Objective: To develop an approach for simultaneous detection of multi-mycotoxins in fresh fruits, so as to provide technical supports for mycotoxins surveillance in fresh fruits. Methods: Fresh fruits were collected from markets and homogenized. Then, 2 g of fresh fruits were added with 10 mL of 0.1% formic acid ( 99∶1, v/v ) in acetonitrile and wortexed for 10 min. Following extraction with 1 g of sodium chloride and 4 g of anhydrous sodium sulfate, samples were centrifuged and 5 mL of the supernatant was cleaned up with 25 mg C18. Following centrifugation, the supernatant was dried under nitrogen. The residue was dissolved in 300 μL of methanol-acetonitrile mixture solution ( 1∶1, v/v ), and mixed evenly in 700 μL of the distilled water. Samples were then eluted in gradient series of 0.1% formic acid and 5 mmol ammonium formate and methanol-acetonitrile mixture solution ( 1∶1, v/v ). The 15 mycotoxins were determined using liquid chromatography-tandem mass spectrometry ( LC-MS/MS ) with electrospray ion source (ESI+/ESI-) under multiple reaction monitoring. In addition, a matrix-matched standard curve was employed for quantitative analysis. Results: There was a good linear relationship for 15 mycotoxins at concentrations of 0.25 to 10 ng/mL ( R2>0.992 ), the LC-MS/MS method showed the detection limits of 0.1-1.0 μg/kg, the spiked recovery rates of 71.68%-117.50%, and the relative standard deviations ( RSDs ) of 0.01%-13.60%. The detection rate of mycotoxins was 27.09% in 203 fresh fruits sold in markets. Conclusions The optimized LC-MS/MS method can be used for simultaneous determination of multi-mycotoxins in fresh fruits.

Objective: To optimize the enzymatic digestion conditions of trypsin, so as to improve the testing capacity of mass spectrometry for shrimp allergens. Methods: The enzymatic digestion test was carried out by response surface methodology for optimizing pH, temperature and time. After enzymatic hydrolysis, the peptides were separated by chromatography and determined by high-resolution mass spectrometry with Q-orbitrap. The allergen protein was identified and quantified by UniProt database and MaxQuant software. Results: Two allergen proteins, tropomyosin and arginine kinase, were isolated from shrimp, and their intensities ranged from 100.2×106 to 436.5×106. Response surface analysis showed that when the digestion time was 4.29 hours, the temperature was 44.15 ℃ and pH value was 6.55, the maximal intensity of the allergen proteins was 457.48×106. The experiment was validated with the digestion time of 4.2 h, pH value of 6.5, and temperature of 44 ℃, then resulted in the average intensity of 448.1×106. The deviation from the predicted value was 2.1%. Conclusions The conditions of enzymatic digestion can be optimized by response surface methodology. The enzyme may have the best performance with the pH value of 6.5, temperature of 44 ℃ and digestion time of 4.2 hours.

Objective: To screen and quantify 16 kinds of β-lactam antibiotics in pork by high performance liquid chromatography-quadrupole/electrostatic field orbit trap mass spectrometry（UPLC-Q-Orbitrap）. Methods: The pork samples were extracted by ultrasound with acetonitrile，then the supernatant was centrifuged and purified by HLB solid phase extraction column. The analytes were separated by Waters HSS T3 column（100 mm×2.1 mm，1.8 μm）with gradient elution. Mass spectrometry adopted positive ion scanning and targeted SIM/dd-MS2 monitoring mode to complete the separation of analytes in samples and mass spectrometry analysis within 10 minutes. The chromatographic retention time and fragments in mass spectrometry were compared with prepared standards to determine whether the samples contained the antibiotics tested，then the positive samples were quantified. Results: The 16 kinds of β-lactam antibiotics had good linear relationship in the range of 5-400 ng/mL（all the correlation coefficients >0.99）. The detection limits ranged from 0.08 μg/kg to 0.41 μg/kg，recovery rate ranged from 85.5% to 116.7%，and relative standard deviation（RSD）ranged from 3.6% to 12.8%. One of twenty pork samples detected was found penicillin G（28 μg/kg）and ampicillin（18.5 μg/kg）. Conclusion UPLC-Q-Orbitrap has high resolution and can reduce matrix interference to improve the accuracy. This method is simple，fast and efficient，thus can be used to screen and quantify β-lactam antibiotics in pork.

Objective : To develop an analytical method of ibotenic acid (IBA) and muscimol (MUS) in wild mushroom by dansyl chloride (DNSCl) derivatization-liquid chromatography-tandem mass spectrometry (LC-MS/MS), and to provide technical support for etiological identification of mushroom poisoning events. Methods : The sample was extracted with hydrochloric acid solution, derived by bimolecular DNSCl, diluted and inorganic salts precipitated with acetonitrile. The extract was separated by a waters XBridgeTM BEH C18 column and measured by LC-MS/MS. Results : The limits of detection for IBA and MUS in wild mushroom were 0.15 mg/kg and 0.1 mg/kg, respectively. Good linear relationship was obtained for IBA and MUS at the range of 0.5-250 mg/kg with the correlation coefficient of 0.997 and 0.999, respectively. The average recoveries at three spiking levels were 84.5%-102.0% with relative standard deviations (RSDs, n=6) of 4.7%-8.6% for IBA. The average recoveries were 88.6%-95.4% with RSDs (n=6) of 4.9%-7.5% for MUS. Conclusion The optimized sample extraction and bimolecular DNSCl derivatization conditions can achieve rapid and accurate analysis of IBA and MUS in wild mushroom poisoning sample.

Objective: To establish the ultra-high performance liquid chromatography coupled with quadrupole orbitrap mass spectrometry ( UPLC-Q-Orbitrap-MS ) for the analysis of allergen protein in Macrobrachium. Methods: Based on the strategy of bottom-up protein analysis, the proteins in Macrobrachium samples were extracted by Tris-HCl, digested by trypsin at 40 ℃ for 6 hours, separated by chromatography, and analyzed by Q-Orbitrap-MS spectrometry ( Full MS/dd-MS2, TopN=10 ) . Allergen proteins were identified with UniProt protein database and Proteome Discoverer software. Results: Four kinds of allergen proteins were obtained, which were tropomyosin, arginine kinase, sarcoplasmic calcium binding protein and hemocyanin. The coverage rates of peptides in proteins were 53%, 36%, 12% and 12%, respectively. Post translation modifications were methylation of aspartic acid (D), deacylylation of aspartic acid ( N ) , glutamyl ammonia ( Q ) and oxidation of methionine ( M ) . Conclusions The UPLC-Q-Orbitrap-MS can identified abundant peptide and fragment information with high sensitivity and resolution, which provides a technical support for the analysis of shrimp allergens.