Determination of Total and A1-Type ß-Casein in Milk and Milk-Derived Ingredients by Liquid Chromatography-Mass Spectrometry Using Characteristic Tryptic Peptides.

BACKGROUND: Gastrointestinal digestion of A1-type ß-casein is conducive to ß-casomorphin-7 with potential adverse digestive health effects. Monitoring of A1-type ß-casein concentration in milk and milk-derived ingredients used in the formulation of A2-type nutritional products with associated health claims is important from a quality standpoint. OBJECTIVE: New analytical methods were developed and validated for total and A1-type ß-casein in milk and milk-derived ingredients. Data on total and A1-type ß-casein concentrations in milk, nonfat dry milk, and whey protein concentrate was generated. METHOD: The methods are based on a bottom-up proteomic approach using tryptic marker peptides and stable isotope dilution liquid chromatography-mass spectrometry. The measurement includes all protein sequences (intact, modified, and partial) which are potential sources of ß-casomorphin-7. RESULTS: Total ß-casein was quantified using a neat calibration curve. Recovery and between-day precision RSD were 98% and 5.8%, respectively. A1-type ß-casein was quantified by the method of standard additions. Between-day precision RSD was 7.2% and limit of quantitation was 0.01% in nonfat dry milk. The mass fraction of A1-type ß-casein in the ß-casein standard was 0.444. Samples manufactured from A2-type milk contained 0.26-5.0% A1-type ß-casein relative to total ß-casein. CONCLUSIONS: The methods described enable the monitoring of the A1-type ß-casein concentration in milk and milk-derived ingredients destined for the manufacture of A2-type products with associated health claims. HIGHLIGHTS: New methods are presented for the analysis of total and A1-type ß-casein in milk and milk-derived ingredients. The mass fraction of A1-type ß-casein in a commercial ß-casein standard was determined to enable its use as a calibrant.

Early Enteral Administration of a Complex Lipid Emulsion Supplement Prevents Postnatal Deficits in Docosahexaenoic and Arachidonic Acids and Increases Tissue Accretion of Lipophilic Nutrients in Preterm Piglets.

BACKGROUND: Preterm delivery and current nutrition strategies result in deficiencies of critical long-chain fatty acids (FAs) and lipophilic nutrients, increasing the risk of preterm morbidities. We sought to determine the efficacy of preventing postnatal deficits in FAs and lipophilic nutrients using an enteral concentrated lipid supplement in preterm piglets. METHODS: Preterm piglets were fed a baseline diet devoid of arachidonic acid (AA) and docosahexaenoic acid (DHA) and randomized to enteral supplementation as follows: (1) Intralipid (IL), (2) complex lipid supplement 1 (CLS1) with an AA:DHA ratio of 0.25, or (3) CLS2 with an AA:DHA ratio of 1.2. On day 8, plasma and tissue levels of FAs and lipophilic nutrients were measured and ileum histology performed. RESULTS: Plasma DHA levels decreased in the IL group by day 2. In contrast, DHA increased by day 2 compared with birth levels in both CLS1 and CLS2 groups. The IL and CLS1 groups demonstrated a continued decline in AA levels during the 8-day protocol, whereas AA levels in the CLS2 group on day 8 were comparable to birth levels. Preserving AA levels in the CLS2 group was associated with greater ileal villus height and muscular layer thickness. Lipophilic nutrients were effectively absorbed in plasma and tissues. CONCLUSIONS: Enteral administration of CLS1 and CLS2 demonstrated similar increases in DHA levels compared with birth levels. Only CLS2 maintained AA birth levels. Providing a concentrated complex lipid emulsion with an AA:DHA ratio > 1 is important in preventing postnatal AA deficits.

Determination of Nitrite in Milk- and Soy-Based Nutritional Ingredients by Derivatization with 2,3-Diaminonaphthalene and Fluorescence Spectrometry.

Nitrite (NO2-) is an inorganic anion that can be found in various powdered milk- and soy-based nutritional ingredients as an incidental contaminant. Reliable determination of NO2- in nutritional ingredients is of paramount importance to ensure the safety of finished products. The derivatization reaction of NO2- with 2,3-diaminonaphthalene with the formation of fluorescent 2,3-naphtotriazole has been adapted to milk- and soy-based nutritional ingredients. The sample preparation consisted of protein precipitation with Carrez solution, simple pass-through cleanup of extracts utilizing a carbon black-based cartridge and derivatization, followed by batch fluorometry. The method was validated in six representative ingredient matrixes-i.e., whole-milk powder, nonfat dry milk, milk protein concentrate, whey protein concentrate, sodium caseinate, and soy protein isolate. Recovery values were 82-109%, whereas within-day and intermediate precision were 0.6-5.2 and 3.6-11% (RSDs), respectively. The method LOQ was 0.1 or 0.2 µg/g sodium nitrite (NaNO2), depending on the ingredient matrix. Surveyed NO2- concentration levels in 25 lots of 10 types of nutritional ingredients ranged from between less than 0.1 to 29 µg/g NaNO2. This method is proposed as a more sensitive and rugged alternative to the widely used ion chromatographic and colorimetric approaches.

The development and validation of a high-throughput LC-MS/MS method for the analysis of endogenous ß-hydroxy-ß-methylbutyrate in human plasma.

A high-throughput, sensitive, and rugged liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the rapid quantitation of ß-hydroxy-ß-methylbutyrate (HMB) in human plasma has been developed and validated for routine use. The method uses 100 µL of plasma sample and employs protein precipitation with 0.1% formic acid in methanol for the extraction of HMB from plasma. Sample extracts were analyzed using LC-MS/MS technique under negative mode electrospray ionization conditions. A 13 C-labeled stable isotope internal standard was used to achieve accurate quantitation. Multiday validation was conducted for precision, accuracy, linearity, selectivity, matrix effect, dilution integrity (2×), extraction recovery, freeze-thaw sample stability (three cycles), benchtop sample stability (6 h and 50 min), autosampler stability (27 h) and frozen storage sample stability (146 days). Linearity was demonstrated between 10 and 500 ng/mL. Inter-day accuracies and coefficients of variation (CV) were 91.2-98.1 and 3.7-7.8%, respectively. The validated method was proven to be rugged for routine use to quantify endogenous levels of HMB in human plasma obtained from healthy volunteers.

Determination of emerging nitrogenous economic adulterants in milk proteins by high-performance liquid chromatography/compact mass spectrometry.

RATIONALE: Milk-derived ingredients are widely used around the world in the manufacturing of nutritional products. They are prone to economically motivated adulteration with nitrogenous compounds such as melamine and its analogs in order to increase the nitrogen content of these ingredients. The need to rapidly screen milk-derived ingredients to detect adulteration is of paramount public health concern. A liquid chromatography/mass spectrometry (LC/MS)-based method using a single quadrupole mass spectrometer has been developed for the rapid frontline analysis of six nitrogenous protein adulterants, i.e. melamine, ammeline, ammelide, amidinourea, cyromazine and cyanuric acid, in three key milk-derived ingredients, i.e. whole milk powder, nonfat milk powder and whey protein concentrate. METHODS: The sample preparation scheme involves both 'dilute and shoot' as well as solid-phase extraction (SPE)-based methods. The 'dilute and shoot' scheme uses a tenfold dilution of sample with water followed by protein precipitation using 2% formic acid in acetonitrile. The SPE scheme involves tenfold dilution of sample with water, followed by protein precipitation using acetonitrile, and further cleanup through Strata Melamine SPE cartridges. Sample extracts were analyzed by hydrophilic interaction chromatography/single quadrupole mass spectrometry (HILIC/MS) in both positive and negative electrospray ionization mode. Accurate quantitation was achieved using stable isotope labeled internal standards. RESULTS: A multi-day method validation study was conducted using three different milk-derived ingredients. Average accuracies, relative standard deviations (RSD) and method detection limits (MDL) for all analytes in whole milk powder were 65-118%, 7-11% and 0.9-30 mg/kg, using the 'dilute and shoot' extraction procedure. The SPE procedure results were 102-111%, 5-13%, and 0.4-2.5 mg/kg, respectively, for melamine, ammeline, ammelide and cyromazine only. CONCLUSIONS: A rugged and simple to use analytical method to screen for the presence of nitrogenous economic adulterants in milk-derived ingredients has been developed for routine frontline use. Copyright © 2016 John Wiley & Sons, Ltd.

Analysis of Glyphosate and Aminomethylphosphonic Acid in Nutritional Ingredients and Milk by Derivatization with Fluorenylmethyloxycarbonyl Chloride and Liquid Chromatography-Mass Spectrometry.

A straightforward analytical method based on derivatization with fluorenylmethyloxycarbonyl chloride and liquid chromatography-mass spectrometry has been developed for the analysis of residues of glyphosate and aminomethylphosphonic acid (AMPA) in a suite of nutritional ingredients derived from soybean, corn, and sugar beet and also in cow's milk and human breast milk. Accuracy and intermediate precision were 91-116% and <10% RSD, respectively, in soy protein isolate. Limits of quantitation were 0.05 and 0.005 µg/g in powdered and liquid samples, respectively. Glyphosate and AMPA were quantified at 0.105 and 0.210 µg/g (soy protein isolate) and 0.850 and 2.71 µg/g (soy protein concentrate, both derived from genetically modified soybean), respectively. Residues were not detected in soy milk, soybean oil, corn oil, maltodextrin, sucrose, cow's milk, whole milk powder, or human breast milk. The method is proposed as a convenient tool for the survey of glyphosate and AMPA in the ingredient supply chain.

Direct Analysis of Leucine and Its Metabolites ß-Hydroxy-ß-methylbutyric Acid, α-Ketoisocaproic Acid, and α-Hydroxyisocaproic Acid in Human Breast Milk by Liquid Chromatography-Mass Spectrometry.

A direct, quantitative, and confirmatory method based on stable isotope dilution liquid chromatography-mass spectrometry was developed and validated for the analysis of leucine and metabolites ß-hydroxy-ß-methylbutyric acid (HMB), α-ketoisocaproic acid (KIC), and α-hydroxyisocaproic acid (HICA) in human breast milk. Chromatographic resolution was achieved between isobaric leucine and isoleucine. Accuracy and intermediate precision were 89-117% and <10% relative standard deviation (RSD) across three validation runs. Limits of quantitation for HMB, KIC, HICA, and leucine in human breast milk were 20 µg/L, 20 µg/L, 10 µg/L, and 1 mg/L. Measured concentrations of HMB, KIC, HICA, and free leucine in human breast milk from six donors at various stages of lactation were 42-164 µg/L, < 20-1057 µg/L, < 10 µg/L, and 2.1-88.5 mg/L. HMB and KIC were confirmed in human breast milk by orthogonal hydrophilic interaction chromatography (HILIC). This work provides a tool for further study of human breast milk composition and its effect on protein turnover in developing infants.

Investigation of the presence of ß-hydroxy-ß-methylbutyric acid and α-hydroxyisocaproic acid in bovine whole milk and fermented dairy products by a validated liquid chromatography-mass spectrometry method.

A simple, rugged, quantitative, and confirmatory method based on liquid chromatography-mass spectrometry was developed and comprehensively validated for the analysis of the leucine metabolites ß-hydroxy-ß-methylbutyric acid (HMB) and α-hydroxyisocaproic acid (HICA) in bovine whole milk and yogurt. Mean accuracy (90-110% for HMB and 85-115% for HICA) and total precision (<10% RSD in most cases, except for <20% RSD for HMB at the limit of quantitation) at four concentration levels across three validation runs have been determined. Limits of quantitation for HMB and HICA in whole milk were 20 and 5 µg/L, respectively. Measured concentrations of HMB and HICA were <20-29 and 32-37 µg/L, respectively, in bovine whole milk and <5 and 3.0-15.2 mg/L, respectively, in yogurt. These concentrations are insufficient by large margins to deliver any musculoskeletal benefits, and fortification of milk and dairy products with HMB and/or HICA appears to be justified.

Liquid chromatography-mass spectrometry method for the quantitative determination of residues of selected veterinary hormones in powdered ingredients derived from bovine milk.

A rugged, quantitative liquid chromatography-tandem mass spectrometry method with modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) sample preparation for 17 selected veterinary hormones in six different powdered ingredients derived from bovine milk was developed and comprehensively validated. A universal post-extraction spiked matrix-matching approach based on whole milk powder has been successfully implemented. Three validation runs based on four levels of pre-extraction spiked quality control (QC) samples have been conducted. Overall accuracy (86-117%), overall precision (<20% RSD), selectivity, absolute extraction recovery (62-82%), matrix effect (<15% for most compounds), limits of detection (0.1-0.8 µg/kg, except for diethylstilbestrol at 3.8 µg/kg), limits of quantitation (0.2-2.0 µg/kg, except for diethylstilbestrol at 10.0 µg/kg), and extract stability (48 h) have been determined. The method is proposed for the routine analysis of hormones potentially present in powdered ingredients derived from bovine milk.

Characterization of three berry standard reference materials for nutrients.

The National Institute of Standards and Technology (NIST) has been working with the National Institutes of Health Office of Dietary Supplements to produce Standard Reference Materials (SRMs) of interest to analysts of dietary supplements. Some of these SRMs are traditional foods including SRM 3281 Cranberry (Fruit), SRM 3282 Low-Calorie Cranberry Juice Cocktail, and SRM 3287 Blueberry (Fruit), which have been characterized for nine nutritional elements and sugars. The blueberries have also been characterized for proximates, two water-soluble vitamins, and amino acids. These new materials are intended for use in method development and validation as well as for quality assurance and traceability in the assignment of values to in-house control materials. Foods can be difficult to analyze because of matrix effects. With the addition of these three new SRMs, it is now possible to more closely match controls to matrices and analyte levels for fruit and vegetable test samples. Several nutritional elements in these three SRMs are present at lower levels than in other food-matrix SRMs.

Analysis of organic acids in fruit juices by liquid chromatography-mass spectrometry: an enhanced tool for authenticity testing.

Organic acid analysis plays a fundamental role in the testing of authenticity of fruit juices. Analytical methods used routinely for organic acids suffer from poor reproducibility, often give false positives/negatives for tartaric acid, and do not offer the possibility of analyte confirmation. There are conflicting reports in the literature on the presence/absence of tartaric acid in pomegranate juice, a potential indicator of adulteration with grape juice. In this work, a method based on stable isotope dilution liquid chromatography-tandem mass spectrometry is described for citric, malic, quinic, and tartaric acid in fruit juices. Validation data including precision and recovery in six types of juice are presented. Tartaric and quinic acids were confirmed in pomegranate juice at concentrations of 1-5 and ∼1 mg/L, respectively. These concentrations are much lower than those resulting from adulteration with grape juice and apple juice, respectively, at the 5% level. A separate method for isocitric acid in orange juice based on the single standard addition method is also described.

Comparison of analytical methods to determine sodium content of low-sodium foods.

U.S. nutrition labeling regulations require the declaration of sodium content on food products. Accurate and reproducible determination of Na in foods with low Na content (< 140 mg/serving) is challenging because of laboratory contamination. Within-laboratory performance of inductively coupled plasma/MS (ICP/MS), flame atomic absorption spectrophotometry (FAAS), ion-selective electrode (ISE), and potentiometric titration of chloride ion were evaluated in 17 low-sodium foods. For 13 types of food, statistically significant differences (P < 0.05) exist between the within-day andlor interday means obtained by ICP/MS, FAAS, and ISE. Median within-day and interday precent RSD values were 2.7 and 6.1, 3.5 and 3.2, and 5.6 and 6.2%, respectively, by ICP/MS, FAAS, and ISE. The fewest matrix effects were found with ICP/MS, followed by FAAS, and ISE. FAAS gave higher results in a variety of matrixes when compared to ICP/MS and/or ISE. ISE did not perform well in fatty foods or at very low Na concentrations. Manufacturers' Nutrition Facts Panel sodium declarations exceeded levels found by analysis in > 70% of the foods. Analysis of chloride content does not produce reliable Na estimates in low-sodium foods, even when added sodium chloride is present. Methodological issues and contamination sources are discussed.

Correlation of acrylamide generation in thermally processed model systems of asparagine and glucose with color formation, amounts of pyrazines formed, and antioxidative properties of extracts.

The relations between the formation of acrylamide and color, pyrazines, or antioxidants in an asparagine/d-glucose browning model system under various conditions were investigated. The highest level of acrylamide was produced in the asparagine/glucose (1:3) system heated at 170 degrees C for 30 min (2629 microg/g asparagine). Color intensity increased with temperature and heating time. The formation of pyrazines increased steadily with an increase of temperature (140-170 degrees C) and heating time (15-60 min). Antioxidant formation varied among the samples heated under different conditions. A clear correlation between formation of acrylamide and browning color was obtained. The formation of acrylamide was linearly correlated with the formation of total pyrazines during the initial stages of the Maillard reaction. No obvious correlation between formation of acrylamide and antioxidants was observed. However, excess amounts of asparagine increased the formation of antioxidants, whereas excess amounts of glucose reduced its formation.

Formation of acrylamide from lipids.

Heating amino acids with dietary oils or animal fats at elevated temperatures produced various amounts of acrylamide. The amount of acrylamide formation corresponded to the degree of unsaturation of the oils and animal fats. The decreasing order of acrylamide formation from dietary oils or animal fats with asparagine was sardine oil (642 microg/g asparagine) > cod liver oil (435.4 microg/g) > soybean oil (135.8 microg/g) > corn oil (80.7 microg/g) > olive oil (73.6 microg/g) > canola oil (70.7 microg/g) > corn oil (62.1 microg/g) > beef fat (59.3 microg/g) > lard (36.0 microg/g). Three-carbon unit compounds such as acrylic acid and acrolein, which are formed from lipids by oxidation also produced acrylamide by heat treatment with amino acids, in particular with asparagine. The results of the present study suggest that acrylamide forms in asparagine-rich foods during deep fat frying in the absence reducing sugars.