Microanalysis And Preliminary Pharmacodynamic Study Of HS203in Beagle Dog Plasma

HS203, a marine oligosaccharide complex of chromium (III) andoligomannuronic acid, which not only had significant inhibitory activity on the fibrilformation of IAPP in vitro，but also significantly improved insulin resistance inDMT2rats and mouse, is a promising candidate for the treatment of type2diabetesmellitus (DMT2).The pharmacokinetic study of HS203is a great challenge due to thelack of a high-sensitivity microdetermination method, especially themicrodetermination method used in biological materials. To detect HS203in beagledog plasma after intravenous injection administration and calculate itspharmacokinetic parameters, a pre-column ultraviolet derivatization HPLC, apre-column fluorescent derivatization HPLC and a post-column fluorescentderivatization HPLC were studied respectively.The optimum degradation condition of HS203was determined by pre-columnderivatization with PMP HPLC as follows: the reaction temperature is110℃, thereaction time is6h and the concentration of TFA is3mol/L. Chromatographicseparation was performed on an Eclipse XDB-C18column with a mobile phase of50mmol/L phosphate buffer (pH6.7) and acetonitrile (83/17, v/v). The endogenoussubstances in plasma did not interfere the detection of the degradation HS203inRP-HPLC analysis under the chromatographic conditions chosen. This method wassuccessfully applied to detect the plasma samples after a single intravenous injectionof60mg/kg of HS203and the pharmacokinetic parameters were calculated. Theresult showed that the plasma concentration-time curve was fit to a two-compartmentmodel.HS203which hydrolyzed into monosaccharide was labelled successfully by2mol/L α-Naphthylamine and1.2mol/L NaBH3CN in acetic acid/methanol (9:1)detected by pre-column derivatization with α-Naphthylamine HPLC. Afterderivatization, the excess reagents were removed by extraction for three times withacetic ether, and then volatilized completely by nitrogen sweeping method with thehelp of ethanol. After plasma protein was removed by a ultrafiltration method, thedegradated HS203, internal standard GalA, and endogenous substances in plasma had a satisfactory resolution monitored by fluorescence detection at249nm (excitation)and435nm (emission) using0.1mol/L phosphate buffer (pH6.7) and acetonitrile(83/17, v/v). These results gave the potential for quantitative microdetermination ofHS203and other marine oligosaccharide drugs.The optimum derivatization condition of HS203was determined by post-columnderivatization with guanidine hydrochloride HPLC as follows: the concentration ofguanidine hydrochloride is200mmol/L, the reaction temperature is125℃and theconcentration of sodium hydroxide is0.5mol/L. The analyte was performed on aShodex Asahipak GS-320HQ column with a mobile phase of50mmol/L phosphatebuffer (pH6.7) and acetonitrile (83/17, v/v), which provided satisfactory resolution ofHS203, internal standard GlcA and endogenous substances in plasma. In thepretreatment procedure, HS203could be released completely from blood plasma byaddition of0.25mol/L sodium chloride solution. The method was successfullyapplied to a pharmacokinetic study of HS203in beagle dog and the data sets were fitto a two-compartment model, which was consistent with that measured by PMPHPLC. This method is highly sensitive, low expenditure of samples, reproducible andhighly accuracy, the limit of detection was found to be0.2μg/mL (more sensitive thansilver staining of HS203in polyacrylamide gel electrophoresis).A pre-column derivatization with PMP HPLC method after HS203degradatedand a post-column derivatization with guanidine hydrochloride HPLC method forHS203was established in this paper, respectively. These two methods weresuccessfully applied to a pharmacokinetic study of HS203in beagle dog. Thepost-column derivatization with guanidine hydrochloride HPLC method whichallowed highly sensitive and selective detection of HS203in plasma samples, was notonly successfully applied to a pharmacokinetic study of HS203in beagle dogs, butalso used to monitor blood glucose level simultaneously after intravenousadministration. These results indicated that post-column derivatization with guanidinehydrochloride HPLC method is very effective for pharmacokinetic andpharmacodynamic study of HS203in blood plasma samples It is also possible to beapplied for the microanalysis of other oligosaccharides in biological samples.