Establishment Of HPLC-ELSD Method For Swainsonine Assay In Locoweeds

Locoweed toxins, swainsonine and nitric oxide swainsonine, are the causation oflocoweed poisoning in China. Swainsonine have three hydroxyl groups at1,2,8positions andit is very hydrophilic and high molecular polarity, so it can not be retained in the conventionalchromatography. It is an azabicyclo chemical compound in the form of a heterocyclic ringwhich contains the piperidine ring and pyrrole ring. There exists some difficulty in applyingswainsonine by using routine detector such as UV-vis detector because it is too weakabsorption in the ultraviolet region for SW to take spectrographic analysis. There is norfluorophore either. A variety of methods are established to apply swainsonine such as gaschromatography with FID detector detects, fluorescence method, enzymatic analysis method,TLC method, enzyme label method, ELISA method, etc. But they are insufficient because ofthe cumbersome measurement step and poor stability. Therefore, this study intended todevelop a sensitive swainsonine detection method which can be easy to use and to providebasic technical support for further purification and preparation research of swainsonine,biological activity, animal locoweed poisoning and non-toxic and harmless research.1. Optimization of HPLC-ELSD chromatographic systemThe samples were extracted with ethyl ethanol, purified with solid phase extractioncolumn and then analyzed by a Waters X-Bredge HILIC column and swainsonine wasdetected by evaporative light scattering detector. The separation property and separationmechanism of the HILIC column to swainsonine were investigated by changing kinds oforganic solvent and concentration, salt concentration and column temperature. Methanol,acetonitrile and other common organic solvents with water or buffered saline are used as themobile phase. We adjust the ratio of buffer concentration, pH value, replace the column typeto find the best HPLC-ELSD chromatographic conditions. The parameters of ELSD are alsoadjusted to find the best testing conditions.2. Method validation of HPLC-ELSDThe method was validated in agreement with the International Conference onHarmonization Guidelines (ICH,1996) with regards to its specificity, linearity, limits of detection (LOD), limits of quantification (LOQ), accuracy and precision. The calibrationcurve was linear within the range of10-100μg/mL (R2=0.997), the detection limit was6μg/mL and the limit of quantitation was20μg/mL. And the recovery rate was over80%.Locoweeds SW was eluted as a defined peak at retention time of7.3min and its purity waschecked by HPLC/MS.3. Optimization of sample pretreatment methodUse different solutions such as methanol, ethanol, acetonitrile, and an acidic aqueoussolution as the extraction solution. Locoweeds samples were extracted by continous refluxsoxhlet extraction method then adsorbed with the cation exchange resin method or solid phaseextraction method such as C18-SPE, PestiCarb-SPE, PCX-SPE, etc. for purification. The bestsample pretreatment method for HPLC-ELSD detection was found to in contrast to thetraditional pre-treatment methods.