Baifen Huang

Direct determination of melamine in dairy products by gas chromatography/mass spectrometry with coupled column separation

A coupled capillary column system was developed for the qualitative and quantitative determination of melamine with isotope internal standard in dairy products by gas chromatography/mass spectrometry (GC/MS) without derivatization. A 30 m of DB-5 ms ((5%-phenyl)-methylpolysiloxane, 0.25 mm i.d., 0.25 microm df) coupled with a 1.5 m of Innowax (polyethylene glycol, 0.32 mm i.d., 0.25 microm df) by a quartz capillary column connector was introduced as separation column. Three advantages were discussed for the coupled system. The sample was fortified with a ring-labeled (13)C(3)(15)N(3)-melamine as an isotope internal standard and extracted by 1% of trichloroacetic acid aqueous solution. 2.2% of lead acetate solution was then added to deposit protein in the sample matrix. After purification by cation exchange cartridge, the sample solution was directly injected and detected by GC/MS. A six-point calibration curve ranging from 0.05 to 2 mg kg(-1) of melamine in sample was used to establish instrument response. The recovery was 93.9-102% with relative standard deviation from 3.1 to 8.7% when isotope internal standard used. The calculated method detection limit was 0.01 mg kg(-1).

Simultaneous determination of aflatoxins B1, B2, G1, G2, M1 and M2 in peanuts and their derivative products by ultra-high-performance liquid chromatography-tandem mass spectrometry.

A reliable ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for simultaneous determination of aflatoxins B1, B2, G1, G2, M1 and M2 in peanuts and their derivative products was developed. The sample was extracted by 84% of acetonitrile aqueous solution and the extract was purified by a reliable solid phase extraction-based clean-up method. Then, the analytes were separated on Acquity UPLC HSS T3 column (100 mm x 2.1 mm, 1.8 microm particle size), and eluted with a mobile phase consisting of (A) water containing 0.1% formic acid and (B) acetonitrile/methanol (50/50, v/v). The separated compounds were detected with a Waters Micromass Quattro Ultima Pt tandem quadrupole mass spectrometer operating in positive electro-spray ionization using multiple reaction monitoring mode. The established method was extensively validated by determining the linearity (R(2) > or = 0.9990), average recovery (74.7-86.8%) and precision (relative standard deviation < or = 10.9%). It was shown to be a suitable method for simultaneous determination of the six aflatoxins in peanuts and their derivative products. Finally, a total of 73 samples randomly collected from different areas in Zhejiang province were screened for aflatoxins with the proposed method. The results showed that 31 samples of peanut butter, 14 samples of fresh peanut and 5 samples of musty peanut were contaminated with aflatoxins. Meanwhile, this was the first report on aflatoxins M1 and M2, which were found in unprocessed peanuts and their derivative products.

Multi‐residue analysis of pesticides in tea by online SEC‐GC/MS

A multi-residue method for the analysis of pesticides in tea was developed by online size exclusion chromatography (SEC)-GC/MS with full scan mode. The sample was fortified with chlorpyrifos-d(10) isotope internal standard and extracted by acetonitrile. After purification by primary secondary amine sorbent and solvent exchange by SEC mobile phase, the sample was detected by online SEC-GC/MS. The purification result of the online system was evaluated by comparing the correlation between Chinese cabbage and tea matrix. The factors for method optimization included sample preparation, matrix effects and the instrument parameters of each online component. Scatter plot was introduced in this study to directly illustrate the results of the condition optimization and matrix effects in the online system. For most of the pesticides, the average recoveries ranged from 70 to 130% and the RSD were below 15%. The feasibility of the application of full scan mode in multi-residue determination of trace amounts of pesticides (LODs below 0.01 mg/kg) in a complex matrix was discussed.

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 Rens 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.

Simultaneous determination of 3-monochloropropane-1,2-diol and acrylamide in food by gas chromatography-triple quadrupole mass spectrometry with coupled column separation.

Both 3-monochloropropane-1,2-diol (3-MCPD) and acrylamide are contaminants found in heat-processed foods and their related products. A quantitative method was developed for the simultaneous determination of both contaminants in food by gas chromatography-triple quadrupole mass spectrometry (GC-MS/MS). The analytes were purified and extracted by the matrix solid-phase dispersion extraction (MSPDE) technique with Extrelut NT. A coupled column (a 3 m Innowax combined with a 30 m DB-5 ms) was developed to separate both compounds efficiently without derivatization. Triple quadrupole mass spectrometry in multiple reaction monitoring mode (MRM) was applied to suppress matrix interference and obtain good sensitivity in the determination of both analytes. The limit of detection (LOD) in the sample matrix was 5 μg kg(-1) for 3-MCPD or acrylamide. The average recoveries for 3-MCPD and acrylamide in different food matrices were 90.5-107% and 81.9-95.7%, respectively, with the intraday relative standard deviations (RSDs) of 5.6-13.5% and 5.3-13.4%, respectively. The interday RSDs were 6.1-12.6% for 3-MCPD and were 5.0-12.8% for acrylamide. Both contaminants were found in samples of bread, fried chips, fried instant noodles, soy sauce, and instant noodle flavoring. Neither 3-MCPD nor acrylamide was detected in the samples of dairy products (solid or liquid samples) and non-fried instant noodles.

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.

Determination of disialoganglioside GD3 and monosialoganglioside GM3 in infant formulas and whey protein concentrates by ultra-performance liquid chromatography/electrospray ionization tandem mass spectrometry.

A method of ultra-performance liquid chromatography combined with electrospray ionization triple quadrupole tandem mass spectrometry (UPLC-ESI-MS/MS) has been established for simultaneous determination of major disialoganglioside 3 (GD3) and monosialoganglioside 3 (GM3) in infant formulas and whey protein concentrates. Gangliosides were extracted by using the technique of Svennerholm and Fredman and then cleaned up with OASIS HLB solid-phase extraction (SPE) cartridges. The various molecular species of gangliosides were separated on an Acquity UPLC BEH C8 column and analyzed under the negative ion mode. GD3 and GM3 were rapidly quantified using internal standard (IS) method. The developed method was further validated by determining the linearity, average recovery, sensitivity (limit of quantification), and precision. The results presented high correlation coefficients (R(2) > 0.993) of the selected 16 gangliosides molecular species and provided the respective linear ranges. The limit of quantification was 0.325-0.734 mg/100 g for eight molecular species of GD3 and 0.008-0.312 mg/100 g for eight molecular species of GM3, respectively. The reasonable average recoveries (81-95%) and precision (relative standard deviation [RSD] ≤15%) were also demonstrated in three different spiked levels. This new method would be very useful in the quantitative determination of gangliosides in infant formulas and whey protein concentrates.

Development and validation of a gas chromatography-mass spectrometry method for determination of sterol oxidation products in edible oils

An efficient gas chromatography-mass spectrometry method was developed and validated for determination of sterol oxidation products (SOPs) in edible oils. The sample preparation involved cold saponification, liquid–liquid extraction, solid-phase extraction on a silica gel cartridge, and trimethylsilylation by N-methyl-N-(trimethylsilyl) heptafluorobutyramide with 5% 1-methyl imidazole. The trimethylsilyl ether derivatives of SOPs were separated by gas chromatography with a 30 m DB-5MS capillary column and quantified by a mass spectrometer in selective ion monitoring mode. 5α-Cholestane and 19-hydroxycholesterol were used as dual internal standards. The calibration curves for each compound showed correlation coefficients (R2) better than 0.98. The detection limits were below 12.9 ng mL−1 (except for epoxides). The intra- and inter-day determination precisions for diversiform SOPs were <10% in relative standard deviations; the recoveries ranged within 89.72% and 117.42%. The developed approach was successfully applied to study the presence of thirty-four different SOPs present at low levels in camellia, olive, sesame, peanut, rapeseed, rice bran, soybean and corn oils.

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.

Authentication of shrimp muscle in complex foodstuff by in-solution digestion and high-resolution mass spectrometry

A method for shrimp muscle identification in complex foods is required to safeguard the shrimp-allergic population. This study described a method for authentication of shrimp in complex foodstuffs (fish balls) by liquid chromatography tandem QTOF mass spectrometry (UPLC-QTOF-MS). The proteins in shrimp muscle were extracted using a Tris–HCl solution and then digested using tryptic protease. The main allergen proteins, tropomyosin (TM) and arginine kinase (AK), were characterized using the ‘bottom up’ MS approach. After analysis of their peptide mass fingerprinting based on the UniProt database, two specific heat-stable peptides, ALSNAEGEVAALNR for TM and VSSTLSSLEGELK for AK, were screened as surrogate (signature) peptides. The detection limit, expressed as shrimp meat per kilogram of food, was 8 g kg−1 (usage of TM) or 5 g kg−1 (usage of AK). The developed method is suitable to screen potential addition of shrimp meat in foodstuffs by detection of allergen proteins.