Establishment Of UPLC-Q-TOF Method For Qualitative And Quantitative Determination Of Alkaloids Compound And Content Analysis Of Alkaloids Compound In Dendrobium Officinale In Yunnan

Objective:Ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry（UPLC-Q-TOF）was used to establish a characteristic spectral library and a qualitative and quantitative detection of alkaloid compounds in Dendrobium officinale.By optimizing the ultrasonic extraction method of 7 alkaloids compounds in Dendrobium officinale,the content detection and analysis of 7alkaloids in 8 prefectures（cities）of Yunnan Province,so as to provide data support for the industrial development of Dendrobium officinale in Yunnan Province.Methods:UPLC-Q-TOF was used to establish the primary and secondary characteristic spectral libraries of seven Dendrobium officinale alkaloids and optimized the MS conditions,further comparing the column,column temperature,flow rate,mobile phase and its elution gradient to establish the detection method.In terms of sample processing,based on single factors,three factors were selected by the extreme difference method,designed orthogonal tests for liquid ratio,ultrasonic time,ultrasonic temperature and other factors,and scored with the total content of alkaloid compounds as the evaluation index to finally determine the best extraction method for the sample.The established UPLC-Q-TOF method was used to quantify the alkaloid compounds in Dendrobium officinale in Yunnan Province,and the seven alkaloid compounds in Dendrobium officinale were analyzed by spss 20.0.Results:1.The MS conditions and the liquid chromatographic separation conditions were determined.UPLC-Q-TOF was established for quantitative detection of dendrobine,lauramide,erucyl amide,N-trans cassia bark benzamide,N-trans-coumaroyl tyramine,N-trans-ferulosyl tyramine,N-feruloyltyramine,Method can be completed within 8minutes,Seven alkaloid compounds showed a good linear relationship in the linear range（r>0.9912）,The detection limit was from 0.05 to 0.2μg·kg^-1,Limit of quantification 0.1 to 0.5μg·kg^-1.The average sample recovery rate（n=6）≥84.62%,the relative standard deviation≤5.76%,and the precision≤5.02%..The proposed method is fast,stable,and reliable.2.Using orthogonal test to compare the extraction efficiency of different material liquid ratio,different ultrasonic time and different ultrasonic temperature,determine the optimal conditions for the sample are:ethanol concentration 100%,ultrasonic time50 min,material liquid ratio 1:40,ultrasonic temperature 40℃,to extract the supernatant over PTFE（PTFE,13 mm diameter,0.2μm）filter membrane.3.In 123 dendrobium officinale in 8 cities of Yunnan Province,dendrobium and lauramide were not detected.The content of erucyl amide was 0.002～0.158 mg·kg^-1,the N-trans cassia bark benzamide was 0.146～1.018 mg·kg^-1,the N-trans-coumaranyl tyramide was 0.182～1.132 mg·kg^-1,the N-trans-ferulosyl tyramine was 0.081～3.573mg·kg^-1,and the N-feruloyltyramine was 0.101～3.613 mg·kg^-1.Conclusions:1.In this study,UPLC-Q-TOF was used to establish a qualitative and quantitative detection method of seven alkaloid kind of compounds in Dendrobium candidum,using 0.1%ammonia-methanol with a flow rate of 0.30 m L·min^-1,column temperature of 30℃,and injection volume of 2μL,and seven compounds were separated in a C₁₈column.In this method,the sample measurement time is only 8 min,which shortens the detection time and improves the efficiency.With high sensitivity,good stability and high recovery rate,this method can meet the determination requirements of seven alkaloid kind of compounds in Dendrobium officinale.2.The study determined the optimal pretreatment conditions for alkaloids in Dendrobium officinale as follow:the ethanol concentration was 100%,the liquid ratio is 1:40;ultrasonic time 50 min;ultrasonic temperature 40℃,extract the supernatant through 0.2μm PTFE membrance.3.For the first time,seven alkaloids were tested and evaluated for Dendrobium officinale in eight different regions in Yunnan Province.According to the statistical analysis results,dendrobium and lauramide were not detected,the total content of alkaloids in Dendrobium officinale was the highest in Wenshan,with the value of2.517 mg·kg^-1,and the lowest total content was in Honghe,with the content of 1.124mg·kg^-1.The highest content of erucyl amide was in Dehong with a value of 0.055mg·kg^-1,and the lowest content was in Kunming with a value of 0.009 mg·kg^-1.The highest content of N-trans cassia bark benzamide was in Wenshan with a value of0.359 mg·kg^-1,which was not detected in Honghe and Pu’er.The highest content of N-trans-coumaranyl tyramide was Wenshan with a value of 0.448 mg·kg^-1,and the lowest was Honghe with a value of 0.104 mg·kg^-1.The highest content of N-trans-feruloyl tyramine was in Dehong with a value of 0.974 mg·kg^-1,and the lowest was in Pu’er with a value of 0.488 mg·kg^-1.The highest content of N-feruloyl tyramine was Dehong with a value of 0.983 mg·kg^-1,and the lowest in Pu’er and Honghe with a value of 0.494 mg·kg^-1.