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Effect of processing on the degradation of ivermectin in milk and milk products
Abstracts / Toxicology Letters 164S (2006) S1–S324
the gastrointestinal tract. The acceptable daily intake (ADI) for nitrites suggested by FAO/WHO (1985) is up to 0.13 mg/kg/day. It was proposed that this value be changed to 0.07 mg NO2 − /kg/day based on a more recent study. JECFA and SCF have proposed an ADI for NO2 − of 0–0.07 mg/kg body weight per day and EPA has set an Rf D of 0.10 mg NO2 − nitrogen per kg, body weight/day. However, these values are not applicable to infants and young children. The present study was aimed to detect whether there was any nitrite contamination in baby foods marketed in Ankara, Turkey and to estimate any possible toxicological risk in this sensitive physiological period. For this purpose, a study was designed to measure the nitrite levels in twenty collected baby foods and infant formulas, which were divided into divided into four groups (milk-, cereal-, vegetable- and fruitbased). An adapted Griess method was used to determine nitrite levels in the following samples. Nitrites reacted with Griess reagent to produce a dye that was detected at 525 nm. An extraction procedure was adapted and the method was applied to twenty baby food samples. The calibration curve was linear from 50 to 1000 ng/ml (y = 0.067x + 0.007, r = 0.998). The detection limit wasa 25 ng/ml. The coefficients of variation at 780 ng/ml concentration were 0.32% and 0.78% for within day and between days precisions, respectively. For supplementation of 1000 ng/ml of nitrite on sample which contained 780 ng/ml concentration, the average recovery value was found as 96%. We found that there were differences of nitrite concentrations between the groups. As it is largely known that babies should be fed with different kinds of food during the first year of life, it is necessary to choose the right baby food for the right month as the baby may not be able to cope with too much burden of chemicals; such as food with nitrite contamination. During manufacturing, nitrite contamination can come from several sources including water, fertilizer contamincation and from milk, fruit and vegetables within; so it is essential to take special care throughout the process of manufacturing food for infants and children. doi:10.1016/j.toxlet.2006.07.227
S273
P22-11 Effect of processing on the degradation of ivermectin in milk and milk products Veronica Lobato 2 , Susanne Rath 1 , Felix G.R. Reyes 1 1 State
University of Campinas, Campinas, S˜ao Paulo, Brazil; 2 Rural Federal University of Rio de Janeiro, Rio de Janeiro, Brazil Ivermectin is the active compound of a veterinary drug with broad-spectrum anti-parasitic action, which is used to control internal and external parasites in bovine, swine and equine livestock. The stability to the thermal treatment, as well as the loss of the ivermectin residue in the yogurt and cheese whey preparation was evaluated in bovine milk obtained from cows (n = 10) treated with a subcutaneous dose of 200 g ivermectin/kg b.w. The milk samples were analyzed before and after the following treatments: pasteurization at 62 ◦ C for 30 min (P); heating at 80 ◦ C for 10 min (H) and boiling at 99 ◦ C for 1 min (B). The levels of ivermectin residue were also determined in the yogurt and whey prepared from the milk of the treated cows. High-performance liquid chromatography with fluorescence detection was used for the quantitation of ivermectin residues in the food sample matrices. After liquid–liquid extraction of ivermectin and purification of the extract, the compound was derivatized with 1-methylimidazol in N,N-dimethyl formamide to form a fluorescent derivative, which was separated by HPLC, using a Merck RP-18 LiChroCart column (125× 4 mm, 5 m). The column was thermostatically controlled at 30 ◦ C. The flow rate for the mobile phase, composed of a mixture of methanol:water (96:4, v/v), was of 0.7 mL min−1 . The excitation and emission wavelengths of the fluorescence detector were 360 nm and 470 nm, respectively. The linearity of the method was in the range of 10–100 ng ivermectin mL−1 . Based on a sample of 5.0 mL, the limit of detection and quantitation for ivermectin in the samples were 0.6 ng mL−1 and 2 ng mL−1 , respectively. The average recovery, at four fortification levels, was 77.9 ± 3.2%. The inter-assay precision of the method was 13% (n = 5). The ivermectin presented stability to the thermal treatments of 83.3 ± 6.4% in P, 70.9 ± 15.3% in H and 63.7 ± 14.3% in B. The residue of ivermectin, in relation to the original level in the milk, verified in yogurt and in the whey were 77.6 ± 17.9% and 26.6 ± 5.6%, respectively. These results demonstrate the high stability of the drug to the principal technological processes to which the milk is submitted, pointing out the presence of ivermectin
S274
Abstracts / Toxicology Letters 164S (2006) S1–S324
residues in the whey, a raw material used in the food industry. doi:10.1016/j.toxlet.2006.07.228 P22-12 Effect of processing on the degradation of tetracyclines in milk and milk products Luiz Severo da Felix G.R. Reyes
Silva
Jr., Susanne
Rath,
State University of Campinas, Campinas, S˜ao Paulo, Brazil Tetracyclines are considered an important group of broad spectrum antimicrobials employed in Brazil in several species of animals producing food, including bovine, swine and fish. A HPLC method for the determination of oxytetracycline (OTC), doxycycline (DC) and tetracycline (TC) residues in milk and yogurt was developed and validated. The method was used to evaluate the degradation of these antimicrobial contaminants during the thermal treatment of milk, as well as during yogurt preparation and storage. Sample extraction was carried out using 30% (v/v) trichloroacetic acid to precipitate the proteins. For the HPLC analyses a Varian C8 analytical column (250× 4.6 mm, 5 m) with a mobile phase: sodium acetate 0.1 mol L−1 + EDTA 25 mmol L−1 + calcium chloride 35 mmol L−1 : methanol (65:35, v/v) and fluorescence detection (λ excitation: 420 nm and λ emission: 530 nm) was used. Tetracycline recovery in milk and yogurt was 92–98% (OTC); 87–83% (DC) and 89–97% (TC). The quantitation limit was 27, 46 and 33 ng mL−1 for OTC, DC and TC, respectively. Different thermal treatments were used to evaluate the heat stability of the tetracyclines in milk: cooking stove treatment, hot plate and microwave oven. Higher degradation occurred using the hot plate, 10.9% (OTC), 27.7% (DC) and 15.0% (TC). With respect to the stability of the tetracyclines during the preparation and shelf life of yogurt, it was verified that 500 ng mL−1 (OTC), 1120 ng mL−1 (DC) and 600 ng mL−1 (TC) did not inhibit yogurt production, and that after 30 days under refrigeration (4 ◦ C), residual levels of the contaminants still remained in the samples. Nonetheless, in relation to the original contamination in the yogurt, the tetracycline levels had diminished by 52%, 61% and 67%, for OTC, DC and TC, respectively. The data indicated that when conducting a risk assessment of tetracyclines in human health, the presence of these contaminants in milk and yogurt should be considered as a potential source. In addition, in order to protect consumer health, the governmental agencies should monitor the application of good practices in the
use of these antimicrobials in the treatment of cows that produce milk for human consumption, as well as conducting health surveillance actions in order to control the residue levels of these substances in commercialized milk and yogurt. doi:10.1016/j.toxlet.2006.07.229 P22-13 Determination of volatile N-nitrosamines in sausages by HS-SPME-GC-TEA Camila B. Dutra, Raquel Andrade, Felix G.R. Reyes, Susanne Rath State University of Campinas, Campinas, S˜ao Paulo, Brazil Nitrosamines are formed in certain foods by the reaction of a nitrosating agent derived from either nitrite salts or nitrogen oxide with a nitrosable amine. The majority of the nitrosamines tested have been carcinogenic in a variety of animal species. Furthermore, these compounds are toxic, mutagenic, teratogenic and act transplacentally. This work describes the development of a simple method using headspace sampling by solidphase microextraction gas chromatography with thermal energy analyzer detector (HS-SPME-GC-TEA) for the determination of N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosopiperidine (NPIP) and N-nitrosopyrrolidine (NPYR) in sausages. Because the SPME efficiency is influenced by several factors such as type of fiber coating, extraction time, ionic strength of the solution and temperature, a fractional factorial design and a central composite design were developed and employed to determine the optimal experimental conditions for the nitrosamine determination in the sausages sample matrices. Fused silica fibers coated with dimethylsiloxane–divinylbenzene, dimethylsiloxane–divinylbenzene carboxen and poliacrylate were evaluated. The chromatographic separations were carried out with a HP-INOWAX megabore column (30 m × 530 m i.d. × 1 m). The CG operating conditions were as follows: injector temperature, 200 ◦ C; oven temperature was held at 100 ◦ C for 3 min, then heated to 140 ◦ C at 40 ◦ C min−1 and then to 160 ◦ C at 5 ◦ C min−1 . The TEA furnace and the GC-TEA interface temperatures were 550 ◦ C and 250 ◦ C, respectively. The helium carrier gas flow-rate was 5 mL min−1 . The validated method is simple, with adequate accuracy, selectivity, sensitivity and precision. In the analyzed hot dog sausages samples (n = 7) volatile nitrosamines were not detected. The results indicate that the absence of nitrosamines in the hot dog sausages possibility occurred
the gastrointestinal tract. The acceptable daily intake (ADI) for nitrites suggested by FAO/WHO (1985) is up to 0.13 mg/kg/day. It was proposed that this value be changed to 0.07 mg NO2 − /kg/day based on a more recent study. JECFA and SCF have proposed an ADI for NO2 − of 0–0.07 mg/kg body weight per day and EPA has set an Rf D of 0.10 mg NO2 − nitrogen per kg, body weight/day. However, these values are not applicable to infants and young children. The present study was aimed to detect whether there was any nitrite contamination in baby foods marketed in Ankara, Turkey and to estimate any possible toxicological risk in this sensitive physiological period. For this purpose, a study was designed to measure the nitrite levels in twenty collected baby foods and infant formulas, which were divided into divided into four groups (milk-, cereal-, vegetable- and fruitbased). An adapted Griess method was used to determine nitrite levels in the following samples. Nitrites reacted with Griess reagent to produce a dye that was detected at 525 nm. An extraction procedure was adapted and the method was applied to twenty baby food samples. The calibration curve was linear from 50 to 1000 ng/ml (y = 0.067x + 0.007, r = 0.998). The detection limit wasa 25 ng/ml. The coefficients of variation at 780 ng/ml concentration were 0.32% and 0.78% for within day and between days precisions, respectively. For supplementation of 1000 ng/ml of nitrite on sample which contained 780 ng/ml concentration, the average recovery value was found as 96%. We found that there were differences of nitrite concentrations between the groups. As it is largely known that babies should be fed with different kinds of food during the first year of life, it is necessary to choose the right baby food for the right month as the baby may not be able to cope with too much burden of chemicals; such as food with nitrite contamination. During manufacturing, nitrite contamination can come from several sources including water, fertilizer contamincation and from milk, fruit and vegetables within; so it is essential to take special care throughout the process of manufacturing food for infants and children. doi:10.1016/j.toxlet.2006.07.227
S273
P22-11 Effect of processing on the degradation of ivermectin in milk and milk products Veronica Lobato 2 , Susanne Rath 1 , Felix G.R. Reyes 1 1 State
University of Campinas, Campinas, S˜ao Paulo, Brazil; 2 Rural Federal University of Rio de Janeiro, Rio de Janeiro, Brazil Ivermectin is the active compound of a veterinary drug with broad-spectrum anti-parasitic action, which is used to control internal and external parasites in bovine, swine and equine livestock. The stability to the thermal treatment, as well as the loss of the ivermectin residue in the yogurt and cheese whey preparation was evaluated in bovine milk obtained from cows (n = 10) treated with a subcutaneous dose of 200 g ivermectin/kg b.w. The milk samples were analyzed before and after the following treatments: pasteurization at 62 ◦ C for 30 min (P); heating at 80 ◦ C for 10 min (H) and boiling at 99 ◦ C for 1 min (B). The levels of ivermectin residue were also determined in the yogurt and whey prepared from the milk of the treated cows. High-performance liquid chromatography with fluorescence detection was used for the quantitation of ivermectin residues in the food sample matrices. After liquid–liquid extraction of ivermectin and purification of the extract, the compound was derivatized with 1-methylimidazol in N,N-dimethyl formamide to form a fluorescent derivative, which was separated by HPLC, using a Merck RP-18 LiChroCart column (125× 4 mm, 5 m). The column was thermostatically controlled at 30 ◦ C. The flow rate for the mobile phase, composed of a mixture of methanol:water (96:4, v/v), was of 0.7 mL min−1 . The excitation and emission wavelengths of the fluorescence detector were 360 nm and 470 nm, respectively. The linearity of the method was in the range of 10–100 ng ivermectin mL−1 . Based on a sample of 5.0 mL, the limit of detection and quantitation for ivermectin in the samples were 0.6 ng mL−1 and 2 ng mL−1 , respectively. The average recovery, at four fortification levels, was 77.9 ± 3.2%. The inter-assay precision of the method was 13% (n = 5). The ivermectin presented stability to the thermal treatments of 83.3 ± 6.4% in P, 70.9 ± 15.3% in H and 63.7 ± 14.3% in B. The residue of ivermectin, in relation to the original level in the milk, verified in yogurt and in the whey were 77.6 ± 17.9% and 26.6 ± 5.6%, respectively. These results demonstrate the high stability of the drug to the principal technological processes to which the milk is submitted, pointing out the presence of ivermectin
S274
Abstracts / Toxicology Letters 164S (2006) S1–S324
residues in the whey, a raw material used in the food industry. doi:10.1016/j.toxlet.2006.07.228 P22-12 Effect of processing on the degradation of tetracyclines in milk and milk products Luiz Severo da Felix G.R. Reyes
Silva
Jr., Susanne
Rath,
State University of Campinas, Campinas, S˜ao Paulo, Brazil Tetracyclines are considered an important group of broad spectrum antimicrobials employed in Brazil in several species of animals producing food, including bovine, swine and fish. A HPLC method for the determination of oxytetracycline (OTC), doxycycline (DC) and tetracycline (TC) residues in milk and yogurt was developed and validated. The method was used to evaluate the degradation of these antimicrobial contaminants during the thermal treatment of milk, as well as during yogurt preparation and storage. Sample extraction was carried out using 30% (v/v) trichloroacetic acid to precipitate the proteins. For the HPLC analyses a Varian C8 analytical column (250× 4.6 mm, 5 m) with a mobile phase: sodium acetate 0.1 mol L−1 + EDTA 25 mmol L−1 + calcium chloride 35 mmol L−1 : methanol (65:35, v/v) and fluorescence detection (λ excitation: 420 nm and λ emission: 530 nm) was used. Tetracycline recovery in milk and yogurt was 92–98% (OTC); 87–83% (DC) and 89–97% (TC). The quantitation limit was 27, 46 and 33 ng mL−1 for OTC, DC and TC, respectively. Different thermal treatments were used to evaluate the heat stability of the tetracyclines in milk: cooking stove treatment, hot plate and microwave oven. Higher degradation occurred using the hot plate, 10.9% (OTC), 27.7% (DC) and 15.0% (TC). With respect to the stability of the tetracyclines during the preparation and shelf life of yogurt, it was verified that 500 ng mL−1 (OTC), 1120 ng mL−1 (DC) and 600 ng mL−1 (TC) did not inhibit yogurt production, and that after 30 days under refrigeration (4 ◦ C), residual levels of the contaminants still remained in the samples. Nonetheless, in relation to the original contamination in the yogurt, the tetracycline levels had diminished by 52%, 61% and 67%, for OTC, DC and TC, respectively. The data indicated that when conducting a risk assessment of tetracyclines in human health, the presence of these contaminants in milk and yogurt should be considered as a potential source. In addition, in order to protect consumer health, the governmental agencies should monitor the application of good practices in the
use of these antimicrobials in the treatment of cows that produce milk for human consumption, as well as conducting health surveillance actions in order to control the residue levels of these substances in commercialized milk and yogurt. doi:10.1016/j.toxlet.2006.07.229 P22-13 Determination of volatile N-nitrosamines in sausages by HS-SPME-GC-TEA Camila B. Dutra, Raquel Andrade, Felix G.R. Reyes, Susanne Rath State University of Campinas, Campinas, S˜ao Paulo, Brazil Nitrosamines are formed in certain foods by the reaction of a nitrosating agent derived from either nitrite salts or nitrogen oxide with a nitrosable amine. The majority of the nitrosamines tested have been carcinogenic in a variety of animal species. Furthermore, these compounds are toxic, mutagenic, teratogenic and act transplacentally. This work describes the development of a simple method using headspace sampling by solidphase microextraction gas chromatography with thermal energy analyzer detector (HS-SPME-GC-TEA) for the determination of N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosopiperidine (NPIP) and N-nitrosopyrrolidine (NPYR) in sausages. Because the SPME efficiency is influenced by several factors such as type of fiber coating, extraction time, ionic strength of the solution and temperature, a fractional factorial design and a central composite design were developed and employed to determine the optimal experimental conditions for the nitrosamine determination in the sausages sample matrices. Fused silica fibers coated with dimethylsiloxane–divinylbenzene, dimethylsiloxane–divinylbenzene carboxen and poliacrylate were evaluated. The chromatographic separations were carried out with a HP-INOWAX megabore column (30 m × 530 m i.d. × 1 m). The CG operating conditions were as follows: injector temperature, 200 ◦ C; oven temperature was held at 100 ◦ C for 3 min, then heated to 140 ◦ C at 40 ◦ C min−1 and then to 160 ◦ C at 5 ◦ C min−1 . The TEA furnace and the GC-TEA interface temperatures were 550 ◦ C and 250 ◦ C, respectively. The helium carrier gas flow-rate was 5 mL min−1 . The validated method is simple, with adequate accuracy, selectivity, sensitivity and precision. In the analyzed hot dog sausages samples (n = 7) volatile nitrosamines were not detected. The results indicate that the absence of nitrosamines in the hot dog sausages possibility occurred