| Plant hormones are widely used in modern agricultural production. But if the plant hormones are used more than normal standards, they will make human and livestock unhealthy, even cause diseases. Three kinds of hormones used in the production of bean sprouts usually are:Gibberellin(GA3, C19H22O6),6-Benzylaminpurine(6-BA, C12H11N5), 2,4-Dichloor-fenoxy(2,4-D, C8H6C12O3). In this experiment, with the conditions of fixed wavelength and column elution, a high efficiency and fast HPLC method was established which could detect three kinds of hormones of bean sprouts at the same time, then we optimized this method, and made the methodological evaluation. On the basis of before, we drew the security fingerprint about the three kinds of hormones detection of bean sprouts, and verified it. The main results are as follows:1. The optimized HPLC conditions to detect three kinds of hormones are as follows:The ZORBAX SB-C18(4.6×150mm,5um) column; detection wavelength of 200nm; mobile phase:methanol:acetonitrile:water(0.08% formic acid solution)=40:10:50(V:V:V); 35℃ tempreture of the column; flow rate of 1.0mL/min. Under these conditions,GA3,6-BA,2,4-D retention times are 3.557min,5.098min,20.798min respectively, and the theoretical plate numbers are 2916.973,3919.87,5419.053 respectively, the resolutions are 0,4.447,18.327 respectively.the peak are normal.2.Optimized the pretreatment conditions of the sample. After the preprocessing steps was determined preliminary, we chosen 9 main factors to make single-factor experiments, and these factors are:the soaking liquid volume, ultrasonic extraction temperature, ultrasonic extraction time, ultrasonic extraction times, ultrasonic extraction power, centrifugal speed, Petroleum ether extraction times, Ethyl acetate extraction times, rotary evaporation temperature. On the basis of single-factor experiments, the analysis of response surface was used to optimize the method. The optimum pretreatment conditions are as follows:soaking liquid volume is 15mL, ultrasonic extraction temperature is 30℃, ultrasonic extraction time is 25min, ultrasonic extraction times are 4, ultrasonic extraction power is 270W(30% of the total power), centrifugal speed is 4500 r/min, and the Petroleum ether extraction times are3, the Ethyl acetate extraction times are 3, rotary evaporation temperature is 40℃. The recovery rate of GA3 is 78.897%,6-BA is 83.712%, 2,4-D is 95.916%.3.Made the methodological evaluation. The results show that:three kinds of hormones have a good linear relationship in the range of 0.025～0.150ug. The linear equation of GA3,6-BA,2,4-D are:Y=3*106X+370.26(R2=0.9999), Y=7*106X-10249(R2=0.999), Y= 1*107X+1019.4(R2=0.9997); The LOD(The minimum detection limit) are 9.34×10-4ug/L, 3.25×10-4ug/L,1.75×10-4ug/L; The RSD(The relative standtard deviation) results of precision experiment are 0.97%,0.40%,0.36%; The RSD results of repeated experiment are1.80%,1.45%,1.96%; The RSD results of intraday stability are 1.20%,0.49%,0.97%. In the case of adding 40,50,60 mg/kg standards, the recovery results of GA3 are 74.96%,77.12%,.50%, RSD are 1.4%,2.9%,1.7%; the recovery results of 6-BA are 80.58%,80.15%,82.07%, RSD are 2.4%,3.1%,1.2%; the recovery results of 2,4-D are 93.14%,91.01%,94.56%, RSD are2.2%,2.0%,1.9%. The experimental results of samples verify that:in the bean sprouts sold in Ya’an market, the hormone GA3,6-BA are not exceed the standard, but 2,4-D exceed the standard seriously.4.Established the security fingerprint of bean sprouts. Imported the HPLC spectrogram of self-made bean sprouts into the similarity calculation software, used the mean value method, and got the relative fingerprint chromatogram, namely the security fingerprint chromatogram of bean sprouts. The similarity between samples and the relative fingerprint chromatogram are all more than 0.9, can meet the requirement. Under the same conditions, the time of chromatographic peaks is invariant, so we can compare between the security fingerprint and the HPLC spectrograms of bean sprouts which need been detected at the peak times of the three kinds of hormones directly without using these hormones standards, and we can get the results that whether the samples of bean sprouts contain the three kinds of hormones. The samples experimental results are:in 11 samples of bean sprouts, four contain the hormone GA3, one contain the hormone 6-BA. The results shows that the security fingerprint of bean sprouts has the practical value in detecting hormones (GA3,6-BA,2,4-D). |
PDF Full Text Request