Determination Of Epigoitrin In Radix Isatidis,Icariin In Ruzengning Capsule And Chlorogenic Acid In Shuanghuanglian Capsule By Solid Phase Extraction-Quantitative Nuclear Magnetic Resonance Spectroscopy

The determination of the active ingredients is a key point to the development of traditional Chinese medicine. Without the high purity standard compound of the test substance, quantitative nuclear magnetic resonance (qNMR) can analyze sample accurately and fastly via using several common internal standards and deuterated reagents. But the detection sensitivity and interference are its shortcomings. Solid phase extraction (SPE), as an effective sample pretreatment technology, with sample enrichment and purification function, is easy to operate and have high recovery rate. Combined SPE with qNMR will extend qNMR application in the low content components in complex sample.In the thesis, the SPE-qNMR method for the determination of active ingredients epigoitrin in Radix Isatidis ,icariin in Ruzengning capsule and chlorogenic acid in shuanghuanglian capsule were established. And the established method was validated. Without the high purity standard compounds, we determination of epigoitrin, icariin and chlorogenic acid in three kinds of traditional Chinese medicine by SPE-qNMR. The SPE is suggested as extender for qNMR application in the low content components in complex sample.1. The method of SPE-qNMR for the determination of epigoitrin in Radix Isatidis was established. The ultrasonic extraction method was double used for fully extracting epigoitrin in sample using pure water as solvent at 60?, and then the extracting solution was enriched and cleaned by Poly-Sery MCX SPE cartridge. The effect of ultrasonic extraction, sample pretreatment conditions via SPE and qNMR experimental conditions were investigated. The qNMR experiment conditions were selected using deuterated DMSO as solvent, calibrated 2,3,5-triiodobenzoate as internal standard, and Pl(pulse width) = 14.4?s, dl(pulse delay time)=5s, NS(number of scan) = 256. The 1H-NMR peaks of 85.365-5.399(H-7b, d, 1H) of epigoitrin was chosen as the quantitative peaks. Method validation was performed including precision (the intra-day precision RSD was 0.47%, and the inter-day precision was 0.80%); linearity (correlation coefficient r > 0.9991); LOD (0.05 mg/g) and LOQ (0.19 mg/g). The recovery of the SPE-qNMR was in 97.4%?101.7%. The determination of epigoitrin in a real Radix Isatidis was in <0.19-1.26 mg/g.2. The SPE-qNMR method for the determination of icariin in Ruzengning capsule was established. The ultrasonic extraction method was used for fully extracting icariin in sample using 10% alcohol as solvent at room temperature, and then the extracting solution was enriched andcleaned by HC-C18 SPE cartridge. The effect of ultrasonic extraction, sample pretreatment conditions via SPE and qNMR experimental conditions were investigated. The qNMR experiment conditions were selected using deuterated DMSO as solvent, calibrated 2,3,5-triiodobenzoate as internal standard, and P1(pulse width) = 14.4?s, dl(pulse delay time)=Is, NS(number of scan) = 256. The 1H-NMR peaks of ?7.890?7.909(2',6'-H,d,2H) of icariin was chosen as the quantitative peaks. Method validation was performed including precision (the intra-day precision RSD was 0.43% , and the inter-day precision was 0.75%); linearity(correlation coefficient r > 0.9999); LOD (0.122 mg/g) and LOQ (0.368 mg/g). The recovery of the SPE-qNMR was in 99.9%?102.9%. The result shows the method stable, accurate and reliabile.The determination of icariin in a real Ruzengning was in 3.947?4.392 mg/g.3. The SPE-qNMR method for the determination of chlorogenic acid in Shuanghuanglian capsule was established. The ultrasonic extraction method was used for fully extracting epigoitrin in sample using pure water as solvent at room temperature, and then the extracting solution was enriched and cleaned by HC-C 18 SPE cartridge. The effect of ultrasonic extraction, sample pretreatment conditions via SPE and qNMR experimental conditions were investigated. The qNMR experiment conditions were selected using deuterated DMSO as solvent, calibrated 1,4-phthalaldehyde as internal standard, and P1 (pulse width) = 14.4?s, d1 (pulse delay time) = Is,NS(number of scan) = 512. The 1H-NMR peaks of 66.149-6.182(H-8' d, 1H) of chlorogenic acid was chosen as the quantitative peaks. Method validation was performed including precision (the intra-day precision RSD was 1.2% , and the inter-day precision was 1.5% ); linearity (correlation coefficient r > 0.9999); LOD (0.0017mg/g) and LOQ (0.0791mg/g). The recovery of the SPE-qNMR was in 101.3%?104.4%. The result shows the method stable, accurate and reliabile.The determination of chlorogenic acid in a real Shuanghuanglian was in 9.68?10.35 mg/g.