Longitudinal Changes in the Concentration of Major Human Milk Proteins in the First Six Months of Lactation and Their Effects on Infant Growth.

Our knowledge related to human milk proteins is still limited. The present study determined the changes in multiple human milk proteins during the first six months of lactation, investigated the influencing factors of milk proteins, and explored the impact of milk proteins on infant growth. A total of 105 lactating women and their full-term infants from China were prospectively surveyed in this research. Milk samples were collected at 1-5 days, 8-14 days, 1 month, and 6 months postpartum. Concentrations of total protein and α-lactalbumin were measured in all milk samples, and concentrations of lactoferrin, osteopontin, total casein, ß-casein, αs-1 casein, and κ-casein were measured in milk from 51 individuals using ultra performance liquid chromatography coupled with mass spectrometry. The concentration of measured proteins in the milk decreased during the first six months of postpartum (p-trend < 0.001). Maternal age, mode of delivery, maternal education, and income impacted the longitudinal changes in milk proteins (p-interaction < 0.05). Concentrations of αs-1 casein in milk were inversely associated with the weight-for-age Z-scores of the infants (1 m: r -0.29, p 0.038; 6 m: r -0.33, p 0.020). In conclusion, the concentration of proteins in milk decreased over the first six months postpartum, potentially influenced by maternal demographic and delivery factors. Milk protein composition may influence infant weights.

Quantitative determination of osteopontin in bovine, buffalo, yak, sheep and goat milk by Ultra-high performance liquid chromatography-tandem mass spectrometry and stable isotope dimethyl labeling.

Osteopontin (OPN) is a multifunctional protein present in different tissues, body fluids and milk. Different milk has different level of OPN content. To determine the amount of osteopontin in bovine, buffalo, yak, sheep and goat milk, we developed an ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method to detect an osteopontin signature peptide. The signature peptides selected by searching Uniprot database for trypsin digested osteopontin. The sample preparation procedure includes trypsin digestion, dimethyl labeling of tryptic peptides, purification and concentration of labeled tryptic peptide with solid phase extraction. The limit of detection and limit of quantification are 0.5 mg L-1 and 2.0 mg L-1, respectively. The method has satisfactory analytical performance with a linearity of R2 ≥ 0.998, recoveries of 103.7-111.0%, and precision of 1.8-6.2%. It is also validated and successfully applied to quantifying osteopontin content in bovine, buffalo, yak, sheep and goat milk.

Risk assessment of dietary exposure to phytosterol oxidation products from baked food in China.

Phytosterols are nutritional phytochemicals that may undergo oxidation and be transformed into phytosterol oxidation products (POPs), thus inducing pathological and toxic effects. This work investigated four main phytosterols and 28 POPs in 104 kinds of commercial baked food by using GC-MS. The dietary exposure and hazard index values (HI) associated with POPs from baked food consumption in China were estimated by using Monte Carlo simulation. Concentrations of the total phytosterols were between 3.39 and 209.80 µg/g. The total concentrations of POPs, including 5α,6α/5ß,6ß-epoxysterols, 7-ketosterol, 7α/7ß-hydroxysterols, 6-hydroxysterols, and triols, ranged from 0.37 to 27.81 µg/g. The median dietary exposure of POP contents in baked food for four age groups in China were 10.91 (children), 6.20 (adolescents), 3.63 (adults), and 3.40 (seniors) mg/(kg×day). Risk assessment of median HI with respect to POPs indicated no risk (HI <1) for people in adolescents, adults, and seniors in the country area of China, while a risk (1 < HI < 10) would refer to the baked food consumption of people in urban area and children in country area of China. Sensitivity and uncertainty analysis showed that the most significant variables for each age group in China were POP concentration, body weight, and ingestion rate.

Quantitative analysis of cow whole milk and whey powder adulteration percentage in goat and sheep milk products by isotopic dilution-ultra-high performance liquid chromatography-tandem mass spectrometry.

The aim of the study was to develop a method for quantification of cow's whey and whole milk powder in goat or sheep milk products including infant formula. A ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was established for simultaneous quantification of four caseins and two major whey proteins by detecting their signature peptides, which were able to act as markers for differentiating goat or sheep from cow whey and whole milk powder in infant formulas. The signature peptides were screened based on the computational prediction by Biolynx software, and confirmed by database searching after analysis of liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry (LC-Q-TOF-MS). The isotopic-labeled signature peptide was used as internal standard to compensate the matrix effect. The limits of quantification were 0.01-0.05 g/100 g for target proteins. The observed recovery rates ranged from 82.3 to 116.6 % and the reproducibility was excellent (RSD <12 %) at different spiking levels. The RSDs of intra- and inter-day precision were 2.8-6.2 and 3.3-9.8 %, respectively. The multiple reaction monitoring method was successfully applied to milk powder with different composition, showing high specificity and accuracy in detection of species involved. The calculating formula was designed to assess the composition of adulteration in the actual detection of infant formulas. These results highlight applicability of this method for the detection of infant formulas with complicated matrix.

Simultaneous quantification of α-lactalbumin and ß-casein in human milk using ultra-performance liquid chromatography with tandem mass spectrometry based on their signature peptides and winged isotope internal standards.

In recent years, there is an increasing need to measure the concentration of individual proteins in human milk, instead of total human milk proteins. Due to lack of human milk protein standards, there are only few quantification methods established. The objective of the present work was to develop a simple and rapid quantification method for simultaneous determination of α-lactalbumin and ß-casein in human milk using signature peptides according to a modified quantitative proteomics strategy. The internal standards containing the signature peptide sequences were synthesized with isotope-labeled amino acids. The purity of synthesized peptides as standards was determined by amino acid analysis method and area normalization method. The contents of α-lactalbumin and ß-casein in human milk were measured according to the equimolar relationship between the two proteins and their corresponding signature peptides. The method validation results showed a satisfied linearity (R(2)>0.99) and recoveries (97.2-102.5% for α-lactalbumin and 99.5-100.3% for ß-casein). The limit of quantification for α-lactalbumin and ß-casein was 8.0mg/100g and 1.2mg/100g, respectively. CVs for α-lactalbumin and ß-casein in human milk were 5.2% and 3.0%. The contents of α-lactalbumin and ß-casein in 147 human milk samples were successfully determined by the established method and their contents were 205.5-578.2mg/100g and 116.4-467.4mg/100g at different lactation stages. The developed method allows simultaneously determination of α-lactalbumin and ß-casein in human milk. The quantitative strategy based on signature peptide should be applicable to other endogenous proteins in breast milk and other body fluids.

A combined tryptic peptide and winged peptide internal standard approach for the determination of α-lactalbumin in dairy products by ultra high performance liquid chromatography with tandem mass spectrometry.

A robust ultra high performance liquid chromatography with tandem mass spectrometry method at peptide level was established for measuring α-lactalbumin in various dairy products. An isotope-labeled winged peptide (VKKILDKVG*INYW*LAHKALCSEKL) with extra amino acids of the sequence of signature peptide concatenated at each end as the internal standard was spiked in samples to participate in the whole tryptic digestion process. The peptide VG*INYW*LAHK that resulted from the isotope-labeled winged peptide was used as the final isotopically labeled internal standard of the α-lactalbumin signature peptide (VGINYWLAHK) during the quantitative analysis. The contents of α-lactalbumin in samples were calculated based on the equimolar relationship between the α-lactalbumin protein and signature peptide. The optimized molar ratio of trypsin to protein (1:60) and enzymatic digestion time (5 h) could not only improve the digestion efficiency and reduce the cost, but also minimize the period of sample pretreatment. Considering the robustness of the current method using the isotopically labeled internal standard and acceptable measurement cost, its application may promote the development of nutrient investigation and quality control of α-lactalbumin in dairy products. This protein analysis method might provide a new reference strategy for food analysis and quantitative protein analysis.

Quantification of bovine ß-casein allergen in baked foodstuffs based on ultra-performance liquid chromatography with tandem mass spectrometry.

The quantification of allergens in food including baked food matrices is of great interest. The aim of the present study was to describe a non-immunologic method to quantify bovine ß-casein using ultra-performance liquid chromatography tandem triple quadrupole mass spectrometry (UPLC-TQ-MS/MS) in multiple reaction monitoring (MRM) mode. Eight of 10 theoretical peptides from ß-casein after tryptic digestion were compared and MRM methods were developed to determine five signature peptides. The peptide VLPVPQK was selected as the signature peptide for bovine ß-casein because of the high sensitivity. A stable isotope-labelled internal standard was designed to adjust the instability of sample pre-treatment and ionisation caused by matrix effect. Using the present suspension digestion method, the native and denatured ß-casein could be digested to release the signature peptide at the maximum extent. The UPLC-TQ-MS/MS method developed based on a tryptic signature peptide led to a reliable determination of bovine ß-casein allergen in baked food matrices at a low quantitation level down to 500 µg kg(-1) with a satisfactory accuracy (< 8.9%) and recovery (98.8% ± 2.6% to 106.7% ± 3.0%).

Determination of bovine lactoferrin in dairy products by ultra-high performance liquid chromatography-tandem mass spectrometry based on tryptic signature peptides employing an isotope-labeled winged peptide as internal standard.

A new and sensitive determination method was developed for bovine lactoferrin in dairy products including infant formulas based on the signature peptide by ultra high-performance liquid chromatography and triple-quadrupole tandem mass spectrometry under the multiple reaction monitoring mode. The simple pretreatment procedures included the addition of a winged peptide containing the isotope-labeled signature peptide as internal standard, followed by an enzymatic digestion with trypsin. The signature peptide was chosen and identified from the tryptic hydrolyzates of bovine lactoferrin by ultra high-performance liquid chromatography and quadrupole-time-of-flight tandem mass spectrometry based on sequence database search. Analytes were separated on an ACQUITY UPLC BEH 300 C18 column and monitored by MS/MS in seven minutes. Quantitative result bias due to matrix effect and tryptic efficiency was corrected through the use of synthetic isotope-labeled standards. The limit of detection and limit of quantification were 0.3 mg/100 g and 1.0 mg/100 g, respectively. Bovine lactoferrin within the concentration range of 10-1000 nmol L(-1) showed a strong linear relationship with a linear correlation coefficient (r) of >0.998. The intra- and inter-day precision of the method were RSD<6.5% and RSD<7.1%, respectively. Excellent repeatability (RSD<6.4%) substantially supported the application of this method for the determination of bovine lactoferrin in dairy samples. The present method was successfully validated and applied to determination of bovine lactoferrin in dairy products including infant formulas.

Multiple reaction monitoring-based determination of bovine α-lactalbumin in infant formulas and whey protein concentrates by ultra-high performance liquid chromatography-tandem mass spectrometry using tryptic signature peptides and synthetic peptide standards.

The determination of α-lactalbumin in various dairy products attracts wide attention in multidiscipline fields because of its nutritional and biological functions. In the present study, we quantified the bovine α-lactalbumin in various infant formulas and whey protein concentrates using ultra-high performance liquid chromatography coupled to tandem mass spectrometer in multiple reaction monitoring mode. Bovine α-lactalbumin was quantified by employing the synthetic internal standard based on the molar equivalent relationship among the internal standard, bovine α-lactalbumin and their signature peptides. This study especially focused on the recovery rates of the sample preparation procedure and robust quantification of total bovine α-lactalbumin in its native and thermally denatured form with a synthetic internal standard KILDKVGINNYWLAHKALCSE. The observed recovery rates of bovine α-lactalbumin ranged from 95.8 to 100.6% and the reproducibility was excellent (RSD<6%) at different spiking levels. The limit of quantitation is 10 mg/100 g for infant formulas and whey protein concentrates. In order to validate the applicability of the method, 21 brands of infant formulas were analyzed. The acquired contents of bovine α-lactalbumin were 0.67-1.84 g/100g in these infant formulas in agreement with their label claimed values. The experiment of heat treatment time showed that the loss of native α-lactalbumin enhanced with an increasing intensity of heat treatment. Comparing with Ren's previous method by analysis of only native bovine α-lactalbumin, the present method at the peptide level proved to be highly suitable for measuring bovine α-lactalbumin in infant formulas and whey protein concentrates, avoiding forgoing the thermally induced denatured α-lactalbumin caused by the technological processing.

Determination of total choline by liquid chromatography-electrospray ionization-tandem mass spectrometry in infant formulas.

Choline is a water-soluble nutrient important for infants' brain and neural development. In infant formulas, choline is one of the important fortified nutrients. A single-laboratory validation study conducted an LC-electrospray ionization-MS/MS to determine total choline in infant formulas. Sample preparation was adopted from AOAC Official Method 999.14, and instrumental running conditions were optimized. The LOQ was 0.2 microg/100 g, which is significant for measuring total choline in infant formulas. Average recoveries for milk-, rice-, soybean-, and hydrolyzed protein-based samples ranged from 86.45 +/- 6.04% to 108.98 +/- 3.68%, with RSD less than 7%. The repeatability RSD (RSD(r)) range was 0.24-3.59% in within-day evaluation and 1.16-3.24% in day-to-day evaluation. Matrix effect was also investigated, and can be effectively eliminated by using an internal standard. Therefore, this method has high credibility, and could be used as a routine method of quality control, or for clinical studies and other research areas.

Simultaneous determination of fumonisins B1, B2 and B3 contaminants in maize by ultra high-performance liquid chromatography tandem mass spectrometry.

The present work developed an analytical method for simultaneous determination of fumonisins B(1), B(2) and B(3) residues in maize by ultra high-performance liquid chromatography combined with electrospray ionization triple quadrupole tandem mass spectrometry (UHPLC-MS/MS) under the multiple reaction monitoring (MRM) mode, and especially focused on the optimization of extraction, clean-up, UHPLC separation and MS/MS parameters. The method involves addition of fumonisins isotope internal standards, extraction with a mixture of acetonitrile and water and clean-up with solid-phase extraction (SPE) cartridges before UHPLC-MS/MS analysis. A single-laboratory method validation was conducted by testing three different spiking levels for repeatability and recovery according to International Union of Pure and Applied Chemistry (IUPAC) guidelines. The LOQ of FB(1), FB(2) and FB(3) were 1.50, 1.65 and 0.4 µg kg(-1), respectively, which were lower than the criteria of EU, USA and other countries regarding minimum residue limits of fumonisins in foods including baby foods and feedstuffs. Recoveries of three fumonisins ranged from 80.9% to 97.0% with RSD values of 2.4-11.1%.The advantages of this method include simple pretreatment, rapid determination and high sensitivity, and it fulfills the requirements for food analysis with respect to minimum residue limits of fumonisins in various countries.