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An isotope-dilution liquid chromatography tandem mass spectrometry method was established for the determination of vitamin B12 in milk and dairy products. The samples were spiked with stable isotope-labeled vitamin B12 and digested by pepsin and amylase. The various forms of cobalamin were transformed to cyanocobalamin by potassium cyanide after they were released from the enzymatically digested samples. Cyanocobalamin was extracted and purified by an immunoaffinity SPE cartridge and then measured in multiple reaction monitoring mode (MRM). The linear correlation coefficient (R2) of this method was greater than 0.999 in the range of 2–100 ng/mL. The detection limit and the quantification limit were 0.5 μg/kg and 1.0 μg/kg, respectively. The spiked recoveries ranged from 92.0% to 99.4% at the three spiked levels with the relative standard deviation (RSD) between 1.89% and 4.51%. The measured results of NIST SRM1849a and NIST SRM1869a by the current method are all within the reference value range. The Z value was 0.8 during participating in the FAPAS proficiency test using the developed method in 2021. The method is simple, rapid, accurate, and sensitive, and it is suitable for the determination of vitamin B12 in different types of milk and dairy products such as whey powder, whole milk powder, pure milk, fermented milk, infant formula, and prescription food for special medical purposes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 β-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 β-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 β-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 β-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 β-casein allergen in baked food matrices at a low quantitation level down to 500 μg kg–1with a satisfactory accuracy (< 8.9%) and recovery (98.8% ± 2.6% to 106.7% ± 3.0%). [ABSTRACT FROM AUTHOR]

• An UHPLC-MS/MS method was developed to determine OPN in milk. • A consensus signature peptide was selected as the representative of OPN from different species' milk. • Stable isotope dimethyl labeling was used to prepare internal standard during determination of OPN. • HLB cartridge was applied to purify and enrich OPN signature peptide. • The method was able to measure OPN content in bovine, buffalo, yak, goat and sheep milk. 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 R 2 ≥ 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. [ABSTRACT FROM AUTHOR]

• UHPLC-MS/MS method was applied to detect casein glycomacropeptide in dairy products. • Characteristic peptide was qualitatively confirmed by high-resolution mass spectrometry. • Labeled amino acid condensation method was used to get isotope-labeled characteristic peptides. • The oxidation conditions (oxidant concentration, temperature, and time) were optimized. The ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS) method was built to quantify the casein glycomacropeptide (CGMP) in bovine dairy products accurately based on targeted proteomics. Qualitative analysis of theoretical peptides was carried out using high-resolution mass spectrometry (HRMS) and protein software. Isotope-labeled characteristic peptides were acquired via the labeled amino acid condensation method to correct the matrix effects. Peptide MAIPPK was the representative characteristic peptide for distinguishing the CGMP from κ -casein through trypsin digestion. After optimizing the pre-treatment conditions, the final 8% oxidant concentration was selected and the 10% formic acid concentration with 2.5 h oxidation time. Moreover, the results of methodological verification showed that the recovery rate was 103.7%, meanwhile the precision of inter-day and intra-day was less than 5%. In conclusion, the research demonstrated the characteristic peptide MAIPPK could quantitatively applied to detect CGMP in dairy products. [ABSTRACT FROM AUTHOR]

• UPLC-MS/MS method was established for determination of acrylamide. • Acrylamide in samples was cleaned up by single-step SPE using mixed-mode sorbents. • Retention capability and resistance to interference was improved by derivatization. • Efficiency of acrylamide derivatization was improved under an acidic condition. • The method is suitable for the determination of acrylamide in coffee its products. This work aimed to establish a novel determination method for acrylamide in coffee and its products by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Acrylamide in samples were prepared by a single-step solid-phase extraction clean-up using mixed mode sorbents. The bromine derivatization efficiency of acrylamide and its internal standard were improved at an acidic condition. After derivation, the retention capability of acrylamide and its resistance to interference were significantly improved. The limit of detection (LOD) and the limit of quantification (LOQ) were 1.2 and 4 μg/kg for roasted and instant coffees, while they were 0.24 and 0.8 μg/kg for ready-to-drink coffees. The average recoveries for acrylamide ranged from 99.3 to 102.2% in coffee and its products. All the results showed that the developed method was simple, quick, specific and suitable for screening and determination of acrylamide in batch samples of coffee and its products. [ABSTRACT FROM AUTHOR]

[Display omitted] • UPLC-MS/MS method was established for determination of Nε-(carboxymethyl)lysine. • Directly pretreatment sample protein without precipitation achieved rapid detection. • Nε-(carboxymethyl)lysine in sterilized milk was purified by MCX SPE cartridges. • No derivatization procedure and ion pair reagents in the isotope dilution method. • The target analyte was separated and analyzed on BEH Amide column. This study developed a sensitive and easy method to directly analyze Nε-(carboxymethyl)lysine (CML) by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Acetonitrile and water were used as the binary mobile phase without ion pair reagents, while BEH Amide column was applied to detect CML. The target substance without derivatization could be well retained and separated after the pretreatment conditions were optimized. Oasis MCX solid phase extraction (SPE) cartridge showed the best recovery rate of CML 96.7 % and purification ability among the tested SPE cartridges. There was no obvious difference in pretreatment abilities with or without protein precipitation on the CML quantitative analysis. The recovery rates 97–98 % for CML were achieved by UPLC-MS/MS, as well as the detection limit and quantification limit were 0.05 mg/kg and 0.15 mg/kg sample, respectively. This method was suitable for quantifying CML content with high recovery rate and low detection and quantification limit in sterilized milk. [ABSTRACT FROM AUTHOR]