Bacterial Diversity of Breast Milk in Healthy Spanish Women: Evolution from Birth to Five Years Postpartum.

The objective of this work was to characterize the microbiota of breast milk in healthy Spanish mothers and to investigate the effects of lactation time on its diversity. A total of ninety-nine human milk samples were collected from healthy Spanish women and were assessed by means of next-generation sequencing of 16S rRNA amplicons and by qPCR. Firmicutes was the most abundant phylum, followed by Bacteroidetes, Actinobacteria, and Proteobacteria. Accordingly, Streptococcus was the most abundant genus. Lactation time showed a strong influence in milk microbiota, positively correlating with Actinobacteria and Bacteroidetes, while Firmicutes was relatively constant over lactation. 16S rRNA amplicon sequencing showed that the highest alpha-diversity was found in samples of prolonged lactation, along with wider differences between individuals. As for milk nutrients, calcium, magnesium, and selenium levels were potentially associated with Streptococcus and Staphylococcus abundance. Additionally, Proteobacteria was positively correlated with docosahexaenoic acid (DHA) levels in breast milk, and Staphylococcus with conjugated linoleic acid. Conversely, Streptococcus and trans-palmitoleic acid showed a negative association. Other factors such as maternal body mass index or diet also showed an influence on the structure of these microbial communities. Overall, human milk in Spanish mothers appeared to be a complex niche shaped by host factors and by its own nutrients, increasing in diversity over time.

Comparison of the fatty acid profile of Spanish infant formulas and Galician women breast milk

The importance of dietary lipids during childhood is evident, as they are necessary for correct growth and development of the newborn. When breastfeeding is not possible, infant formulas are designed to mimic human milk as much as possible to fulfill infant’s requirements. However, the composition of these dairy products is relatively constant, while human milk is not a uniform bio-fluid and changes according to the requirements of the baby. In this study, breast milk samples were donated by 24 Spanish mothers in different lactation stages and different infant formulas were purchased in supermarkets and pharmacies. Gas chromatography coupled to flame ionization detection was used for the fatty acid determination. Compared to breast milk, first-stage formulas are apparently very similar in composition; however, no major differences were observed in the fatty acid profiles between formulas of different lactation stages. The Galician women breast milk has a fatty acid profile rich in oleic acid, linoleic acid, arachidonic acid, and docosahexaenoic acid. When comparing human milk with formulas, it becomes evident that the manufacturers tend to enrich the formulas with essential fatty acids (especially with α-linolenic acid), but arachidonic and docosahexaenoic acid levels are lower than in breast milk. Additionally, the obtained results demonstrated that after 1 year of lactation, human milk is still a good source of energy, essential fatty acids, and long-chain polyunsaturated fatty acids for the baby

Comparison of the fatty acid profile of Spanish infant formulas and Galician women breast milk.

The importance of dietary lipids during childhood is evident, as they are necessary for correct growth and development of the newborn. When breastfeeding is not possible, infant formulas are designed to mimic human milk as much as possible to fulfill infant's requirements. However, the composition of these dairy products is relatively constant, while human milk is not a uniform bio-fluid and changes according to the requirements of the baby. In this study, breast milk samples were donated by 24 Spanish mothers in different lactation stages and different infant formulas were purchased in supermarkets and pharmacies. Gas chromatography coupled to flame ionization detection was used for the fatty acid determination. Compared to breast milk, first-stage formulas are apparently very similar in composition; however, no major differences were observed in the fatty acid profiles between formulas of different lactation stages. The Galician women breast milk has a fatty acid profile rich in oleic acid, linoleic acid, arachidonic acid, and docosahexaenoic acid. When comparing human milk with formulas, it becomes evident that the manufacturers tend to enrich the formulas with essential fatty acids (especially with α-linolenic acid), but arachidonic and docosahexaenoic acid levels are lower than in breast milk. Additionally, the obtained results demonstrated that after 1 year of lactation, human milk is still a good source of energy, essential fatty acids, and long-chain polyunsaturated fatty acids for the baby.

A facile method for the fabrication of magnetic molecularly imprinted stir-bars: A practical example with aflatoxins in baby foods.

A fast and facile method for the fabrication of magnetic molecularly imprinted stir-bars (MMIP-SB) has been developed, using a combination of imprinting technology and magnetite. Magnetite was prepared in the laboratory from the raw and embedded into molecularly imprinted polymers through a process of bulk polymerization. This novel design was applied to the analysis of aflatoxins, one of the most important groups of mycotoxins in terms of occurrence and toxicity. In the context of food safety, molecularly imprinted polymers are a promising tool to achieve selective and accessible methods of extraction for different residues and contaminants. Considering the toxicity of aflatoxins, a dummy template was preferred for the synthesis of the imprinted polymers. A rapid and affordable extraction method for isolating five different aflatoxins that may be present in food was developed. The MMIP-SB was used as a conventional stir-bar and combined with high performance liquid chromatography and mass spectrometry for the determination of aflatoxin M1 in milk powder (infant formulas) and aflatoxins B1, B2, G1 and G2 in cereal-based baby foods. The results showed an average recovery of 60%, 43, 40, 44 and 39%, respectively, and RSD below 10%. These in-house prepared stir-bars featured good stirring and extraction performance, and recognition abilities, offering a good alternative to more complicated.

Analysis of Naturally Occurring Steroid Hormones in Infant Formulas by HPLC-MS/MS and Contribution to Dietary Intake.

Milk is a natural fluid and as such contains small amounts of naturally occurring steroids. Human milk is recommended as the optimal source of nutrients for infants and young children, and it has been associated to several short- and long-term benefits. For this reason, its composition is used as a reference for designing infant formulas. However, the available information on the hormonal levels of these dairy products is scarce, and it is usually limited to estradiol and estrone. In the present study, six natural sex hormones (pregnenolone, progesterone, estrone, dehydroepiandrosterone, testosterone and androstenedione) have been extracted from sixteen milk-based infant formulas and analyzed with liquid chromatography tandem mass spectrometry (HPLC-MS/MS). The purpose of this research was to quantify natural steroid hormones in various infant formulas, to provide food and nutrition practitioners with information to estimate intakes in children. In addition, data found in the literature was used for comparison. The findings suggest that there are certain similarities between bovine milk and dairy products for infants. Furthermore, the detected levels were in general lower than those observed in human milk and/or colostrum. The reported results represent a valuable addition to the current knowledge on natural hormone content of infant foods.

Evaluation of the discriminative potential of a novel biomarker for estradiol treatments in bovine animals.

The endogenous occurrence of natural hormones obstructs the application of classical targeted methods as confirmatory options. In the case of estradiol, the ultimate confirmation of its exogenous administration relies on gas chromatography coupled to combustion/isotope ratio mass spectrometry (GC-C/IRMS). A serum dipeptide composed of pyroglutamic acid and phenylalanine was identified as a potential biomarker of estradiol treatments in adult cows. To evaluate its potential to pinpoint suspicious samples, samples from prepubertal females under different estrogenic treatments have been analyzed. The results confirmed the up-regulation of the dipeptide in adult bovines. The 2-week-old females exhibited short-lasting responses only in a few animals. The 6-month-old female showed a delayed but clear increase on the biomarker level. The composition of the anabolic preparations, the dose, and/or the administration route are possible additional reasons for the reduced response in young animals. A comparison to previous results reported by various researchers is included.

A new approach for identifying patients with ovarian epithelial neoplasms based on high-resolution mass spectrometry.

We investigated the serum profiles of patients with ovarian neoplasm using high-performance liquid chromatography (HPLC) and high-resolution mass spectrometry (HRMS) to obtain serum "fingerprints" for use in identifying patients with these neoplasms. We used HPLC-HRMS to analyze serum samples from patients with ovarian neoplasms and control subjects. Serum samples from 145 patients were analyzed, including 85 with ovarian epithelial neoplasms. We also compared the results of this serum-fingerprinting approach with the results of the CA-125 test and imaging. Fingerprinting successfully permitted the separation of control patients and patients with ovarian neoplasms. The sensitivity and specificity of the test were between 96% and 100%. When the results of this test were concordant with the results of the CA-125 test, 99% of serum samples were correctly classified as being from a patient with an ovarian neoplasm or with no ovarian neoplasm. We found that a metabolite of molecular weight 472 is the main metabolite in the separation of patients with ovarian neoplasms from control subjects. HPLC-HRMS serum profiling could become a screening test for ovarian neoplasms.

Development and validation of LC-MS/MS method for the determination of cyproheptadine in several pharmaceutical syrup formulations.

A rapid and sensitive liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the qualitative and quantitative assay of cyproheptadine (CP) in pharmaceutical samples. Diphenylpyraline hydrochloride (DPP) was used as an internal standard (IS). Two multiple reaction-monitoring (MRM) transitions for each analyte were observed: 288.1/96.1 and 288.1/191.2 for CP and 282.1/167.2 and 282.1/116.3 for DPP. The retention time of the drug was 7.29 min. The analytical method was successfully validated for linearity (1-100 ng/ml), intra-day precision, inter-day precision, and accuracy. The limit of detection (LOD) and limit of quantification (LOQ) were 0.86 and 0.98 ng/ml, respectively. The proposed method was applied to analyse the cyproheptadine content from seven different syrup formulations.

Application of dummy molecularly imprinted solid-phase extraction in the analysis of cyproheptadine in bovine urine.

Due to the difficulty of monitoring trace levels of cyproheptadine (CYP) residues in complicated biological matrices, specific adsorption materials for the preconcentration and clean-up of CYP are indispensable. In this work, CYP was extracted from urine using dummy molecularly imprinted SPE (DMISPE) to avoid leakage of the imprinting molecules during the desorption phase. For synthesis of DMISPE, azatadine (AZA) was employed as the dummy template, methacrylic acid (MAA) as the monomer, ethylene glycol dimethacrylate (EGDMA) as the cross-linker, 2,2'-azobis(2-methylpropionitrile) (AIBN) as the initiator, and dichloromethane as the porogen solvent. An LC-MS/MS method was used to analyze CYP. Two MRM (multiple reaction monitoring) transitions for each analyte were monitored using diphenylpyraline hydrochloride (DPP.HCl), which was used as an internal standard. The advantages of DMISPE include obtaining less complex chromatograms and reducing ion suppression in ESI. The process efficiencies for DMISPE and SPE were 80% and 12%, respectively. In addition, the demonstrated reusability of the DMISPE cartridges is an advantage compared with single-use SPE cartridges or immunoaffinity materials.

Development and validation of an LC-MS/MS confirmatory method for residue analysis of cyproheptadine in urine of food-producing animals.

The possible off-label and illegal use of cyproheptadine (CYP) as an appetite stimulant for food-producing animals creates the need for methods capable of detecting it. A high-performance liquid chromatography tandem mass spectrometry method (LC-MS/MS) was developed to identify CYP in bovine urine, according to Commission Decision 2002/657/EC. Two multiple reaction monitoring (MRM) transitions for each analyte were monitored: 288.1/96.1 and 288.1/191.2 for CYP and 282.1/167.2 and 282.1/116.3 for diphenylpyraline hydrochloride (DPP), which was used as an internal standard. The solid phase extraction technique without a liquid-liquid step gives good results in urine samples from treated animals. The analytical method was successfully validated for linearity (0.15-10 ng/mL), with intraday precision of 9.4%, interday precision of 20.4%, and accuracy of 96.7%. The decision limit (CCalpha) and detection capability (CCbeta) were 0.48 and 0.82 ng/mL, respectively.

Development of a rapid and confirmatory procedure to detect 17beta-estradiol 3-benzoate treatments in bovine hair.

A high-performance liquid chromatography-tandem mass spectrometry method (LC-MS/MS) was developed for efficient and confirmatory surveillance of illegal use of estradiol benzoate, even when this substance is used in reproductive control. After cryogenic grinding, estradiol benzoate was extracted from hair with acetonitrile for 24 h on a rocking table. The validation of the method was based on Commission Decision 2002/657/EC using the deuterated analogue of estradiol benzoate as internal standard. Decision limit (0.81 ng/g), detection capability (1.38 ng/g), repeatability CV% (13.7), within in laboratory reproducibility CV% (15.6%), and trueness (99.3%) were calculated. Using the proposed methodology the presence of estradiol benzoate in samples obtained from animals treated to synchronize their estrous cycles can be confirmed.

Cryogenic grinding pre-treatment improves extraction efficiency of fluoroquinolones for HPLC-MS/MS determination in animal tissue.

An efficiency extraction of fluoroquinolones in chicken muscle was achieved by pulverizing it in a freezer mill before treatment with NaOH (10mM)/MeCN (1:1). The improvement of cryogenic grinding in the extraction was demonstrated for the same piece (whole leg) of four chickens treated with enrofloxacin in equal doses. A confirmatory method based on high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) was used to analyze the extracts. The chromatographic separation was achieved in 5 min with a Synergi Fusion-RP 80A (50 x 2 mm, 4 microm) column filled with a hybrid polymer. The HPLC was coupled with a detector based in a quadrupole-linear ion trap Q-TRAP that allows a confirmatory detection according to the European legislation. The specificity of the method was assessed by testing a number of representative blank muscle samples (n = 10) to verify the absence of potential interfering compounds. The limits of detection and quantitation were 2 and 5 ng g(-1) of quinolones in muscle samples, respectively. The chromatographic method was demonstrated to be linear for the range studied (5-500 ng g(-1)) with the P value for lack-of-fit in the ANOVA table greater or equal to 0.10 (calibration coefficient 0.9998 and 0.9996 for ciprofloxacin and enrofloxacin, respectively). The mean intra-day relative standard deviation (RSD) (n = 6, c = 50 ng g(-1)) was 6%; inter-day assay gave a RSD of 12%. The extraction and clean-up were carried out in one step with very satisfactory recovery data (between 65 and 101%).

Comparison of extraction methods for the recovery, amplification and species-specific analysis of DNA from bone and bone meals.

We report the effect of several parameters on the efficiency of recovery of DNA from animal bones. The effects of preheating the samples (at either 60 degrees C or 100 degrees C) at different intervals (from 1 h to overnight) in different media (water, 0.5 M ethylenediaminetetraacetic acid (EDTA), or 0.5 M EDTA + 0.05% sodium dodecyl sulfate (SDS) were investigated. The effect of slight (5 min) or intense (30 min) pretreatments with ultrasound was also evaluated. Several different treatments with proteinase K (ranging from 200 to 800 microg, and lasting from 1 to 3 h) at 65 degrees C were also considered. Additionally, two different DNA extraction methods (based on silica resins and purification columns, respectively) were evaluated. The recovery of DNA from the samples was 40% higher when the bones were preheated in 0.5 M EDTA at 60 degrees C for 1 h, this being followed by treatment with 800 microg of proteinase K for 3 h. The DNA thus obtained was successfully amplified by polymerase chain reaction (PCR) using a set of primers specific to a 359 bp region of the mitochondrial cytochrome b gene, and the species of origin were identified by visualizing the restriction fragment length polymorphism (RFLP) with the endonucleases PalI and MboI.