Comprehensive characterization and detection of nut allergens in bakery foods using Q-TOF mass spectrometry and bioinformatics

Food allergy is a growing health issue worldwide and the demand for sensitive,robust and high-throughput analytical methods is rising.In recent years,mass spectrometry-based methods have been established for multiple food allergen detection.In the present study,a novel method was developed for the simultaneous detection of almond,cashew,peanut,and walnut allergens in bakery foods using liquid chromatography–mass spectrometry.Proteins unique to these four ingredients were extracted,followed by trypsin digestion,quadrupole time-of-flight(Q-TOF)mass spectrometry and bioinformatics analysis.The raw data were processed by de-novo sequencing module plus PEAKS DB(database search)module of the PEAKS software to screen peptides specific to each nut species.The thermal stability and uniqueness of these candidate peptides were further verified using triple quadrupole mass spectrometry(QQQ-MS)in multiple reaction monitoring(MRM)mode.Each nut species was represented by four peptides,all of which were validated for label-free quantification(LFQ).Calibration curves were constructed with good linearity and correlation coefficient(r 2)greater than 0.99.The limits of detection(LODs)were determined to range from 0.11 to 0.31 mg/kg,and were compared with the reference doses proposed by Voluntary Incidental Trace Allergen Labelling(VITAL).The recoveries of the developed method in incurred bakery food matrices ranged from 72.5%to 92.1%with relative standard deviations(RSD)of<5.2%.The detection of undeclared allergens in commercial bakery food samples confirmed the presence of these allergens.In conclusion,this method provides insight into the qualitative and quantitative detection of trace levels of nut allergens in bakery foods.

Lysophosphatidylcholine Biomarkers of Lung Cancer Detected by Ultra-performance Liquid Chromatography Coupled with Quadrupole Time-of-flight Mass Spectrometry

Centrifugal ultrafiltration after methanol extraction of whole plasma was used as an optimal condition for the preparation of blood plasma before metabonomic studies. The plasma samples from 102 lung cancer patients and 34 healthy volunteers were prepared with this approach. With ultra-performance liquid chromatography（UPLC） coupled with quadrupole time-of-flight mass spectrometry（Q-TOF MS） analysis, the samples were investigated in order to find potential disease biomarkers. After data acquisition, orthogonal signal correction partial least squares models were built to differentiate the healthy volunteers from lung cancer patients and to identify metabolites that showed significantly different expression between the two groups. Several metabolite ions were identified as potential biomarkers according to the variable importance in the project（VIP） value in both ion modes. Five lysophosphatidylcholines were further identified as specifically lysoPC 16：0, isomer of lysoPC 16：0, lysoPC 18：0, lysoPC 18：1 and lysoPC 18：2. These results suggest that UPLC coupled with Q-TOF MS is an effective technique for the analysis of plasma metabolites in metabonomic studies.

Analysis of Phytochemical Constituents of Zuogui Wan in Rat Serum and its Effects on Early Embryonic Development of Mice

Objective:Zuogui Wan(ZGW)has been used as a typical prescription for tonifying kidney essence in traditional Chinese medicine.The objective of this study is to elucidate the phytochemical constituents of ZGW-treated rat serum(ZGWRS)using ultra-performance liquid chromatography-electrospray ionization/quadrupole-time-of-flight high-definition mass spectrometry(UPLC-ESI-Q-TOF-MS).Methods:ZGW was administered to rats,and the phytochemical constituents in rat serum were determined using UPLC-ESI-Q-TOF-MS.MetaboLynx analysis in negative ion mode was adopted to characterize the chemical constituents of ZGWRS.Orthogonal partial least squares discriminant analysis was applied for the discovery of constituents of ZGW that entered the serum of rats.The fertilized eggs collected from the same experiment were randomly divided into four groups,including the normal,40 mmol/L glucose,40 mmol/L glucose 5%control rat serum,and 40 mmol/L glucose 5%ZGWRS groups.They were cultivated at 37°C and 5%CO2 in a saturated humidity CO2 incubator.The blastocyst rate and two-cell rate were used to evaluate the effects of ZGWRS on embryonic development.Results:Thirteen constituents were identified in the ZGWRS,among which sweroside,loganin,morroniside,loganic acid,and 8-epiloganic acid were from Fructus Corni(Shan Zhu Yu).5-Hydroxymethyl-2-furfural-glucuronide,2-H-5-hydroxymethyl-2-furfural-glucuronide,3-hydroxy-2,6,6-trimethyl-1-cyclohexene-1-carboxylic acid,andβ-D-ribofuranuronic acid methyl ester triacetate were from Radix Rehmanniae Preparata(Shu Di Huang).Coumaric acid was from Fructus Lycii(Gou Qi Zi).Kaempferol-3-beta-O-glucuronide and cuscutamine were from Semen Cuscutae(Tu Si Zi).The embryonic development was significantly inhibited using 40 mmol/L glucose.Compared with the normal group,the blastocyst rate of the glucose group was decreased.The blastocyst rate of the 40 mmol/L glucose 5%ZGWRS group was significantly higher than that of the glucose group,indicating that ZGWRS negates the effect of glucose on mouse embryonic development.Conclusion:The results verified that a rapid and robust UPLC-ESI-Q-TOF-MS-based platform had been successful for identifying multiple constituents of ZGW.ZGWRS is rich in active constituents of iridoid glycosides.The results of this study also showed that ZGWRS could negate the effect of glucose on mouse embryonic development.