Clinical Applications Of Capillary Electrophoresis Based On The On-line Field-amplified Sample Stacking

Clinical bio-sample analysis including metabolomics, therapeutic drug monitoring(TDM), pharmacokinetics and biochemical sample analysis is an important assistingmeans of clinical drug therapy. The method of bio-sample analysis compriseschromatography, spectroscopy, microbiological assay and immunoassay. Comparing withother three, chromatography combining their advantages is a more effective and accurateanalysis method. Common chromatography for the bio-sample assay is based on gaschromatography (GC), liquid chromatography (LC) or capillary electrophoresis (CE).Recently, capillary electrophoresis (CE) has become a very important instrumentaltechnique in pharmaceutical and biological analysis because of its high separationefficiency in a short time and extremely low solvent consumption, while, the relativelypoor concentration sensitivity of CE when using UV detection due to the limited samplevolume and the short optical path length, limits CE application for sensitive detection ofbio-analysis. To solve the problem, field-amplified sample stacking (FASS) is usuallyemployed as a simple and efficient technique in capillary zone electrophoresis. It is basedon a mismatch between the electric conductivity of the sample and that of the runningbuffer. And when compared to injection from a nonstacking sample, conventional FASSprovided a significant sensitivity enhancement.In our experiment, ambroxol, amlodipine and mirtazapine were selected as researchsubjects, scince all of them are effective in low dosage with a long half-life, and theirplasma concentration are very low. We try to establish capillary methods combined withon-line sample stacking for determination of ambroxol, amlodipine (enantiomers) andmirtazapine (enantiomers and those of the active metabolites) in human plasma. Theconditions of CE such as separation voltage, concentrations and pH of borate-phosphatebuffer were optimal to get the best running time. In addition, key parameters that affected-4- the stacking efficiency including injection modes, time and voltage, composition ofsample solvent were investigated to achieve higher sensitivity. Furthermore, type andconcentration of cyclodextrin were also studied to improve peak shape, resolution andcolumn efficiency or be employed as chiral selectors.Pharmacokinetics is the study of the time course of a drug within the body andincorporates the processes of absorption, distribution, metabolism and excretion. To betterinterpret the effect and toxicity of the drug, an analytical method should be established todetermine the drug and its metabolites quantitatively, which help us to know about theirmechanisms of action and their pharmacokinetic properties during the initial state of drugdevelopment and in clinical therapy. In this paper, a β-cyclodextrin enhancedfield-amplified sample stacking (FASS) and capillary zone electrophoresis (CZE) methodis described for the quantification of ambroxol hydrochloride in human plasma，followingliquid-liquid extraction in the96-well format. The separation was carried out at25℃in a31.2cm×75μm fused-silica capillary with an applied voltage of15kV. The backgroundelectrolyte (BGE) was composed of6.25mM borate-25mM phosphate (pH3.0) and1mMβ-cyclodextrin. The sample was introduced by using electrokinetic (7.5kV×15s) injectionmodes. The detection wavelength was210nm. Under the above conditions the retentiontime of ambroxol hydrochloride and diphenhydramine (IS) was3.5and4.2min,respectively. Compared with those reported methods, the assay was time-saving andsensitive, and no difference was found between results of our method and the others.In fact, the enantiomers of a chiral drug may differ significantly in their bioavailability,rate of metabolism, metabolites, excretion, potency and selectivity for receptors,transporters and/or enzymes, and toxicity. As a racemic mixture, racemic amlodipinecontains (R)-and (S)-amlodipine isomers in a1:1ratio, but only (S)-amlodipine as theactive moiety possesses therapeutic activity. Therefore, information about thepharmacokinetic behavior of the amlodipine enantiomers is necessary for understandingrelationships between drug levels and therapeutic response. In this study, a simple,sensitive and low-cost method using capillary electrophoresis coupled withhydroxypropyl-β-cyclodextrin interaction assisted acetonitrile field-amplified samplestacking technique for the quantitative enantioselective determination of trace drug (amlodipine, AML) was established for clinical applications. The separation wasperformed at25℃in a31.2cm×75μm fused-silica capillary with an applied voltage of15kV. The background electrolyte (BGE) was composed of6.25mM borate-25mMphosphate (pH2.5) and3mg/mL hydroxypropyl-β-cyclodextrin. The sample wasintroduced by using electrokinetic (7.5kV×15s) injection modes. The detectionwavelength was200nm. The retention time of amlodipine enantiomers anddiphenhydramine (IS) were5.7,6.2and6.7min, respectively.Therapeutic drug monitoring (TDM) plays an important part in the optimal use of aselect few drugs. With the individualisation of drug therapy through measuredconcentrations the aim is to achieve maximum therapeutic response with minimal adverseeffects. And TDM is a constantly expanding practice in psychiatric clinics, mainly forantipsychotic agents. Mirtazapine is a chiral compound, however it is administeredclinically as a racemic mixture of S (+)-and R(-)-enantiomers. The two enantiomersdiffer in both pharmacokinetic and pharmacodynamic parameters. In this study, a simple,sensitive and environment-friendly method using capillary electrophoresis coupled withcarboxymethyl-β-cyclodextrin interaction assisted acetonitrile field-amplified samplestacking technique for the quantitative enantioselective determination of mirtazapine andits active demethylated metabolite in human plasma was established for clinicalapplications. The separation was performed at25℃in a40.2cm×75μm fused-silicacapillary with an applied voltage of17kV. The background electrolyte (BGE) wascomposed of6.25mM borate-25mM phosphate (pH2.5) and6.5mg/mLcarboxymethyl-β-cyclodextrin. The sample was introduced by using electrokinetic(7.5kV×10s) injection modes. The detection wavelength was200nm. The running timewas8.5min.