Evolution of breast milk fatty acids in Spanish mothers after one year of uninterrupted lactation.

The WHO recommends exclusive breastfeeding of infants for the first sixth months of life and advises that it shall continue for up to two years of age or beyond in combination with complementary foods. However, the image of a woman breastfeeding a toddler or a preschooler is unusual in western societies. Exploring the nutritional properties of milk during prolonged lactation can help normalizing prolonged breastfeeding. Human milk fatty acid composition was determined in sixteen lactating mothers practicing prolonged lactation (≥12 months) and sixteen women on their first twelve months of lactation. Breast milk after one year is richer in saturated fatty acids, particularly lauric and myristic, showing a tendency towards lower levels of oleic acid, and higher of arachidonic, α-linolenic and docosahexaenoic acids, in comparison to early milk (< 1 year). The age and body condition of the mother, parity, sex of the baby, and diet influence also the fattyacidome of milk.

Fatty acid composition of breast milk in Galicia (NW Spain): A cross-country comparison.

Based on the premise that the fatty acid composition of human milk can be substantially altered by diet, the current study investigated the fatty acid profile (fattyacidome) of breast milk in Galicia, a small region located in the north-west of Spain and characterized by the Southern European Atlantic Diet (SEAD). A cross-country comparison was also performed to assess worldwide variety and diet impact, reviewing the profiles reported various European, North and South American, Asian and African countries and Australia. Galician human milk appeared similar to the rest of Europe, with some particular features related to the SEAD (dairy, pork, beef and sunflower and olive oils), such as relatively high levels of linoleic acid and lower α-linolenic acid. The results also showed the existence of woman-specific profiles and significant changes over lactation in some fatty acids. Worldwide, the fatty acid profiles were similar, with the clear exception of Asiatic breast milk. The impact of fatty acids on infant health warrants further investigation.

Simultaneous determination of sulfonamides, penicillins and coccidiostats in pork by high-performance liquid chromatography-tandem mass spectrometry.

Veterinary drugs are widely and legally used to treat and prevent disease in livestock. However, drugs are also used illegally as growth-promoting agents. To protect the health of consumers, maximum residue limits (MRL) in food of animal origin have been established and are listed in Regulation 37/2010. According to this regulation, more than 300 drugs need to be controlled regularly in laboratories for residues of veterinary drugs. A cost-effective analytical method is very important and explains why the development of multi-residual methods is becoming popular in laboratories. The aim of this work is to describe a simple, rapid and economical high-performance liquid chromatography-tandem mass spectrometry method for the simultaneous identification and quantification of 21 veterinary drugs in pork muscle samples. The sample clean-up procedure is performed with acidified dichloromethane and does not require solid phase extraction. The method is applicable to nine sulfonamides and seven coccidiostats identified within 36 min. Calculated relevant validation parameters such as recoveries (from 72.to 126 %), intra-precision and intermediate precision (relative standard deviation below 40 %) and decision limits (below 7 µg Kg(-1)) were within acceptable range and in compliance with the requirements of Commission Decision 2002/657/EC.

Development of an LC-MS/MS method to quantify sex hormones in bovine milk and influence of pregnancy in their levels.

Hormones work in harmony in the body, and this status must be maintained to avoid metabolic disequilibrium and the subsequent illness. Besides, it has been reported that exogenous steroids (presence in the environment and food products) influence the development of several important illnesses in humans. Endogenous steroid hormones in food of animal origin are unavoidable as they occur naturally in these products. The presence of hormones in food has been connected with several human health problems. Bovine milk contains considerable quantities of hormones and it is of particular concern. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, based on hydroxylamine derivatisation, has been developed and validated for the quantification of six sex hormones in milk [pregnenolone (P5), progesterone (P4), estrone (E1), testosterone (T), androstenedione (A) and dehydroepiandrosterone (DHEA)]. This method has been applied to real raw milk samples and the existence of differences between milk from pregnant and non-pregnant cows has been statistically confirmed. Basing on a revision of existing published data, it could be concluded that maximum daily intakes for hormones are not reached through milk ingestion. Although dairy products are an important source of hormones, other products of animal origin must be considered as well for intake calculations.

Determination of the hormonal growth promoter 17alpha-methyltestosterone in food-producing animals: bovine hair analysis by HPLC-MS/MS.

This paper describes the development, validation and application of a confirmatory method to detect 17alpha-methyltestosterone (MT) in bovine hair, to aid in controlling the administration of this growth promoter in meat-producing animals. After cryogenic grinding, MT was removed from the hair matrix using a single step extraction procedure with acetonitrile. Hydroxylamine derivatisation was used to enhance analyte determination with an electrospray ionisation (ESI) source. Determination was carried out using a triple quadrupole liquid chromatography tandem mass spectrometer (LC-MS/MS) in multiple reaction monitoring mode (MRM). The method was validated in accordance with the criteria defined in Commission Decision 2002/657/EC and using deuterated testosterone (T-d(3)) as the internal standard. The decision limit (CCalpha) was 0.07 ng g(-1) and the detection capability (CCbeta) was 0.12 ng g(-1). Repeatability was CV% (7%), within-laboratory reproducibility was CV% (11.0%), and trueness was (87%). Applicability of the method was demonstrated in an animal study. Samples obtained from animal experiments were analyzed and the presence of MT was confirmed.

Quantitative LC-MS/MS method for the sensitive and simultaneous determination of natural hormones in bovine serum.

We have developed a liquid chromatography tandem mass spectrometric (LC-MS/MS) method for the simultaneous quantitative analysis of several free forms of steroid hormones in bovine serum [pregnenolone (P(5)), progesterone (P(4)), 17hydroxyP(5), 17hydroxyP(4), testosterone (T), dehydroepiandrosterone (DHEA), androstenedione (A), estrone (E(1)), 2, 4 and 16 hydroxyE(1), 2 and 4 methoxyE(1)]. Deuterated analogs were used as internal standards. Serum proteins were eliminated with acetonitrile. Oxime derivatives of steroids were extracted with tert-butylmethylether and analyzed in positive MRM mode. Methodology was validated in accordance with the European Commission Decision 2002/657/EC. Performance characteristics permit the use of this methodology for steroid determination in animal serum samples.

Influence of farming methods on microbiological contamination and prevalence of resistance to antimicrobial drugs in isolates from beef.

The presence of Escherichia coli, Staphylococcus aureus, Listeria monocytogenes and Salmonella spp. was determined in 75 samples of conventional beef and in 75 samples of organic beef. All samples came from cattle slaughtered and processed in the same slaughterhouse and quartering room. A total of 180 E. coli, 180 S. aureus and 98 L. monocytogenes strains were analyzed by an agar disk diffusion assay for their resistance to 11 antimicrobials, for the case of E. coli and S. aureus, or 9 antimicrobials, for the case of L. monocytogenes. Salmonella spp. were not isolated from any of the beef samples. No significant differences in prevalence were obtained for any of the bacterial species tested between organic and conventional beef. E. coli isolated from organic beef exhibited significant differences in antimicrobial resistance against 5 of the 11 antimicrobials tested as compared to isolates recovered from conventional beef. In the case of S. aureus, these differences were only found for 3 of the 11 antimicrobials tested and for L. monocytogenes, no differences were obtained between isolates obtained from organic or conventional beef. Although no significant differences were obtained in microbiological contamination, E. coli and S. aureus isolates from organically farmed beef samples showed significantly lower rates of antimicrobial resistance in E. coli and S. aureus isolates.

Syntheses of molecularly imprinted polymers: Molecular recognition of cyproheptadine using original print molecules and azatadine as dummy templates.

The use of custom-made polymeric materials with high selectivities as target molecules in solid-phase extraction (SPE), known as molecularly imprinted solid-phase extraction (MISPE), is becoming an increasingly important sample preparation technique. However, the potential risk of leakage of the imprinting molecules during the desorption phase has limited application. The use of a mimicking template, called a dummy molecular imprinting polymer (DMIP), that bears the structure of a related molecule and acts as a putative imprinting molecule may provide a useful solution to this problem. In the current study, cyproheptadine (CPH) and azatadine (AZA) were used as templates in the development of an MIP and DMIP for acrylic acid and methacrylic acid monomers. Our results indicate that DMIPs have equal recognition of CPH, avoiding the problem of leakage of original template during the desorption phase relative to MIPs synthesized in presence of the print molecule CPH. Examination of the surface structure of the two polymer products by SEM shows appreciable differences in structural morphology and function of the monomers employed. These results are well supplemented by data obtained for swelling ratios and solvent uptake. Molecular modelling of CPH and AZA suggests that both substrates are similar in shape and volume.

Evolution of resistance in poultry intestinal Escherichia coli during three commonly used antimicrobial therapeutic treatments in poultry.

The resistance rates of intestinal Escherichia coli populations from poultry were determined during treatment and withdrawal period with 3 antimicrobial agents commonly used as therapeutics in poultry medicine. A total of 108 chickens were considered: 18 were treated orally with enrofloxacin, 18 with doxycycline, and 18 with sulfonamides, whereas another 18 chickens were maintained as controls for each antimicrobial group. Fecal samples were taken during the treatment and after the withdrawal period, and E. coli were isolated through Fluorocult media plating. A total of 648 E. coli strains (216 per antimicrobial tested) were isolated and identified though biochemical methods. Minimal inhibitory concentrations to the antimicrobials used were also determined using a broth microdilution method. The resistance rates of intestinal E. coli to all of the antimicrobials tested significantly increased during the course of the therapeutic treatment. In addition, significant differences (P = 0.0136) in resistance rates persisted between the intestinal E. coli of the enrofloxacin-treated and control batches until the end of the withdrawal period, but this difference was not observed for the cases of doxycycline or sulfonamides treatments. Antimicrobial use in poultry medicine seems to select for antimicrobial-resistant strains of pathogenic bacterial species such as E. coli. In some cases, the higher frequencies of resistant strains may persist in the avian intestinal tract until the end of the withdrawal period, when it is legal to use these animals for human consumption.

Comparison of antimicrobial resistance in Escherichia coli, Staphylococcus aureus, and Listeria monocytogenes strains isolated from organic and conventional poultry meat.

The presence of Escherichia coli, Staphylococcus aureus, and Listeria monocytogenes was determined in 55 samples of organic poultry meat and in 61 samples of conventional poultry meat. A total of 220 E. coli, 192 S. aureus, and 71 L. monocytogenes strains were analyzed by an agar disk diffusion assay for their resistance to ampicillin, cephalothin, chloramphenicol, ciprofloxacin, doxycycline, fosfomycin, gentamicin, nitrofurantoin, streptomycin, and sulfisoxazole (E. coli); chloramphenicol, ciprofloxacin, clindamycin, doxycycline, erythromycin, gentamicin, nitrofurantoin, oxacillin, and sulfisoxazole (S. aureus); and chloramphenicol, doxycycline, erythromycin, gentamicin, sulfisoxazole, and vancomycin (L. monocytogenes). The results indicated a significantly higher (P < 0.0001) prevalence of E. coli but not of S. aureus and L. monocytogenes in organic poultry meat as compared with conventional poultry meat. E. coli isolated from organic poultry meat exhibited lower levels of antimicrobial resistance against 7 of the 10 antimicrobials tested as compared with isolates recovered from conventional meat. In the case of S. aureus and L. monocytogenes isolated from conventional poultry, antimicrobial resistance was significantly higher only for doxycycline as compared with strains isolated from organic poultry. In the case of E. coli, the presence of multiresistant strains was significantly higher (P < 0.0001) in conventional poultry meat as compared with organic poultry meat. Organically farmed poultry samples showed significantly lower development of antimicrobial resistance in intestinal bacteria such as E. coli.

Liquid chromatography-electrospray ionization-mass spectrometry method in multiple reaction monitoring mode to determine 17alpha-ethynylestradiol residues in cattle hair without previous digestion.

A liquid chromatography-mass spectrometric (LC-MS/MS) method has been developed for determination of ethynylestradiol residues in cattle hair. Hair samples were pulverized with a cryogenic mill followed by a simple extraction with acetonitrile. A dansyl derivatization procedure to improve ethynylestradiol detection was used before the LC-MS/MS analysis in multiple reaction monitoring (MRM) mode using alpha-estradiol as an internal standard. The method was validated following the latest EU guidelines using blank hair samples spiked at 2 ng g(-1). The detection capability (CCbeta) was less than 2 ng g(-1), and the decision limit (CCalpha) was 1 ng g(-1). Incurred samples obtained 56 days after cow treatment with ethynylestradiol were analyzed, and the presence of ethynylestradiol in the hair was confirmed in all cases.

Antimicrobial resistance in Enterococcus spp. strains isolated from organic chicken, conventional chicken, and turkey meat: a comparative survey.

The mean counts of Enterococcus spp. were determined for 30 samples each of organic chicken meat, conventional chicken meat, and turkey meat, and differences for Enterococcus contamination in meat were determined. Two enterococci strains from each sample were isolated to obtain a total of 180 strains, and resistance to ampicillin, chloramphenicol, doxycycline, ciprofloxacin, erythromycin, gentamicin, nitrofurantoin, and vancomycin was determined by a disk diffusion method. Average counts obtained showed that Enterococcus mean counts from organic chicken meat (3.18 log CFU/g) were significantly higher than those obtained from conventional chicken meat (2.06 log CFU/g) or conventional turkey meat (1.23 log CFU/g). However, the resistance data obtained showed that isolates from organic chicken meat were less resistant than enterococci isolates from conventional chicken meat to ampicillin (P = 0.0067), chloramphenicol (P = 0.0154), doxycycline (P = 0.0277), ciprofloxacin (P = 0.0024), erythromycin (P = 0.0028), and vancomycin (P = 0.0241). In addition, isolates from organic chicken were less resistant than conventional turkey meat isolates to ciprofloxacin (P = 0.001) and erythromycin (P = 0.0137). Multidrug-resistant isolates were found in every group tested, but rates of multidrug-resistant strains were significantly higher in conventional chicken and turkey than those obtained from organic chicken meat. Enterococcus faecalis was the most common species isolated from organic chicken (36.67%), whereas Enterococcus durans was the most common species isolated from conventional chicken (58.33%) and turkey (56.67%). The rates obtained for antimicrobial resistance suggest that although organic chicken meat may have higher numbers of Enterococcus, these bacteria present a lower level of antimicrobial resistance.

Study of a room temperature phosphorescence phenomenon to allow the detection of aflatoxigenic strains in culture media.

A novel screening method based on room temperature phosphorescence (RTP) for the visual detection of aflatoxigenic strains from Aspergillus genus is described. Strains were cultured on media widely used in food mycology to which methyl-beta-cyclodextrin plus bile salts (0.6% sodium deoxycholate) were added. Aflatoxin production was readily detectable after 3 days of incubation at 28 degrees C by RTP emission from the mycelium of aflatoxigenic strains observed after exposure to UV light. The method was tested on thirty-two Aspergillus sp. strains. The phosphorescence phenomenon was reproduced in vitro by immobilizing aflatoxin B1 on ion exchange resin beads.

Effect of cooking on enrofloxacin residues in chicken tissue.

The aim of this study was to determine the effect of different cooking processes (microwaving, roasting, boiling, grilling and frying) on naturally incurred enrofloxacin residues in chicken muscle. Enrofloxacin and its metabolite, ciprofloxacin, were analysed using a validated LC-MS method with limits of detection (LOD) and quantification (LOQ), respectively, of 2 and 5 ng g-1 quinolones in muscle samples. The method was shown to be linear over the range 5-500 ng g-1. Mean intra-day relative standard deviation (RSD) at a concentration of 50 ng g-1 (n = 6) was 6%; inter-day RSD was 12%. A recovery study demonstrated that 65-101%, of the drug and metabolite could be recovered from the tissue. The RSD with naturally incurred roasted chicken breast was 9.18% at a concentration of 11 +/- 1.01 ng g-1 (n = 6). In water, enrofloxacin remained stable for 3 h when heated at 100 degrees C. It was concluded that residue data from raw tissue are valid for estimation of consumer exposure to this drug, as well as the ADI calculations because cooking procedures did not affect enrofloxacin residues, which remained stable during heating. However, there was an apparent decrease in quinolone concentration in tissue because some was lost by exudation into the liquid used for cooking. Conversely, for a cooking procedure with water loss, there was an apparent increase in residue concentration.

Evaluation of Chromocult enterococci agar for the isolation and selective enumeration of Enterococcus spp. in broilers.

AIMS: To investigate the productivity and specificity of a new chromogenic enterococci selective medium (Chromocult enterococci agar) recently developed by Merck. METHODS AND RESULTS: The study was carried out comparing Chromocult enterococci agar with MRS agar (Merck), a basal lactic acid bacteria medium in current use. A total of 216 faecal samples from poultry were collected and enterococci populations were counted. Likewise, 100 randomly selected strains were identified for each medium. The differences found between the two media were analysed and discussed. CONCLUSIONS: A good sensitivity of 98% was obtained for Chromocult agar and all false-positive isolates obtained were identified as Leuconostoc spp. However significant differences (P<0.01) were obtained between the enterococci species isolation rates identified from these two media, suggesting the poor growth of some species in Chromocult enterococci agar. Viable counts of Enterococcus spp. obtained with MRS agar were significantly higher than those obtained with Chromocult enterococci agar. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of chromogenic media for microbiological analysis is increasing. Independent studies are important to evaluate newly developed chromogenic media.

Detection of synthetic corticosteroids in bovine urine by chemiluminescence high-performance liquid chromatography.

The development of a black market of chemical cocktails for illegal growth promotion in food-producing animals includes substances that are potentially dangerous for human health, such as synthetic corticosteroids. The potential presence of these residues in food makes it necessary to develop rapid and sensitive analytical methodologies to detect such substances, preferably in live animals before they arrive at the market. A chemiluminescence (CL) detection method for the determination of four synthetic corticosteroids (prednisolone, betamethasone, dexamethasone and flumethasone) in bovine urine has been developed. The proposed system, which does not need any derivatization procedure, offers an easy method well suited for routine research. Urine samples were homogenized with methanol:water (50:50, v/v) and centrifuged. The upper layer was collected and Strata X cartridges were used for cleaning up. The purified residues were evaporated to dryness and then redissolved in the mobile phase. Analysis of the extracts was performed using high-performance liquid chromatography with chemiluminescence detection, employing luminol as the CL reagent. The recovery curves, obtained at four spiking levels (different for each corticosteroid), showed that recoveries of at least 70% could be obtained for urine. The chemiluminescence detection procedure afforded satisfactory results with respect to sensitivity and the LOD and LOQ, taken as the first point of the regression curve, ranged from 4 ppb to 65 ppb. The maximum mean RSD was below 13% and below 15% for intra- and inter-day assay, respectively, in all cases.

Study of enrofloxacin depletion in the eggs of laying hens using diphasic dialysis extraction/purification and determinative HPLC-MS analysis.

A study of the depletion of enrofloxacin residues in eggs was carried out using a diphasic dialysis procedure for the extraction of fluoroquinolone residues from the matrix. Enrofloxacin was administered to laying hens through the intramuscular route (15 mg/day) and orally (12 mg/day). After daily collection, the egg albumen and the egg yolk were separated, and the residue levels were determined using an HPLC-MS (API-ESI) method. The enrofloxacin and ciprofloxacin peaks gradually increased until the fifth day, because the drug was employed for 5 days. However, differences were observed in the depletion curves of enrofloxacin and its metabolite when both parts of the egg and the mode of administration were considered.

Development of a method for direct visual determination of aflatoxin production by colonies of the Aspergillus flavus group.

This report describes a simple, rapid and reliable method for screening the aflatoxin production by moulds of the Aspergillus flavus group. Strains were cultivated on yeast extract agar to which methylated beta-cyclodextrin derivative plus sodium desoxycholate was added. Production of aflatoxins was readily detectable by direct visualisation of a beige ring surrounding colonies after an incubation time of 3 days at 28 degrees C. When this ring was examined under UV light, it exhibited blue fluorescence. The presence of aflatoxins was confirmed by extracting the medium with chloroform and examining the extracts by HPLC with fluorescence detection.

Application of a modified culture medium for the simultaneous counting of molds and yeasts and detection of aflatoxigenic strains of Aspergillus flavus and Aspergillus parasiticus.

Molds and yeasts from 91 samples of feed and raw materials used in feed formulation were enumerated on a new culture medium to which a beta cyclodextrin (beta-W7M 1.8-cyclodextrin) had been added. This medium was compared with other media normally used in laboratories for the routine analysis of fungi, such as Sabouraud agar, malt agar supplemented with 2% dextrose, and potato dextrose agar. When a t test for paired data (0.05 significance level, 95% confidence interval) was applied, no statistically significant differences between the results obtained with the new culture medium and those obtained with the other media used to enumerate molds and yeasts were found. For the evaluation of contamination due to aflatoxin for all of the samples, Sabouraud agar and yeast extract agar, both supplemented with 0.3% beta-W7M 1.8-cyclodextrin, and APA (aflatoxin-producing ability) medium were used. Aflatoxin was detected in 21% of the feed samples and in 23% of the raw-material samples analyzed, with maximal amounts of 2.8 and 6.0 microg of aflatoxin B1 per kg, respectively, being detected. In any case, the aflatoxin contents found exceeded the legally stipulated limits. The t test for paired data (0.05 significance level, 95% confidence interval) did not show statistically significant differences between the results obtained with the different culture media used for the detection of aflatoxins. The advantage of the new medium developed (Sabouraud agar with 0.3% beta-W7M 1.8-cyclodextrin) is that it allows simultaneous fungal enumeration and determination (under UV light) of the presence of aflatoxin-producing strains without prior isolation and culture procedures involving expensive and/or complex specific media and thus saves work, time, and money.

Development of a diphasic dialysis method for the extraction/purification of residues of ethinylestradiol in hair of cattle, and determination by gas chromatography-tandem mass spectrometry.

A confirmatory method for the analysis of ethinylestradiol extracted from cattle hair was developed. After the extraction of the xenobiotic from the hair, by using alkaline digestion, the purification of the extract was carried out by employing diphasic dialysis. For the optimization of the technique several parameters was evaluated such as pH, extraction solvents, temperatures, times and agitation speeds. The detection and confirmation of the steroid was accomplished by using a GC-MS2 ion trap system after trimethylsilylation. The calibration curve was linear over the range of 4-20 ng/g. The detection and quantification limit were 0.52 and 0.80 ng/g respectively; with recoveries up to 94%.