| Chinese herbal medicine (CHM) is easy to be contaminated by pesticides, mycotoxins and heavy metals during planting, harvesting, processing and storage. Especially in the process of storage, CHM is likely to be infected by fungal to produce mycotoxins under the poor storage conditions. Contamination with pesticides, mycotoxins and heavy metals in CHM bring security risks to its use and become a bottleneck restricting the development of traditional Chinese Medicine. Extrinsic harmful residues in CHM have attracted the attention of some countries and regions. Establish a simple and accurate analysis method for the detection of exogenous pollutants in CHM and scientific maintenance for some easy moldy CHM has become the primary problem in the quality control and safety study of CHM.Alpinia oxyphylla (A. oxyphylla), the dried fruit of Alpinia oxyphylla Miq, is widely cultivated in southern China (Hainan and Guangdong province) as one of the most frequently used crude drugs and healthy foods. As a perennial herb, A. oxyphylla is widely cultivated in tropical and subtropical regions, which means that it is much vulnerable to be polluted by exogenous pollutants. However, there are few studies on the pesticides, mycotoxins and heavy metals residues in A. oxyphylla. In this research, firstly, a series of experimental were carried out to establish the analytical methods of pesticide residues, mycotoxins and heavy metals residues in A. oxyphylla. Secondly, after sterilizing, A. oxyphylla was contaminated by artificial inoculation of Aspergillus flavus spores and cultured based on "trans-culture". Main chemical components and mycotoxins in A. oxyphylla were determined at different time. Central Composite Design-response surface methodology was used to study the effects of different temperature and humidity of storge on the fungal growth in A. oxyphylla and accumulation of mycotoxins, and screening the best storage conditions of A. oxyphylla. It provided the theoretical basis for establishing the mildew prevent system during A. oxyphylla storage.1. One step extraction for gas chromatography-flame photometric detection method was firstly established for simultaneous multi-component determination of 31 organophosphorus pesticides (OPPs) residues in A. oxyphylla, and the positive samples were confirmed by GC-MS/MS.Some crucial parameters, including extraction solvent and time were optimized to improve the extraction efficiency.The samples were extracted by the acetone/ethyl acetate (1:1, v/v). Sample preparation was completed in a single step without any clean-up procedure. All pesticides expressed good linear relationships between 0.004 and 1.0 μg/mL with correlation coefficients higher than 0.9973. he limits of detection varied from 1.0μg/kg to 10μg/kg, and the limits of quantification (LOQs) were between 4μg/kg and 30μg/kg. For the majority of the tested pesticides, the recoveries were in acceptable range (between 70%and 110%) and the average recovery from 57.6%to 104.7% with RSD between 0.7%and 13.7%. The proposed method was successfully applied to 55 commercial samples purchased from different areas. Seven pesticide residues were detected in eight (7.27%) samples with the maximum residual amount was 138.2 μg/kg. The positive samples were confirmed by gas chromatography with tandem mass spectrometry (GC-MS/MS). Compared with other reported OPPs analytical methods, the proposed method was simple, cost-effective and easy, which means it could be used for the pesticides residues analysis in the fruit of A. oxyphylla.2. A simplified ultrasound-assisted solid phase extraction and acid clean-up coupled gas chromatography with electron capture detector (GC-ECD) method was developed for the analysis of 26 organochlorine pesticides (OCPs) residues in A. oxyphylla.Different clean-up procedures including QuEChERs, SPE, sulphuric acid and the combination of SPE and sulphuric acid were compared in this research. The target pesticides were extracted by sonication and cleaned up with florisil solid phase extraction and sulphuric acid. Some crucial parameters, including extraction solvent and time, sorbent type, elute solvent and concentration of sulphuric acid were optimized to improve the performance of sample preparation procedure. The optimized method gave high sensitivity with detection limit ranging from 0.1 to 2.0 μg/kg. Matrix-matched calibration was employed for the quantification, and a wide linear range (from 1.0 to 1000 μg/kg) with r2 values ranging from 0.9971 to 0.9998 was obtained. For the majority of the tested pesticides, the average recoveries were in acceptable range (between 70% and 110%) with relative standard deviation values below 15.0%. Matrix effect was evaluated for target compounds through the study of ratio of peak area obtained in the solvent and blank matrix. The proposed method was applied to simultaneously analyze 26 pesticides in 55 batches of A. oxyphylla samples.3 samples were found to be positive with four pesticides (a-BHC, quintozene, trans-chlordane and op’-DDD), which were confirmed by gas chromatography-mass spectrometry (GC-MS) in selective ion monitoring (SIM) mode.3. A solid-liquid extraction (SLE) procedure without any clean-up step based ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) was developed for simultaneous quantitation of 11 mycotoxins from A. oxyphylla.Three different extraction procedures (SLE, solid-phase extraction and modified QuEChERS) were evaluated based on the recovery and matrix effect. Some crucial factors including extraction solvent, time and temperature, were carefully optimized. The proposed SLE-UPLC-MS/MS method was applied to determine 11 mycotoxins (AFB1, AFB2, AFG1, AFG2, OTA, HT-2, T-2, FB1, FB2, ZEA and DON) in 42 batches of A. oxyphylla. Significant matrix effect was compensated using the matrix-matched calibration. Under the optimized conditions, good linearities were obtained, with correlation coefficients (R2) above 0.9958. The limits of detection varied from 0.03 to 6μg/kg, and the limits of quantification (LOQs) were between 0.1 and 20μg/kg. The spiked recoveries ranged from 59.69%(T-2 toxin) to 111.34%(HT-2 toxin), with RSD of less than 20%. Three mycotoxins (AFB1, ZEA, OTA) were detected in 4 samples. The established method was simple and rapid for the analysis of multiple mycotoxins in A. oxyphylla.4. A microwave-digestion and inductively coupled plasma-mass spectrometry (ICP-MS) method was proposed for determination of 20 metal elements in A. oxyphylla.The micro-wave digestion was optimized and the digestion solvent was HNO3 and H2O2. Certified standard reference material was used to assess the accuracy of the method. The analysis results agreed with the certified values. The developed method was applied to determine 20 metal elements (Mg, K, Ca, Na, Fe, Al, Zn, Ba, Mn, Cu, Mo, Cr, Ni, As, Se, Cd, Hg, Tl, Pb and V) in 16 batches fruits, different parts (roots, stem, leaves and fruits) and the extractum of decoctions of A. oxyphylla. Precision between sample replicates was better than 5.0% for all the determination. All the elements were detected in fruits, leaves, stems and roots. The concentration of elements in fruits expresses wide variations. The mean concentration of Cu was highest in heavy metals (6.74 mg/kg) and the content Cd was the lowest (25.82 μg/kg). The highest concentration of Mg、Ca、Mn and Se were found in leaves, while heavy metals concentration (Cd, Cr, Cu, As, Pb, et al) in roots were highest. The transfer rate (Tr) of all elements present in the decoctions were calculated. All elements in A. oxyphylla were also released into the decoctions at different percentage (5.65%-93.96%). Especially for heavy metals, the transfer rate was lower than 30.0%. The results showed that the decoction can reduce the intake of heavy metals.5. Using the Central Composite Design-response surface methodology (CCD-RSM) combined with "trans-culture" mode to optimal storage conditions of A. oxyphylla, and provide the basis for the development of storage specification.Based on "trans-culture", after sterilizing, A. oxyphylla was contaminated by artificial inoculation of Aspergillus flavus spores and cultured. Main chemical components and mycotoxins in A. oxyphylla were determined at different time. Central Composite Design-response surface methodology was used to study the effects of different temperature and humidity of storge on the fungal growth in A. oxyphylla and accumulation of mycotoxins. The results showed that the lowest total concentration (<LOQ) of AFs (AFB1+AFB2+AFG1+AFG2) in A. oxyphylla when the temperature below 25℃ and the humidity was lower than 85%. Additionally, the content and composition of volatile oil changed little. However, the polysaccharides content reduced remarkably. Furthermore, the polysaccharides content of no inoculation A. oxyphylla reduced remarkably under appropriate temperature and humidity. The best storage condition for A. oxyphylla to avoid Aspergillus flavus were temperature and humidity below 25℃ and 85%. Furthermore, the A. oxyphylla could also avoid the content reduce of polysaccharides under the optimized condition. The results in chapter provided the theoretical basis for establishing the mildew prevent system during A. oxyphylla storage. |
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