The Establishment And Application Of Method For Detecting Bioactive Substance Of Micro-quantity By Capillary Electrophoresis

The capillary electrophoresis (CE) is a separating andanalyzing method, which can be applied to detect organiccompound, inorganic ion, neutral molecule, protein andpolypeptide, DNA and nucleic acid fragments and so on. Thismethod is characterized by high automation, resolution andsensitivity. In present study, the methods for detectingmicro-quantity protein and the effectiveingredients----benzofuranone of traditional Chinese drug wereestablished by different separation models and applied to detectpractical samples.1 The establishment and application of the method fordetecting microprotein by non-gel sieving capillaryelectrophoresisThe method for detecting microprotein was established bynon-gel sieving capillary electrophoresis (NGSCE), usinguncoated fused-silica capillary and polyethlene glycol20000(PEG20000) as sieving medium. Osteopontin (OPN)secreted by vascular smooth muscle cell (VSMC) was detectedby this method.1.1 The establishment of NGSCEThe nature, pH and concentration of electrode buffer andsieving medium, running voltage, quantity of injecting sampleand the effective length of uncoated fused-silica capillary wereoptioned by experiments in order to detect OPN. The PDAdetector was used at 214 nm. 150 mmol/L borate buffercontaining 40 g/L PEG20000 was used as electrode buffer andpressure injection from positive electrode was employed for 5seconds, the electrophoresis was performed at 25℃and 23 kVrunning voltage [(+)→(-)] applied to 57cm×75μm inwarddiameter (50cm effective length) uncoated capillary. Theexperimental results indicated that PEG20000 was excellentsieving medium and elevated pH value and ionic strength ofelectrode buffer can effectively eliminate adhesion of protein onuncoated capillary wall.1.2 Assessment of methodThe reproducibility test indicated that the relative standarddeviation (RSD) of OPN migration time (MT) was from 2.02%to 2.3% in three intra-batches, and RSD of OPN MT ininter-batch was 2.16%, which was less than 5%. Sensitivity testand linear relative analysis suggested that linear ranges were0.079-2.5 mg/ml, that linear regression of OPN concentrationand peak area was y=39019x+1193.8, that the coefficient ofproduct-moment correlation was 0.996 and that the limits ofdetection were 0.079 mg/ml. The recovery test proved that therecovery rate was more than 95%. All the results demonstratedthat reproducibility was good, the detection limits and recoveryrate could satisfy detection of microprotein.1.3 Effect of serum deprivation on OPN secreted by VSMCsDedifferentiated VSMCs were induced to redifferentiate byserum deprivation. The change of OPN synthesized and secretedby VSMCs was determined by NGSCE during redifferentiating.The result suggested that OPN secreted by VSMCs reached thehighest peak at 24 h after serum was withdrawed, thendecreased with time prolonged, and restored to control leveluntil 72 h.1.4 Effect of angiotensinⅡ(AngⅡ) on OPN secreted byVSMCsThe culture medium was collected after VSMCs werestimulated by AngⅡfor 3 h, 6 h, 12 h, 24 h and 48 h. OPN inthe culture medium was determined by NGSCE. The resultsshowed that AngⅡstimulation dramatically induced theexpression of OPN. OPN synthesized and secreted by VSMCsreached high peak at 24 h, and maintained the high level until 48h after stimulation by AngⅡ.2 Detection of Flos Inulae ingredients and its dynamicchange in vivoThe micellar electrokinetic capillary chromatography(MECC) is a kind of capillary electrophoresis which cansimultaneously separate the neutral and charge substances. Themethod was established to detect the benzofuranone compoundby MECC, in which the sample stacking technique was appliedto elevate the limits of the detection. This method was applied todetect ingredients in the effective part of Flos Inulae and itsdynamic change in vivo.2.1 The establishment of MECCAn ideal condition of MECC was established byinvestigating the effect of the concentration and pH of electrodebuffer, concentration of SDS, and running voltage on sampleresolution. The optional condition of MECC was as follows:The PDA detector was used at 195 nm. The sample buffer waspH 9.5, 10 mmol/L boric acid-borate. 50 mmol/L borate buffercontaining 50 mmol/L SDS was used as electrode buffer, andpressure injection from positive electrode was carried out for 5seconds, the electrophoresis was performed at 25℃and 23 kVrunning voltage [(+)→(-)] applied to 57cm×75μm inwarddiameter (50cm effective length) uncoated fused-silica capillary.The results demonstrated that pH of electrode buffer and SDSconcentration were key factors that affected sample stacking.The sweeping technique can dramatically elevate the sensitivityof detection.2.2 Assessment of methodThe reproducibility test indicated that the relative standarddeviation (RSD) of ABL migration time (MT) was 2.82%,2.94% and 3.90%, respectively, in three intra-batches, and RSDof ABL MT in inter-batch was 3.27%, which was less than 5%.Sensitivity test and linear relative analysis suggested that linearranges were 0.005-1.5 mg/ml, that linear regression ofacetylbritannilactone (ABL) concentration and peak area wasy=9220099x+21894, that the coefficient of product-momentcorrelation was 0.997, and that the limits of detection were0.005 mg/ml ABL. The recovery test proved that the recoveryrate was more than 95%. All the results demonstrated thatreproducibility was excellent, and that the detection limits andrecovery rate could satisfy detection of microquantity organiccompound.2.3 Detection of ingredients in the effective part of Flos Inulaeand its dynamic change in vivoThere were 5 peaks detected by MECC in the effectivepart of Flos Inulae (inulicin). Peak 3 was identified to be ABLby intra-standard and spectrum analysis. Concentration of ABLwas 2.97 mg/ml by standard curve for ABL concentration andpeak area.Dynamic change of ABL was detected by MECC after ratswere administered the effective part of Flos Inulae. The resultsshowed that the concentration of ABL in blood reached highpeak at 1.5 h, then progressively declined in blood as the timeprolonged after intragastric administration with innulicin. Theform of peak varied dramatically after 6 h, suggested that ABLin rat's body had been converted.Conclusions1. A rapid and simply method for detecting miroprotein wasestablished by non-gel sieving capillary electrophoresis. Inpresent study, this method was applied to determine the effect ofdifferent stimulating factors on osteopontin synthesis and...