Determination of phospholipid concentrations in breast milk and serum using a high performance liquid chromatography–mass spectrometry–multiple reaction monitoring method

Phospholipids (PLs), including sphingomyelin, play important roles in cell membrane integrity, neural and brain development, and inflammatory responses. They are found in tissues and biological fluids, including human breast milk. In this cross-sectional study using high performance liquid chromatography–mass spectrometry, the average total PL concentrations in Malaysian mothers' colostrum and transitional milk were determined as 331 and 266 mg L⁻¹, respectively. In mature milk, the average total PL concentrations were 170, 210 and 220 mg L⁻¹ at 2, 6 and 12 months, respectively, with a strong correlation between the total PL concentration and the fat concentration (p < 0.001). The dominant PL class in mature milk was sphingomyelin (36–38%), followed by phosphatidylethanolamine (27–37%). The average maternal serum PL concentrations were higher in the third trimester (2089 mg L⁻¹) than in the second (1667 mg L⁻¹), with phosphatidylcholine predominant at 66% and with sphingomyelin at 22–24% of total PLs.     

Chemical changes of thermoxidized virgin olive oil determined by excitation–emission fluorescence spectroscopy (EEFS)

The fluorophores present in virgin olive oil evolve during any thermoxidation process and thereby fluorescence spectroscopy can be used as a straightforward approach to study the oxidative degree of oils. Samples of heated virgin olive oils collected from a fryer every two hours up to 94 h were analyzed to study their excitation–emission matrix (EEM) and the evolution of the fluorescent composition during the thermoxidation process. The potential usefulness of EEM to study thermoxidized virgin olive oils has been explored. For this purpose, parallel factor analysis (PARAFAC) was used to interpret the results. The emission profile obtained from three-factor PARAFAC model calculated for 47 samples of thermoxidized oil in the range of λ_ex = 250–298 nm and λ_em = 300–700 nm allows the decomposition of the fluorescence landscape in specific information about phenols, pigments and oxidation products respectively. The chemical interpretation from the PARAFAC model was checked with chemical analysis of phenols, tocopherols and chlorophyll pigments.     

Determination of Nϵ-carboxymethyllysine in milk products by a modified reversed-phase HPLC method

A modified reversed-phase-HPLC method with o-phthalaldehyde pre-column-derivatisation for determination of N^ϵ-carboxymethyllysine in food samples is presented. It is shown, that the method has to be modified if applied to milk products, including specific modifications in sample preparation and chromatographic separation conditions. The increased selectivity of a double endcapped RP 18 phase is necessary for reliable separation of N^ϵ-carboxymethyllysine in hydrolysates of complex products like cheese. With a detection limit of 0.5 pMol the method shows high sensitivity and a very good reproducibility (s=2.81%). In total, several different milk products (n=50) as well as fresh, processed and ripened cheese samples (n=50) were analysed. The highest amounts of N^ϵ-carboxymethyllysine were found in a whey cheese (1016 mg/kg protein), evaporated milk (1691 mg CML/kg protein), coffee cream (613 mg CML/kg protein) and cocoa milk (413 mg CML/kg protein). N^ϵ-carboxymethyllysine could not be detected in UHT milk, fresh, processed and ripened cheese. The results show that N^ϵ-carboxymethyllysine can give valuable information on lysine damage in severely heat-treated milk products and in products, with added sugar, pre-damaged constituents or stabilising agents. ©     

Multiresidue method for determination of 88 pesticides in berry fruits using solid-phase extraction and gas chromatography–mass spectrometry: Determination of 88 pesticides in berries using SPE and GC–MS

A method using solid phase extraction (SPE) cleanup followed by gas chromatography–mass spectrometry (GC–MS) has been established for quantitative determination of 88 pesticide residues in berry fruits including raspberry, strawberry, blueberry and grape. Based on an appraisal of the characteristics of GC–MS, validation experiments were conducted for 88 pesticides. In the method, solid-phase extraction was carried out using Envi-Carb cartridge coupled with NH₂-LC cartridge with acetonitrile–toluene (3:1, v/v) as the eluted solvent. In the linear range of each pesticide, the correlation coefficient was R² ? 0.99. At the low, medium and high three fortification levels of 0.05–0.5 mg kg⁻¹, recoveries fell within 63–137%. The relative standard deviation was between 1% and 19% for all 88 pesticides. Low limits of detection (0.006–0.05 mg kg⁻¹) and quantification (0.02–0.15 mg kg⁻¹) were readily achieved with this method for all tested pesticides.     

Determination of ionophores in raw bovine milk using LC–MS/MS: Application to residue surveillance

Residues of four ionophores (lasalocid, monensin, narasin, and salinomycin) in raw milk samples were extracted with acetonitrile and subsequently determined using liquid chromatography–tandem mass spectrometry. Ionophores could be determined down to 0.1 ng g⁻¹ level, without additional cleanup or concentration of the resulting extract. The analysis of a series of raw milk samples fortified at analyte concentrations ranging from 1 to 20 ng g⁻¹ yielded average accuracies ranging from 60.7% to 118.3% with percent relative standard deviations below 13%. During six months of a surveillance program, 1072 raw milk samples were collected from the transport chain of dairy producers in Alberta and analysed. Monensin was detected in 736 of 1072 samples tested at concentrations ranging from 0.10 to 0.53 ng g⁻¹ which is well below the current Canadian maximum residue limit of 10 ng g⁻¹.     

Determination of alkylphenol residues in breast and commercial milk by solid-phase extraction and gas chromatography–mass spectrometry

This study presents a feasible and sensitive method to determine alkylphenol residues (i.e., 4-t-octylphenol (4-t-OP) and the isomers of 4-nonylphenols (4-NPs)) in breast milk samples and commercial cow milk products. The matrix interference associated with the constituents in the milk was reduced by extraction with n-hexane and dilution with 50% methanolic solution (v/v, methanol/water), then followed by the Oasis-HLB SPE extraction. The analytes were determined by a GC–MS system in full-scan and selected ion monitoring modes simultaneously. Limit of quantitation was less than 0.05 ng/g in a 20 g (wet weight) milk sample. The 4-NPs were detected in 19 of the 20 breast milk samples at concentrations ranging from 1.7 to 11.6 ng/g, while 4-t-OP was detected in 8 samples at concentrations ranging from 0.4 to 1.1 ng/g. The 4-NPs were detected in all the testing commercial cow milk products at concentrations ranging from 2.9 to 8.8 ng/g.     

Determination and validation of D-values for Listeria monocytogenes and Shiga toxin–producing Escherichia coli in cheese milk

Certain cheeses can be legally produced in the United States using raw milk, but they must be aged for at least 60 d to reduce pathogen risks. However, some varieties, even when aged for 60 d, have been shown to support growth of Listeria monocytogenes or survival of Shiga toxin–producing Escherichia coli (STEC). Thermization, as a subpasteurization heat treatment, has been proposed as a control to reduce the risk of pathogens in raw cheese milk while retaining some quality attributes in the cheese. However, the temperature and time combinations needed to enhance safety have not been well characterized. The objective of this research was to determine and validate decimal reduction values (D-values) for L. monocytogenes and STEC at thermization temperatures 65.6, 62.8, and 60.0°C; a D-value at 57.2°C was also determined for L. monocytogenes only. Nonhomogenized, pasteurized whole-milk samples (1 mL) were inoculated with 8-log cfu/mL L. monocytogenes or STEC (5- or 7-strain mixtures, respectively), vacuum-sealed in moisture-impermeable pouches, and heated via water bath submersion. Duplicate samples were removed at appropriate intervals and immediately cooled in an ice bath. Surviving bacteria were enumerated on modified Oxford or sorbitol MacConkey overlaid with tryptic soy agar to aid in the recovery of heat-injured cells. Duplicate trials were conducted, and survival data were used to calculate thermal inactivation rates. D_65.6°C-, D_62.8°C-, and D_60.0°C-values of 17.1 and 7.2, 33.8 and 16.9, and 146.6 and 60.0 s were found for L. monocytogenes and STEC, respectively, and a D_57.2°C-value of 909.1 s was determined for L. monocytogenes. Triplicate validation trials were conducted for each test temperature using 100 mL of milk inoculated with 3 to 4 log cfu/mL of each pathogen cocktail, A 3-log reduction of each pathogen was achieved faster in larger volumes than what was predicted by D-values (D-values were fail-safe). Data were additionally compared with published results from 21 scientific studies investigating L. monocytogenes and STEC in whole milk heated to thermization temperatures (55.0–71.7°C). These data can be used to give producers of artisanal raw-milk cheese flexibility in designing thermal processes to reduce L. monocytogenes and STEC populations to levels that are not infectious to consumers.     

Determination of patulin in apple and quince products by GC–MS using C5–7 patulin as internal standard

A reliable gas chromatographic mass spectrometric method has been validated for the determination of trace levels (<10 μgL⁻¹) of patulin in apple products and quince jam. The method was based on extraction of patulin with ethyl acetate-hexane, alkalinisation and silylation with N,O-bis-trimethylsilyltrifluoroacetamide with 1% of trimethylchloro-silane. The accurate determination of patulin was achieved by employing commercial ¹³C_5–7 patulin labelled as an internal standard, which allowed compensating target analyte losses and enhancement or suppression matrix effects. Limits of detection and quantification of method using real samples were 0.4 and 1.6 μgkg⁻¹, respectively. Recoveries of patulin from samples spiked at 8–50 μgkg⁻¹ levels ranged between 71% and 89%. The repeatability of measurements (expressed as relative standard deviation) was lower than 16%. The method was successfully applied to the determination of patulin in apple fruit and apple products including juice, cider and baby food, and also in quince fruit and quince jam. A new PCR system for the detection of Penicillium expansum in samples containing highly degraded DNA was developed which permitted the detection of the mould in 2/3 of the samples containing patulin, including juices and jams.     

Determination of acrylamide in food using a UPLC–MS/MS method: results of the official control and dietary exposure assessment in Cyprus

ABSTRACT

The determination of acrylamide in potato products, bakery products and coffee, and the human dietary exposure is reported. The method reported is based on a single extraction step with water, followed by the clean-up of the extract using solid phase extraction columns and finally, the determination of acrylamide using UPLC–MS/MS. The MS/MS detection was carried out using an ESI interface in positive ion mode. Internal calibration was used for the quantification of acrylamide, because of the suppression/enhancement matrix effects due to the complex nature of the samples. The method performance characteristics were determined after spiking blank samples. The mean recoveries in spiked coffee samples, potato chips, breakfast cereals and crispbread ranged from 93% to 99%, with RSDs lower than 5% for both repeatability and reproducibility conditions. The estimated limits of detection and quantification of the method were 10 and 32 μg kg^?1, respectively. The method was used for monitoring acrylamide in 406 samples. Acrylamide amounts ranged from <32 to 2450 μg kg^?1. A total of 360 samples (89%) were contaminated with acrylamide, but only 14% of the samples exceeded the benchmark levels of the EU legislation. Foods with the highest mean acrylamide amounts were potato crisps (642 μg kg^?1), French fries (383 μg kg^?1) and biscuits (353 μg kg^?1). The mean and 95th percentile acrylamide exposures of adolescents in Cyprus were 0.8 and 1.8 μg kg^?1 body weight per day, respectively. The estimated levels of dietary exposure to acrylamide are not of concern with respect to neurotoxicity. However, the margins of exposure (MOEs) indicate a concern for carcinogenicity. Potato fried products (45%), fine bakery ware (21%) and potato chips (14%) contributed the most to overall acrylamide exposure.     

Determination of phthalate esters in wine using solid-phase extraction and gas chromatography–mass spectrometry

A method for the determination of six phthalate esters in wine samples has been developed. The phthalates were extracted from wine samples with an optimised solid-phase extraction method on C18 column and quantification was achieved via gas chromatography coupled with a mass spectrometer. The method was linear between 0.015 and 5.000 μg mL⁻¹ for DMP, DEP and DEHP and between 0.018 and 5.000 μg mL⁻¹ for iBP, DBP and BBP. The LOQs of DMP, DEP and DEH were 0.024 μg mL⁻¹ while those of iBP, DBP and BBP were 0.029 μg mL⁻¹. The intra-day method repeatability was between 10% and 15% RSD, whereas the inter-day method repeatability was between 13% and 21% RSD. A survey was performed on white and red wines (n = 62) from the market, winemakers and an experimental pilot plant. All the analysed samples were phthalate contaminated. Commercial wine showed higher detection frequency and level of total phthalate, DBP and BBP than those produced in a pilot plant. iBP and DEHP concentrations were similar in all the groups of samples. iBP concentration was higher in red wines than in white ones.     

Determination of ochratoxin A in eggs and target tissues of experimentally drugged hens using HPLC–FLD

Ochratoxins are fungal secondary metabolites that may contaminate various foods and beverages. The intake of ochratoxins by humans may result in typical syndromes (nefrotoxicity, carcinogenity, teratogenicity and immunotoxicity) and has been associated with Balkan Endemic Nephropathy (BEN). In this study we evaluated the effects of accumulation of ochratoxin A throughout the chain production of eggs, by investigating the dynamics of OA accumulation in eggs placed by laying hens experimentally exposed to OA.     

HPLC-Chip-Multiple Reaction Monitoring (MRM) method for the label-free absolute quantification of γ-conglutin in lupin: Proteotypic peptides and standard addition method

In food science there is a growing demand of methods for the absolute quantification of proteins, such as allergens or bioactive proteins, and shotgun proteomics based on mass spectrometry is a promising analytical tool in this area. This paper describes an innovative label-free method for the absolute quantification of γ-conglutin, one of the most relevant lupin seed proteins, which is hypoglycaemic and a major allergen. The main features of the method are: (a) the chromatographic separation was performed on an HPLC-chip system coupled with an ion trap mass spectrometer; (b) five proteotypic peptides of γ-conglutin were selected and analysed with a multiple reaction monitoring (MRM) method; (c) absolute quantification was obtained by the standard addition method after purification of a reference sample of γ-conglutin from lupin seed; (d) the matrix effect was overcome by spiking with an exogenous protein, i.e. BSA, as internal standard.     

Determination of 66 pesticide residues in livestock products using QuEChERS and GC–MS/MS

Food Science and Biotechnology - Determinations of 66 pesticide residues in different matrices including beef, pork, chicken, eggs, and milk were conducted using GC–MS/MS combined with the...     

Study of the acid and thermal stability of β-lactoglobulin–ligand complexes using fluorescence quenching

The major protein in bovine milk whey, β-lactoglobulin (β-LG), has several binding sites for ligands. Its interactions with folic acid (a hydrophilic compound), resveratrol (amphiphilic) and α-tocopherol (hydrophobic) at neutral and acidic pH and after heating to 85 °C were studied using fluorescence quenching. Binding of folic acid occurs in a hydrophobic pocket in the groove between the α-helix and the β-barrel and is disturbed by decreasing the pH from 7.0 to 2.0. Resveratrol binds to the outer surface of β-LG near Trp19–Arg124 to form complexes that are stable at acidic pH. Acidification caused the release of α-tocopherol bound to the internal cavity but had no influence on that bound to a site at the surface of β-LG. The β-LG/folic acid complex was thermally stable. Thermal denaturing improved the affinity of the protein for resveratrol but decreased somewhat its affinity for α-tocopherol. These results should help guide the development of formulations based on β-LG as a carrier of a wide range of bioactive nutrients.     

Predicting enteric methane emission of dairy cows with milk Fourier-transform infrared spectra and gas chromatography–based milk fatty acid profiles

The objective of the present study was to compare the prediction potential of milk Fourier-transform infrared spectroscopy (FTIR) for CH₄ emissions of dairy cows with that of gas chromatography (GC)–based milk fatty acids (MFA). Data from 9 experiments with lactating Holstein-Friesian cows, with a total of 30 dietary treatments and 218 observations, were used. Methane emissions were measured for 3 consecutive days in climate respiration chambers and expressed as production (g/d), yield (g/kg of dry matter intake; DMI), and intensity (g/kg of fat- and protein-corrected milk; FPCM). Dry matter intake was 16.3 ± 2.18 kg/d (mean ± standard deviation), FPCM yield was 25.9 ± 5.06 kg/d, CH₄ production was 366 ± 53.9 g/d, CH₄ yield was 22.5 ± 2.10 g/kg of DMI, and CH₄ intensity was 14.4 ± 2.58 g/kg of FPCM. Milk was sampled during the same days and analyzed by GC and by FTIR. Multivariate GC-determined MFA–based and FTIR-based CH₄ prediction models were developed, and subsequently, the final CH₄ prediction models were evaluated with root mean squared error of prediction and concordance correlation coefficient analysis. Further, we performed a random 10-fold cross validation to calculate the performance parameters of the models (e.g., the coefficient of determination of cross validation). The final GC-determined MFA–based CH₄ prediction models estimate CH₄ production, yield, and intensity with a root mean squared error of prediction of 35.7 g/d, 1.6 g/kg of DMI, and 1.6 g/kg of FPCM and with a concordance correlation coefficient of 0.72, 0.59, and 0.77, respectively. The final FTIR-based CH₄ prediction models estimate CH₄ production, yield, and intensity with a root mean squared error of prediction of 43.2 g/d, 1.9 g/kg of DMI, and 1.7 g/kg of FPCM and with a concordance correlation coefficient of 0.52, 0.40, and 0.72, respectively. The GC-determined MFA–based prediction models described a greater part of the observed variation in CH₄ emission than did the FTIR-based models. The cross validation results indicate that all CH₄ prediction models (both GC-determined MFA–based and FTIR-based models) are robust; the difference between the coefficient of determination and the coefficient of determination of cross validation ranged from 0.01 to 0.07. The results indicate that GC-determined MFA have a greater potential than FTIR spectra to estimate CH₄ production, yield, and intensity. Both techniques hold potential but may not yet be ready to predict CH₄ emission of dairy cows in practice. Additional CH₄ measurements are needed to improve the accuracy and robustness of GC-determined MFA and FTIR spectra for CH₄ prediction.     

Determination of sulbactam in raw bovine milk by isotope dilution–ultra-high-performance liquid chromatography–tandem mass spectrometry

We developed a sensitive and selective isotope dilution ultra-high-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) method for the determination of sulbactam residue in raw bovine milk. Sulbactam and internal standard, sulbactam-d₅, were extracted from raw bovine milk via liquid-liquid extraction and enriched with strong anion exchange solid-phase extraction cartridges and finally analyzed by using UPLC-MS/MS with multiple reaction monitoring mode. The method was validated according to European regulations. The calibration curve showed good linearity, with a correlation coefficient of 0.9998. Decision limit and detection capability of sulbactam were determined by matrix calibration curve and were 0.0445 and 0.0517 μg/L, respectively. The recoveries of sulbactam in fortified raw bovine milk ranged from 72.1 to 91.5%, with the intra- and interday relative standard deviations ranging from 3.0 to 18.9%. Furthermore, the developed method was applied to analyzing real raw bovine milk samples collected from dairy farms in Beijing, China. Sulbactam was not determined in all samples. The proposed method could ultimately serve as a methodological foundation for the determination of sulbactam in different types of raw milk and dairy products.