, Medellin, Colombia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 December 2014 Received in revised form 12 February 2015 Accepted 10 March 2015 Available online 18 March 2015
In this work, a highly sensitive method was developed for the selective determination of aflatoxin M1 mycotoxin in ice cream samples using ultra high performance liquid chromatographyetandem mass spectrometry (UHPLC-MS/MS). The low detection values required, of mg/kg, made necessary sample concentration and cleaning using highly targeted immunoaffinity columns for this compound after extraction from matrix using a phosphate buffered saline solution (PBS). Method validation was carried out for ice cream matrix by means of recovery experiments using fortified samples near to the lower end of the calibration curve and a higher level. All recoveries were found in the range of 70e120 % with % RSD less than 25%. Quantitation was performed using external calibration with standards prepared in mobile phase. Validated method was used to determine the content of AFM1 in samples of ice cream bought in local stores. Compound identification was performed using the quantitation/qualification ratios for monitored transitions (Q/q1 and Q/q2). © 2015 Elsevier Ltd. All rights reserved.
Keywords: Aflatoxin M1 Ice cream Immunoaffinity columns UHPLC-MS/MS Chemical compounds studied in this article: Aflatoxin M1 (PubChem CID: 15558498)
1. Introduction Mycotoxins are toxic secondary metabolites produced by fungi. These types of substances have very different chemical structures, but all are generally chemical compounds of relatively low molecular mass. Mycotoxins are distributed in a wide range of food and feed, and constitute a risk for human and animal health, to induce potent and diverse biological effects (Peraica, Radic, Luci c, & Pavlovi c, 1999). Some of these substances exhibit carcinogenic, teratogenic, mutagenic and neurotoxic effects,etc; while others show antitumor capacity, cytotoxic and antimicrobial properties (Kumar, Basu, & Rajendran, 2008; Steyn, 1995). The effects on human and animal health, known as mycotoxicosis, depend among other factors on the toxicity of the mycotoxin, the degree of exposure, age, the amount consumed and nutrition status of the individual (Steyn, 1995). Human ingestion of mycotoxins is due to the consumption of foods prepared from contaminated sources with these substances and the consumption of animal source foods with metabolites residues of this kind of toxins (as aflatoxin M1 in
* Corresponding author. Tel.: þ57 4 2196689; fax: þ57 4 2196571. E-mail address: [email protected] (D.E. Hoyos Ossa). http://dx.doi.org/10.1016/j.foodcont.2015.03.011 0956-7135/© 2015 Elsevier Ltd. All rights reserved.
milk) (Steyn, 1995). These toxins are produced mostly by 5 fungi genera: Aspergillus, Fusarium, Alternaria, Penicillium and Claviceps (Steyn, 1995). Aflatoxins, ochratoxins, trichothecenes, zearalenone and fumonisins are the most important groups of mycotoxins often found in foodstuffs and agricultural commodities (Kumar et al., 2008; Zain, 2001). Due to the risk of contamination by mycotoxins, products like peanuts, cereals (barley, maize, rice, sorghum and oats), spices (like black pepper) and chili are considered of global significance over human health beings (Kumar et al., 2008). The Aflatoxins are produced mainly by Aspergillus flavus and Aspergillus parasiticus, A. flavus produces only B aflatoxins, and A. parasiticus produces B and G aflatoxins (Peraica et al., 1999). The oxidative metabolites of aflatoxin B1 and B2 are respectively the aflatoxin M1 and M2, which can be found in milk or milk products obtained from animals that have consumed contaminated feed (Peraica et al., 1999). By eating foods contaminated with aflatoxin B1, mammals excrete hydrolyzed aflatoxin M1 (AFM1) in the urine, faeces and milk. The AFM1 excreted in cow milk has toxic properties similar to that of aflatoxin B1, the most carcinogenic of aflatoxins (Meerdink & Abvt, 2002). Cow milk is one of the key ingredients for ice cream preparation, and is used for its contribution of fat and non-fat solids. In ice cream
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preparation, milk can be added both liquid and powder, and therefore the presence of AFM1 residues in milk is transferred to the finished product, since it has been demonstrated that thermal treatment used in dairy industry, like pasteurization and sterilization, cannot deactivate this type of mycotoxin (Aguilera-Luiz, Plaza~ os, Romero-Gonza lez, Vidal, & Frenich, 2011; Prandini et al., Bolan 2009). In order to protect public health, maximum levels of mycotoxins have been established in certain foods (European Commission, 2006), including AFM1 in milk and dairy products with a value of 0.050 mg/kg. There are several methodologies for the determination of mycotoxins in dairy products, such as ELISA (Enzyme-linked inmunosorbent assay), thin layer chromatography (TLC) and solid phase extraction using immunoaffinity columns and subsequent quantification by liquid chromatography, either high performance liquid chromatography (HPLC) or ultra-high performance liquid chromatography (UHPLC) (Shephard et al., 2013). For ice cream, different studies have been conducted in recent years in order to evaluate the presence of this type of mycotoxin in this kind of product, either using thin layer chromatography (Fallah, 2010), ELISA (Nilchian & Rahimi, 2012) or HPLC (Iqbal, Asi, & Jinap, 2013) but the combination of immunoaffinity column and UHPLC for the determination of AFM1 in ice cream has not been reported (Shephard et al., 2013). HPLC hyphenated to different detection methods has been, after ELISA, one of the most important techniques in the determination of mycotoxins in food matrices and within these, the fluorescence detection has been preferred (Shephard et al., 2013). In recent years, the introduction of UHPLC allowed to carry out this analysis in shorter run times and better chromatographic resolution than HPLC, and coupled to mass spectrometry has become a highly sensitive and selective technique to quantify and confirm the presence of such contaminants in food (Beltr an et al., 2011). The aim of this work was to develop a highly sensitive liquid chromatography method which could be able to quantify and confirm the presence of aflatoxin M1 in ice cream samples at the level required by European Legislation. Particular attention was placed on the sample preparation, in order to eliminate interferences and concentrate the analyte. Immunoaffinity columns Aflaprep M were selected due to their capacity to selectively clean the ice cream matrix and concentrate the mycotoxin. The optimized method using immunoaffinity columns and UHPLC-MS/MS technology was validated with satisfactory results and applied to the analysis of commercial ice cream samples.
2. Materials and methods 2.1. Chemicals , AFM1 standard of 2000 mg/L was bought to Micotox (Bogota Colombia). Working solutions of 100, 10 and 1 mg/L were prepared in LC-MS grade methanol obtained from Merck (Darmstadt, Germany) and stored at 20 C in amber vials until use. Immunoaffinity columns Aflaprep M were purchased from R-Biopharm (Glasgow, Scotland). Deionized water used for the preparation of
35
mobile phases and the buffer solution was supplied by a UV Synergy equipment obtained from Millipore (Bedford, USA). Monobasic anhydrous potassium phosphate was purchased from Sigma Aldrich (Steinheim, Germany). Sodium chloride, potassium chloride and dibasic potassium phosphate were purchased from Merck (Darmstadt, Germany) and were used for the preparation of phosphate buffered saline solution (PBS). Ammonium formate (purity > 99.995%) was used for mobile phase preparation and was supplied by Sigma Aldrich (Germany). The PBS buffer was prepared as follows: For 1 L of solution, 8 g of sodium chloride, 0.2 g of potassium chloride, 1.44 g of dibasic potassium phosphate and 0.24 g of monobasic anhydrous potassium phosphate were weighed and diluted with deionized water. 2.2. Apparatus For UHPLC e MS/MS analysis, an ACQUITY UPLC H-Class equipment (Waters, USA) was used. An ACQUITY UPLC BEH C18 column (2.1 mm (ID) 50 mm, 1.7 mm particle size) was used for the chromatographic separation. A Waters XEVO TQD mass spectrometer (Manchester, UK) equipped with an electrospray ionization source (ESI) was used for all experiments. Equipment control and data acquisition were performed with Masslynx v. 4.1 software (Waters, USA). In addition, a BOECO U-320 centrifuge (Hamburg, Germany) equipped with temperature control was used for the sample preparation. A New Brunswick Scientific Excella E2 shaker was used to perform the mechanical agitation of the samples. 2.3. Extraction and cleanup Ten grams of ice cream melted at room temperature were weighed into a 125 mL e Erlenmeyer flask, then 100 mL of PBS solution were added and the mixture was mechanically stirred for 30 min at 270 rpm. Then, the sample was evenly divided into two centrifuge tubes of 50 mL and centrifuged at 5000 rpm for 5 min at 5 C. Following this, the supernant fat was discarded and the process repeated 2 more times. For solid phase extraction using immunoaffinity columns, the free e fat sample was passed through an Aflaprep M column at a flow not greater than 5 mL/min. Once the sample has passed, the column was washed with about 20 mL of PBS solution. Finally, the toxin was eluted from column using 1.5 mL of a methanol: acetonitrile (40:60 v/v) solution and collected on a 25 mL e clear vial. The extract was concentrated to dryness using nitrogen and then diluted to 5.0 mL using a water: methanol (95:5 v/v) 5 mM in ammonium formate solution. This solution was filtered through 0.22 mm PVDF filters and about 1 mL of sample was deposited on a 2 mL amber vial and then was injected in the UHPLC-MS/MS equipment. 2.4. UHPLC conditions Waters ACQUITY UPLC H-Class equipment coupled to a XEVO TQD mass spectrometer was used for the analysis. The mobile phase consisted of water:methanol (95:5 v/v) 5 mM in ammonium formate (A) and methanol:water (95:5 v/v) 5 mM in ammonium
Table 1 Monitored transitions for AFM1. Compound
Formula
Transition
Parent ion (m/z)
Cone voltage (V)
Daughter ions (m/z)
Collision energy (V)
Ionization mode
Aflatoxin M1
C17H12O7
1 2 3
328.96 328.96 328.96
42 42 42
273.01 258.98 229.03
26 24 48
ESI þ ESI þ ESI þ
36 D.E. Hoyos Ossa et al. / Food Control 56 (2015) 34e40 Fig. 1. Comparison of AFM1 standards chromatograms obtained by HPLC-FLD and UHPLC-MS/MS. The chromatogram (a) represents a 0.100 mg/L AFM1 standard obtained by HPLC-FLD (AFM1 at 5.40 min) and the chromatogram (b) represents a 0.050 mg/L AFM1 standard obtained by UHPLC-MS/MS (AFM1 at 2.98 min).
D.E. Hoyos Ossa et al. / Food Control 56 (2015) 34e40
37
Table 2 ANOVA for response surface (quadratic model, Box e Behnken design). Factors
Sum of squares
Mean square
F Value
p- value
A: Source temperature B: Desolvation temperature C: Desolvation gas flow D: Cone gas flow AA AB AC AD BB BC BD CC CD DD Lack-of-fit Random error
56581.3 1.0286 107 43320.1 625177 16689.9 83521.0 289.0 11236.0 96584.4 15376.0 45796.0 29466.6 32580.3 66628.0 110398. 36658.8
56581.3 1.0286 107 43320.1 625177 16689.9 83521.0 289.0 11236.0 96584.4 15376.0 45796.0 29466.6 32580.3 66628.0 11039.8 9164.7
6.17 1122.35 4.73 68.22 1.82 9.11 0.03 1.23 10.54 1.68 5.00 3.22 3.55 7.27 1.20
0.0679 0.0000 0.0954 0.0012 0.2485 0.0392 0.8677 0.3303 0.0315 0.2649 0.0891 0.1474 0.1324 0.0543 0.4644
R2 ¼ 98.7327%. R2 (adjusted) ¼ 97.4654%. DurbineWatson statistic ¼ 2.24485 (p ¼ 0.6668).
formate (B). The run was carried out in gradient mode according to the following program: at 0 min 100% A, 0.5 min 100% A, 1 min 100% B, 4 min 100% B, 5 min 100% A, 8 min 100% A; for a total run time of 8.0 min. The mobile phase flow was set at 0.35 mL/min and the injection volume was 50 mL. Column temperature was set at 40 C. The capillary voltage of Waters Xevo TQD was set to 3.6 kV for ESI þ. The source temperature was set at 120 C and desolvation gas temperature (N2) at 600 C to a flow of 800 L/h and a cone gas flow of 29 L/h. Xevo TQD was used in selected reaction monitoring mode (SRM). The collision energy (CE) and the cone voltage (CV) were optimized for the monitored compound via AFM1 standard infusion. Table 1 shows the transitions monitored for AFM1 compound.
of ice cream at two different levels, 0.050 and 0.150 mg/kg. Specificity was assessed by comparing the signals obtained at the expected retention time for a reagent blank and fortified ice cream samples. From the chromatograms of the ice cream samples spiked at 0.050 mg/kg, the LOQ and LOD were estimated regarding a signal to noise ratio of 10 and 3 respectively. Q/q ratios were evaluated from reference standards in initial mobile phase and compared to those experimentally obtained from spiked samples.
3. Results and discussion 3.1. Optimization of analysis conditions for UHPLC-MS/MS
2.5. Validation In order to ensure the method quality for the determination of AFM1 in ice cream the following parameters were evaluated: linearity, accuracy, precision, specificity, limit of quantification (LOQ), limit of detection (LOD) and Q/q ratios of the SRM transitions monitored, which were used for quantification (Q/q1) and confirmation (Q/q2) of positive findings. Linearity was estimated by analysis of six calibration standards prepared in initial mobile phase in range from 0.050 to 1.0 mg/L. Accuracy and precision were estimated by apparent recovery experiments of the target analyte, spiking five contaminated samples
Exploratory tests showed better differentiation between the signals obtained for AFM1 compound and the baseline using UHPLC-MS/MS compared to the signal provided by HPLC-FLD, even in lower concentrations. Fig. 1 shows the chromatograms obtained in the exploratory tests for the compound AFM1 using HPLC-FLD for a 0.100 mg/L AFM1 standard (a) and UHPLC-MS/MS chromatogram (TIC mode) for a 0.050 mg/L AFM1 standard (b). According to the Commission Decision of August 12th of 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results, liquid chromatography with fluorescence or spectrometric detection can be used for the
Fig. 2. Peak area residuals vs. run number.
38
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Table 3 Optimized values for analyzed factors. Factor
Low
High
Optimum
Source temperature Desolvation temperature Desolvation gas flow Cone gas flow
120.0 400.0 800.0 20.0
150.0 600.0 1000.0 100.0
120.0 600.0 800.02 29.015
analysis of mycotoxins residues, but the use of chromatography without spectrometric detection are not suitable on their own as confirmatory methods, due the incapacity to provide information on the chemical structure of the analyte (The Commission of the European Communities, 2002). The unequivocal identification of the compound and the clear distinction between the analyte signal and the baseline were the key features to choose the technology UHPLC-MS/MS for this work. Also, the sample cleaning obtained just with immunoaffinity columns was not enough to observe a well e resolved AFM1 peak in HPLC analysis of ice cream samples using a fluorescence detector. The use of ultra-high performance liquid chromatography for methodology development provides superior chromatographic resolution, higher sensitivity and shorter run times as compared with conventional liquid chromatography (Swartz, 2005). The total run time obtained was just 8.0 min that includes 2.0 min for column stabilization.
Two mobile phase pairs were initially evaluated in order to proceed to a subsequent optimization. The pairs tested were: 1) H2O:MeOH 95:5 (v/v) 5 mM ammonium formate and MeOH:H2O 95:5 (v/v) 5 mM ammonium formate solution, 2) H2O:MeOH 95:5 (v/v) 5 mM ammonium acetate and MeOH:H2O 95:5 (v/v) 5 mM ammonium acetate solution. The results showed a better response for mobile phase pair 1 evaluated under the same type of gradient, flow of 0.35 mL/min and 50 mL of injection. The operating conditions of XEVO TQD mass spectrometer were optimized using a BoxBehnken experimental design (Ferreira et al., 2007), which was performed using the following factors: source temperature, desolvation temperature, desolvation gas flow and cone gas flow (Nitrogen). A total of 29 runs were carried out by injecting an AFM1 standard prepared in methanol at a concentration of 1.0 mg/L. Statistical analysis of each factor is shown in Table 2. Results showed that the second-order model used to fit data explained 98.73% of the variability. The adjusted R2 (97.46%) indicated that none model terms could be discarded. The DurbineWatson statistic (p ¼ 0.6668) shows no significant correlation between the residuals, lack of fit was not significant (p ¼ 0.4644) and the residuals showed no trend in their behavior by plotting them against the runs as shown in Fig. 2. All this confirmed the model validity to adjust the response. Finally, the desirability function of DerringerSuich was used to calculate the optimum analysis conditions regarding all factors, which are shown in Table 3 and were used for UHPLC analysis for each one of samples processed.
Fig. 3. UHPLC e MS/MS chromatograms for AFM1 in ice cream matrix: (a) Reagent blank, b) Non e spiked sample and c) Spiked sample with 0.150 mg/kg by addition of 150 mL of a 10 mg/L AFM1 standard solution.
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3.2. Sample treatment The sample preparation and therefore mycotoxin extraction from the food matrix was the most critical and the most time consuming step. Foods are complex sample matrices which make highly selective extraction methods necessary and because of the low concentrations at which it is common to find the compound AFM1, methods such as solid phase extraction using immunoaffinity columns are one of the options. Several studies have been conducted in recent years in order to assess the presence of this type of mycotoxin in this kind of product, either by using thin layer chromatography (Fallah, 2010), ELISA (Nilchian & Rahimi, 2012) or HPLC (Iqbal et al., 2013). The results showed presence of AFM1 in ice cream at 69.4, 29.0 and 43.0% of the ice cream samples analyzed respectively. The QuEChERS methodology and immunoaffinity columns were tested in order to assess both the AFM1 recovery from ice cream and cleaning of the obtained extract. Immunoaffinity columns, given the ability of the methodology to selectively clean the matrix and n et al., 2011), were selected as concentrate the compound (Beltra the most suitable technique for sample preparation. The results showed satisfactory percentages of recovery in the range of 72e102 % for spiked ice cream samples. Due to the high fat and solids contents in ice cream samples, solid phase extraction using immunoaffinity columns was difficult.
39
Centrifugation at 5 C for 5 min was used to precipitate the greater amount of solids as possible and to extract fat from the top of the 50 mL centrifuge tubes. This was done after the mechanical stirring.
3.3. Methodology validation The decision of the European Union Commission of 2002 concerning to the performance of analytical methods and the interpretation of results (The Commission of the European Communities, 2002) is applicable to analytical methodologies for the determination of certain residues in products of animal origin, such as ice cream, in whose preparation milk is used. The decision of the European Union Commission in 2002 aimed to fulfill Directive 96/23/EC of April 29th, 1996, within which mycotoxins as substances to be measured and monitored in animal products are included. This document was used as a basis for validation of the methodology developed. The developed method was validated prior to be used for the analysis of commercial ice cream samples. A correlation coefficient (R2) of 0.9979 for compound AFM1 using weighted linear calibration by external standard method was obtained in the range of 0.050e1.0 mg/L (Calibration standards of 0.050, 0.100, 0.200, 0.300, 0.600 and 1.0 mg/L were prepared). Individual residuals deviation was below 20% for each one of the calibration standards.
Fig. 4. Confirmation of samples with AFM1 using retention time and Q/q1 and Q/q2 ratios. Chromatogram (a) represents one of the spiked samples and chromatogram (b) one of the analyzed commercial samples.
40
D.E. Hoyos Ossa et al. / Food Control 56 (2015) 34e40
Method accuracy was estimated at 2 levels, one near to the lowest concentration level of the calibration curve and the other at an intermediate level. For this, 2 samples contaminated with AFM1 were analyzed and subsequently spiked (each one by fivefold) in order to calculate recovery percentages. This was done because the absence of an AFM1 mycotoxin e free matrix during the exploratory trials. Samples used for the recovery experiments were spiked to 0.050 and 0.150 mg/kg adding 50 and 150 mL of a 10 mg/L AFM1 standard solution respectively. Satisfactory recoveries were obtained in the range from 76 to 102 % with relative standard deviations (% RSD) of 18.85 for the low level (n ¼ 5) and 22.90 for the other level (n ¼ 5). These were satisfactory results regarding the low concentration working range and the complexity of the matrix evaluated. Recovery for each sample with a known added amount of AFM1 was calculated according to the Equation (A).
Recovery ð%Þ ¼ ððC2 C1 Þ=Cadd Þ*100
(A)
where: C2: Concentration calculated for spiked sample. C1: Concentration calculated for contaminated sample without spiking. Cadd: Amount of AFM1 added in terms of concentration. Using signal to noise ratio calculated from the chromatograms of the ice cream samples spiked at 0.050 mg/kg, the LOQ and the LOD were estimated. The LOD varied between 0.0004 and 0.0009 mg/kg and the LOQ had values between 0.001 and 0.003 mg/kg. The method was considered to be highly specific as no interferences were observed in reagent blank at AFM1 retention time. Fig. 3 shows a chromatogram for a reagent blank (a), a sample contaminated with AFM1 (b) and for this same sample spiked with AFM1 compound (c). Q/q ratios deviations below 20% were obtained for each one of the spiked and commercial samples analyzed in relation to the values measured for calibration solutions. Fig. 4 shows the Q/q ratios and the deviation obtained for a spiked (a) and a commercial sample (b) of ice cream analyzed with the developed method. 3.4. Application to commercial ice cream samples The developed and optimized method was applied to the analysis of commercial ice cream samples from different trademarks and flavors obtained from local stores. For 7 of the 15 samples analyzed, the presence of the mycotoxin was confirmed using AFM1 retention time and Q/q ratios obtained from calibration curve. For three of these positive samples the concentration measured was above the lowest calibration level (0.025 mg/kg in sample, equivalent to 0.050 mg/L in the calibration curve according to the sample treatment) and just one sample (0.058 mg/kg) exceeded the limit fixed by the European Union regulation (0.05 mg/ kg; LMR for AFM1 in raw milk, heat-treated milk and milk for the manufacture of milk-based products) (European Commission, 2006). 4. Conclusions An efficient and highly selective method for the determination of aflatoxin M1 in ice cream samples was developed, optimized and validated. The methodology was focused on ice cream products given that previous studies have shown residuality of this mycotoxin in milk used in their manufacture. Both the conventional liquid chromatography with fluorescence detection (HPLC-FLD)
and ultra-high performance liquid chromatography coupled to tandem e mass spectrometry (UHPLC-MS/MS) were tested. The first one showed poor resolution because of the interference of the matrix, while the second successfully enabled the development of a highly sensitive method, which in turn enabled compound presence confirmation by mean of selected reactions monitoring (SRM). Methodology was validated in ice cream matrix at 2 different levels and applied to the quantification and confirmation of AFM1 in different commercial samples.
Acknowledgment This project was conducted with funding from the Departan “COLmento Administrativo de Ciencia, Tecnología e Innovacio CIENCIAS” within the Call 566 e 2012: Young Researchers and Innovators Program 2012. Funding and infrastructure necessary to perform the experiments described in the project were supplied by the Grupo Diag stico y Control de la Contaminacio n (GDCON), which provided its no materials and equipment for this research work.
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