Identifying adulteration of raw bovine milk with urea through electrochemical impedance spectroscopy coupled with chemometric techniques.

This study aimed to evaluate the applicability of electrochemical impedance spectroscopy to identify raw bovine milk adulteration with urea. Three batches of raw milk adulterated with urea were studied. Hierarchical clustering indicated that the samples could be split in three groups corresponding to low adulteration (less than 7 wt%), medium adulteration (between 8 and 16 wt%) and high adulteration (over than 16 wt%). A linear discriminant analysis was performed resulting in 90% of accuracy in classifying between groups. Besides, a partial least squares model containing three directions provided good accuracy in quantitatively predicting the urea mass fraction added to raw bovine milk. Finally, calculations using an approximated electric circuit model suggested the formation of urea aggregates that hinder charge transportation within the milk thus diminishing the solution conductivity. Results indicate that electrochemical impedance spectroscopy can be a useful, low cost and rapid tool to identify milk adulteration with urea.

Online fully automated SPE-HPLC-MS/MS determination of ceftiofur in bovine milk samples employing a silica-anchored ionic liquid as sorbent.

Solid-phase extraction coupled online with high performance liquid chromatography and tandem mass spectrometry was successfully applied to determine low concentrations of ceftiofur antibiotic in bovine milk samples. A silica-anchored ionic liquid was applied as sorbent material to be used as extraction phase in the proposed online system. The material was characterized by Fourier transform infrared spectroscopy and scanning electron microscopy. In order to improve the system reproducibility, the following experimental parameters were optimized: organic solvent percentage, time and sample loading flow rate. Subsequently, the method was validated presenting satisfactory results as adequate selectivity, good linearity and correlation coefficient higher than 0.98. The limit of detection and quantification were 0.1 and 0.7 µg/L, respectively. The precision of the methodology was evaluated as repeatability and intermediate precision, with relative standard deviation values lower than 15%. The accuracy of the method ranged from 72.8 to 137% and the minimum and maximum recovery values were 73.4 and 111.3%, respectively. After the validation, seven milk samples were analyzed and although ceftiofur was not detected in any of them the method was demonstrated to be efficient when applied to the analysis of milk samples fortified with the pollutant of interest.

Recent advances on determination of milk adulterants.

Milk adulteration is a current fraudulent practice to mask the quality parameters (e.g. protein and fat content) and increase the product shelf life. Milk adulteration includes addition of toxic substances, such as formaldehyde, hydrogen peroxide, hypochlorite, dichromate, salicylic acid, melamine, and urea. In order to assure the food safety and avoid health risks to consumers, novel analytical procedures have been proposed for detection of these adulterants. The innovations encompass sample pretreatment and improved detection and data processing, including chemometric tools. This review focuses on critical evaluation of analytical approaches for assay of milk adulteration, with emphasis on applications published after 2010. Alternatives for fast, environmentally friendly and in-situ detection of milk adulterants are highlighted.

Multiresidue analysis of cephalosporin antibiotics in bovine milk based on molecularly imprinted polymer extraction followed by liquid chromatography-tandem mass spectrometry.

This work reports the preparation of molecularly imprinted polymers (MIPs) selective to cephalosporin (CF) antibiotics, and their application as molecularly imprinted solid-phase extraction (MISPE) sorbents for the determination of these antimicrobials in milk samples. Several functional monomers and cross-linkers have been screened to select the best combination that provides high selectivity for the simultaneous multiresidue extraction of cefthiofur (THIO), cefazolin (AZO), cefquinome (QUI), cephapirin (API), cephalexin (ALE) and cephalonium (ALO) from the samples. The novel MIPs were prepared by a non-covalent imprinting approach in the form of spherical microparticles using the synthetic surrogate molecule sodium 7-(2-biphenylylcarboxamido)-3-methyl-3-cepheme-4-carboxylate, N-3,5-bis(trifluoromethyl)phenyl-N'-4-vinylphenyl urea (VPU) as functional monomer, and divinylbenzene (DVB) as crosslinking agent in a 1:2:20 molar ratio. The optimized MISPE method allowed the extraction of the target antimicrobials from raw cow milk samples using a selective washing with 5mL methanol/2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) buffer (0.1M, pH 7.5) (2:98, v/v) to remove the non-specifically retained compounds, followed by elution with 1mL of trifluoroacetic acid (TFA) in methanol (0.1:99.9, v/v). The extracts have been analysed by UHPLC-MS/MS and the analytical method has been validated according to EU guideline 2002/657/EC. The limits of quantification (S/N=10) were in the 1.7-12.5µgkg-1 range, well below the maximum residue limits (MRLs) currently established for the quantified cephalosporins in milk samples. The developed MIP allows mutiresidual determination of the six cephalosporin antibiotics mentioned above, significantly broadening the application to food analysis of MISPE methods.

Environmentally-friendly in situ plated bismuth-film electrode for the quantification of the endocrine disruptor parathion in skimmed milk.

An in situ bismuth-film electrode (BiFE) together with square-wave cathodic voltammetry (SWCV) was used to determine the concentration of the endocrine disruptor parathion in skimmed milk. The experimental conditions (deposition time, deposition potential and Bi (III) concentration) were optimized for the preparation of the BiFE. A glassy carbon electrode was used as the substrate. The selection of the chemical composition of the supporting electrolyte and the solution pH was aimed at improving the reduction of parathion at the BiFE surface. In addition, the parameters of the square-wave cathodic voltammetry were adjusted to improve the sensor performance. A cathodic current identified at -0.523 V increased linearly with the parathion concentration in the range of 0.2-2.0 µmol L(-1) (R=0.999). The sensitivity of the calibration curve obtained was 4.09 µA L µmol(-1), and the limits of detection (LOD) and quantification (LOQ) were 55.7 nmol L(-1) and 169.0 nmol L(-1), respectively. The performance of the sensor was tested using a sample of skimmed milk with parathion added. The same determination was carried out by UV-vis spectroscopy and the results obtained were used for the statistical evaluation of the data obtained.

Single-step solubilization of milk samples with N,N-dimethylformamide for inductively coupled plasma-mass spectrometry analysis and classification based on their elemental composition.

A single-step procedure for trace elements analysis of milk samples is presented. Solubilization with small amounts of dymethylformamide (DMF) was assayed prior to inductively coupled plasma mass spectrometry (ICPMS) detection with a high efficiency sample introduction system. All main instrumental conditions were optimized in order to readily introduce the samples without matrix elimination. In order to assess and mitigate matrix effects in the determination of As, Cd, Co, Cu, Eu, Ga, Gd, Ge, Mn, Mo, Nb, Nd, Ni, Pb, Pr, Rb, Sm, S, Sr, Ta, Tb, V, Zn, and Zr, matrix matching calibration with (103)Rh as internal standard (IS) was performed. The obtained limits of detection were between 0.68 (Tb) and 30 (Zn) µg L(-1). For accuracy verification, certified Skim milk powder reference material (BCR 063R) was employed. The developed method was applied to trace elements analysis of commercially available milks. Principal components analysis was used to correlate the content of trace metals with the kind of milk, obtaining a classification according to adults, baby or baby fortified milks. The outcomes highlight a simple and fast approach that could be trustworthy for routine analysis, quality control and traceability of milks.

The effects of oral contraceptive use on iron and copper concentrations in breast milk.

OBJECTIVE: To determine the effects of oral contraceptive (OC) treatment on maternal iron and copper metabolism during lactation. DESIGN: Observational study. SETTING: Private and public clinics in Brazil. PATIENT(S): Lactating mothers attending the family planning clinic of the University of Brasilia. INTERVENTION(S): The OCs used were a combination pill (0.15 mg of levonorgestrel and 0.03 mg of ethinyl estradiol) and a minipill (0.35 mg of norethidrone). Blood and breast milk samples were collected before and after a measured period of OC treatment. MAIN OUTCOME MEASURE(S): Iron and copper concentrations were studied in the serum and breast milk of 54 breast-feeding mothers who had been advised by their physicians to use an OC, and in a control group. RESULT(S): Regression analysis was performed using a model that took into consideration socioeconomic status, number of children, duration of previous lactation, type of OC, length of treatment, and age. Repeated measurements (before and after OC treatment) showed that only copper concentrations in breast milk were significantly affected by stage of lactation. During the study period, which varied from 2-15 weeks, the decline in iron concentrations in breast milk was negligible, whereas copper concentrations decreased by 50%. CONCLUSION(S): The use of OCs does not appear to affect significantly the secretion of iron and copper in breast milk during the first 6 months of lactation.

PIP: This study examines the effects of oral contraceptive (OC) treatment on maternal iron and copper metabolism during lactation. The sample included lactating mothers attending the family planning clinic at the University of Brasilia. Participants were divided into 3 groups: 1) mothers who were taking combination pills (0.15 mg levonorgestrel and 0.03 mg ethinyl estradiol); 2) those who were taking minipills (0.35 mg norethindrone); and 3) a control group. Blood and breast milk samples were collected before and after a measured period of OC treatment. Results showed no significant effects of OC use on either iron or copper concentrations in serum during lactation. However, copper concentrations were significantly affected with the stage of lactation (iron, P = 0.9619; copper, P = 0.0002) as reflected in the multivariate model analysis. Therefore, the use of OCs does not appear to affect significantly the secretion of iron and copper in breast milk during the first 6 months of lactation.

Calcium and phosphorus in milk of Brazilian mothers using oral contraceptives.

OBJECTIVE: Oral contraceptives (OC) are the most efficient method of contraception and it is the most prescribed by doctors in developing countries. Therefore we studied the effects of combination pill and mini-pill on calcium and phosphorus in milk of breast-feeding mothers at different stages of lactation. METHODS: Fifty-four breast-feeding mothers made up three study groups: 33 mothers who had been advised by their doctors to use either combination pill (12), or mini-pill (21), as well as a control group of 21 mothers that used no hormonal contraceptives. All mothers completed a questionnaire and provided samples of milk before and after a measured period of observation. Mean duration of study was 76, 120, and 101 days, respectively for users of mini-pill, combination pill, and controls. Determination of calcium and phosphorus was done by inductively coupled plasma-atomic absorption spectrometry. RESULTS: Overall the decrease in milk concentrations of phosphorus (6%) and calcium (26.3%) during the study period was not influenced by OC treatment. Regression analyses which took into consideration length of treatment, socioeconomic status, number of children, duration of previous lactation, type of contraceptive, and age of mothers and repeated measurements (before and after OC) showed that milk calcium was significantly affected by stage of lactation (p=0.0013). CONCLUSION: The use of hormonal contraceptive such as the combination pill (levonorgestrel 0.15 mg+ethynilestradiol 0.03 mg) and mini-pill (norethindone 0.35 mg) does not seem to affect the secretion of calcium and phosphorus in milk of mothers.

PIP: This article is based on a study of the effects of oral contraceptives (OCs) on the concentration of calcium and phosphorus in breast milk among 54 lactating Brazilian women. Confounding constitutional variables were considered which include maternal age, previous lactation, length of breast-feeding, and variables associated with contraception, such as type and length of use. The subjects were divided into 3 groups: 12 using combination pills, 21 using mini-pills, and a control group of 21 mothers that used no hormonal contraceptives. Milk was sampled before and after a measured period of observation. The mean durations of study were 76, 120, and 101 days, respectively, for users of mini-pills, combination pills, and controls. The determination of calcium and phosphorous was done by inductively coupled plasma-atomic absorption spectrometry. The results indicate that a mean stage of lactation at start of treatment for the 3 groups ranged from 2.5 to 4 months. Both calcium and phosphorous declined in concentration with time, confounded with OC treatment. There was no significant difference for calcium concentration due to OC treatment, only stage of lactation, per se, was a significant source of variation for calcium concentration. As lactation progressed, the calcium and phosphorous concentration decreased for all mothers. Therefore, short-term use of OCs containing estrogen do not affect calcium and phosphorous concentrations in breast milk.

Nutritional factors in milk from Brazilian mothers delivering small for gestational age neonates.

The composition of breast milk from brazilian mothers delivering low birthweight infants and its adequacy as a source of nutrients for this group has not yet been fully elucidated. A total of 209 milk samples from 66 women were analysed. The mothers were divided into three groups: G1, mothers delivering term babies of low birthweight (TSGA, n = 16); G2, mothers delivering preterm babies of appropriate birthweight (PTAGA, n = 20); G3, mothers delivering term babies of appropriate birthweight (TAGA, n = 30). The following factors were analysed: osmolarity, total proteins and protein fractions, creamatocrit, sodium, potassium, calcium and magnesium. Milk samples were collected 48 h and 7, 15, 30 and 60 days after delivery. The groups did not differ significantly in terms of osmolarity, total proteins and fractions, creamatocrit, calcium, magnesium or potassium throughout the study period. Sodium levels were higher in all samples from mothers of TSGA infants and in samples from mothers of PTAGA infants on the 7th, 15th and 30th days than in milk from the TAGA group. The authors consider the needs of the low birthweight and TAGA infants and that these high sodium levels may be necessary for growth of low birthweight infants.

Lactoferrin levels and unsaturated iron-binding capacity in colostrum of Brazilian women of two socioeconomic levels.

The objective of this study was to compare lactoferrin (LF) levels and unsaturated iron-binding capacity (UIBC) of colostrum from Brazilian women aged 20-38 years of low (N = 28) and high (N = 15) socioeconomic level. Both groups received a similar amount of supplemental iron (25-65 mg/day) during pregnancy. We also determined the relationship between LF levels and UIBC, and between these components and maternal iron status. Both groups (low and high socioeconomic level) showed adequate and similar mean hematocrit (35.0 +/- 1.3% and 35.9 +/- 1.3%, respectively) and serum ferritin (64 +/- 12 micrograms/l and 57 +/- 12 micrograms/l, respectively) levels. Furthermore, LF levels (5.7 +/- 0.9 mg/ml and 5.5 +/- 0.6 mg/ml) and UIBC (3.8 +/- 0.3 micrograms/ml and 3.1 +/- 0.3 micrograms/ml) were not significantly different in the two groups of women, nor were they related to maternal hematocrit or serum ferritin. UIBC was significantly (P = 0.009), although weakly positively correlated (r = 0.45) with LF levels, suggesting that other colostrum ligands may contribute substantially to the capacity of colostrum to bind exogenous iron.

Protection of breast-fed infants against Campylobacter diarrhea by antibodies in human milk.

To investigate the role of breast-feeding in preventing diarrhea caused by Campylobacter jejuni, we followed 98 Mexican children prospectively for 2 years beginning at their birth. Attack rates of diarrhea in children less than 6 months of age who were not fed human milk were 2.3 times greater than those in children of the same age who were fed human milk. Breast-fed children remained free of diarrhea for a longer time than non-breast-fed children (p less than 0.0005). The diarrhea attack rate caused by C. jejuni for non-breast-fed infants was significantly greater (p less than 0.005) than that in the breast-fed group. Secretory IgA milk antibody titers against glycine acid-extractable antigen of C. jejuni were high in colostrum, decreased during the first month of breast-feeding, and generally persisted throughout lactation. Human milk consumed by children in whom Campylobacter diarrhea developed did not contain secretory IgA antibodies to the glycine acid-extractable common antigen of Campylobacter. This study shows an association between Campylobacter antibodies in human milk and prevention of diarrhea caused by Campylobacter.

Iron, zinc, folate and vitamin B12 nutritional status and milk composition of low-income Brazilian mothers.

The purpose of this study was to assess the nutritional status of low-income Brazilian mothers, who were supplemented with iron and vitamin B12 during pregnancy, in terms of iron, zinc, folate and vitamin B12, in different stages of lactation and to determine the influence of the maternal nutritional status on milk composition. The effect of folate supplementation during pregnancy on folate status of the nursing mothers and milk composition was investigated. The effect of partial weaning on maternal status and milk composition was also studied. In general, the nutritional status of iron, zinc, folate and vitamin B12 of the mothers appears adequate. However, some of the mothers had indices of status lower than normal limits for non-pregnant women. These values, particularly after 30 d post-partum, indicate that these mothers might be at nutritional risk and that the nutrient supplementation received during pregnancy was insufficient to meet demands. There was an increase with the stage of lactation for haematocrit, serum vitamin B12, serum zinc, serum albumin, milk folate and saturation of its binding protein, but there was a decrease for milk protein, total and whey-bound iron and zinc, and lactoferrin. Mothers who took folate supplements during pregnancy had higher serum folate levels immediately after birth than those not taking the supplements but no differences were found at later stages of lactation. Milk composition was not affected. Partial weaning did not affect the maternal nutritional status or the milk composition except for iron which was higher in milk from mothers who were partially breastfeeding.(ABSTRACT TRUNCATED AT 250 WORDS)

Breast milk anti-Escherichia coli heat-labile toxin IgA antibodies protect against toxin-induced infantile diarrhea.

A prospective study to assess whether milk IgA antibodies against Escherichia coli heat labile-toxin protect breast-fed children against labile toxin-induced gastroenteritis was carried out among infants of a marginal urban area in Guatemala. One hundred and thirty children were kept under surveillance for diarrhea by periodic home visits. Stool specimens were collected from each child routinely every 2-3 weeks and during diarrheal episodes, to study the excretion of labile toxin-producing Escherichia coli. Milk samples from the children's mothers were obtained concomitantly with the fecal specimens of the infants to be analyzed for anti-labile toxin antibodies. Twenty infections by heat-labile toxin-producing Escherichia coli as a sole agent were documented among breast-fed infants. Nine of these infections resulted in gastroenteritis, while the remaining 11 were asymptomatic. At the time of infection children who became sick were ingesting breast milk with significantly (p = 0.028) lower titers of antilabile toxin IgA than those who remained healthy. Only one of the 8 infected children receiving breast milk with high titers (greater than or equal to 256) of anti labile toxin IgA developed diarrhea, compared to 8 of the 12 subjects being fed milk with low titers (less than or equal to 64) (p = 0.025). This is the first report documenting protection by IgA antibodies in milk against labile toxin-induced gastroenteritis in infected breast-fed infants.

Concentration and distribution pattern of selected micronutrients in preterm and term milk from urban Brazilian mothers during early lactation.

The purpose of this study was to assess the concentration and binding pattern of zinc, iron, folate and vitamin B12 in milk of Brazilian women of low socioeconomic status giving birth at term or preterm, during early lactation. Protein, fat, total solids and ash concentrations were also determined. Protein and zinc concentrations decreased significantly as lactation proceeded whereas milk fat and folate increased with the lactation period. Total solids, ash, iron and vitamin B12 remained unchanged. Zinc was present mainly in the whey fraction while less than half of the total iron was present in this fraction, with no significant change due to stage of lactation. Unsaturated folate and vitamin B12 binding capacities and percentage of saturation of the folate binding protein increased with the stage of lactation. The vitamin B12 binding protein was highly unsaturated in all samples. There was no significant difference between term and preterm samples in the parameters investigated. Correlation analysis between milk components indicated significant relationships between total solids and fat, total zinc and whey zinc, folate and total folate binding capacity, unsaturated and total folate binding capacity, and unsaturated and total B12 binding capacity. In general, the nutrient concentrations found in this study are in good agreement with published data on milk composition of women from developed countries, with the possible exceptions of folate, which was lower, and iron, which was higher. The correlation of folate concentration with its binding protein found in this work supports the hypothesis of a regulatory role for folate levels in milk exerted by the folate binding protein in the mammary gland.

Isotope dilution measurement of breast-milk production in Chilean urban mothers.

A heavy-water dilution technique has been used to measure mean daily breast-milk output in a group of nursing mothers from an urban community in Santiago, Chile. Infant milk intake was found to correlate significantly with weight (r = 0.646, P less than 0.005) and with weight-for-age (r = 0.640, P less than 0.005), but a much stronger and highly significant correlation was found with infant birth-weight (r = 0.802, P less than 0.001). Milk output was also found to depend on the mother's nutritional status at the beginning of pregnancy and was significantly higher in overweight mothers. An even more marked difference was obtained if mothers were divided into two subgroups, above and below normal, according to their weight-for-height values at the first antenatal check-up (x+ = 1148.1 ml/d, x- = 814.2 ml/d, P less than 0.02). Relationships between milk output and duration of lactation and parity were also apparent from the study but the latter was probably mediated through maternal nutritional status.