Simultaneous Screening of Six Families of Antibiotic Residues in Milk Samples by Biochip Multi-array Technology.

Background: Antimicrobial compounds are used in animal husbandry to prevent and treat bacterial diseases and as illegal growth-promoting agents. Due to the excessive and inappropriate use of antibiotics, the antibiotic residues in milk can cause allergic reactions and antibiotic resistance. A rapid biochip-based method for the multi-analyte screening of 6 families of antibiotic residues (quinolones, ceftiofur, florfenicol, streptomycin, tylosin, and tetracyclines) in milk was validated based on Commission Decision 2002/657 and the European guidance for screening methods for veterinary medicinal products. Methods: This methodology allows the 6 antibiotic families to be detected simultaneously, increasing the screening capacity and reducing costs in test settings. The method's applicability was shown by screening 38 UHT cow milk samples taken from Tehran province, IR Iran. Results: The results showed that the positive threshold T was above Fm, and the CCß was below the European Commission's Maximum Residue Limit (MRL) (100 ppb for ceftiofur and tetracycline and 50 ppb for tylosin in milk). Norfloxacin was detected in about 8% of the samples and tylosin in 2.63%. The total antibiotic concentration in UHT cow milk samples was lower than the European Commission's MRL. Conclusions: This study showed that the biochip technique is valid for screening tylosin, ceftiofur, streptomycin, tetracycline, norfloxacin, and florfenicol in milk. It was found that the method was easy, quick, and capable of detecting 6 families of antibiotic residues simultaneously from a single milk sample without sample preparation.

Performance Evaluation of Biochip Chemiluminescent Immunoassay for Screening Seven Mycotoxins in Wheat Flour Simultaneously.

Background: Wheat grains are susceptible to mycotoxins, toxic natural secondary metabolites generated by certain fungi on agricultural produce in the field during growth, harvest, transportation, or storage. Therefore, wheat flour can be contaminated with mycotoxins, which seriously threaten human health. Methods: A rapid method for screening seven mycotoxins in wheat flour was validated in accordance with Commission Decision 2002/657/EC. With this multi-analytical screening method, 7 prevalent mycotoxins (fumonisin B1, ochratoxin A, aflatoxin G1, deoxynivalenol, T-2 toxin, aflatoxin B1, and zearalenone) can be determined simultaneously. The method's applicability was demonstrated by screening 7 mycotoxins in 39 wheat flour samples collected from different bakeries in Tehran province, Iran. Results: The validation results indicated that for all 7 mycotoxins, the positivity threshold (T) was above the cut-off value (Fm), and no false positive results were obtained for any of the mycotoxins. The screening results of 12 packaged and 27 bulk wheat flour samples indicated that the concentrations of all mentioned mycotoxins were higher than the cut-off (in the relative light unit [RLU]), and all the samples were compliant. Conclusions: The present study revealed that the biochip-based technique is valid for identifying and assessing the levels of 7 mycotoxins in grain samples, such as wheat flour, at the measured validation concentrations. The method was simple, fast, and able to screen 7 mycotoxins simultaneously. The test process of the kit is easy to conduct, and the results are straightforward to interpret.

Health risk assessment of process-related contaminants in bread.

Bread constitutes a popular and even daily component of human diet world-wide, Iran included. However, there are concerns that various processing methods such as frying and baking could result in the production of potentially a source of known carcinogens such as acrylamide (AA) and benzo(a)pyrene (BaP). The present study tried to perform a risk assessment on seven categories of bread consumers, based on age and gender, calculating the Target Hazard Quotient (THQ), Incremental Lifetime Cancer Risk (ILCR), and Margin of Exposure (MOE) related to the dietary intake of AA and BaP. AA and BaP were analyzed in 87 bread samples using LC-MS/MS and GC-MS/MS, respectively. The results indicated that more than 94% (mean concentration: 25.9 ng/g) and about 20% (mean concentration: 1.98 ng/g) of the samples were contaminated with AA and BaP, respectively. The THQ of AA intake through bread consumption for seven categories was in the following decreasing order: semi-industrial Sangak bread of Shiraz (SIS-Sh)> traditional Sangak bread of Shiraz (TS-Sh)> traditional Sangak bread of Tehran (TS-Th)> commercial bread of Tehran (C-Th). The non-neoplastic and neoplastic MOE for AA (˂10,000) indicates a high risk of exposure for all people in Tehran and Shiraz through the consumption of all tested bread. Due to the consumption of TS, SIS, and C bread, the BaP MOE for all people in Tehran and Shiraz was >10 000, which shows a low health risk for consumers. Our findings showed that ILCR for AA in seven classes of people who had TS-Sh and TS-Th was remarkably higher than ILCR for all categories that consumed the C-Th. BaP ILCR for the people who ingested TS-Th, TS-Sh, SIS-Th, and SIS-Sh was about 2-4 times higher than people who had C-Th. This study indicates that bread is the main source of AA and BaP intake in Iran, which might elevate the cancer risk.

Simultaneous Screening of Nitrofuran Metabolites in Honey Using Biochip Array Technology: Validation Study According to the Decision 2002/657/EC of the European Union.

Background: Although no authorization is available for antibiotics to treat bee diseases, some veterinary compounds are used by beekeepers, and each country sets its own thresholds. Inappropriate and excessive use of these drugs can cause allergic reactions and antibiotic resistance in humans who consume the remaining antibiotic residues in honey and its products. It is, therefore, relevant to monitor the presence of antibiotic residues in this matrix. Objectives: A rapid method for the simultaneous screening of nitrofuran metabolite residues in honey was validated according to Commission Decision 2002/657/EC (C.D 657) and the European guideline for the validation of screening methods for veterinary medicines. Methods: This multi-analytical screening method enables the simultaneous determination of four nitrofuran metabolites [3-amino-2-oxazolidone (AOZ), 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ), 1-Aminohydantoin HCl (ADH), and semicarbazide (SEC)] from a single honey sample. Thirty-five honey samples were collected randomly as real samples for screening from Tehran, IR Iran, Germany, and the Netherlands in 2018. Results: For all four antibiotic residues, the positivity threshold T was higher than the cut-off value Fm, and no false-positive results were obtained for three antibiotics (AOZ, AMOZ, and SEC). Detection capabilities (CCß) of all compounds were under the minimum required performance limit (MRPL) authorized by the European Commission (currently 1 µg/kg). The screening results of 15 domestic and 20 imported honey samples showed that the levels of AOZ in 6.66% and 10% of the samples, the level of AMOZ in 13.33% and 0% of the samples, and the level of SEC in 33.33% and 40% of the samples were less than the cut-off ([in relative light units (RLUs)], respectively. Conclusions: This study found that this technique is valid for detecting and quantifying three antibiotic residues in honey samples at the measured validation levels. This method was simple, rapid, and capable of simultaneously screening three nitrofuran metabolites from a single honey sample.

Validation of a Multiclass Method for the Screening of 15 Antibiotic Residues in Milk Using Biochip Multi-array Technology and Its application to Monitor Real Samples.

Veterinary drugs are extensively and legally consumed to treat and prevent disease in chattels but some are also used illegally as growth-stimulating agents. Inappropriate or intensive use of antibiotics can cause allergic reactions and, above all, antibiotic resistance. A multiclass approach for the screening of antimicrobial substances in milk was validated in consonance with Commission Decision 2002/657/EC and to the European guideline for the validation of screening methods for veterinary medicines. This biochip-based approach enables the simultaneous determination of a total of 13 sulphonamide, dapsone and trimethoprim. For monitoring of antibiotic residues, 53 UHT milk samples collected from Tehran, IR Iran were screened applying this technology. The result showed that for all antibiotic residues, the positivity threshold T was much more than the cut-off value Fm. A false positive rate of less than 5% was found for all antibiotics which are satisfactory. All detection capabilities (CCß) were well below the Maximum Residue Level (MRL) set by the European Commission (100 µg/kg for the sum of all sulphonamides and 50 µg/kg for trimethoprim in milk). The screening results of 53 milk samples showed that 71.7% of samples were compliant and all positive samples were below the MRL set by European Commission. This study showed that the biochip-based technique is valid to identify and quantify antibiotic residues in milk at the studied validation levels. The method was rapid, easy, safe, and able to screen 13 sulphonamide, dapsone and trimethoprim from a single milk sample simultaneously with no sample preparation procedure (or just one-step centrifugation).

Exposure to multiple mycotoxins in domestic and imported rice commercially traded in Tehran and possible risk to public health.

Mycotoxins are secondary fungi metabolites that induce acute and chronic toxic effects in humans and animals. In the present study, nine mycotoxins including aflatoxins (AFB1, AFB2, AFG1 and AFG2), fumonisins (FB1 and FB2), Ochratoxin A (OTA), deoxynivalenol (DON), and zearalenone (ZEN) were determined in one hundred rice samples collected from Tehran using high performance liquid chromatography (HPLC) with fluorescence or photodiode array detector. In addition, possible risk to public health was investigated by assessing dietary exposure through rice consumption, the margin of exposure (MOE), respective risk of cancer and hazard index (HI) of the monitored mycotoxins in children and adults. The higher mean levels were determined for DON (102.22 µg.Kg-1), followed by FB1 (85.00 µg.Kg-1). For the rests of mycotoxins the levels did not exceed 20 µg.Kg-1. The estimated AFB1 intake for the adults and children through rice consumption exceeds the safe levels established for both carriers and non-carriers of hepatitis B virus. The mean and median determined exposure levels of OTA, DON ZEN and FB1, were found lower than the Provisional Maximum Tolerable Daily Intake (PMTDI) value for both adults and children of Tehran that consuming domestic and imported rice. The mean HI for adults and median HI for adults and children were below one, and mean HI for children was close to one. All the mean, median and maximum MoE values were <10,000 in adults and children, indicating a risk due to AFB1 exposure through rice consumption in Tehran. In addition, the calculated mean cancer risk in adult and child populations of Tehran were 0.27 and 0.64 cases per year per 105 individuals, respectively, that shows population in Tehran could be at risk of cancer due to AFB1 exposure through rice consumption as calculated. So further studies are necessary for the monitoring mycotoxins in rice and different food products as well as estimating average dietary exposure and cumulative exposure assessment of mycotoxins for main foods in IR Iran.

Occupational exposures and respiratory symptoms and lung function among hairdressers in Iran: a cross-sectional study.

OBJECTIVE: Exposures at hairdressers' work have been reported to lead to an increased risk of several health outcomes. The present study aimed to investigate the relations between occupational exposures and respiratory symptoms and lung function among hairdressers in Iran. METHODS: We conducted a cross-sectional study to compare potential respiratory effects among 140 women working as hairdressers to such effects among 140 women working as office workers (administrative personnel). Both groups worked in Shiraz, Iran. Respiratory symptoms were assessed by a standard respiratory questionnaire. The questionnaire also inquired about substances used and workspace conditions, including ventilation type. Lung function levels were measured by spirometry. RESULTS: Respiratory symptoms, including cough, wheezing, shortness of breath, and chest tightness were significantly more frequent in hairdressers compared to the reference group (p < 0.05). After controlling for potential confounders, hairdressers had a prevalence ratio (PR) of 2.18 (95% CI 1.26-3.77) for cough, 9.59 (95% CI 1.004-91.73) for wheezing, 2.06 (95% CI 1.25-3.39) for shortness of breath, and 3.31 (95% CI 1.84-5.97) for chest tightness compared to the reference group. Lung function parameters (including VC, FVC, and FEV1) were significantly reduced in hairdressers (p < 0.001). Absence of air conditioning predicted greater reduction in lung function (p < 0.05) in the exposed. Decrease in FVC with normal FEV1/FVC in the exposed group suggested existence of restrictive lung function. CONCLUSIONS: This study provides evidence of increased prevalence of respiratory symptoms and restrictive lung function impairment among hairdressers in Iran.

Validation of Simultaneous Biochip-based Method for Screening of 3 Beta-Lactam Families Residues in Cow's Milk in Accordance with the European Union Decision 2002/657/EC and its Application on Real Samples.

Illegal and excessive use of veterinary antibiotics as a food additive for growth promotion in livestock can lead to allergic reactions and antibiotic resistance, which is a worldwide concern. A biochip-based semi-quantitative screening method of antimicrobial residues in milk was validated based on Commission Decision 2002/657/EC and the European guideline to validate screening methods for veterinary medicines. This multi-analytical screening method enables to determine of 3 beta-lactams (cefalexin, ampicillin, and cefuroxime) simultaneously. Analysis of 20 blank and 20 spiked milk samples showed that for all 3 antibiotic residues, the positivity threshold T was above cut-off value Fm, and no false-positive results were obtained for all 3 antibiotics. All detection capabilities (CCß) were below Maximum Residue Level (MRL) authorized by European Commission. 47 UHT cow's milk samples collected from Tehran province, IR Iran, were screened, and compliance was found in 100% of samples. This study found that the biochip method is valid to determine antibiotic residues in milk samples at the measured validation levels. The method was fast, simple, and able to simultaneous screen three families of beta-lactams from a single milk sample with almost no sample preparation.

Development of a Sensitive and Rapid Method for Determination of Acrylamide in Bread by LC-MS/MS and Analysis of Real Samples in Iran IR.

A new sample preparation procedure and a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method were developed for the quantitative analysis of acrylamide in bread. The method is based on sample extraction in methanol, purification with Carrez solutions and clean- up with Primary Secondary Amine (PSA).The developed method offers an efficient, inexpensive, easy sample preparation and very sensitive procedure for determination of acrylamide in bread. The use of spiked calibration curves for constructing the calibration curve substantially reduced adverse matrix-related effects. Recoveries were between 96 and 105.3%. Good results were obtained with respect to repeatability (RSDs <11%). The limit of detection and quantification of the method was 0.3 and 1 ng/g, respectively, which shows the method is very sensitive. The developed method was used for the determination of acrylamide in 26 traditional bread samples (Sangak) collected from Shiraz. The results showed that about 96% of Sangak bread samples were contaminated with acrylamide that 64.3 and 33.3 of semi-industrial and traditional Sangak bread were higher than benchmark levels (50 µg/kg), respectively. There are a few reports concerning contamination of Sangak bread samples with acrylamide in Iran. Therefore, this method could be used for a comprehensive survey of acrylamide in Sangak bread samples in the country.

Health risk assessment of aflatoxin M1 in infant formula milk in IR Iran.

In this study, two accurate, precise, selective and sensitive methods were developed for determining aflatoxin M1 (AFM1) in infant formula milk using immunoaffinity column clean-up followed by high performance liquid chromatography (HPLC) with fluorescence detection. The validated methods were used for determination of AFM1 in 29 samples of 6 different infant formula milk brands and the risk of AFM1 in infants aged zero to 6 months old was assessed using cancer risk, Margin of Exposure (MOE) and Hazard Index (HI). Only one sample (3.4%) was contaminated with AFM1. Although the results showed that MOE values for the mean and median exposure to AFM1 was <10,000 in infants, the additional cancer risk due to mean and median exposure to AFM1 in infant <6 months were 0.00010 and 0.00012 additional cases per year per 105 individuals, respectively, which indicates no health concern. In addition, HI values for the mean and median exposure to AFM1 for infants were quite below one which indicates no health concern. To the best of our knowledge, this is the first report on risk assessment of AFM1 in infant formula milk consumed by Iranian infants <6 months old, presenting a low risk for the evaluated groups.

Health risk assessment of acrylamide in bread in Iran using LC-MS/MS.

Acrylamide is a chemical, often present in bread, legally classified as carcinogen, mutagen and reproductive toxicant. Since bread is consumed both world-wide and in Iran, determination of acrylamide in different types of breads is of high interest. In the present study, acrylamide was monitored in 56 Sangak and 30 industrial bread samples collected from Tehran and Shiraz, using LC-MS/MS (LOQ = 1 ng/g). In addition, the noncarcinogenic risk (target hazard quotient-THQ) and carcinogenic risk (incremental lifetime cancer risk-ILCR) due to ingestion of acrylamide through bread consumption in children and adults were assessed. Acrylamide was detected in more than 90% of the samples tested. The average daily intake of acrylamide in Iran based on exclusive consumption of Sangak bread, was estimated at 145 ng/kg bw/day. Based on the THQ for bread acrylamide in adults and children, the decreasing risk order was: Shiraz semi-industrial Sangak, Shiraz traditional Sangak, Tehran traditional Sangak, Tehran industrial bread. The ILCR of bread acrylamide calculated for adults and children was higher than the permissible lifetime carcinogenic risk value established by USEPA (1.00E-5). Results show that bread is a major source of acrylamide intake by people in Iran and all consumers regardless of age could be at elevated carcinogenic risk.

Validation of an Analytical Method for Determination of 13 priority polycyclic aromatic hydrocarbons in mineral water using dispersive liquid-liquid microextraction and GC-MS.

Dispersive liquid-liquid microextraction (DLLME) combined with gas chromatography-mass spectrometry (GC-MS) was used for the extraction and determination of 13 polycyclic aromatic hydrocarbons (PAHs) in mineral water samples. In this procedure, the suitable combination of extraction solvent (500 µL chloroform) and disperser solvent (1000 µL acetone) were quickly injected into the water sample (10.00 mL) by Hamilton syringe. After centrifugation, 500 µL of the lower organic phase was dried under a gentle stream of nitrogen, re-dissolved in chloroform and injected into GC-MS. Chloroform and acetone were found to be the best extraction and disperser solvent, respectively. Validation of the method was performed using spiked calibration curves. The enrichment factor ranged from 93 to 129 and the recovery ranged from 71 to 90%. The linear ranges for all the PAHs were 0.10-2.80 ngmL(-1). The relative standard deviations (RSDs) of PAHs in water by using anthracene-d10 as internal standard, were in the range of 4-11% for most of the analytes (n = 3). Limit of detection (LOD) for different PAHs were between 0.03 and 0.1 ngmL(-1). The method was successfully applied for the analysis of PAHs in mineral water samples collected from Tehran.

Validation of an Analytical Method for Determination of Benzo[a]pyrene Bread using QuEChERS Method by GC-MS.

A fast and simple modified QuEChERS (quick, easy, cheap, rugged and safe) extraction method based on spiked calibration curves and direct sample introduction was developed for determination of Benzo [a] pyrene (BaP) in bread by gas chromatography-mass spectrometry single quadrupole selected ion monitoring (GC/MS-SQ-SIM). Sample preparation includes: extraction of BaP into acetone followed by cleanup with dispersive solid phase extraction. The use of spiked samples for constructing the calibration curve substantially reduced adverse matrix-related effects. The average recovery of BaP at 6 concentration levels was in range of 95-120%. The method was proved to be reproducible with relative standard deviation less than 14.5% for all of the concentration levels. The limit of detection and limit of quantification were 0.3 ng/g and 0.5 ng/g, respectively. Correlation coefficient of 0.997 was obtained for spiked calibration standards over the concentration range of 0.5-20 ng/g. To the best of our knowledge, this is the first time that a QuEChERS method is used for the analysis of BaP in breads. The developed method was used for determination of BaP in 29 traditional (Sangak) and industrial (Senan) bread samples collected from Tehran in 2014. These results showed that two Sangak samples were contaminated with BaP. Therefore, a comprehensive survey for monitoring of BaP in Sangak bread samples seems to be needed. This is the first report concerning contamination of bread samples with BaP in Iran.

Simultaneous Determination of 13 Priority Polycyclic Aromatic Hydrocarbons in Tehran's Tap Water and Water for Injection Samples Using Dispersive Liquid-Liquid Micro Extraction Method and Gas Chromatography-Mass Spectrometry.

Polycyclic aromatic hydrocarbons (PAHs) are classified as persistent and carcinogenic organic pollutants. PAHs contamination has been reported in water. Many of relevant regulatory bodies such as EU and EPA have regulated the limit levels for PAHs in drinking water. In this study, 13 priority polycyclic aromatic hydrocarbons (PAHs) were determined in tap water samples of Tehran and water for injection. Dispersive liquid-liquid microextraction procedure combined with gas chromatography-mass spectrometry was used for the extraction and determination of PAHs in the samples. Under the optimized conditions, the range of extraction recoveries and relative standard deviations (RSDs) of PAHs in water using internal standard (anthracene-d10) were in the range of 71-90% and 4-16%, respectively. Limit of detection for different PAHs were between 0.03 and 0.1 ngmL(-1). The concentration of PAHs in all tap water as well as water for injection samples were lower than the limit of quantification of PAHs. This is the first study addressing the occurrence of PAHs in water for injection samples in Iran using dispersive liquid-liquid micro extraction procedure combined with gas chromatography-mass spectrometry.

Restrictive Pattern of Pulmonary Symptoms among Photocopy and Printing Workers: A Retrospective Cohort Study.

BACKGROUND: According to the growth of photocopier usage in workplaces, the potential risk of occupational exposure to the airborne chemicals has been raised up. Hence, monitoring the photocopy worker's respiratory functions seems to be necessary. We aimed to evaluate the respiratory health on photocopy and printing workers so that a reliable description can be made about their occupational hygiene. METHODS: This study was performed in Shiraz, southwest Iran in 2014 and a group of 150 photocopy and printing workers were surveyed as exposed group in addition to a group of 114 office staff as unexposed group. The respiratory standard questionnaire was used to evaluate the prevalence of respiratory symptoms among the selected staff. Pulmonary function indexes including VC, FVC, FEV1 and the FEV1/FVC ratio were calculated. Finally, t-test, Chi Square and multiple logistic regressions were conducted. RESULTS: VC, FVC and FEV1 in photocopy and printing workers were lower than the unexposed group of which these differences for FVC and FEV1 were statistically significant (P<0.05). Moreover, the prevalence of all respiratory symptoms, except the shortness of breath, in exposed group was more than the unexposed group and the prevalence of coughing and wheezing was statistically significant (P<0.05). There was a significant difference in respiratory symptoms (cough and wheezing) between two groups after controlling for confounding variables, OR: 2.61 (95% CI: 1.21, 5.62) and 2.92 (95% CI: 1.25, 6.84), respectively. CONCLUSIONS: The prevalence of excess respiratory symptoms along with pattern of pulmonary restrictive sings in photocopy and printing workers revealed that the workplace conditions can result in occupational respiratory diseases.

Determination of Benzo[a]pyrene in Traditional, Industrial and Semi- industrial Breads Using a Modified QuEChERS Extraction, Dispersive SPE and GC-MS and Estimation of its Dietary Intake.

A fast and simple modified QuEChERS extraction method was developed for determination of Benzo[a]pyrene (BaP) in 137 traditional (Sangak), semi-industrial (Sangak) and industrial bread samples using spiked calibration curves by GC/MS. Sample preparation includes extraction of BaP into acetone followed by cleanup with dispersive solid phase extraction. The limit of detection and limit of quantification were 0.3 ng/g and 0.5 ng/g, respectively. The values for recoveries and RSD were calculated as 110.5-119.85% and <11.6% respectively. Average concentration of BaP in Sangak bread samples of Shiraz and Tehran was 0.59 and 0.60 ng/g, respectively. 35.5% of samples of breads collected in Tehran were contaminated with BaP at the amount higher than maximum levels regulated in processed cereal-based foods and baby foods by European Commission (1 ng/g). Seventeen percent of samples of breads collected in Shiraz were contaminated with BaP which 13 % of total samples were >1 ng/g. BaP content in all industrial samples was lower than LOQ. Assuming the consumption of bread in Tehran and Shiraz is limited to these kinds of breads, the daily intake of BaP in Tehran and Shiraz population through bread consumption was estimated to be 170.6 and 168.7 ng/day, respectively. This is the first report concerning contamination of bread samples with BaP in Iran.