The Application Of Original Technique In Capillary Electrophoresis For Pharmaceutical Analysis

Capillary electrophoresis (CE) and high performance liquid chromatogram (HPLC) are recent segregation analysis techniques and each is the complement for the other to a certain extent. However, on separation mechanism, demand for sample preparation and on-line concentration, as the complement for HPLC, CE exhibits distinct superiorities, settles problems which HPLC is hard to solve and obtains extensive application in pharmaceutical analysis. For compounds with strong complexation groups, because they can complex with metal cations left in chromatographic column, peak broadens, tails and even separation effect is weakened. The separation mechanism of CE is different from HPLC and it is the combination of classic electrophoresis and modern microcolumn technique. The mechanism of CE is based on the fast and efficient separation of charged particles with different mobilities in capillary column under the effect of electric field. The drive of electric field can efficiently decrease diffusion of target analyte in capillary, increase separation efficiency and improve peak tailing. The separation efficiency of HPLC relays on particle size, uniformity and porous structure and sample preparation has a great effect on chromatographic column, therefore, a careful and complicate sample preparation procure is usually needed when using HPLC. Separation by CE mainly depends on sample characteristic and the separate channel is the hollow quartz capillary, so there is a low demand on sample pretreatment. In addition, there is no on-line concentration by HPLC and high sensitivity is obtained by concentration with off-line pretreatment. However, there are many on-line concentration modes with CE that not only educe the advantages of CE, such as high separation efficiency and fast analytical speed, but also can realize dual concentrations with on-line and off-line. CE with on-line concentration possesses a comparative or even predominant sensitivity compared to HPLC and is used widely in trace analysis.The purpose of our study is to adopt original CE techniques, such as micellar electrokinetic capillary chromatography, separation using hollow fiber combinding with capillary zone electrophoresis and field-amplified sample stacking with CE to settle problems which traditional methods are hard to solve. Many shortcomings raised by conventional methods are overcome and experiment procedures are simplified. These techniques provide new, convenient and pragmatic analytical means in pharmaceutical quality control. Specific studies are as follows: (1) When HPLC is used for the determination of ciclopirox olamine, peak tails badly because of the strong chelating effect between hydroxyl groups in molecular constitution and metal cations in solvent, reagent and chromatographic column. Peak tailing can not be improved even though the chromatographic column is rinsed with abluent and mobile phase for 20 h. Therefore, HPLC can only be used for determination of ciclopirox olamine related compounds, however, ultraviolet spectrophotometry is usually employed for the assay of ciclopirox olamine. In our previous work, a micellar electrokinetic capillary chromatography method is described for the simultaneous analysis of the drug and its forced degradation products of ciclopirox olamine based on the difference in separation mechanism between CE and HPLC. This method is simple, rapid with high separation efficacy and it provides a new, convenient and pragmatic analytic means for ciclopirox olamine in fast quality control in process of pharmaceutical production. (2) Separation with hollow fiber and determination benzalkonium bromide in Hhypromellose Eye Drop by capillary zone electrophoresis is developed by combining original off-line sample pretreatment using hollow fiber with on-line detection employing capillary zone electrophoresis. The hollow fiber with U-shape configuration which contains sample solution was placed in the centrifuge tube vertically. Under the effect of centrifugal force, the hollow fiber allows micromolecule benzalkonium bromide pass through membranous wall, while macromolecule hypromellose is interrupted at the outside of the membranous wall and macromolecule filtrate is removed. Capillary zone electrophoresis is directly employed to determinate the assay of benzalkonium bromide in Hhypromellose Eye Drop. Some disadvantages caused by traditional methods, such as tedious, time-consuming, poor selectivity and short column life are overcome and it provides a new, simple and green analytic means for the separation and analysis of benzalkonium bromide. (3) Field-amplified sample stacking with CE is developed for the determination of trace effective toxical composition in commonly used Chinese drugs preparation by simple regulations to sample solution, running buffer and injection procedure. The detection sensitivity increases by above 400 folds. This method is economic, fast and possessing comparative sensitivity compared to HPLC. (4) Field-amplified sample stacking with CE is developed for simultaneous determination of trace strychnine and brucine residues in human urine. This method is simple, fast and economic with high enrichment efficiency and it can be used for preliminary screening of drug abuse in the doping control and forensic toxicology laboratories.PART 1 Fast and simple method for assay of ciclopirox olamine by micellar electrokinetic capillary chromatographyObjective: to establish a micellar electrokinetic capillary chromatography method for fast separation ciclopirox olamine and its degradation products and determination of ciclopirox olamine in pharmaceutical formulations.Methods: An uncoated fused-silica capillary column (50 μm i.d.×45 cm, effective length 38 cm) was used. The running buffer contained 200 mmol·L^-1 borate, 20 mmol·L^-1 SDS and 2 mg·mL^-1 EDTA at pH 8.0. A constant voltage of 20 kV was applied and the UV detector was set at 298 nm. Samples were injected by liquid differential pressure with a height of 10 cm for 5 s.Results: A good separation between the drug and its forced degradation products of ciclopirox olamine was achieved. The total analysis time was less than 10 min with good peak shape for ciclopirox olamine, which eluted at 3.6 min. The standard curve of ciclopirox olamine was linear over the range of 31.3-2.00×103μg·mL^-1 (r=0.9999), with the limit of detection 9.39μg·mL^-1. The average recovery of ciclopirox olamine in ciclopirox olamine suppository was 99.7% with RSD of 0.9%. The average recovery of ciclopirox olamine in ciclopirox olamine cream was 100.3% with RSD of 1.4%.Conclusion: The method is simple and rapid with high separation efficiency and it can be used for fast quality control in process of pharmaceutical production.PART 2 Separation with hollow fiber and determination benzalkonium bromide in Hhypromellose Eye Drop by capillary zone electrophoresis Objective: A simple and economic pretreatment method using hollow fiber was developed for direct determination of benzalkonium bromide in Hhypromellose Eye Drop by capillary zone electrophoresis.Methods: The hollow fiber with U-shape configuration which contains sample solution was placed in the centrifuge tube vertically and ultrafiltration was performed under the action of centrifugal force that allowed the separation between macromolecule hypromellose and micromolecule benzalkonium bromide in Hhypromellose Eye Drop. The separation was carried out on an uncoated fused-silica capillary column of 50μm i.d.×43 cm (effect length 35 cm). The method was developed by utilizing a runnig buffer of 50 mmol·L^-1 of phosphate electrolyte solution (pH=3.0)-acetonitrile (7: 3), an injection by liquid differential pressure with a height of 10 cm for 10 s and a running voltage of 20 kV. The detection length was 214 nm.Results: An excellent separation was achieved between hypromellose and benzalkonium bromide. The standard curve of benzalkonium bromide was linear over the range of 24.9³.99×102μg·mL^-1 (r= 0.9996), with the limit of detection 3.11μg·mL^-1, and the average recovery was 98.4% with RSD of 2.0%.Conclusion: The method is simple, rapid, accurate and specific and it provides a new reliable means for segregation analysis of benzalkonium bromide in Hhypromellose Eye Drop. PART 3 Concentration and detection of trace and effective toxical ingredient in commonly used Chinese drugs preparation(1) Rapid determination of trace toxical aconitine alkaloid in Wenweishu Particle by CE with field-amplified sample stackingObjective: To establish a CE with field-amplified sample stacking method for rapid determination of trace, effective and toxical aconitine alkaloid in Wenweishu Particle.Methods: An uncoated fused-silica capillary column (50μm i.d.×43 cm, effective length 35 cm) was used. The running buffer was 60 mmol·L^-1 phosphate electrolyte solution (pH=6.0)-acetonitrile (8: 2). The running voltage was 10 kV and detection wavelength was 235 nm. Field-amplified sample stacking was employed and the samples were dissolved in 20% acetonitrile. Enrichment procedure was performed as follow: the capillary inlet was dipped in water for 5 s by liquid differential pressure with a height of 10 cm prior to electrokinetic injection (12 kV, 30 s).Results: This method allowed selective concentration of aconitine alkaloid and about 400 fold enrichment was achieved. A good linear relation was obtained in the range of 37.5².40×103 ng·mL^-1 (r=0.9995), with the detection limit of 9.38 ng·mL^-1. The average recovery was 97.7% with the RSD of 2.2%.Conclusion: The method is simple, rapid and specific with high stacking efficiency; it provides a new reliable means for quality control and clinical drug monitoring of Wenweishu Particle.(2) Repaid and limited determination of trace toxical aconitine alkaloid in Xiaohuoluo PillsObjective: to establish a CE with field-amplified sample stacking method for the separation and determination of trace toxical aconitine alkaloid in Xiaohuoluo Pills.Methods: An uncoated fused-silica capillary column (50μm i.d.×43 cm, effective length 35 cm) was used. The running buffer was 60 mmol·L^-1 phosphate electrolyte solution (pH=6.0)-acetonitrile (8: 2). The running voltage was 10 kV and detection wavelength was 235 nm. Field-amplified sample stacking was employed and the samples were dissolved in 20% acetonitrile. Enrichment procedure was performed as follow: the capillary inlet was dipped in water for 5 s by liquid differential pressure with a height of 10 cm prior to electrokinetic injection (12 kV, 30 s).Results: This method allowed about 400 fold enrichment of aconitine alkaloid. A good linear relation was obtained in the range of 37.5².40×103 ng·mL^-1 (r=0.9995), with the detection limit of 9.38 ng·mL^-1. The average recovery was 97.6% with the RSD of 1.9%.Conclusion: The method is simple, rapid, specific and selective with high stacking efficiency; it provides a new reliable means for production and quality control of Xiaohuoluo Pills.(3) Determination of toxical strychnine and brucine in Gujinwan Capsules by CE with field-amplified sample stackingObjective: To establish a CE with field-amplified sample stacking method for the separation and determination of effective toxical strychnine and brucine in Gujinwan Capsules simultaneouly.Methods: An uncoated fused-silica capillary column (50μm i.d.×57 cm, effective length 50 cm) was used. The running buffer was 20 mmol·L^-1 sodium acetate (pH 3.8, adjusted by acetic acid). The running voltage was 20 kV and detection wavelength was 260 nm. Field-amplified sample stacking was employed and the samples were dissolved in 20% methanol. Enrichment procedure was performed as follow: the capillary inlet was dipped in water for 3 s by liquid differential pressure with a height of 10 cm prior to electrokinetic injection (20 kV, 25 s).Results: This method allowed about 600 and 400 fold enrichments for strychnine and brucine, respectively. The standard curve of strychnine was linear over the range of 14.8⁹.40×10² ng·mL^-1 (r= 0. 9998), with the limit of detection 1.85 ng·mL^-1, and the average recovery was 98.5% with RSD of 1.9%. The standard curve of brucine was linear over the range of 17.2～1.09×103 ng·mL^-1 (r= 0. 9999), with the limit of detection 2.15 ng·mL^-1, and the average recovery was 98.5% with RSD of 1.8%.Conclusion: The method is simple and rapid with high stacking efficiency and it provides a new reliable means for quality control and clinical drug monitoring of Gujinwan Capsules.PART 4 Rapid and sensitive determination of strychnine and brucine in human urine by CE with field-amplified sample stackingObjective: To establish a CE with field-amplified sample stacking method for the determination of strychnine and brucine in human urine simultaneouly.Methods: An uncoated fused-silica capillary column (50μm i.d.×57 cm, effective length 50 cm) was used. The running buffer was 20 mmol·L^-1 sodium acetate (pH 3.8, adjusted by acetic acid). The running voltage was 20 kV and detection wavelength was 260 nm. Field-amplified sample stacking was employed and the samples were dissolved in 20% methanol. Enrichment procedure was performed as follow: the capillary inlet was dipped in water for 3 s by liquid differential pressure with a height of 10 cm prior to electrokinetic injection (20 kV, 25 s).Results: This method allowed 550 and 350 fold enrichments for strychnine and brucine, respectively. The standard curve of strychnine was linear over the range of 8.00-2.56×102 ng·mL^-1 (r= 0. 9995), with the limit of detection 2.00 ng·mL^-1. The standard curve of brucine was linear over the range of 10.0-3.20×102 ng·mL^-1 (r= 0. 9999), with the limit of detection 2.50 ng·mL^-1. The average recovery of strychnine was 98.2% with RSD of 3.6%. The average recovery of brucine was 98.9% with RSD of 4.0%. The urine levels of the volunteer could be detected even at 24 h after administration.Conclusion: The method is simple, rapid and economical with high stacking efficiency and it provides a new reliable means for quality control and clinical drug monitoring of Gujinwan Capsules.