Establishment Of Highly Sensitive Analytical Method For Chondroitin Sulfate And Preliminary Study Of Pharmacokinetics

Chondroitin sulfate is a type of glycosaminoglycans formed by a chain of repeated disaccharide units consisting of glucuronic acids and N-acetylgalactosamine,which is covalently attached to proteins and widely distributed on cartilage tissue and cell membranes.Researches found that chondroitin sulfate has a variety of biological activities and pharmacological effects.Now it is mainly used as a health product for oral or injectable use,and is mainly used in the treatment of osteoarthritis internationally.Due to the large and wide range in molecular weight,and the lack of chromophores in the structure of chondroitin sulfate,the detection and analysis for chondroitin sulfate is always difficult and a bottleneck problem.Limited by analytical techniques in vivo,the pharmacokinetics process for chondroitin sulfate is ambiguity at present,which restricts partly chondroitin sulfate to be developed into more dosage form to exert its more pharmacological activities.Therefore,it is great significant to establish a highly sensitive analytical method in vivo and to ascertain the pharmacokinetics process for the further development and application and rational use of chondroitin sulfate in clinical.The specific contents of this study are as follows:1.A capillary electrophoresis method for analyzing chondroitin sulfate has been established based on online and offline enrichment techniques.The optimal capillary electrophoresis conditions:the capillary was a fused silica capillary with an inner diameter of 75 ?m,an effective length of 40 cm and a total length of 49 cm.The detection wavelength was 200 nm and the electrophoresis temperature was 25?.The buffer solution was 20 mmol/L sodium dihydrogen phosphate-90 mmol/L ethylenediamine(pH 3.0).The separation voltage was-15 kV.The online enrichment using field amplification and large sample injection combining with offiine enrichment using cetyltrimethylammonium bromide was used,the enrichment effect is good and the method validation meet the requirements.2.A flow injection chemiluminescence method for the determination of chondroitin sulfate was established.The optimum conditions were as follows:pump speed was 3 ml/min,pipeline length was 30 cm,the concentration of luminol,sodium hydroxide and hydrogen peroxide was 0.2 mmol/L,0.02 mol/L and 0.05mol/L respectively.The luminol stock solution was used after standing time for 7 days.Under optimal conditions,the concentration range of chondroitin sulfate in standard curve was 25 ng/mL?250 ng/mL,the limit of detection was 5 ng/mL,and the precision was 2.5%(n = 6).Compared with the pharmacopoeia method,the method is simple,rapid,accurate,sensitive and specific,and is suitable for the determination of chondroitin sulfate.3.The pharmacokinetics of chondroitin sulfate was studied using established high-sensitivity method.Optimal plasma and tissue sample processing methods were determined.The plasma concentration-time curve was established and the pharmacokinetic parameters were obtained.The results showed that chondroitin sulfate was rapidly absorbed into the blood after intragastric administration in rats,and reached a peak in half an hour.The half-life of chondroitin sulfate was prolonged with the increase of the dose,and the bioavailability increased with the dose.The pharmacokinetic parameters were accorded with the two-compartment model.The kinetics of chondroitin sulfate in vivo is non-linear within the range of dosage.The results of tissue distribution experiment showed that the drug was quickly distributed to knee cartilage,heart,liver and spleen within 1 hour,and then gradually distributed to the lung and kidney tissues.The content of drugs in tissues and organs all showed a downward trend after 4-12 h.Excretion test results showed that the drug was mainly excreted by feces at 0-12 h,and was mainly excreted by urine at 12-24 h.After 24 h,only a small amount was excreted through urine and feces.In summary,the innovations of this study are as follows:1.A capillary electrophoresis method for the determination of chondroitin sulfate based on on-line and off-line enrichment techniques was established for the first time.The enrichment factor was 130 times and the limit of detection was 50 ng/mL.2.It was first discovered that the products from enzymatic hydrolysis of chondroitin sulfate can enhance the luminescence intensity of luminol-hydrogen peroxide system.A simple,rapid and highly sensitive flow injection-chemiluminescence method was developed.3.The preliminary research results about pharmacokinetic process and characteristics of chondroitin sulfate were obtained using the established high-sensitivity method,which provides an experimental basis for the clinical rational use of drugs and the further investigation of chondroitin sulfate pharmacokinetics.