毛细管区带电泳在临床药理学和药物治疗学中的应用
作者：曹成喜 何友昭 钱逸泰 周书林
单位：曹成喜 何友昭 钱逸泰(中国科学技术大学化学系，合肥 230026)；周书林(皖南医学院变态反应研究所，芜湖241001)
关键词：毛细管区带电泳；药物代谢；药物；治疗学；临床药理学
中国临床药理学与治疗学000231
摘要 介绍了毛细管区带电泳(capillary zone electrophoresis,CZE)的原理，以及CZE在药物治疗浓度监测、代谢性疾病的诊断、药物及其代谢产物在体内代谢等方面的应用,并简要地讨论了CZE在药理学及临床医学中的应用前景。
Application of capillary zone electrophoresis in
, 百拇医药
clinical pharmacology and therapeutics
CAO Cheng-Xi
(Department of Chemistry, University of Science and Technology of China, Hefei 230026)
HE You-Zhao
(Department of Chemistry, University of Science and Technology of China, Hefei 230026)
QIAN Yi-Tai
(Department of Chemistry, University of Science and Technology of China, Hefei 230026)
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ZHOU Shu-Lin
(Institute of Allergy Research, Wannan Medical College, Wuhu 241001)
1 Introduction
Capillary electrophoresis(CE),also called as high-performance capillary electrophoresis (HPCE),is one of the most powerful analytical and separative techniques[1].It was in 1967 that Hjerten[2] invented the first apparatus of free zone electrophoresis, viz, the prototype of CE. But it is only within recent 20 years that CE attracts increasing interest in multi-sciences. Today CE possesses wide applications in analytical chemistry, food analysis, earth science, clinical analysis and biochemistry, including important uses in clinical pharmaceutical analysis and clinical medicine.
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According to the principle, CE can be classified into capillary zone electrophoresis(CZE), capillary gel electrophoresis (CGE),micellar electro-kinetic capillary chromatography(MEKC),capillary electro-chromatography(CEC), capillary isotachophoresis (CITP) and capillary isoelectric focusing (CIEF)[3]. The former four techniques are the most widely used techniques of CE in pharmaceutical and metabolite analysis, clinical diagnosis and therapeutics, while CIEF has more and more applications in drug analysis since more proteins and peptides-zwitterionic materials thatcan be analyzed with CIEF efficientlyare used in clinical medicine. Therefore, we try our best to introduce the former four of CE into the fields of clinical pharmacology and therapeutics. In this paper, we introduce the principle of CZE and review the applications of CZE in clinical pharmacology and therapeutics.
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2 Instrumentation
Fig 1 is the basic set-up of CE equipment[3,4]. In brief, a CE instrument consists of an injection system of sample, a separative capillary tube(20～ 100 μm ID,20～100 cm length),a high voltage power supply(delivering up to 30 kV and up to 200～250(μA),two electrodes,two electrode jars and a detector.
Fig 1 The diagram of capillary electrophoresis
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HV=high power supply; the symbols+and-imply the anode and cathode respectively.
Because the capillary tube used in CE is very find, the volume of a capillary is very small, for example, the volume of a 100 cm long, 50 μm ID capillary is about 2 μl. Thus in order not to influence the separative efficiency of CE greatly, the injection volume ought not to exceed 1 %～2 % of the total volume, viz, 20～40 nl. Handing with accuracy these tiny volumes faces great difficulties, but modern instruments may assure precision better than 5 %.
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In CE run, two injection techniques of sample,viz,hydrodynamic and electro-kinetic mode, are currently used. The former, which uses pressure or vacuum method while the injection end of the capillary is dipped into the sample solution, is non-selective, whereas the latter, which uses the application of potential, is selective, due to the dependence of injection on the mobility and charge(s) of ions in the sample analyzed and on the electro-osmostic flow(EOF).
In Fig 1, the capillary is the compartment where the separation and detection take place. The capillary should be chemically and physically resistant, accurately and precisely produced with narrow internal diameters, transparent to UV radiation and able to dissipate Joule's heat through good thermal conductivity. Fused-silica capillary fits these requirements, but tends to adsorb analytes, particularly proteins. At present, silica capillary is by far the first choice. Fused-silica capillary is externally protected by a layer of poly-amide. The internal surface can be coated with polymer to diminish the EOF and the adsorption to proteins.
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A high power supply is needed to supply at least deliver voltages up to 30 KV and current up to 200～300 μA .Because of the usual movement of EOF towards the cathode, the common polarity is the anode at the injection end of capillary and the cathode at the opposite side, a few centimeters after the detector placed “on-column”. However, for particular purposes, the polarity can be reversed.
The detection in CE is of many methods[3，4], for example,UV-Vis-absorption detection,multi- wavelength detection of diode-array, laser-induced fluorescence,electrochemical ampero-metric, conductimetric detection and so on. One of the most popular detection in CE is the UV-Vis absorption.
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Besides those detection methods above, a variety of other detection techniques have been introduced into CE including mass spectrometry(MS), laser-based thermo-optical,refractive index, radioisotope and indirection detection modes[3,4].
3 Principles of CZE
The separation of CZE relies principally on the pH controlled dissociation of acidic groups on the solute or the protonation of basic functions on the solute. These ionic species are separated based on differences in their charge-to-mass ratios. For basic drugs, the separation is performed at low pH value due to analytes existing as cations whilst for acidic drugs the separation is carried out at high pH due to as anions.In CZE, all neutral compounds are swept, unresolved, through the detector together. Thus in CZE, the neutrals can not be separated, but can be isolated by MEKC that will be discussed in other place.
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Under an electric field, cations move towards the cathode and anions migrate towards the anode. The speed of ionic movement is governed by ionic size and charge (s). Smaller molecular with a large number of charge(s) will move more quickly than larger or less charged compounds. The apparent speed of movement, known as the apparent mobility(μ app), is characteristic of the solute and can be derived from the following expression:
μapp=μact+μEOF (1)
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where, μact is the actual mobility and μEOF the mobility caused by EOF. In Eqn.(1) the actual is given as
μact=q/(6πηr) (2)
where q is the number of ionic charges,η the solution viscosity and r the ionic radius. And μEOF is
μEOF=εξ/(4πη) (3)
where ε is the dielectric constant of internal solution, ξ the zeta potential of capillary wall.
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Clearly, under the electric field, the ionic actual velocity is
(4)
where, E is the strength of electric field, V the voltage between the two ends of capillary and L the length of capillary. And the EOF velocity should be formulated as
(5)
Therefore, in the presence of the EOF, the migration velocity, viz, the apparent velocity, of the analytes follows the equation:
(6)
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Obviously, a mixture of compounds with different mobilities can be separated according to their mobilities.As shown in Eqn( 4), the higher an ionic mobility is, the faster the ionic velocity is, and the more ahead the ionic movement and the shorter the detection time. Thus by comparing the electromigration time of an ion with standard electrophogram, one marks a series of compounds from their mixture.
The theoretical plate number(N), which presents the separative efficiency, can be calculated with
(7)
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where, D is the diffusional coefficient.This equation shows the higher the voltage and EOF are，the better the N,viz, separative efficiency is. Therefore, with high voltage used in CE, one can obtain very high plate number(over 105), much higher than that of HPLC
Tab 1 The comparisons of drug quantitative analyses by HPLC and CZE Salbutamol
HPLC
CZE
run1=,run2=a)
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Mean
RD/%
run1=,run2=a)
Mean
RD/%
Tablet /4mg.tablet-1
4.05,4.00
4.03
0.75
3.94,3.76
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3.85
3.75
Influxion/1mg.ml-1
1.04,1.02
1.03
3.00
0.99,1.00
1.00
1.00
Syrup/0.4mg.ml-1
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0.40,0.40
0.40
0.00
0.41,0.39
0.40
2.50
4 Applications
Traditionally, HPLC and GC are very powerful analytical tools in drug and its metabolism analyes both in quality control and in bio-fluids. But with the great development of capillary electrophoresis, CE has widely been used in biomedicine.
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As compared with HPLC,CE has the following particular advantages[3,4].Firstly,the analysis times can be short(1～5 min, even less than 30 s[45～47]).Secondly,the separation can be performed until the peak(s) of interest has been detected and after this point the separation can be stopped and a rinse step can be used to remove un- quantified peak(s) remaining in the capillary; thus a lot of time can be saved.Thirdly,the run of CE is very cheap,usually less than 1/10 of HPLC run cost.Fourthly, the injection volumes are in the order of 10～50 nl which is a fraction of the volumes in HPLC(10～100 μl), this means that several injections are possible in CE from tiny sample volumes that can lead to significant savings. Fifthly, the separative efficiency is high(over 105 theoretical plate number). Sixthly, the detecting is on-line as shown in Fig 1. Additionally, the performance is automatic. As shown in Tab 1,the results of drug analyses by CZE are almost the same as those by HPLC, if appreciate conditions are chosen for CZE.
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Generally, there are four kinds of applications of CZE in clinical medicine, viz, therapeutic monitoring,pheno-typing disease diagnosis, drug metabolism and analysis as displayed in Tab 2.
Tab2 Applications fo CZE in the rapeutic monitoring ,disease diagnosis drug metabolism and analyses Application
Eelectrolyte System
Ref.
Therapeutic monitoring
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Climetidine in Plasma
phosphate,pH2.0
6
Cyclic guanine mono-phosphate phosphodiesterase inhibitor in rat serum
phosphate,pH6.0
7
Cytosine-β-D-arabinsoine in plasma
Acetate,pH2.5
8
Fosfomycin in serum
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Borate and phenyl phosphonic acid
9
Heproxen mimetics in human and rat plasma
Citrate-methanol buffer ,PH4.0
10
Iohexol in serum
Borate,pH8.0
11
Naproxen in serum
Tricine pH8.0
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12
Pentobarbital in serum
Borate,pH8.5
13
Piracetamin humanp;asma
Borate and alpha-CD
14
Suramin in serum
CAPSO,pH9.7
15
Phenotyping disease diagnosis
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Dextromethorphan in urine
Borate ,pH9.3
16
Hypoxanthine adn xanthine in urine
Phosphate,pH8.0
17
Methylanonic acid in serum
Teis-citrate,pH6.4
18
Drug metablism
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Cefixime adn metabolites in urine
Phosphate,pH6.8
19
Coumarin metabolites in urine and serum
Phosphate,pH7.5
20
Famotidien
Polyethylene oxide&dextran,pH4.5
21
Haloperidol and 10synthetic metabolite
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pH4.5with 10%MeOH
22
Nitrite and nitrate in bio-fluids
Sulfate ,pH8.0
23
Oxprenolol and metabolites in human urine
Phosphate pH2.5 with PH-β-CD
24
Pyrazolacridine metabolites in urine
NH4 acetate MeOH,acetic acid
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25
Zolpiddem and metabolites in urine
Phosptate pH5.6
26
Zopiclone adn its metabolites in urine and saliva
Phosptate pH2.8with -β-CD
27
Druganalyses
10 acidic drugs and excipients
Borate ,pH9.5
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28
13 aminoglycoside antibiotics
Imidazole-acetate ,PH5.0
29
Aspartyl dipeptides
Phosphate,PH3.3with CM-β-CD
30
12 basic drugs and excipients
Phosphate,PH2.5
31
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22 basic drugs in urine
Phosphate,PH22.0
32
17 basic drugs
Phosphate,PH2.4
33
10 beta-blockers
Citrate buffer,low pH
34
Cardiovascular drugs
Various separations
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35
Chiral drug:clorprenalinge ,benzhexol,esmolol,terazosin
Phosphate ,pH2.5 with neutral CD
36
Chlortetracycline and relative substances
Teraborate,Ph8.5with EDTA
37
15 diuretics in blood and urine
60mmol.L-1 CAPS ,pH10.6
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38
Diuretic drugs
Various modes
39
18 common abused drugs
Phosphate-borate,pH8.5
40
Glucose ,Galactose adn Lactose
75 mmol.L-1 borate
41
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Pyridinyl-methyl-sulfiyl-benzimidazoles
Non-aqueous solution
42
Quinolone antibiotics
HEPES,pH 8.5with acetonitrile
43
16 sulphonamides
Imidazole-acetate,pH7.0
44
CAPS=cyclohexaneamino suphonic acid;CAPSO=zwiterionic buffer;CD=cyclodextrin;HEPES=2[N-hydroxylethyl]piperazine-N'-thanesup-phonic acid+sodium hydroxyide
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The therapeutic monitoring by CZE possesses great significances in clinical medicine.At first, the method can be used for the control of therapeutic blood concentration of drug used, this is of obvious significance for real individual therapeutics. With the monitoring of drug in blood, one can in time adjust individual therapeutics to prevent toxic effect of drugs(like anti-humor, cardiovascular, calmative and anesthetic drugs, to name but just a few).This is also important for the patients with low kidney/or liver function exhaustion.In Tab 2, we have listed some drugs used in clinical medicine.
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Gout is a kind of metabolizing disease, also a phenotyping disease. The diagnosis of the disease needs to detect the concentration level of hypoxanthine and xanthine in both urine and blood. Tab 2 shows the method of monitoring hypoxanthine and xanthine in urine for the phenotyping disease diagnosis(details see Ref 17),coupled with the dextromethorphen in urine and Methylmanonic acid in serum.
Drug metabolism in body is one of the most important content in pharmacology, pharmacokinetics and toxicology.Tab 2 implies that by using CZE method, numerous medicines, including their metabolites, have been detected. As shown in Tab 2, the electrolyte systems are very simple and astounding cheap as compared with flow phases used in HPLC. Thus, the procedures based on CZE for drug and its metabolites are of apparent importance for pharmacology and pharmacokinetics, including toxicology.
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Drug analysis is the most basic work not only in quality control, but also in pharmacology and in clinical therapeutics. The last content in Tab 2 displays numerous drugs have been analyzed by the procedures based upon CZE. In fact, more drug analyses, including those in the former three contents in Tab 2, can not be listed here due to the print limit. The drugs analyzed with CZE concern a wide range from antibiotics, to vitamins, to cardiovascular drugs, to steroids, to diuretics, to common abused drugs as well. This also shows CZE is a versatile tool of analysis for pharmacology and therapeutics.
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5 Outlook
A series of new great developments in CZE have been achieved in recent years. The achievements that hold great significance in biomedicine are the CE based on microchip[45～47] and single- cell analyses[48]with CZE. With the microchip based CE, including CZE, the capillary is eroded in a very small glass chip, the separative length is less than 30 mm, and the run can be completed within 30 seconds,even within 20 seconds. Thus, the technique is of great potential of becoming very powerful and efficient tool of analysis. It will also open a wide way for drug and metabolite analyses in clinical pharmacology and therapeutics.
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Single-cell analyses with CZE have been reported since 1995[48]. With the development of micro-injection, one can introduce a single cell into capillary and then run it. Clearly, with the further development in other techniques, one can study pharmacology at cell-level, and analyze drug effect and metabolite with single cell.The cell analyses with CZE not only show great attraction to biochemist, but also exhibit exciting enticement to pharmacologist and therapeutist.
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This project is supported by the National Natural Scientific Foundation (№ 29775014, 29975026) and The Scientific Foundation of Chinese Health Committee(№98-2-334).
Correspondence to:HE You-Zhao,Tel:0551-3607072,e-mail: yzhe@mail. ustc.edu.cn
References
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15，Garcia LL,Shihabi ZK.Sample matrix effects in capillary electrophoresis.J Chromatogr,1993;652: 465
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17，Bory C, Chantin C, Boulieu R. Comparison of capillary electrophoretic and liquid chromatographic determination of hypoxanthine and xanthine for the diagnosis of xanthinuria.J Chromatogr A,1996;730: 329
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18，Schneede J,Ueland PM.Application of capillary electrophoresis with laser-induced fluorescence detection for routine determination of methylmalonic acid in human serum.Anal Chem,1995;67:812
19，Honda S, Taga A, Kakehi K, et al. Determination of cefixime and its metabolites by high-performance capillary electrophoresis.J Chromatogr, 1992; 590: 364
20，Bogan DP, Deasy B, O'Kennedy R, et al. Determination of free and total 7-hydroxyl-coumarin in urine and serum by capillary electrophoresis. J Chromatogr B, 1995; 663: 371
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21，Soini H, Riekkola ML, Novotny MV. Mixed polymer networks in the direct analysis of pharmaceuticals in urine by capillary electrophoresis.J Chromatogr A, 1994; 680: 623
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23，Bories PN, Scherman E, Dziedzic L. Analysis of nitrite and nitrate in biological fluids by capillary electrophoresis.Clin Biochem,1999;32:9
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24，Li F, Cooper SF, Mikkelsen SR. Enantioselective determination of oxprenolol and its metabolites in human urine by cyclodextrin-modified capillary zone electrophoresis. J Chromatogr B,1995;674:277
25，Tomlinson AJ, Benson LM, Gorrod JW, et al. In vestigation of the in vitro metabolism of the H-2 antagonist mifentidine by on-line capillary elec trophoresis mass spectrometry using non-aqueous separation conditions. J Chromatogr B, 1994; 657:
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26，Hempel G, Blaschke G. Direct determination of zolpidem and its main metabolites in urine using capillary electrophoresis with laser-induced fluorescence detection. J Chromatogr B,1996;675: 131
27，Hempel G, Blaschke G. Enantioselective determination of zopiclone and its metabolites in urine by capillary electrophoresis.J Chromatogr B, 1996; 675: 139
28，Altria KD, Bryant SM, Hadgett T. Validated capillary electrophoresis method for the assay of a range of acidic drugs and excepients.J Pharm Biomed. Anal,1997;15:1091
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29，Ackermans MT, Everaerts FM, Beckers JL. Determination of aminogycloside antibiotics in pharmaceuticals by capillary zone electrophoresis with indirect UV detection coupled with micellar electro-kinetic capillary chromatography. J Chromatogr,1992; 606: 229
30，Verleysen K, Sabah S, Scriba G, et al. Enantioseparation of aspartyl dipeptides by CE: comparison between 18-crown-6-tetracarboxylic acid and carboxylmethyl-β-cyclodextrin as chiral selector.Chromatographia, 1999; 49: 215
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31，Altria KD, Frake P, Gill I, et al. Validated capillary electrophoresis method for the assay of a range of basic drugs and excipients. J Pharm Biomed Anal,1995;13:951
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33，Chee GL, Wan TSM. Reproducible and high-speed separation of basic drugs by capillary zone electrophoresis. J Chromatogr, 1993; 612: 172
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34，Lin CE, Chang CC, Lin WC, et al. Capillary zone electrophoresis separation of β-blockers using citrate buffer at low pH. J Chromatogr A, 1996; 753: 133
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36，Ren XQ, Dong YY, Huang AJ, et al. Separation of the enantiomers of four chiral drugs by neutral cyclodextrin-mediated capillary zone electrophoresis. Chromatographia,1999;49:411
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37，Li YM, Moons H, Schepdael AV, et al. Analysis of chlortetracycline and related substances by capillary zone electrophoresis: development and validation. Chromatographia,1998;48:576
38，Jumppanen J, Siren H, Riekkola ML. Screening for diuretics in urine and blood serum by capillary zone electrophoresis. J Chromatogr,1993;652: 441
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41，Altrai K, Lao W, Wang G. Quantitative and selective analysis of lactose by capillary electrophoresis.Chromatographia, 1999; 49: 406
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43，Perez-Ruiz T, Martinez-Lozano C, Sanz A, et al. Separation and simultaneous determination of quinolone antibiotics by capillary zone electrophoresis. Chromatographia, 1999; 49: 419
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2000-04-10 received, 百拇医药
单位：曹成喜 何友昭 钱逸泰(中国科学技术大学化学系，合肥 230026)；周书林(皖南医学院变态反应研究所，芜湖241001)
关键词：毛细管区带电泳；药物代谢；药物；治疗学；临床药理学
中国临床药理学与治疗学000231
摘要 介绍了毛细管区带电泳(capillary zone electrophoresis,CZE)的原理，以及CZE在药物治疗浓度监测、代谢性疾病的诊断、药物及其代谢产物在体内代谢等方面的应用,并简要地讨论了CZE在药理学及临床医学中的应用前景。
Application of capillary zone electrophoresis in
, 百拇医药
clinical pharmacology and therapeutics
CAO Cheng-Xi
(Department of Chemistry, University of Science and Technology of China, Hefei 230026)
HE You-Zhao
(Department of Chemistry, University of Science and Technology of China, Hefei 230026)
QIAN Yi-Tai
(Department of Chemistry, University of Science and Technology of China, Hefei 230026)
, http://www.100md.com
ZHOU Shu-Lin
(Institute of Allergy Research, Wannan Medical College, Wuhu 241001)
1 Introduction
Capillary electrophoresis(CE),also called as high-performance capillary electrophoresis (HPCE),is one of the most powerful analytical and separative techniques[1].It was in 1967 that Hjerten[2] invented the first apparatus of free zone electrophoresis, viz, the prototype of CE. But it is only within recent 20 years that CE attracts increasing interest in multi-sciences. Today CE possesses wide applications in analytical chemistry, food analysis, earth science, clinical analysis and biochemistry, including important uses in clinical pharmaceutical analysis and clinical medicine.
, http://www.100md.com
According to the principle, CE can be classified into capillary zone electrophoresis(CZE), capillary gel electrophoresis (CGE),micellar electro-kinetic capillary chromatography(MEKC),capillary electro-chromatography(CEC), capillary isotachophoresis (CITP) and capillary isoelectric focusing (CIEF)[3]. The former four techniques are the most widely used techniques of CE in pharmaceutical and metabolite analysis, clinical diagnosis and therapeutics, while CIEF has more and more applications in drug analysis since more proteins and peptides-zwitterionic materials thatcan be analyzed with CIEF efficientlyare used in clinical medicine. Therefore, we try our best to introduce the former four of CE into the fields of clinical pharmacology and therapeutics. In this paper, we introduce the principle of CZE and review the applications of CZE in clinical pharmacology and therapeutics.
, 百拇医药
2 Instrumentation
Fig 1 is the basic set-up of CE equipment[3,4]. In brief, a CE instrument consists of an injection system of sample, a separative capillary tube(20～ 100 μm ID,20～100 cm length),a high voltage power supply(delivering up to 30 kV and up to 200～250(μA),two electrodes,two electrode jars and a detector.
Fig 1 The diagram of capillary electrophoresis
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HV=high power supply; the symbols+and-imply the anode and cathode respectively.
Because the capillary tube used in CE is very find, the volume of a capillary is very small, for example, the volume of a 100 cm long, 50 μm ID capillary is about 2 μl. Thus in order not to influence the separative efficiency of CE greatly, the injection volume ought not to exceed 1 %～2 % of the total volume, viz, 20～40 nl. Handing with accuracy these tiny volumes faces great difficulties, but modern instruments may assure precision better than 5 %.
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In CE run, two injection techniques of sample,viz,hydrodynamic and electro-kinetic mode, are currently used. The former, which uses pressure or vacuum method while the injection end of the capillary is dipped into the sample solution, is non-selective, whereas the latter, which uses the application of potential, is selective, due to the dependence of injection on the mobility and charge(s) of ions in the sample analyzed and on the electro-osmostic flow(EOF).
In Fig 1, the capillary is the compartment where the separation and detection take place. The capillary should be chemically and physically resistant, accurately and precisely produced with narrow internal diameters, transparent to UV radiation and able to dissipate Joule's heat through good thermal conductivity. Fused-silica capillary fits these requirements, but tends to adsorb analytes, particularly proteins. At present, silica capillary is by far the first choice. Fused-silica capillary is externally protected by a layer of poly-amide. The internal surface can be coated with polymer to diminish the EOF and the adsorption to proteins.
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A high power supply is needed to supply at least deliver voltages up to 30 KV and current up to 200～300 μA .Because of the usual movement of EOF towards the cathode, the common polarity is the anode at the injection end of capillary and the cathode at the opposite side, a few centimeters after the detector placed “on-column”. However, for particular purposes, the polarity can be reversed.
The detection in CE is of many methods[3，4], for example,UV-Vis-absorption detection,multi- wavelength detection of diode-array, laser-induced fluorescence,electrochemical ampero-metric, conductimetric detection and so on. One of the most popular detection in CE is the UV-Vis absorption.
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Besides those detection methods above, a variety of other detection techniques have been introduced into CE including mass spectrometry(MS), laser-based thermo-optical,refractive index, radioisotope and indirection detection modes[3,4].
3 Principles of CZE
The separation of CZE relies principally on the pH controlled dissociation of acidic groups on the solute or the protonation of basic functions on the solute. These ionic species are separated based on differences in their charge-to-mass ratios. For basic drugs, the separation is performed at low pH value due to analytes existing as cations whilst for acidic drugs the separation is carried out at high pH due to as anions.In CZE, all neutral compounds are swept, unresolved, through the detector together. Thus in CZE, the neutrals can not be separated, but can be isolated by MEKC that will be discussed in other place.
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Under an electric field, cations move towards the cathode and anions migrate towards the anode. The speed of ionic movement is governed by ionic size and charge (s). Smaller molecular with a large number of charge(s) will move more quickly than larger or less charged compounds. The apparent speed of movement, known as the apparent mobility(μ app), is characteristic of the solute and can be derived from the following expression:
μapp=μact+μEOF (1)
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where, μact is the actual mobility and μEOF the mobility caused by EOF. In Eqn.(1) the actual is given as
μact=q/(6πηr) (2)
where q is the number of ionic charges,η the solution viscosity and r the ionic radius. And μEOF is
μEOF=εξ/(4πη) (3)
where ε is the dielectric constant of internal solution, ξ the zeta potential of capillary wall.
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Clearly, under the electric field, the ionic actual velocity is
(4)where, E is the strength of electric field, V the voltage between the two ends of capillary and L the length of capillary. And the EOF velocity should be formulated as
(5)Therefore, in the presence of the EOF, the migration velocity, viz, the apparent velocity, of the analytes follows the equation:
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Obviously, a mixture of compounds with different mobilities can be separated according to their mobilities.As shown in Eqn( 4), the higher an ionic mobility is, the faster the ionic velocity is, and the more ahead the ionic movement and the shorter the detection time. Thus by comparing the electromigration time of an ion with standard electrophogram, one marks a series of compounds from their mixture.
The theoretical plate number(N), which presents the separative efficiency, can be calculated with
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where, D is the diffusional coefficient.This equation shows the higher the voltage and EOF are，the better the N,viz, separative efficiency is. Therefore, with high voltage used in CE, one can obtain very high plate number(over 105), much higher than that of HPLC
Tab 1 The comparisons of drug quantitative analyses by HPLC and CZE Salbutamol
HPLC
CZE
run1=,run2=a)
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Mean
RD/%
run1=,run2=a)
Mean
RD/%
Tablet /4mg.tablet-1
4.05,4.00
4.03
0.75
3.94,3.76
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3.85
3.75
Influxion/1mg.ml-1
1.04,1.02
1.03
3.00
0.99,1.00
1.00
1.00
Syrup/0.4mg.ml-1
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0.40,0.40
0.40
0.00
0.41,0.39
0.40
2.50
4 Applications
Traditionally, HPLC and GC are very powerful analytical tools in drug and its metabolism analyes both in quality control and in bio-fluids. But with the great development of capillary electrophoresis, CE has widely been used in biomedicine.
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As compared with HPLC,CE has the following particular advantages[3,4].Firstly,the analysis times can be short(1～5 min, even less than 30 s[45～47]).Secondly,the separation can be performed until the peak(s) of interest has been detected and after this point the separation can be stopped and a rinse step can be used to remove un- quantified peak(s) remaining in the capillary; thus a lot of time can be saved.Thirdly,the run of CE is very cheap,usually less than 1/10 of HPLC run cost.Fourthly, the injection volumes are in the order of 10～50 nl which is a fraction of the volumes in HPLC(10～100 μl), this means that several injections are possible in CE from tiny sample volumes that can lead to significant savings. Fifthly, the separative efficiency is high(over 105 theoretical plate number). Sixthly, the detecting is on-line as shown in Fig 1. Additionally, the performance is automatic. As shown in Tab 1,the results of drug analyses by CZE are almost the same as those by HPLC, if appreciate conditions are chosen for CZE.
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Generally, there are four kinds of applications of CZE in clinical medicine, viz, therapeutic monitoring,pheno-typing disease diagnosis, drug metabolism and analysis as displayed in Tab 2.
Tab2 Applications fo CZE in the rapeutic monitoring ,disease diagnosis drug metabolism and analyses Application
Eelectrolyte System
Ref.
Therapeutic monitoring
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Climetidine in Plasma
phosphate,pH2.0
6
Cyclic guanine mono-phosphate phosphodiesterase inhibitor in rat serum
phosphate,pH6.0
7
Cytosine-β-D-arabinsoine in plasma
Acetate,pH2.5
8
Fosfomycin in serum
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Borate and phenyl phosphonic acid
9
Heproxen mimetics in human and rat plasma
Citrate-methanol buffer ,PH4.0
10
Iohexol in serum
Borate,pH8.0
11
Naproxen in serum
Tricine pH8.0
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12
Pentobarbital in serum
Borate,pH8.5
13
Piracetamin humanp;asma
Borate and alpha-CD
14
Suramin in serum
CAPSO,pH9.7
15
Phenotyping disease diagnosis
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Dextromethorphan in urine
Borate ,pH9.3
16
Hypoxanthine adn xanthine in urine
Phosphate,pH8.0
17
Methylanonic acid in serum
Teis-citrate,pH6.4
18
Drug metablism
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Cefixime adn metabolites in urine
Phosphate,pH6.8
19
Coumarin metabolites in urine and serum
Phosphate,pH7.5
20
Famotidien
Polyethylene oxide&dextran,pH4.5
21
Haloperidol and 10synthetic metabolite
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pH4.5with 10%MeOH
22
Nitrite and nitrate in bio-fluids
Sulfate ,pH8.0
23
Oxprenolol and metabolites in human urine
Phosphate pH2.5 with PH-β-CD
24
Pyrazolacridine metabolites in urine
NH4 acetate MeOH,acetic acid
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25
Zolpiddem and metabolites in urine
Phosptate pH5.6
26
Zopiclone adn its metabolites in urine and saliva
Phosptate pH2.8with -β-CD
27
Druganalyses
10 acidic drugs and excipients
Borate ,pH9.5
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28
13 aminoglycoside antibiotics
Imidazole-acetate ,PH5.0
29
Aspartyl dipeptides
Phosphate,PH3.3with CM-β-CD
30
12 basic drugs and excipients
Phosphate,PH2.5
31
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22 basic drugs in urine
Phosphate,PH22.0
32
17 basic drugs
Phosphate,PH2.4
33
10 beta-blockers
Citrate buffer,low pH
34
Cardiovascular drugs
Various separations
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35
Chiral drug:clorprenalinge ,benzhexol,esmolol,terazosin
Phosphate ,pH2.5 with neutral CD
36
Chlortetracycline and relative substances
Teraborate,Ph8.5with EDTA
37
15 diuretics in blood and urine
60mmol.L-1 CAPS ,pH10.6
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38
Diuretic drugs
Various modes
39
18 common abused drugs
Phosphate-borate,pH8.5
40
Glucose ,Galactose adn Lactose
75 mmol.L-1 borate
41
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Pyridinyl-methyl-sulfiyl-benzimidazoles
Non-aqueous solution
42
Quinolone antibiotics
HEPES,pH 8.5with acetonitrile
43
16 sulphonamides
Imidazole-acetate,pH7.0
44
CAPS=cyclohexaneamino suphonic acid;CAPSO=zwiterionic buffer;CD=cyclodextrin;HEPES=2[N-hydroxylethyl]piperazine-N'-thanesup-phonic acid+sodium hydroxyide
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The therapeutic monitoring by CZE possesses great significances in clinical medicine.At first, the method can be used for the control of therapeutic blood concentration of drug used, this is of obvious significance for real individual therapeutics. With the monitoring of drug in blood, one can in time adjust individual therapeutics to prevent toxic effect of drugs(like anti-humor, cardiovascular, calmative and anesthetic drugs, to name but just a few).This is also important for the patients with low kidney/or liver function exhaustion.In Tab 2, we have listed some drugs used in clinical medicine.
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Gout is a kind of metabolizing disease, also a phenotyping disease. The diagnosis of the disease needs to detect the concentration level of hypoxanthine and xanthine in both urine and blood. Tab 2 shows the method of monitoring hypoxanthine and xanthine in urine for the phenotyping disease diagnosis(details see Ref 17),coupled with the dextromethorphen in urine and Methylmanonic acid in serum.
Drug metabolism in body is one of the most important content in pharmacology, pharmacokinetics and toxicology.Tab 2 implies that by using CZE method, numerous medicines, including their metabolites, have been detected. As shown in Tab 2, the electrolyte systems are very simple and astounding cheap as compared with flow phases used in HPLC. Thus, the procedures based on CZE for drug and its metabolites are of apparent importance for pharmacology and pharmacokinetics, including toxicology.
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Drug analysis is the most basic work not only in quality control, but also in pharmacology and in clinical therapeutics. The last content in Tab 2 displays numerous drugs have been analyzed by the procedures based upon CZE. In fact, more drug analyses, including those in the former three contents in Tab 2, can not be listed here due to the print limit. The drugs analyzed with CZE concern a wide range from antibiotics, to vitamins, to cardiovascular drugs, to steroids, to diuretics, to common abused drugs as well. This also shows CZE is a versatile tool of analysis for pharmacology and therapeutics.
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5 Outlook
A series of new great developments in CZE have been achieved in recent years. The achievements that hold great significance in biomedicine are the CE based on microchip[45～47] and single- cell analyses[48]with CZE. With the microchip based CE, including CZE, the capillary is eroded in a very small glass chip, the separative length is less than 30 mm, and the run can be completed within 30 seconds,even within 20 seconds. Thus, the technique is of great potential of becoming very powerful and efficient tool of analysis. It will also open a wide way for drug and metabolite analyses in clinical pharmacology and therapeutics.
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Single-cell analyses with CZE have been reported since 1995[48]. With the development of micro-injection, one can introduce a single cell into capillary and then run it. Clearly, with the further development in other techniques, one can study pharmacology at cell-level, and analyze drug effect and metabolite with single cell.The cell analyses with CZE not only show great attraction to biochemist, but also exhibit exciting enticement to pharmacologist and therapeutist.
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This project is supported by the National Natural Scientific Foundation (№ 29775014, 29975026) and The Scientific Foundation of Chinese Health Committee(№98-2-334).
Correspondence to:HE You-Zhao,Tel:0551-3607072,e-mail: yzhe@mail. ustc.edu.cn
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2000-04-10 received, 百拇医药