Study On The Fidelity Determination Of Trace Aflatoxins And Plant Growth Regulators Based On LC-MS

In crop tissues or agro-products, concentrations of aflatoxins and plant growth regulators are usually very low, and both of them belong to trace-compounds, however, their activity are much strong. Aflatoxins are highly toxic and carcinogenic, which are serious threat to the safe consumption of peanuts, corns, rice and other agricultural products. Plant growth regulators take actions in promotion or inhibition in the growth of plant in very low concentrations, whose residues also threat to the safe consumption of fruits and vegetables. For their low concentrations, aflatoxins and plant growth regulators'fidelity determination are hotspot and difficult issues in the field of quality and safty inspection of agricultural products. Therefore it is very important to develop fidelity method in determination of aflatoxins and plant growth regulators in agricultural products based on high performance liquid chromatography-tandem mass spectrometry, which play key role in protection safe consumption of agricultural products and in promoting the development of relateive disciplines.Based on linear ion trap mass spectrometry, chromatographic elution conditions of aflatoxins were researched; mass spectrometry determination parameters also were confirmed; a high performance liquid chromatography with fluorescence detection (FLD) method was used as a comparison in accuracy, precision and sample determination. Finally, a liquid chromatography- tandem mass spectrometer was established to determine the aflatoxins in peanut, corn and rice. Based on triple stape quadrupole mass spectrometry, chromatographic elution conditions of 13 plant growth regulators were verified and the mass spectrometry parameters in determination also were ascertained; the matrix effect of rape tissues was also evaluated; all quantitative parameters of the method were determined and confirmed. Then a liquid chromatography-tandem mass spectrometry method was developed in determination of 13 plant growth regulators in rape tissues.Main conclusions were as follows.1. To solve the technical problem of laboratory arbitration of aflatoxins, fidelity determination method based on liquid chromatography-tandem mass spectrometry (linear ion trap mass spectrometry) was established. Four aflatoxins were separated completely in 10 min by HPLC equipped with a reversed-phase column whose internal diameter is 2.0 mm. The optimum chromatographic condition was isocratic eluted with methanol and water containing 0.1% formic acid (50/50, v/v) and the confirmed quantitative ions of AFB₁, AFB₂, AFG₁ and AFG₂ were 313/285, 315/287, 329/311 and 331/313. Limit of detection (LOD) (the concentration of analyte when the ratio of signal/noise is three) of AFB₁, AFB₂, AFG₁ and AFG₂ determined by LC-MS were in the range of 0.02-0.04μg/kg, the spiked recoveries in peanut, corn and rice were in the range of 82.09-109.36%, 70.97-121.27% and 50.38-120.61%, respectively. As a comparison, LOD of AFB₁, AFB₂, AFG₁ and AFG₂ determined by HPLC-FLD were in the range of 0.1-0.3μg/kg, and the spiked recoveries in peanut, corn and rice were in the range of 63.80-112.25%, 53.53-121.04% and 50.29-126.54%, respectively. The results showed that liquid chromatography-ion trap mass spectrometry method was more sensitive than HPLC-FLD, and the sensitivity was enhanced nearly 20 times, the selectivity of LC-MS was mach better than HPLC-FLD. The work provided critical techniques support for aflatoxin laboratory arbitration.2. To solve the prolem of fidelity determination of plant growth regulators, a liquid chromatography-tandem mass spectrometry (triple stape quadrupole mass spectrometry) was developed. The optimum chromatographic conditions were gradient eluted by methanol and water both containing of 0.1% formic acid. Under the optimum separation conditions, all compounds were successfully separated within 28 min. The quantitative ions of indole-3-acetic acid,α-naphthaleneacetic acid, 2-chlorobenzoic acid, 4-chlorobenzoic acid, indole-3-butyric acid, gibberellic acid, 2, 4-dichlorophenoxyacetic acid, 2-naphthoxyacetic acid, abscisic acid, 2, 3, 5-triiodobenzoic acid, uniconazole, paclobutrazol and 2, 4-epibassinolide were 174/130.4, 185/141.3, 155/111.4, 155/111.3, 202/158.5, 345/143.2, 219/161.4, 201/143.3, 263/153.3, 498.8/455.1, 292/70.5, 294/70.4 and 481.3/445.7, respectively. LOD of indole-3-acetic acid,α-naphthaleneacetic acid, 2-chlorobenzoic acid, 4-chlorobenzoic acid, indole-3-butyric acid, gibberellic acid, 2, 4-dichlorophenoxyacetic acid, 2-naphthoxyacetic acid, abscisic acid, 2, 3, 5-triiodobenzoic acid, uniconazole, paclobutrazol and 2, 4-epibassinolide was 0.03, 0.1, 0.1, 0.04, 0.06, 0.01, 0.01, 0.01, 0.01, 0.1, 0.001, 0.001 and 0.02μg/mL, respectively. The spiked recoveries of rape root, stem, leaf, flower, immature pod and seed were in the range of 85.8-99.8%, 85.5-99.7%, 77.9-99.9%, 78.5-99.9%, 87.4-99.9%, 81.5-99.2%, respectively.Matrix effect of rape tissues was determined. Molecules originating from the sample matrix that coelute with the compounds of interests can interfere with the ionization process in the mass spectrometry, causing ionization suppression or enhancement, which is the so-called matrix effect. The value of matrix effect > 100 indicates ionization enhancement, and a value of < 100 indicates ionization suppression. For specific plant growth regulator, such as indole-3-acetic acid, the matrix effect of seed extracts is the most strong, the ratio of ionization suppression is over 20%. For specific matrix, such as root extracts, the matrix effect toα-naphthaleneacetic acid is the most seriously, the ratio of ionization suppression is more 23%. So the matrix effect was induced by both the characters of analyte and the sample matrix. Matrix effect influenced on the spiked recovery, and then on the accuracy.So the LC-MS/MS method could be used to simultaneously determine thirteen plant growth regulators in rape tissues, the ratio of ionization suppression is less than 30%, the ratio of ionization enhancement is less than 20%, and the spiked recovery in the range of 77.9-99.9%, the accuracy in the range of 67.0-118.2%.