Determination Of Alkaloid In Three Natural Product By Liquid Chromatography-Tandem Mass Spectrometry

In this thesis, we elaborate the pharmacological action of effective constituent of alkaloid compounds from six aspects, and also comment the extraction and separation technology of effective constituent of alkaloids. The advantages and disadvantages of different extraction methods has been pointed out, moreover, the analysis research of the alkaloids has been summarized, at last, the defects of current testing method has been pointed out by comparing of characteristic of different detection methods.In view of the present determination of active components in alkaloids, and on the basis of their chemical structures, we successfully combined high performance liquid chromatogrphy (HPLC) analysis with mass spectrum (MS) detection technology, depending on the high performance and resolution of HPLC, and high sensitivity and selectivity of MS. we established several new methods of HPLC-MS/MS with electrospray ionization interface (ESI) for the determination of alkaloids. The methods were proved to be sensitive and selective, and could be used for the quality control of Chinese traditional medicines containing alkaloids in the future.In this research, the optimal mass spectrometry and chromatography analysis conditions have been discussed in details. The chromatography has been selected by the research of stationary phase, mobile phase, column temperature and flow rate. The mass spectrometry has been selected by the research of ionization mode, spray needle voltage, drying gas temperature, capillary voltage and collision energy. Finally, the corresponding alkaloid compounds separation analysis methods has been established as follows:(1) Research on alkaloids of coptis chinensis. Object:A liquid chromatography-tandam mass spectrometric (LC-MS/MS) method was developed for determination of a jatrorrhizine, coptisine, palmatine and berberine in coptis chinensis. Method:The separation was performed on a Ultimate XB-C18(150mm×2.1mm,3micron) column by using the elution with10mM ammonium acetate solution (2%ammonium acetate) and acetonitrile. Flow rate was0.2mL·min-1and column temperature was35℃. The alkaloids were determined by electrospray ionization-tandem mass spectrometry analyzed in multiple reaction monitoring (MRM) mode. The following parameters of mass spectrometry:positive ion mode; spray needle voltage,4500V; drying gas temperature,300℃; drying gas voltage,35psi; collision gas(Ar) pressure,1.5mT. Results:Quasi-molecular ion peak [M+H]+=339.1for jatrorrhizine;320.9for coptisine;353.1for palmatine and337for berberine. The calibration curves showed good linearity in0.01～50.0mg·L-1for coptisine;0.05～50.0mg·L-1for palmatine;0.01～20.0mg·L-1for palmatine and0.05～50.0mg·L-1for berberine. For regression equations with the correlation coefficients of jatrorrhizine, coptisine, palmatine and berberine as follows:y=139718x-101208, R2=0.9993; y=53117x-4296, R2=0.9994: y=1598570x-933627, R2=0.9993; y=127291x-2514, R2=0.9999. The limits of detection were1μg·L-1、6μg·L-1、0.2μg·L-1and0.1μg·L-1. The recoveries of samples were98.6%、99.8%、98.5%and101.0%. The relative standard deviation were2.58%,1.96%,2.22%and3.72%(n=6). Conclusion:the established method has high selectivity, rapid qualitative and quantitative analysis, and also it has been applied to determine alkaloids in coptis chinensis with satisfactory results.(2) Research on alkaloids of tea. Object:A liquid chromatography-tandam mass spectrometric (LC-MS/MS) method was developed for determination of a theophylline. theobromine and caffeine in tea. Method:The separation was performed on a Ultimate XB-C18(150mm×2.1mm,3micron) column by using the elution with0.5%formic acid aqueous solution and acetonitrile. Flow rate was0.2mL·min-1and column temperature was35℃. The alkaloids were determined by electrospray ionization-tandem mass spectrometry analyzed in multiple reaction monitoring (MRM) mode. The following parameters of mass spectrometry:positive ion mode; spray needle voltage,4500V; drying gas temperature,300℃; drying gas voltage,35psi; collision gas(Ar) pressure,1.5mT. Results:Quasi-molecular ion peak [M+H]+=180.9for theobromine;180.9for theophylline and194.9for caffeine. The calibration curves showed good linearity in0.05～50.0mg·L-1for theobromine;0.01～20.0mg·L-1for theophylline and0.005～20.0mg·L-1for caffeine. For equations with the correlation coefficients of theobromine, theophylline and caffeine as follows:y=154107x-90789, R2=0.9991; y=145178x-77282, R2=0.9989; y=112287x+693, R2=0.9996. The limits of detection were27μg·L-1and0.8μg·L-1. The recoveries of samples were99.3%,99.9%and98.9%. The relative standard deviation were2.42%,2.78%and2.48%(n=6). Conclusion:the established method has high selectivity, rapid qualitative and quantitative analysis, and also it has been applied to determine alkaloids in tea with satisfactory results.(3) Research on alkaloids of radix sophorae flavescentis. Object:A liquid chromatography-tandam mass spectrometric (LC-MS/MS) method was developed for determination of matrine and oxymatrine in radix sophorae flavescentis. Method:The separation was performed on a Ultimate XB-C18(150mm×2.1mm,3micron) column by using the elution with10mM ammonium acetate(pH=3with formic acid) solution and acetonitrile. Flow rate was0.2mL·min-1and column temperature was30℃. The alkaloids were determined by electrospray ionization-tandem mass spectrometry analyzed in multiple reaction monitoring (MRM) mode. The following parameters of mass spectrometry:positive ion mode; spray needle voltage,4000V; drying gas temperature,300℃; drying gas voltage,35psi; collision gas(Ar) pressure,1.5mT. Results:Quasi-molecular ion peak [M+H]+=249.1for matrine and265.1for oxymatrine. The calibration curves showed good linearity in0.005～20.0mg·L-1for matrine and0.01～20.0mg·L-1for oxymatrine. For regression equations with the correlation coefficients of matrine and oxymatrine as follows:y=260663x+33205, R2=0.9994, and y=1598570x+236468, R2=0.9990. The limits of detection were0.7μg·L-1and0.8μg·L-1. The recoveries of samples were100.2%and99.8%. The relative standard deviation were2.29%and2.22%(n=6). Conclusion:the established method has high selectivity, rapid qualitative and quantitative analysis, and also it has been applied to determine alkaloids in radix sophorae flavescentis with satisfactory results.