Capillary electrophoresis and the pharmaceutical industry: a new era.
To increase the confidence of results determination, two or more complementary techniques are usually required for qualitative and quantitative assays. Protein, peptide and oligonucleotide separations primarily employ HPLC and slab-gel electrophoresis techniques. Therapeutic drug monitoring studies commonly use immunoassays, TLC, GC and HPLC.
The development of capillary electrophoresis (CE) has proven to be an effective alternative to many of the techniques listed above. Often touted as a hybrid between liquid chromatography and electrophoresis, CE appears to be ideally suited for the analysis of a large spectrum of sample types using several different selectivities.
Together, high-performance liquid chromatography (HPLC) and high-performance capillary electrophoresis (HPCE) appear to be ideal complementary techniques. HPLC and HPCE can both be used for automated, high resolution separations with fast analysis times (typically less than thirty minutes), while each technique retains its own unique mechanism of separation.
High-Performance Capillary Electrophoresis
HPLC has undergone tremendous growth in the last two decades. This growth can be attributed to the development of high performance packing materials, along with advances in instrumentation and a much better theoretical understanding of the separation mechanisms.
Similar developments are now being made in the field of capillary electrophoresis. Research into open-tube capillary electrophoresis with on-column detection is approximately 10-years old, while commercial instrumentation has been available for over three years. Like HPLC, CE is capable of using several different selectivities for the separation process. The different modes of capillary electrophoresis include capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), micellar electrokinetic capillary chromatography (MECC), isoelectric focussing and isotachophoresis (ITP).
The principles of operation in capillary electrophoresis are relatively simple. A representation of the experimental setup is illustrated in Figure 1. In general, separations are carried out in fused-silica microbore (25-200 um ID) capillary tubes. A small (nanolitre volume) plug of sample solution is introduced into one end of a capillary tube equilibrated with an appropriate electrolyte. Sample components then migrate at different rates under the action of a large electrical field typically 10000 to 30000 volts), and detected on-column' as they approach the other end of the capillary by one of several detection schemes.(1) The utilization of extremely high-voltage potentials allows for very rapid separations, with very high separation efficiencies. Typical efficiencies range from 100000 to 200000 TPs while separations exceeding 1 million plates have been reported.(2) The efficient dissipation of heat, provided by the capillary tube, nearly eliminates convective mixing so that separated components approach the theoretical limit of being broadened only by longitudinal diffusion.(3,4) Since detection is 'on-column', band broadening associated with the transport of the solute to an off column detector is eliminated.
The most common format of CE involves the separation of molecules in free solution. Here, the basis of separation is due to differences in electrophoretic mobilities, a function of both the mass and exposed charge of a molecule. Manipulation of both pH and ionic strength may influence the net charge of the molecule and thus affect its separation. Conversely the addition of a pseudostationary phase to the separation medium may combine chromatographic interaction with electrophoretic migration which is the basis behind electrokinetic capillary chromatography EKC).(5) A popular form of EKC has been micellar electrokinetic capillary chromatography (MECC). In MECC, ionic surfactants are added to the separation electrolyte above their critical micelleforming concentrations. Separation of neutral and ionic compounds may be achieved on the basis of differential solute distribution and the differential migration of the micellar phase.(6,7) The further addition of bile salts to the electrolyte has been successful for the separation of very lipophilic compounds.(8) The resolving of racemic mixtures has also been achieved through the incorporation into the electrolyte of chiral additives, which serve to create mobility differences through diastereomeric interaction with analyte molecules.(9,10,11)
Intrinsic within free-solution capillary electrophoresis is a built-in noise free pump generated by electroosmotic flow. The electroosmotic pump plays an important role in allowing the simultaneous analysis of complex mixtures containing anions, cations and neutral compounds. However, in certain instances it has proven beneficial to reduce the magnitude or even reverse the direction of the electroosmotic flow through the use of charged surfactants, which alter the zeta potential of the capillary inner walls.(12,13) Alternatively, for techniques like capillary isoelectric focussing and capillary gel electrophoresis the electroosomotic flow is eliminated.
Capillary Electrophoresis of Pharmaceuticals
Stringent guidelines exist to ensure that pharmaceutical compounds are of high quality. Metabolic studies must be carried out to determine the fate of the pharmaceuticals in animals and man, while quality control is concerned with purity determination and the assay of active ingredients. Confidence in the results from these assays usually require a minimum of two complementary analytical techniques. HPLC has been used routinely for these analyses because of the high efficiency and many different selectivities it employs.
Recently, CE has gained popularity in the analysis of pharmaceutical preparations. The attributes of CE that have made it appealing include, automation (throughput), speed of analysis, minimal sample preparation and methods development, very high-separation efficiencies (exceeding HPLC), multiple selectivities and reduced operational costs." Reduced operational costs have been realized through decreased solvent usage and disposal costs (a few millilitres rather than litres), as well as reduced sample preparation. An example of reduced sample preparation is described in work by Altria and Rogan.(15) In this example the two diastereoisomeric forms of the antibiotic cefuroxime axetil ester are separated from their major hydrolysis product (cefuroxime). Due to the complex nature of the axetil formulation, considerable sample pretreatment is required prior to HPLC analysis. However, using MECC, the sample is injected directly from a methanolic solution. Figure 2 illustrates this separation.
MECC of Pharmaceutical Preparations
The combined selectivity provided by micellar electrokinetic capillary chromatography has made it very appealing for the separation of a wide range of molecules. The following are a few examples of samples already examined with MECC.
Nucleic Acids and Analogs
Analogs of nucleic acid derivatives have considerable interest as therapeutic agents and as tags of modifiers in biochemical studies. The agent 3'-azido-2'-deoxythymidine (AZT), used in the treatment of acquired immune deficiency syndrome (AIDS), is one such substance for which a convenient method of monitoring at the therapeutic level in serum would be desirable. Figure 3 illustrates the separation of AZT from other nucleic acid analogs using MECC.
Opiates by MECC
The QC of Morphine and other opiates have previously been analyzed with HPLC procedures combined with electrochemical detection for trace analysis. However, quite often the resolution of similarly structured compounds is inadequate. For example, the separation of morphine from codeine (which differ only by methylation of the phenolic group) is notoriously difficult by HPLC. Capillary electrophoresis, in its MECC format, has shown great potential for the separation of a wide variety of neutral and charged pharmaceuticals. Figure 4 illustrates the separation of morphine, codeine, pholcodine and dextromethorphan as well as ephedrine (commonly included in opiate based antitussive preparations).
The understanding of the metabolic fate of a given pharmaceutical preparation, in either man or animal, is of great importance. Metabolic drug studies require assays of both the product and its phase I and phase 11 metabolites. Phase I metabolites often have physiochemical characteristics very similar to the starting product, and can usually be isolated and assayed with the same procedure (eg. RP-HPLC). Phase II metabolites are enzymatically catalyzed conjugation products that are generally hydrophylic as well as being thermally unstable. Often they cannot be easily assayed directly by RPHPLC (due to lack of retention) or GC (due to thermal degradation). Since MECC combines both hydrophobic interaction and charged-based electrophoretic migration in one separation, it offers great potential for the co-analysis of drugs and their phase I and phase II metabolites. Figure 5 illustrates work by Kerr and Jung where the relatively lipophilic and basic drug metoclopramide is separated from its metabolites and hydrophilic conjugates.
Chiral isomer separation is an important area of research for the pharmaceutical industry. Although enantiomers may appear to have identical physiochemical properties, they may behave differently when exposed to an optically discriminating environment, such as the human body.(16) Although there have been many successful separations of enantiomers with HPLC, problems of column expense, lifetime and limited applicability are regularly encountered.
MECC can provide a suitable, fast alternative to HPLC for the analysis of chiral compounds. Chiral selectivity is imposed through the addition of additives to the buffer. Figure 6 illustrates the separation of the enantiomers of cicietanine in less than three minutes using an SDS micellar buffer.
In this example, the differential rate of partitioning of the two entantiomers into the SDS micelle is great enough to allow them to be resolved. There are also several examples of cyclodextrins and bile salts providing chiral selectivity when added to the separation electrolyte.
The examples presented in this article represent only a very small portion of the breadth of work already reported on capillary electrophoresis. However, these examples do point out several advantages that CE has over other analytical techniques. The technique is rapid in both analysis and methods development, the applicability is widespread and the potential for cost savings is high. Additionally, CE has excelled in application areas that have historically proven to be extremely difficult. Whether its use is to separate compounds previously unresolved or simply as a complement to HPLC, CE is a very powerful tool for the pharmaccutical laboratory.
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|Publication:||Canadian Chemical News|
|Date:||Sep 1, 1991|
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