Food Additives & Contaminants: Part B, 2014 Vol. 7, No. 4, 302–308, http://dx.doi.org/10.1080/19393210.2014.933269
Determination of moulds and mycotoxins in dry dog and cat food using liquid chromatography with mass spectrometry and fluorescence detection A. Błajet-Kosicka*, R. Kosicki, M. Twarużek and J. Grajewski Division of Physiology and Toxicology, Faculty of Natural Science, Institute of Experimental Biology, Kazimierz Wielki University, Bydgoszcz, Poland (Received 25 April 2014; accepted 6 June 2014) In this study moulds and 12 mycotoxins in dry pet food samples (25 for dogs and 24 for cats) were determined. Primary moulds identified were Aspergillus, Mucor and Penicillium, found in 55% of the samples. Deoxynivalenol and zearalenone (ZEN) were detected in all samples with mean respective concentrations being 97.3 and 38.3 µg kg−1 in cat food and 114 and 20.1 µg kg−1 in dog food. T-2 and HT-2 toxins were present in 88% and 84% of the samples, respectively. Two samples contained fumonisins, with a maximum concentration of 108 µg kg−1. Aflatoxin B1 and ochratoxin A were detected in 8% and 45% of the samples, respectively. The measured mould and mycotoxin levels were consistent with results obtained by other studies. However, potential exposure to relatively high concentrations of an oestrogen mycotoxin as is ZEN, especially when in combination with other mycotoxins, needs attention. Keywords: mycotoxins; moulds; pet food; dog; cat; liquid chromatography; fluorescence detection; mass spectrometry
Introduction Mycotoxins are toxic metabolic products of fungi that can be present in raw materials, including cereals and their byproducts that are often the main components of food for animals. Contamination of pet food caused by mycotoxins can occur during production, if poor quality materials are used, and during product processing, packaging and storage. Mycotoxins can be produced after the purchase of food, especially if it is not properly stored. Presently, there are over 400 known chemically different mycotoxins that can cause several diseases (mycotoxicoses). Typical clinical symptoms for mycotoxicoses depend on the type of mycotoxin and its concentration, the duration of exposure, and the species, gender, age and health of the host animal. The most common secondary metabolites of moulds are: Fusarium mycotoxins (trichothecenes, ZEN and fumonisins (FBs)), ochratoxin A (OTA) and aflatoxins (AFs) (Richard & Payne 2003). Trichothecenes of group A, that include T-2 toxin, HT2 toxin and diacetoxyscirpenol, are primarily produced by Fusarium poae and Fusarium sporotrichioides species, while trichothecenes of group B, including nivalenol, deoxynivalenol (DON) and fusarenon X, are produced primarily by Fusarium graminearum, Fusarium avenaceum and Fusarium culmorum. Typical clinical signs of trichothecene toxicity are loss of appetite, vomiting, diarrhoea, gastrointestinal bleeding, ataxia and interferences with the immune system. F. graminearum, F. culmorum and F. sporotrichioides are also the primary fungal species that produce ZEN, which even at low doses can cause *Corresponding author. Email: [email protected] © 2014 Taylor & Francis
infertility, swelling of the vulva, vaginal prolapse, testicular and ovarian atrophy. It can also adversely affect ovulation, development of the foetus and newborn vitality (Boermans & Leung 2007; Richard 2007; Gliński et al. 2011). FBs are produced by various Fusarium species (e.g. F. verticillioides, F. proliferatum). They can cause liver and kidney cancer and their action can lead to the refusal of food intake, depression, ataxia and blindness (Richard 2007). Despite mycotoxins produced by Fusarium fungi are widespread in cereals, few studies on pet food are published. There are many reports of the presence of AFs and OTA in dog and cat food. OTA can be produced by the moulds Penicillium (P. verrucosum, P. viridicatum) and Aspergillus (A. ochraceus), even after the purchase of food and feed if it is stored improperly. Clinical symptoms of ochratoxicosis are nephrotoxicity, hepatotoxicity, anorexia, vomiting, excessive thirst, fatigue and interference with the immune system (Richard 2007). It was reported that six dogs died in Germany in 1987, one in Scotland in 1991 and three in Korea in 2006 as a result of renal failure after consumption of feed containing OTA (Gareis et al. 1987; Little et al. 1991; Jeong et al. 2006). AFs are among the most potent naturally occurring carcinogenic compounds known and are produced by Aspergillus (A. flavus and A. parasiticus) and Penicillium (P. puberulum) species. Primary symptoms of aflatoxicosis are liver diseases (hepatotoxic and hepatocarcinogenic action), bleeding (from kidneys and gastrointestinal tract), reduced growth rate and malfunctions of
Food Additives & Contaminants: Part B the immune system (Böhm & Razzazi–Fazeli 2005; Richard 2007). Aflatoxicosis in dogs was first reported in 1952 in the south-eastern United States (Bailey & Groth 1959). Veterinarians discovered a liver disease called “hepatitis X” that was diagnosed in dozens of dogs fed mouldy food. In 1974, in connection with the consumption of contaminated maize, dead dogs were reported in about 200 villages in India (Krishnamachari et al. 1975). In 1998, in Texas, 55 dogs died from acute or chronic symptoms after eating commercially available feed (Garland & Reagor 2001). Only a few research studies have focused on moulds and mycotoxins in pet foods, possibly because productivity of such animal feed is of less interest. The existing studies involved evaluation only of a single mycotoxin. The present study was conducted to give information about mould contamination and levels of the most important mycotoxins, including DON, ZEN, T-2 and HT-2 toxins, FBs, OTA and AFs in pet food for dogs and cats that are available in retail trade.
Materials and methods Samples Forty-nine commercially available dry pet foods for dogs and cats, of different trademarks and places of production, were purchased in pet stores in the central part of Poland. The package size ranged between 190 g and 5 kg. The samples were picked from shelves and when the package sizes were smaller than 1 kg, more packages were bought to have a representative sample of a lot. The samples were kept in their original packaging until analysis. Before analysis, all samples were milled (ZM 200, Retsch, Haan, Germany) and divided into 200 g subsamples. The subsamples were stored in a dark and dry place at 4°C until analysis. All samples were analysed within the shelf life of the product. The study involved testing 24 samples for cats (9 for kittens and 15 for adult cats) and 25 samples for dogs (5 for puppies and 20 for adult dogs). Twentythree pet foods were produced in Poland, 23 in other EU countries and 3 outside the EU. The primary components of the pet foods tested were: wheat, maize, soybean, rice, meat and animal-origin products (chicken and fish) and dairy products.
Chemicals AFs B1, B2, G1, G2, OTA, DON, T-2 toxin, HT-2 toxin, ZEN, FBs B1, B2, B3, 13C-DON and zearalanone (ZAN) were purchased from Biopure (Tulln, Austria). DG18 and YGC media, lactophenol, potassium bromide, sodium chloride, ammonium acetate, acetic acid, acetonitrile (gradient grade) and methanol (gradient grade) were supplied by Merck (Darmstadt, Germany). Deionised water was
303
obtained using a Simplicity 185 water purification system (Millipore, Bedford, USA).
Mycological analysis A sample portion (20 g) was placed in a sterile bag within a Stomacher-type homogeniser (BagMixer 400, Interscience, France); 180 ml sterile dilution fluid was added and the mixture was homogenised for 90 seconds. The number of fungi (cfu g−1) was determined according to PN-ISO-7954 (1999) (surface inoculation of 1 ml and 0.1 ml in triplicate). A series of dilutions was made using the original homogenised suspension (1:10 dilution). Surface inoculation was performed on YGC and DG18 media to isolate xerophilic fungi. Incubation was for 5– 7 days (10–14 days for xerophilic fungi) at 25°C. After incubation, 10–100 colonies were counted. Results were expressed as the number of colony-forming units per 1 g of sample (cfu g−1) fungi (yeast + anamorphic fungi). Anamorphic fungi and yeasts were identified by microscopy. The colonies grown on YGC and DG18 substrate were prepared for microscopy in lactophenol. Dominant types of anamorphic fungi were determined based on colony morphology and the type of sporulation.
Mycotoxin analysis Fusarium mycotoxins Sample preparation for trichothecenes and zearalenone analysis. A sample portion (12.5 g) was homogenised with 50 ml of ACN:H2O (80:20) for 3 min. The extract was filtered. To 4 ml of the extract, 40 µl of ZAN (c = 1000 ng ml−1) solution was added and the mixture was applied to a Bond Elut® Mycotoxin column (Agilent, Santa Clara, CA, USA). Subsequently, to 2 ml of purified extract, 50 µl of internal standard solution (13C-DON; c = 2500 ng ml−1) was added and the mixture was evaporated to dryness using nitrogen. MeOH:H2O (1:4) (495 µl) was added to the vial and the sample was vortexed. Sample preparation for fumonisins analysis. NaCl (2.5 g) was added to 25 g sample. The mixture was homogenised with 50 ml of MeOH:H2O (80:20) for 5 min. The extract was filtered and 10 ml were added to 40 ml phosphatebuffered saline (PBS) solution and filtered. The diluted extract (10 ml) was applied to a FumoniTestTM column (Vicam, Watertown, USA). The column was washed twice with 10 ml of H2O and the FBs were eluted using 1.5 ml of MeOH. The eluate was collected in a sample vial and evaporated to dryness with nitrogen; 1 ml of ACN:H2O (1:1) solution was added to the vial and the sample was vortexed.
304
A. Błajet-Kosicka et al.
Chromatographic analysis. Trichothecenes, ZEN and FBs were determined using high-performance liquid chromatography (HPLC) with MS/MS detection. HPLC: Agilent 1200 (Agilent Technologies Inc., Santa Clara, CA, USA), mass spectrometer: 3200 QTRAP (AB Sciex, Foster City, CA, USA), chromatographic column: Gemini C18 (150x4.6mm, 5 µm) (Phenomenex Inc., Torrance, CA, USA), mobile phase: A: H2O + 5 mM CH3COONH4 + 1% CH3COOH, B: MeOH + 5 mM CH3COONH4 + 1% CH3COOH, flow rate: 0.7 ml min−1 (for trichothecenes and ZEN) and 0.5 ml min−1 (for FBs), injection volume: 20 µl (for trichothecenes and ZEN) and 15 µl (for FBs). Ochratoxin A Sample preparation. A sample portion (12.5 g) was homogenised with 50 ml of ACN:H2O (60:40) for 2 min. The extract was centrifuged at 4000 rpm for 20 min. A 5 ml aliquot of supernatant was added to 55 ml PBS solution and the mixture was filtered; 48 ml of diluted extract was applied to an OCHRAPREP® column (Rhone Diagnostic Technologies Ltd, Glasgow, UK) at a flow rate of 2– 3 ml min−1. The column was washed with 20 ml of H2O and dried with air. OTA was eluted using 1.5 ml of MeOH: CH3COOH (98:2). The eluate was collected in a sample vial, 1.5 ml of H2O was passed through the column and the sample was vortexed.
KBr + 100 µl 65%HNO3, flow rate: 1 ml min−1, injection volume: 50 µl. Method validation Quantification was performed by comparing sample peak areas to calibration curve standards. Recovery and precision of the methods, expressed as repeatability (% RSD), was evaluated by analysing samples in triplicate fortified at different concentrations. Correlation coefficients were determined to estimate linearity of peak areas relative to standards. Internal standards (13C-DON and ZAN) were used to correct for analytical recovery and for matrix effects in MS/MS detection. The limit of detection (LOD, signal-to-noise ratio of 3) and limit of quantification (LOQ = 3x LOD), respectively, were calculated by spiking samples at low concentrations and subjecting them to sample preparation and clean-up procedures. Descriptive statistics The number of samples, mean value and median of positive and all analysed samples, maximum and minimum mycotoxin content in positive samples were calculated using Statistics 9 for Windows (Analytical Software, version 9.0). Results and discussion
Chromatographic analysis. OTA was determined using HPLC with fluorescence detection (FLD). HPLC: LaChrom ELITE (Merck-Hitachi, Darmstadt, Germany), chromatographic column: LiChrospher 100 RP-18 (250 × 4.0 mm, 5 µm), mobile phase: ACN:2%CH3COOH (70:30), flow rate: 1 ml min−1, injection volume: 50 µl. Aflatoxins Sample preparation. To 25 g of pet food sample, 2.5 g of NaCl were added and homogenised with 50 ml of MeOH: H2O (80:20) for 1 min. The extract was filtered and 10 ml was added to 40 ml of H2O, shaken and filtered again; 10 ml of diluted extract was applied to an AflaTest® column (Vicam, Watertown, USA). The column was washed twice with 10 ml of H2O. AFs were eluted using 1 ml of MeOH. The eluate was collected in a sample vial, 1 ml of H2O was added and the sample was vortexed. Chromatographic analysis. AFs were determined using HPLC with FLD preceded by post-column derivatisation. HPLC: pump L-7100, autosampler L-7250, oven L-7300, FLD L-7480, chromatographic column: LiChroCART 250–4, LiChrospher 100 RP-18 (250 × 4.0 mm, 5 µm), obtained from Merck-Hitachi (Darmstadt, Germany), mobile phase: ACN:MeOH:H2O (20:20:60) + 119 mg
Dogs and cats are the most popular pets in Europe. According to the World Society for the Protection of Animals (WSPA) report, there were more than 7 million dogs and 5.5 million cats owned in Poland in 2007 (Batson 2008) and this number has been increasing. A human–animal bond between pets and their owners make the health problems of pets more of an emotional concern as compared to a mainly financial issue in farm animals. Therefore, pet food quality and safety seem to be a subject of great importance (Aquino & Corrêa 2011). Mycotoxin determination methods for dry pet food were validated and the results are presented in Table 1. The LOD ranged from 0.02 µg kg−1 (AF B2) to 7.00 µg kg−1 (DON). Correlation coefficients of the calibration curves were higher than 0.995 in all cases, indicating good linearity for both HPLC-MS/MS and HPLCFLD methods for quantification of the target mycotoxins in the studied range. Recovery values varied from 79.7 ± 7.6% for AF B1 by HPLC-FLD to 104.5 ± 2.3% for FB1 by HPLC-MS/MS. The results of mycological examinations are shown in Table 2. The most common moulds were: Aspergillus (35% of the samples), Mucor (10% of the samples) and Penicillium (8% of the samples). These results are consistent with studies by Martins et al. (2003), who also identified three genera of
Food Additives & Contaminants: Part B Table 1.
Method validation results.
Method and mycotoxin HPLC-MS/MS DON T-2 Toxin HT-2 Toxin ZEN FB1 FB2 FB3 HPLC-FLD OTA AF B1 AF B2 AF G1 AF G2
Table 2.
305
n
LOD (µg kg−1)
LOQ (µg kg−1)
Linearity range (µg kg−1)
Spiking level (µg kg−1)
3 3 3 3 3 3 3
7.00 0.50 1.60 0.10 1.60 1.60 1.60
20.0 1.50 5.00 0.30 5.00 5.00 5.00
15.5–1030 11.4–379 11.5–383 2.45–163 21.0–1260 15.5–928 11.2–674
250 100 100 50.0 250 100 100
3 3 3 3 3
0.13 0.05 0.02 0.25 0.08
0.40 0.15 0.06 0.75 0.24
0.50–25.0 0.40–20.2 0.10–5.00 0.41–20.3 0.11–5.40
Fungal contamination in analysed pet food samples.
Number of pet food type
Kitten Cat Puppy Dog (n = 9) (n = 15) (n = 5) (n = 20)
Fungi Contamination rate (%) Min total amount (cfu g−1) Max total amount (cfu g−1) Moulds Contamination rate (%) Min total amount (cfu g−1) Max total amount (cfu g−1) Yeast Contamination rate (%) Min total amount (cfu g−1) Max total amount (cfu g−1)
77.8 n.e. 90
60.0 n.e. 268
40.0 n.e.
Determination of moulds and mycotoxins in dry dog and cat food using liquid chromatography with mass spectrometry and fluorescence detection A. Błajet-Kosicka*, R. Kosicki, M. Twarużek and J. Grajewski Division of Physiology and Toxicology, Faculty of Natural Science, Institute of Experimental Biology, Kazimierz Wielki University, Bydgoszcz, Poland (Received 25 April 2014; accepted 6 June 2014) In this study moulds and 12 mycotoxins in dry pet food samples (25 for dogs and 24 for cats) were determined. Primary moulds identified were Aspergillus, Mucor and Penicillium, found in 55% of the samples. Deoxynivalenol and zearalenone (ZEN) were detected in all samples with mean respective concentrations being 97.3 and 38.3 µg kg−1 in cat food and 114 and 20.1 µg kg−1 in dog food. T-2 and HT-2 toxins were present in 88% and 84% of the samples, respectively. Two samples contained fumonisins, with a maximum concentration of 108 µg kg−1. Aflatoxin B1 and ochratoxin A were detected in 8% and 45% of the samples, respectively. The measured mould and mycotoxin levels were consistent with results obtained by other studies. However, potential exposure to relatively high concentrations of an oestrogen mycotoxin as is ZEN, especially when in combination with other mycotoxins, needs attention. Keywords: mycotoxins; moulds; pet food; dog; cat; liquid chromatography; fluorescence detection; mass spectrometry
Introduction Mycotoxins are toxic metabolic products of fungi that can be present in raw materials, including cereals and their byproducts that are often the main components of food for animals. Contamination of pet food caused by mycotoxins can occur during production, if poor quality materials are used, and during product processing, packaging and storage. Mycotoxins can be produced after the purchase of food, especially if it is not properly stored. Presently, there are over 400 known chemically different mycotoxins that can cause several diseases (mycotoxicoses). Typical clinical symptoms for mycotoxicoses depend on the type of mycotoxin and its concentration, the duration of exposure, and the species, gender, age and health of the host animal. The most common secondary metabolites of moulds are: Fusarium mycotoxins (trichothecenes, ZEN and fumonisins (FBs)), ochratoxin A (OTA) and aflatoxins (AFs) (Richard & Payne 2003). Trichothecenes of group A, that include T-2 toxin, HT2 toxin and diacetoxyscirpenol, are primarily produced by Fusarium poae and Fusarium sporotrichioides species, while trichothecenes of group B, including nivalenol, deoxynivalenol (DON) and fusarenon X, are produced primarily by Fusarium graminearum, Fusarium avenaceum and Fusarium culmorum. Typical clinical signs of trichothecene toxicity are loss of appetite, vomiting, diarrhoea, gastrointestinal bleeding, ataxia and interferences with the immune system. F. graminearum, F. culmorum and F. sporotrichioides are also the primary fungal species that produce ZEN, which even at low doses can cause *Corresponding author. Email: [email protected] © 2014 Taylor & Francis
infertility, swelling of the vulva, vaginal prolapse, testicular and ovarian atrophy. It can also adversely affect ovulation, development of the foetus and newborn vitality (Boermans & Leung 2007; Richard 2007; Gliński et al. 2011). FBs are produced by various Fusarium species (e.g. F. verticillioides, F. proliferatum). They can cause liver and kidney cancer and their action can lead to the refusal of food intake, depression, ataxia and blindness (Richard 2007). Despite mycotoxins produced by Fusarium fungi are widespread in cereals, few studies on pet food are published. There are many reports of the presence of AFs and OTA in dog and cat food. OTA can be produced by the moulds Penicillium (P. verrucosum, P. viridicatum) and Aspergillus (A. ochraceus), even after the purchase of food and feed if it is stored improperly. Clinical symptoms of ochratoxicosis are nephrotoxicity, hepatotoxicity, anorexia, vomiting, excessive thirst, fatigue and interference with the immune system (Richard 2007). It was reported that six dogs died in Germany in 1987, one in Scotland in 1991 and three in Korea in 2006 as a result of renal failure after consumption of feed containing OTA (Gareis et al. 1987; Little et al. 1991; Jeong et al. 2006). AFs are among the most potent naturally occurring carcinogenic compounds known and are produced by Aspergillus (A. flavus and A. parasiticus) and Penicillium (P. puberulum) species. Primary symptoms of aflatoxicosis are liver diseases (hepatotoxic and hepatocarcinogenic action), bleeding (from kidneys and gastrointestinal tract), reduced growth rate and malfunctions of
Food Additives & Contaminants: Part B the immune system (Böhm & Razzazi–Fazeli 2005; Richard 2007). Aflatoxicosis in dogs was first reported in 1952 in the south-eastern United States (Bailey & Groth 1959). Veterinarians discovered a liver disease called “hepatitis X” that was diagnosed in dozens of dogs fed mouldy food. In 1974, in connection with the consumption of contaminated maize, dead dogs were reported in about 200 villages in India (Krishnamachari et al. 1975). In 1998, in Texas, 55 dogs died from acute or chronic symptoms after eating commercially available feed (Garland & Reagor 2001). Only a few research studies have focused on moulds and mycotoxins in pet foods, possibly because productivity of such animal feed is of less interest. The existing studies involved evaluation only of a single mycotoxin. The present study was conducted to give information about mould contamination and levels of the most important mycotoxins, including DON, ZEN, T-2 and HT-2 toxins, FBs, OTA and AFs in pet food for dogs and cats that are available in retail trade.
Materials and methods Samples Forty-nine commercially available dry pet foods for dogs and cats, of different trademarks and places of production, were purchased in pet stores in the central part of Poland. The package size ranged between 190 g and 5 kg. The samples were picked from shelves and when the package sizes were smaller than 1 kg, more packages were bought to have a representative sample of a lot. The samples were kept in their original packaging until analysis. Before analysis, all samples were milled (ZM 200, Retsch, Haan, Germany) and divided into 200 g subsamples. The subsamples were stored in a dark and dry place at 4°C until analysis. All samples were analysed within the shelf life of the product. The study involved testing 24 samples for cats (9 for kittens and 15 for adult cats) and 25 samples for dogs (5 for puppies and 20 for adult dogs). Twentythree pet foods were produced in Poland, 23 in other EU countries and 3 outside the EU. The primary components of the pet foods tested were: wheat, maize, soybean, rice, meat and animal-origin products (chicken and fish) and dairy products.
Chemicals AFs B1, B2, G1, G2, OTA, DON, T-2 toxin, HT-2 toxin, ZEN, FBs B1, B2, B3, 13C-DON and zearalanone (ZAN) were purchased from Biopure (Tulln, Austria). DG18 and YGC media, lactophenol, potassium bromide, sodium chloride, ammonium acetate, acetic acid, acetonitrile (gradient grade) and methanol (gradient grade) were supplied by Merck (Darmstadt, Germany). Deionised water was
303
obtained using a Simplicity 185 water purification system (Millipore, Bedford, USA).
Mycological analysis A sample portion (20 g) was placed in a sterile bag within a Stomacher-type homogeniser (BagMixer 400, Interscience, France); 180 ml sterile dilution fluid was added and the mixture was homogenised for 90 seconds. The number of fungi (cfu g−1) was determined according to PN-ISO-7954 (1999) (surface inoculation of 1 ml and 0.1 ml in triplicate). A series of dilutions was made using the original homogenised suspension (1:10 dilution). Surface inoculation was performed on YGC and DG18 media to isolate xerophilic fungi. Incubation was for 5– 7 days (10–14 days for xerophilic fungi) at 25°C. After incubation, 10–100 colonies were counted. Results were expressed as the number of colony-forming units per 1 g of sample (cfu g−1) fungi (yeast + anamorphic fungi). Anamorphic fungi and yeasts were identified by microscopy. The colonies grown on YGC and DG18 substrate were prepared for microscopy in lactophenol. Dominant types of anamorphic fungi were determined based on colony morphology and the type of sporulation.
Mycotoxin analysis Fusarium mycotoxins Sample preparation for trichothecenes and zearalenone analysis. A sample portion (12.5 g) was homogenised with 50 ml of ACN:H2O (80:20) for 3 min. The extract was filtered. To 4 ml of the extract, 40 µl of ZAN (c = 1000 ng ml−1) solution was added and the mixture was applied to a Bond Elut® Mycotoxin column (Agilent, Santa Clara, CA, USA). Subsequently, to 2 ml of purified extract, 50 µl of internal standard solution (13C-DON; c = 2500 ng ml−1) was added and the mixture was evaporated to dryness using nitrogen. MeOH:H2O (1:4) (495 µl) was added to the vial and the sample was vortexed. Sample preparation for fumonisins analysis. NaCl (2.5 g) was added to 25 g sample. The mixture was homogenised with 50 ml of MeOH:H2O (80:20) for 5 min. The extract was filtered and 10 ml were added to 40 ml phosphatebuffered saline (PBS) solution and filtered. The diluted extract (10 ml) was applied to a FumoniTestTM column (Vicam, Watertown, USA). The column was washed twice with 10 ml of H2O and the FBs were eluted using 1.5 ml of MeOH. The eluate was collected in a sample vial and evaporated to dryness with nitrogen; 1 ml of ACN:H2O (1:1) solution was added to the vial and the sample was vortexed.
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A. Błajet-Kosicka et al.
Chromatographic analysis. Trichothecenes, ZEN and FBs were determined using high-performance liquid chromatography (HPLC) with MS/MS detection. HPLC: Agilent 1200 (Agilent Technologies Inc., Santa Clara, CA, USA), mass spectrometer: 3200 QTRAP (AB Sciex, Foster City, CA, USA), chromatographic column: Gemini C18 (150x4.6mm, 5 µm) (Phenomenex Inc., Torrance, CA, USA), mobile phase: A: H2O + 5 mM CH3COONH4 + 1% CH3COOH, B: MeOH + 5 mM CH3COONH4 + 1% CH3COOH, flow rate: 0.7 ml min−1 (for trichothecenes and ZEN) and 0.5 ml min−1 (for FBs), injection volume: 20 µl (for trichothecenes and ZEN) and 15 µl (for FBs). Ochratoxin A Sample preparation. A sample portion (12.5 g) was homogenised with 50 ml of ACN:H2O (60:40) for 2 min. The extract was centrifuged at 4000 rpm for 20 min. A 5 ml aliquot of supernatant was added to 55 ml PBS solution and the mixture was filtered; 48 ml of diluted extract was applied to an OCHRAPREP® column (Rhone Diagnostic Technologies Ltd, Glasgow, UK) at a flow rate of 2– 3 ml min−1. The column was washed with 20 ml of H2O and dried with air. OTA was eluted using 1.5 ml of MeOH: CH3COOH (98:2). The eluate was collected in a sample vial, 1.5 ml of H2O was passed through the column and the sample was vortexed.
KBr + 100 µl 65%HNO3, flow rate: 1 ml min−1, injection volume: 50 µl. Method validation Quantification was performed by comparing sample peak areas to calibration curve standards. Recovery and precision of the methods, expressed as repeatability (% RSD), was evaluated by analysing samples in triplicate fortified at different concentrations. Correlation coefficients were determined to estimate linearity of peak areas relative to standards. Internal standards (13C-DON and ZAN) were used to correct for analytical recovery and for matrix effects in MS/MS detection. The limit of detection (LOD, signal-to-noise ratio of 3) and limit of quantification (LOQ = 3x LOD), respectively, were calculated by spiking samples at low concentrations and subjecting them to sample preparation and clean-up procedures. Descriptive statistics The number of samples, mean value and median of positive and all analysed samples, maximum and minimum mycotoxin content in positive samples were calculated using Statistics 9 for Windows (Analytical Software, version 9.0). Results and discussion
Chromatographic analysis. OTA was determined using HPLC with fluorescence detection (FLD). HPLC: LaChrom ELITE (Merck-Hitachi, Darmstadt, Germany), chromatographic column: LiChrospher 100 RP-18 (250 × 4.0 mm, 5 µm), mobile phase: ACN:2%CH3COOH (70:30), flow rate: 1 ml min−1, injection volume: 50 µl. Aflatoxins Sample preparation. To 25 g of pet food sample, 2.5 g of NaCl were added and homogenised with 50 ml of MeOH: H2O (80:20) for 1 min. The extract was filtered and 10 ml was added to 40 ml of H2O, shaken and filtered again; 10 ml of diluted extract was applied to an AflaTest® column (Vicam, Watertown, USA). The column was washed twice with 10 ml of H2O. AFs were eluted using 1 ml of MeOH. The eluate was collected in a sample vial, 1 ml of H2O was added and the sample was vortexed. Chromatographic analysis. AFs were determined using HPLC with FLD preceded by post-column derivatisation. HPLC: pump L-7100, autosampler L-7250, oven L-7300, FLD L-7480, chromatographic column: LiChroCART 250–4, LiChrospher 100 RP-18 (250 × 4.0 mm, 5 µm), obtained from Merck-Hitachi (Darmstadt, Germany), mobile phase: ACN:MeOH:H2O (20:20:60) + 119 mg
Dogs and cats are the most popular pets in Europe. According to the World Society for the Protection of Animals (WSPA) report, there were more than 7 million dogs and 5.5 million cats owned in Poland in 2007 (Batson 2008) and this number has been increasing. A human–animal bond between pets and their owners make the health problems of pets more of an emotional concern as compared to a mainly financial issue in farm animals. Therefore, pet food quality and safety seem to be a subject of great importance (Aquino & Corrêa 2011). Mycotoxin determination methods for dry pet food were validated and the results are presented in Table 1. The LOD ranged from 0.02 µg kg−1 (AF B2) to 7.00 µg kg−1 (DON). Correlation coefficients of the calibration curves were higher than 0.995 in all cases, indicating good linearity for both HPLC-MS/MS and HPLCFLD methods for quantification of the target mycotoxins in the studied range. Recovery values varied from 79.7 ± 7.6% for AF B1 by HPLC-FLD to 104.5 ± 2.3% for FB1 by HPLC-MS/MS. The results of mycological examinations are shown in Table 2. The most common moulds were: Aspergillus (35% of the samples), Mucor (10% of the samples) and Penicillium (8% of the samples). These results are consistent with studies by Martins et al. (2003), who also identified three genera of
Food Additives & Contaminants: Part B Table 1.
Method validation results.
Method and mycotoxin HPLC-MS/MS DON T-2 Toxin HT-2 Toxin ZEN FB1 FB2 FB3 HPLC-FLD OTA AF B1 AF B2 AF G1 AF G2
Table 2.
305
n
LOD (µg kg−1)
LOQ (µg kg−1)
Linearity range (µg kg−1)
Spiking level (µg kg−1)
3 3 3 3 3 3 3
7.00 0.50 1.60 0.10 1.60 1.60 1.60
20.0 1.50 5.00 0.30 5.00 5.00 5.00
15.5–1030 11.4–379 11.5–383 2.45–163 21.0–1260 15.5–928 11.2–674
250 100 100 50.0 250 100 100
3 3 3 3 3
0.13 0.05 0.02 0.25 0.08
0.40 0.15 0.06 0.75 0.24
0.50–25.0 0.40–20.2 0.10–5.00 0.41–20.3 0.11–5.40
Fungal contamination in analysed pet food samples.
Number of pet food type
Kitten Cat Puppy Dog (n = 9) (n = 15) (n = 5) (n = 20)
Fungi Contamination rate (%) Min total amount (cfu g−1) Max total amount (cfu g−1) Moulds Contamination rate (%) Min total amount (cfu g−1) Max total amount (cfu g−1) Yeast Contamination rate (%) Min total amount (cfu g−1) Max total amount (cfu g−1)
77.8 n.e. 90
60.0 n.e. 268
40.0 n.e.
