Investigation and Application of High Performance Liquid Chromatography with Chemiluminescence Detection in Pharmaceutical Analysis

Abstract: This thesis describes the application of high performance liquidchromatography (HPLC) with chemiluminescence (CL) detection in pharmaceuticalanalysis, which mainly contains two parts.The first part was a review that introduced high performance liquidchromatography technique, chemiluminescence technique and HPLC-CL technique.Among them, technique of HPLC coupling with CL detection was mainly reviwed, inwhich much information about principles, equipment, the main chemiluminescencereaction systems, the application and development of HPLC-CL was summarized.The second part is research reports, which are composed of four parts as follows.(一) Determination of icariin using high-performance liquid chromatographywith chemiluminescence detectionIn alkaline solution, the complex of cobalt (Ⅱ) with icariin can greatly catalyze thesignal produced by the chemiluminescent reaction (CL) between luminol and H_2O_2.Based on this, a novel HPLC-CL detection method for determination of icariin wasdeveloped. The proposed method has been applied satisfactorily to the determination oficariin in Anshenbunaoye. The linear range of icariin concentration was 0.5-20μg/mL(r=0.9986) and the detection limit was 2×10~(-7) g/mL (3σ). The relative standarddeviation was 4.5 %(c=2×1010~(-6) g/mL, n=11).(二) Determination of propylthiouracil and methylthiouracil in human serumusing high-performance liquid chromatography with chemiluminescence detectionBased on the sensitizing effect of formaldehyde on the chemiluminescence (CL)reaction of propylthiouracil (PTU) and methylthiouracil (MTU) with acidic potassiumpermanganate and the combination technique of high-performance liquidchromatography (HPLC), a sensitive, selective and simple post-column CL detectionmethod for determining PTU and MTU is described. The optimal conditions for the CLdetection and HPLC separation were carried out. The linear ranges are: 0.2-20μg mL~(-1)for MTU, 0.2-10μg mL~(-1) for PTU, respectively, the detection limit of MTU and PTUwere 0.08μgmL~(-1) respectively, and the relative standard deviation with a concentrati on of 1.0×10~(-6) g/mL was 4.3% for MTU and 3.9% for PTU, respectively (n=11).Themethod has been satisfactorily applied to the determination of MTU and PTU inpharmaceutical preparations and human serum samples.(三) Simple method for determination of arbutin and L-ascorbie acid inwhitening cosmetics by high-performance liquid chromatography withchemiluminescence detectionThe simultaneous determination of arbutin (ART) and L-ascorbic acid (AA) bymeans of HPLC with CL detection is proposed for the first time. This method is basedon the CL reaction of the acidic potassium permanganate with ART and AA in thepresence of formaldehyde as an enhancer. The separation was carried out on NucleosilRP-C_(18) column using a mixture of 0.02 M phosphate buffer and methanol (90:10, v/v)as a mobile phase. The effects of several parameters on the HPLC resolution and CLemission were studied systematically. The linear ranges were: 0.5-50μg mL~(-1) for ART,1-200μg mL~(-1) for AA, respectively.The detection limits are: 0.2μg mL~(-1) for ART, 0.3μg mL~(-1) for AA, respectively and the relative standard deviation with a concentration of1.0×10~(-6) g/mL was 3.7% for ART and 4.5 % for AA, respectively (n=11). The methodwas applied successfully to the determination of ART and AA in whitening cosmetics.(四) Determination of Piperazine ferulate using high-performance liquidchromatography with chemiluminescence detectionIn alkaline solution, Piperazine ferulate can greatly inhibited the signal producedby the chemiluminescent reaction between luminol and NaIO_4.Based on this, a novelmethod for determination of Piperazine ferulate was developed. The separation wascarried out on Nucleosil RP-C_(18) column using a mixture of methanol and water (35:65,v/v) as a mobile phase. The linear range of Piperazine ferulate concentration was 1-20mg/L and the detection limit was 3×10~(-7) g/mL (3σ). The relative standard deviation was3.0 %(c=2.0×10~(-6) g/mL, n=11)…
Key words: High Performance Liquid Chromatography ; Chemiluminescence; Pharmaceutical Analysis

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