Objective: To establish an on-line column switching liquid chromatography for quantitative determination of vitamin D3 in poultry eggs and their products. Methods: Antioxidant-protected samples were saponified, extracted with a mixture of ethyl acetate-n-hexane mixture (6︰4, v/v) and subjected for determination using two-dimensional liquid chromatography. One-dimensional chromatography was performed with the UniChiral® OD-5H column (4.6 mm × 100 mm, 5 μm), acetonitrile: methanol (75︰25, v/v) and water as a mobile phase for gradient elution at a flow rate of 1.00 mL/min to complete vitamin D purification, and two-dimensional chromatography was performed with the Agilent Eclipse PAH column (2.1 mm × 100 mm, 3.5 μm, column temperature of 35 ℃), detection wavelength of 264 nm for separation and determination of vitamin D3 and internal standard vitamin D2 with the internal standard method. Results: Vitamin D3 had a good linear relationship within the range of 2.5 to 100 ng/mL, with r2 of >0.999. The mean spiked recovery rate of vitamin D3 was 101.23% to 102.08% in egg yolk powder, with relative standard deviation (RSD) of 3.91% to 5.85%, detection limit of 1.5 μg/100 g, and quantitative limit of 4.9 μg/100 g, and the limits of detection and quantitation of vitamin D3 were 0.15 μg/100 g and 0.49 μg/100 g in whole egg liquids. Conclusions The method is simple, rapid, highly accurate, sensitive and reproducible, which is suitable for rapid and quantitative determination of vitamin D3 in poultry eggs and their products.

A method for the determination of B6-pyridoxamine (PAM), pyridoxal (PAL) and pyridoxine (POL) by RPLC was proposed. The procedure included the addition of 0.1M H2SO4 to the sample, hydrolysis for 30 min at 120℃, centrifuging, filtration and direct analysis by ODS Cl8 column. PAM, PAL and POL were completely separated, when the flow rate of the mobile phase (pH2.0-2.1) was 1.0-1.5ml/min. Quantitation by a fluorescent detector was performed with Exc: 293 nm and Em: 395 nm. The recovery ranged from 99 to 103% with CV 5.0-5.3%. The method was simple, rapid, sensitive and reproducible.

Objective: A high-performance liquid chromatographic (HPLC) procedure with a fluorescence detector was developed to rapidly separate ?,?,?,?- tocopherol isomers in human serum Methods: The HPLC system consisted of Inertsil silica column (100-A, 3?m,4.6mm?250mm) and 7% (v/v) methyl-tert- butyl ether in n-hexane as mobile phase . Prior to HPLC, the serum sample wa deproteined by ethanol (BHT 0.0625%) and the tocopherol isomers were efficiently extracted in thei original isomeric conformations using n-hexane-ethyl acetate (5:1) in the presence of 2,6-bi-buty p-methylphenol (BHT). Result: The quantification limits, defined as the lowest quantitatively measurable concentration of the different compounds (ng/ml) are calculated according to the experiment:?-tocophero 1.0,?-tocopherol 1.0,?-tocopherol 0.5,?-tocopherol 0.5. The recovery rates are between 95%~105% Correlation coefficients are over 0.999 when the concentration is between 5 ng/ml~5 ?g/ml. Conclusion This technique is suitable for assay of tocopherol isomers in human serum at all ages.

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.

There are eight forms of vitamin E in human blood, including α-, β-, γ-, δ-tocopherols and α-, β-, γ-, δ-tocotrienols. As the most abundant and active form of vitamin E, α-tocopherol is widely accepted as a reliable indicator for nutritional assessment of body vitamin E status across the world. Considering that different vitamin E forms have diverse biological activities, separation and detection of different vitamin E forms in human blood facilitates the understanding of the association between vitamin E and diseases. In this review, the advances in sample-pretreatment techniques and detection techniques for vitamin E in human blood were presented. Currently, the sample-pretreatment techniques include solid-phase extraction, liquid-liquid extraction, dispersive liquid-phase microextraction, supported liquid extraction and direct protein precipitation; the detection techniques include automatic biochemical analysis, enzyme-linked immunosorbent assay, gas chromatography, liquid chromatography and ultra-high performance supercritical fluid chromatography mass spectrometry. This review summarizes the characteristics and scope of above-mentioned techniques used for detection of vitamin E in human blood, so as to provide insights into the selection of an appropriate method for inspection technicians.

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 quickly determine bongkrekic acid（BKA）in plasma qualitatively and quantitatively by liquid chromatography-tandem mass spectrometry（LC-MS/MS）,and to provide technical support for etiological identification of food poisoning events. Methods: The plasma sample was protein precipitated with acetonitrile,diluted with water and purified with anion exchange solid phase extraction cartridge of PAX. The sample extract was separated by an XBridgeTM BEH C18 chromatographic column. Gradient elution was conducted with the mobile phase of 0.01 %（v/v）ammonia and methanol. Then BKA was detected by LC-MS/MS. Results: The equation of linear regression was y=16 509x+3 134.3. Good linear relationship was obtained for BKA at a range from 1 to 400 ng/mL in plasma,with the correlation coefficient of 0.999 3. The limit of detection（LOD）was 0.5 ng/mL and the limit of quantitation（LOQ）was 1 ng/mL. The average recoveries were 76.0%-96.7% with relative standard deviations（RSDs,n=6）of 5.2%-12.8% at three spiking levels of 1（LOQ）,10（10 times of LOQ）and 200 ng/mL（medium of linear range）. The concentrations of BKA in plasma obtained from two patients suffering from food poisoning were 394 and 92.3 ng/mL. Conclusion The optimized sample pretreatment and chromatographic separation conditions can achieve rapid,accurate,qualitative and quantitative analysis of BKA in plasma.

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.