Infants have a more restricted diet and they generally consume more food on a body weight basis than adults. Therefore, the significance and potential health risk of any contaminant in foods consumed by infants is increased and diligent attention must be paid to this particular area. The present study aims to determine the occurrence of aflatoxin M 1 (AFM 1 ), aflatoxin B 1 (AFB 1 ) and ochratoxin A (OTA) in processed cereal-based foods (flours) and infant formulae (milk powder) available in the Portuguese market, both sold as conventional and organic origin. Mycotoxin determination was carried out using a method previously applied to duplicate diet samples. This method employed chloroform extraction, liquid-liquid extraction, immunoaffinity column (IAC) cleanup and HPLC analysis with fluorescence detection after postcolumn derivatisation. Quantification limits were 0.014, 0.004 and 0.028 μg kg −1 for AFM 1 , AFB 1 and OTA, respectively. These toxins could only be quantified in 12 of 27 analysed samples (15 positive results): two samples with AFM 1 , two samples with AFM 1 and OTA, one sample with AFB 1 and OTA and seven samples with OTA. Positive results concerned four for AFM 1 (26%), one for AFB 1 (7%) and ten for OTA (67%). For these samples, contents ranged between 0.017-0.041 μg AFM 1 kg −1 , 0.034-0.212 μg OTA kg −1 , and one sample had a value of 0.009 μg AFB 1 kg −1 . Considering the presented results, we could provisionally conclude that the presence of these mycotoxins in baby foods does not constitute a public health problem. These are the first results concerning the occurrence of mycotoxins in marketed baby foods in Portugal and this is the first study using the HPLC method, proposed for duplicate diets, in baby food sample analysis.
A liquid chromatographic (LC) method for the determination of fumonisins B1 (FB1) and B2 (FB2) in corn and corn flakes was collaboratively studied by 23 laboratories, which analyzed 5 blind duplicate pairs of each matrix to establish the accuracy, repeatability, and reproducibility characteristics of the method. Fumonisin levels in the corn ranged from <0.05 (blank) to 1.41 μg/g for FB1 and from <0.05 to 0.56 μg/g for FB2, whereas in the corn flakes they ranged from <0.05 to 1.05 μg/g for FB1 and from <0.05 to 0.46 μg/g for FB2. The method involved double extraction with acetonitrile–methanol–water (25 + 25 + 50), cleanup through an immunoaffinity column, and LC determination of the fumonisins after derivatization with o-phthaldialdehyde. Relative standard deviations for the within-laboratory repeatability (RSDr) of the corn analyses ranged from 19 to 24% for FB1 and from 19 to 27% for FB2; for the corn flakes analyses, RSDr ranged from 9 to 21% for FB1 and from 8 to 22% for FB2. Relative standard deviations for the between-laboratories reproducibility (RSDR) of the corn analyses ranged from 22 to 28% for FB1 and from 22 to 30% for the FB2; for corn flakes analyses, RSDR ranged from 27 to 32% for FB1 and from 26 to 35% for FB2. Mean recoveries of FB1 and FB2 from corn spiked with FB1 at 0.80 μg/g and with FB2 at 0.40 μg/g were 76 and 72%, respectively; for corn flakes spiked at the same levels recoveries were 110 and 97% for FB1 and FB2, respectively. HORRAT ratios for the analyses of corn ranged from 1.44 to 1.53 for FB1 and from 0.96 to 1.48 for FB2, whereas for corn flakes they ranged from 1.60 to 1.82 for FB1 and from 1.39 to 1.68 for FB2.
In the spring and autumn of 1994, a total diet study, in which 123 participants collected duplicates of their 24-hour diets, was carried out. The goal of this study was to determine the mass fractions of a number of analytes in these duplicate diets, so as to be able to establish oral daily intake values. After measurements were carried out for pesticides, PCBs, elements, sterols, nitrate and nitrite, and fatty acids, the duplicate diet study was concluded with analyses for aflatoxin M1, aflatoxin B1 and ochratoxin A. For this purpose a method of analysis was developed, that could simultaneously determine these mycotoxins at very low levels. The method involved chloroform extraction, liquid-liquid extraction, immunoaffinity cleanup and liquid chromatography. The method was supplemented with a procedure to confirm the identity of chromatographic peaks, assumed to represent aflatoxin M1, aflatoxin B1 and ochratoxin A. The method was in-house validated. Recoveries ranged from 68-74% for aflatoxin M1 (at spiking levels from 30-120 ng/kg, c.v. 7.6%), from 95-97% for aflatoxin B1 (at spiking levels from 50-200 ng/kg, c.v. 2.8%), and from 75-84% for ochratoxin A (at spiking levels from 150-600 ng/kg, c.v. 4.3%). Limits of quantitation (defined as signal/noise = 10) were estimated to be 24, 5 and 16 ng/kg lyophilised material for aflatoxin M1, aflatoxin B1 and ochratoxin A respectively. The newly developed method was used to analyse 123 samples of 24-hour diets. Aflatoxin M1 was detectable in 48% of the samples; the toxin contents remained below the limit of quantitation in all samples. Aflatoxin B1 could be detected in 42% of the samples; in 25% of the samples the levels were above the limit of quantitation. Ochratoxin A could be quantified in all samples. The analytical results were further processed to estimate levels of intake. Intake levels for the aflatoxins were very low, and could not reliably be established. The mean ochratoxin A intake was estimated to be 1.2 ng/kg body weight per day. This is well below the tolerable daily intake established by JECFA at 14 ng/kg body weight per day. The current dietary intake of ochratoxin A in the Netherlands is concluded to pose no appreciable health risk.
A collaborative study was conducted to evaluate the effectiveness of an immunoaffinity column cleanup liquid chromatographic method for determination of aflatoxin M1 in milk at proposed European regulatory limits. The test portion of liquid milk was centrifuged, filtered, and applied to an immunoaffinity column. The column was washed with water, and aflatoxin was eluted with pure acetonitrile. Aflatoxin M1 was separated by reversed-phase liquid chromatography (LC) with fluorescence detection. Frozen liquid milk samples both naturally contaminated with aflatoxin M1 and blank samples for spiking, were sent to 12 collaborators in 12 different European countries. Test portions of samples were spiked at 0.05 ng aflatoxin M1 per mL. After removal of 2 noncompliant sets of results, the mean recovery of aflatoxin M1 was 74%. Based on results for spiked samples (blind pairs at 1 level) and naturally contaminated samples (blind pairs at 3 levels) the relative standard deviation for repeatability (RSDr) ranged from 8 to 18%. The relative standard deviation for reproducibility (RSDR) ranged from 21 to 31%. The method showed acceptable within- and between-laboratory precision data for liquid milk, as evidenced by HORRAT values at the low level of aflatoxin M1 contamination.
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