Capillary Electrophoresis For The Analysis of Small-Molecule Pharmaceuticals

Electrophoresis 2006, 27, 22632282
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Kevin Altria1 Alex Marsh1 Cari Snger-van de Griend2

1
Review
GlaxoSmithKline Research & Development, Harlow, Essex, UK 2 AstraZeneca Pharmaceutical and Analytical R&D, Sdertlje, Sweden
Capillary electrophoresis for the analysis of small-molecule pharmaceuticals

This paper reviews the application of CE to the analysis of small-molecule pharmaceuticals. The areas of pharmaceutical analysis covered are enantiomer separation, the analysis of small molecules such as amino acids or drug counter-ions, pharmaceutical assay, determination of related substances and physicochemical measurements such as log P and pKa of compounds. The different electrophoretic modes available and their advantages for pharmaceutical analysis are described. Recent applications of CE for each subject area are tabulated with electrolyte details. Keywords: Capillary electrophoresis / Chiral separation / Log P determination / Pharmaceuticals / pKa determination DOI 10.1002/elps.200600030
Received January 17, 2006 Revised March 16, 2006 Accepted March 17, 2006
1 Introduction
The use of CE methods for pharmaceutical analysis has become increasingly popular in recent years. The wide range of applications for which its use has proved successful includes [1] assay of drugs, determination of drugrelated impurities, physicochemical measurements of drug molecules, chiral separation and the analysis of pharmaceutical excipients. Pharmaceutical analysis is dominated by HPLC. Other separative techniques used include TLC and GC, but the range of applications for which they can be used and their quantitative capabilities are not as widespread as HPLC. Other techniques include IR and UV spectroscopy, which are often used for identity testing of pharmaceuticals, and a range of flask-based methods, e.g. titration, which are used for physicochemical parameter determinations. The advantages of CE for pharmaceutical analysis include its speed and cost of analysis, reductions in solvent consumption and disposal, and the possibility of rapid method development. CE also offers the possibility that a single set of separation conditions can be applicaCorrespondence: Dr. Kevin Altria, GlaxoSmithKline R&D, Building H89, New Frontiers Science Park South, Harlow, Essex, CM19 5AW, UK E-mail: kevin.d.altria@gsk.com Fax: 144-1279-643860 Abbreviations: API, active pharmaceutical ingredients; FSCE, free solution capillary electrophoresis; MEEKC, microemulsion electrokinetic chromatography
ble for a wide range of analyses, giving efficiency savings. CE instruments can be coupled to a variety of detector types, including mass spectrometers, for special applications and more detailed analysis.
1.1 Electrophoretic modes

There are several electrophoretic modes that can be used to analyse pharmaceuticals. Free-solution capillary electrophoresis (FSCE) is very popular and involves the use of simple buffered aqueous electrolytes. Separation of ionic drugs is achieved in FSCE through pH control. The addition of reagents to the electrolyte, such as organic solvents or ion-pair reagents, can also be used to achieve the required separation selectivity. For the separation of drug enantiomers, FSCE with chirally selective agents, such as CDs, added to the electrolyte to facilitate chiral resolution is the most common method used [2]. For the separation of water-insoluble or sparingly soluble pharmaceuticals, NACE, which employs electrolytes composed of organic solvents, has been used successfully [37]. NACE is also useful for the resolution of watersoluble charged solutes as the selectivity obtained can be different to aqueous-based separations. MEKC and microemulsion electrokinetic chromatography (MEEKC) employ electrolytes that contain surfactant molecules which form micelles (MEKC) and microemulsion droplets (MEEKC). These micelles or droplets add a chromatographic element to the separation that enables separation of acidic, basic and neutral drugs.
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MEEKC is the most flexible of all the separation modes, offering the greatest selectivity to the widest range of compounds and can be considered the separation method of choice when performing CE analysis.
1.1.3 MEKC and MEEKC

MEKC or MEEKC can be employed to achieve separation of mixtures of acidic, basic and neutral pharmaceutical compounds. Both techniques enhance electrophoretic separations by providing a chromatographic partitioning mechanism into the micelles (MEKC) or microemulsion droplets (MEEKC). This partitioning allows resolution of neutral compounds as well as charged species. Neutral solutes are separated solely based on their solubility. Charged solutes are separated based on their electrophoretic movement, solubilitybased partitioning and potential ion-pair interaction with the charged micelle or droplet. MEEKC has been found to provide superior separation efficiency to MEKC, probably due to improved mass transfer between the microemulsion droplet and aqueous phase [18]. Solutes also penetrate the MEEKC droplet more easily than the rigid MEKC micelle, which allows MEEKC to be applied to a wider range of analytes. MEEKC also offers greater separation capability for water-insoluble compounds than MEKC [19] and a larger separation window [18, 20]. The use of MEEKC to determine compound solubility (log P) has been successfully shown for many types of drugs [2129] and is an important pharmaceutical application of the technique. Many reports have been published [22, 3042] detailing the use of MEEKC for pharmaceutical applications. Table 1 details selected pharmaceutical applications using MEEKC and the composition of the microemulsion used. Readers are referred to two recent reviews [43, 44] for further examples of MEEKC applications and descriptions of operating parameter effects.
1.1.1 FSCE
The majority of drugs are basic and thus ionised at low pH. FSCE using low-pH buffer systems has been employed to separate a range of basic drugs; separations are based on analyte size and number of positive charges, but neutral compounds do not migrate through the detector. This offers an advantage over HPLC analysis of formulated pharmaceutical products where the analyte peak can be masked by the co-elution of neutral excipient or flavouring compounds. A low-pH phosphate buffer has been used to analyse 550 different basic drugs [8] and a similar method has been validated [9] for analysis of a variety of basic drugs, excipients and raw materials. To separate mixtures of acidic and basic drugs (or cationic and anionic species), FSCE at high pH can be utilised [10]. At pH 7 or greater, the EOF generated by the applied current is sufficiently strong to sweep anions to the detector. Analyte migration time is dependent on solute charge type and density; strongly cationic species migrate first while small, highly charged anions attempt to migrate against the EOF and are detected last. Neutral species migrate unresolved at the EOF solvent front.
1.1.2 NACE
Water-insoluble basic drugs can be difficult to separate using FSCE methods, and NACE can be applied for such analyses. [36, 11]. NACE electrolytes do not contain water, with organic solvents such as methanol and ACN being used instead. Selectivities which are difficult to obtain using aqueous buffers, even when using surfactants or complexing agents, may be easily obtained with nonaqueous systems [12]. Separation selectivity can be altered in NACE by changing the composition of organic solvents in the electrolyte. The compatibility of MS coupling is increased due to the solvent volatility and the use of organic buffers [3]. NACE is starting to be more frequently considered and its use for the analysis of basic drugs has been reported. These reports include the separation of a number of opium alkaloids [12], a mixture of cationic drug substances [13], a range of tropane alkaloids [6], a range of b-blockers [14], tricyclic antidepressants [7] and different basic drugs [5, 15]. NACE has also been successfully used to separate polar acidic and basic drugs [16], to perform chiral separation of pharmaceutical amines [17] and to calculate pKa* values of basic analytes in methanol [15].
1.2 CE-MS
There is an increased utility of CE-MS in pharmaceutical analysis as the methodology and equipment skills/reliability/robustness have all improved in recent years. CE-MS methods can offer sufficient sensitivity to be operational methods. For example, 0.05% galantamine impurities were monitored [45] in Reminyl capsules using CE-MS (ESI). Standard CE-MS methods have been developed and applied to a wide range of drug types. For example, Vassort et al. [46] reported the successful application of standard CE-MS method conditions to 22 drugs and their process impurities.
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Table 1. Selected pharmaceutical applications of MEEKC Application Analysis of betamethasone and derivatives Microemulsion composition 1.44% w/w SDS, 0.81% w/w octane, 6.61% w/w butan-1-ol, 91.14% 20 mM sodium phosphate pH 7.5 1.9% w/w IPM, 2.0% w/w SC/SDC, 3.5% w/w PC, 0.81% w/w octane, 7.5% w/w butan-1-ol, 85.1% 20 mM sodium phosphate pH 7.5 3.3% w/w SDS, 0.81% w/w octane, 6.6% w/w butan-1-ol, 89.3% w/w 50 mM borate pH 9.5 3.31% w/w SDS, 0.81% w/w n-octane, 6.61% w/w butan-1-ol, 89.27% w/w 50 mM phosphate buffer pH 2.1 3.31% w/w SDS, 0.81% w/w n-octane, 6.61% w/w butan-1-ol, 89.27% w/w 10 mM borate buffer pH 9.2 3.97% w/w SDS, 0.81% w/w n-octane, 6.61% w/w butan-1-ol, 10% w/w propan-2-ol, 78.61% w/w 10 mM borate buffer pH 9.2 1.8% w/w SDS, 0.48% w/w n-octane, 3.96% w/w butan-1-ol, 93.76% w/w 10 mM borate buffer pH 9.2 1.44% w/w SDS, 0.81% w/w octane, 6.61% w/w butan-1-ol, 91.14% 20 mM sodium phosphate pH 7.5 2.0% w/w SDC, 3.5% w/w PC, 1.9% w/w IPM, 0.81% w/w octane, 7.5% w/w butan-1-ol, 85.1% 20 mM sodium phosphate pH 7.5 2.0% w/w SC, 3.5% w/w PC, 1.9% w/w IPM, 0.81% w/w octane, 7.5% w/w butan-1-ol, 85.1% 20 mM sodium phosphate pH 7.5 23.3 mM SDS, 16.4 mM n-heptane, 180.85 mM butan-1-ol, 8% ACN, 20 mM borate 2.12% w/w SDS, 0.52% w/w heptane, 4.21% w/w butanol, 84 mM borate pH 8.4 3.3% w/w SDS, 0.8% w/w octane, 6.6% w/w butan-1-ol, 89.29% w/w 10 mM, sodium tetraborate pH 9.15 6.0% w/w SDS, 0.8% octane, 6.6% butanol, 20.0% propan-2-ol, 66.6% 25 mM phosphate pH 2.75 80 mM SDS, 1% w/w octane, 5% v/v butanol, 40 mM borate pH 8.5 6.0% w/w SDS, 0.8% octane, 6.6% butanol, 20.0% propan-2-ol, 66.6% 25 mM phosphate pH 2.75 2.25% w/w SDS/0.75% w/w Brij35, 0.8% w/w n-alkane, 6.6% w/w 1-butanol, 17.5% w/w 2-propanol, 72.1% w/w 10 mM borate buffer pH 9.2 Ref. [22]
Determination of nine preservatives in pharmaceutical and cosmetic products Analysis of 4-hydroxybenzoate preservatives in pharmaceuticals Analysis of formulated drug products Insoluble ingredients of pharmaceutical ointment Levetiracetam from other antiepileptic drugs Separation of immunosuppressive drugs
[30]
[31]
[32] [33] [34] [35]
Analysis of ephedrine and pseudoephedrine Amino acid derivatives using LIFD detection Nicotine-related alkaloids Analysis of vitamins Analysis of water and fat-soluble vitamins Separation of fat-soluble vitamins UV filters in sunscreen lotions
[36] [37] [38] [39] [40] [41] [42]
IPM = isopropylmyristate, PC = phosphatidylcholine, SC = sodium cholate, SDC = sodium deoxycholate
2 Chiral CE for pharmaceutical analysis

The resolution of enantiomers is one of the most studied areas in CE. It has been shown that CE is a powerful analytical technique for separating chiral compounds and fast, efficient, sensitive and selective methods have been developed. Extensive research has shown that chiral analysis by CE can be superior compared to conventional techniques such as LC and GC. Advantages include reduced costs as the chiral selector is added to the BGE
instead of using expensive chiral columns. The possibility of low-UV wavelength detection for CE also allows the separation and detection of analytes with poor chromophores which are difficult to detect by LC. CE has become the technique of choice for enantiomeric separations [47 49] in some companies. Several companies [46, 5054] have published generic approaches for chiral method development. The importance of using orthogonal methods and techniques in method evaluation and validation has been stressed [46].
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There are many types of chiral selectors that can be applied to achieve enantioseparation, but the most common are native and derivatised CDs. (Table 2 contains a listing of CD examples and abbreviations.) An illustrative example from AstraZeneca [55, 56] is presented in Fig. 1, which shows chiral resolution of a series of five local anaesthetics using heptakis(2,6-di-O-methyl)-b-CD in a low-pH buffer. Recently, the enantiomeric purity determination of ropivacaine hydrochloride based on these results was published in the US Pharmacopeia [57]. Other
chiral selectors that have been applied to CE separations include natural and synthetic chiral micelles, crown ethers, chiral ligands, proteins, carbohydrates and macrocyclic antibiotics. Readers are referred to textbooks [5860] and recently published reviews on the fundamental aspects and applications of chiral CE [6173]. Scriba [61] specifically reviewed the use of chiral CE techniques for pharmaceutical and bioanalysis. Other reviews are more general (chiral CE and/or CEC [62, 64], CE for pharmaceuticals [63, 66], chiral CE-MS [65]).
Table 2. Applications of chiral CE for pharmaceutical analysis Application R209130 BIX SLV307 (antipsychotic) Calcium levofolinate Methotrexate Ragaglitazar, arginine Citalopram Moxiflaxin Nicardipine Propanolol Adrenaline Butorphanol, cycloamine
L-DOPA
Substance/product Substance Substance Capsules Substance Tablets, injection solutions Substance, tablets Tablets Substance, ophthalmic/ otic solutions Substance-CD complexes Local anaesthetic solutions Substance, intermediate Tablet Substance
Electrolyte 5 mM a-CD, 2% S-b-CD, 10 mM phosphoric acidtriethanolamine buffer pH 3.0 2.5% w/v CM-b-CD, 25 mM NaH2PO4 buffer pH 4.0 50 mM HP-a-CD, 100 mM Trisphosphoric acid pH 2.5 20 mg/mL DM-b-CD, 40 mM sodium tetraborate pH 9.9 45 mM b-CD, 100 mM SDS, 50 mM borate buffer pH 9.30 containing 25% methanol 90% (2% SB-b-CD, 0.7% DM-b-CD, 25 mM phosphate buffer pH 8.0) 10% ACN 0.15% CM-g-CD, 20 mM NaH2PO4 pH 7.0 (with NaOH) 5% HS-g-CD, 12.5 mM triethylammonium phosphate buffer pH 2.5, 6% ACN 3% S-b-CD, 2% TM-b-CD, 20 mM triethanolamine phosphate pH 3.0 40 mM DM-b-CD, 100 mM phosphoric acid, 50 mM triethanolamine 0.02% HS-b-CD, 250 mM Tris, 350 mM phosphoric acid 5 mM HP-b-CD, 100 mM borate pH 8.0, 120 mM SDS 20 mM g-CD, 100 mM Tris, 140 mM phosphoric acid 10 mM TM-b-CD, 50 mM phosphate buffer pH 2.5 15 mM CM-b-CD, 120 mM a-CD, 100 mM borate pH 9.2 2 mM sulphated b-CD, 50 mM phosphate buffer pH 2.5 20 mM HP-b-CD, 100 mM phosphate buffer pH 7 3% w/v highly S-b-CD, 25 mM phosphate buffer pH 2.5 2% S-b-CD, 25 mM phosphate buffer pH 2.2 14 mM HP-b-CD, 100 mM Tris-phosphate pH 3.0 90% (2% SB-b-CD, 0.7% DM-b-CD, 25 mM phosphate buffer pH 8.0) 10% ACN 7.5 mM b-CD, 75 mM triethanolammonium phosphate pH 2.5
Ref. [51] [53] [75] [76] [77] [78] [80] [81] [83] [84] [86] [87] [88] [89] [90] [91] [92] [93] [94] [95] [96] [97] [98]
Tablets, injection solutions 100 mM HSA, 67 mM phosphate buffer pH 7.4
Lisuride
Ketoconazole, terconazole Substance, tablets, syrup, gel 3-(4-Methylbenzylidene)camphor Aziridine-type protease inhibitors Etodolac Baclofen Substituted imidazole p38 MAP kinase inhibitor Apomorphine Cream Substance Tablets Substance Substance Substance
Balaglitazone, pioglitazone, Substance, tablets thiaglitazone Rivastigmine Substance
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Table 2. Continued Application Pheniramine Cefoperazone Simendan Organic disulphates Sertraline Anticholinergic drugs Omeprazole Substance/product Granulate Substance, solutions Substance Substance Substance Substance Capsules Electrolyte 2.5 mg/mL CE-b-CD, 0.2% MHEC, 20 mM e-aminocaproic acid pH 4.5 (with acetic acid) 0.04 mM b-CD 75 mM NaH2PO4 pH 4.0 50% (12 mM b-CD, 20 mM borate pH 11.0), 50% methanol 0.5 mM QA-b-CD, 10 mM glycine pH 2.4 20 mM HP-b-CD, 30 mM sodium deoxycholate, 35 mM borate pH 11.5 10 mM HDMS-b-CD, 20 mM phosphoric acid, 10 mM NaOH, in methanol 30 mM M-b-CD, 5 mM sodium disulphide, 40 mM phosphate buffer pH 2.2 Ref. [99] [100] [101] [102] [103] [104] [105]
Abbreviations: a-CD b-CD g-CD CE-b-CD CM-b-CD CM-g-CD DM-b-CD HDMS-b-CD HP-a-CD HP-b-CD HS-b-CD HS-g-CD QA-b-CD M-b-CD S-b-CD SB-b-CD TM-b-CD
a-cyclodextrin b-cyclodextrin g-cyclodextrin carboxyethyl-b-cyclodextrin carboxymethyl-b-cyclodextrin carboxymethyl-g-cyclodextrin dimethyl-b-cyclodextrin heptakis(2,3-di-O-methyl-6-O-sulphato)-b-cyclodextrin hydroxypropyl-a-cyclodextrin hydroxypropyl-b-cyclodextrin highly sulphated b-cyclodextrin highly sulphated g-cyclodextrin quaternary ammonium b-cyclodextrin methyl-b-cyclodextrin sulphated-b-cyclodextrin sulphobutylether-b-cyclodextrin trimethyl-b-cyclodextrin
Lmmerhofer [69, 70] recently reviewed chiral NACE [69] and CEC [70]. In the chiral CE review the focus was on solvent effects on the chiral separation, while the CEC review discussed potentials and peculiarities of chiral nonaqueous CEC. NACE considerably expands the potential for chiral CE, since both analytes and chiral selectors that are poorly soluble in water can be employed. But the number of publications is still modest, probably due to the dominant role of CDs as chiral selector [69]. Mangelings et al. [71] have recently reviewed the use of chiral CEC for pharmaceutical analysis, highlighting potential practical pharmaceutical applications published in the past 10 years. Chiral stationary phases based on macrocyclic antibiotics and polysaccharide selectors are most frequently used, but more recently monolithic stationary phases have gained interest.
The usefulness of chiral CEC has been proven many times. But in order to survive as a separation technique within pharmaceutical industry, it is important that the between-column reproducibility problems and technical issues (e.g. sample loop injections) reportedly encountered [71] are resolved. Mertzman and Foley [72] evaluated the use of CD-modified MEEKC in different modes, thereby opening possibilities for resolution of enantiomeric hydrophobic compounds. A series of overviews sorted by chiral selectors and technique was recently published in Methods in Molecular Biology [73]. Patel reviewed the use of CE techniques for the enantiomeric resolution of a group of NSAIDs, the 2-arylpropionic acids, with the application to real samples of pharmaceutical and biomedical interest [67]. They concluded that
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Figure 1. Achiral (A) and chiral (B) separation of local anaesthetics. BGE: 0.10 M phosphoric acid, 0.09 M triethanolamine, with (B) and without (A) 10 mM heptakis(2,6-di-O-methyl)-b-CD. Reproduced with permission from [55, 56].
the majority of the reports have been focused on the mechanism of resolution and the evaluation of the effect of parameters on the separation, while only a limited amount of reports describe fully validated methods that have subsequently been applied to pharmaceutical or bioanalytical problems. The various modes of chiral CE for the analysis of pollutants have been reviewed by Hernndez-Borges et al. [68]. They concluded that CE is an impressive tool for this purpose, but some effort needs to be put into on-line and off-line concentration techniques [68].
the pH of the BGE is aligned to the nature of the compound. Fine-tuning of the experimental parameters is then performed. An example was also described in a subsequent paper [51]. A BoxBehnken experimental design was used [51] in which four factors were optimised to obtain separation of the eight stereoisomers of the compound of interest (three chiral centres). Finally, the method development was continued with optimising the method for QC performance, i.e. appropriate sensitivity and stability. The method was then validated according to the demands during phase II clinical studies. Another illustration of their approach was presented [52]. Sokoliess [54] (Boehringer Ingelheim Pharma) published a similar generic approach for the initial screening, which was illustrated with the enantiomeric separation of an active pharmaceutical ingredient. Particular attention was paid to the conditioning of the capillary whilst optimising the method. The inlet and outlet buffer vials were swapped between injections to diminish buffer depletion and chiral selector concentration changes. This allowed the same 1-mL buffer vials to be used for up to 20 runs. Vassort and Szcs et al. [46] (Pfizer) included an evaluation of CE vs. LC and LC vs. CE orthogonality, as well as the compatibility to CE-ESI-MS [46] in their generic
2.1 Pharmaceutical applications of chiral CE: generic approaches

Several pharmaceutical companies [46, 4954] have recently published generic approaches for chiral method development. Jimidar [49] (Johnson & Johnson) described a widely applicable strategy for screening through a number of CDs for the separation of basic, acidic or neutral components. The type of CD, the CD concentration, buffer electrolyte concentration and the amount of organic modifier in the BGE is evaluated in these screens. In the first instance,
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screen. They conclude that their screen offers significant time and resource savings and will speed up the process of pharmaceutical analysis. A logical further advance would be to evaluate additional techniques such as GC, SFC, NACE and CEC to extend the versatility of the approach. Rocheleau [53] (Bristol-Myers Squibb) used only sulphated b-CDs in her screen, and additionally evaluated the different suppliers and batches of CD. The approach has been successfully applied to more than a dozen of the companys compounds in different phases of development. Matthijs [74] compared the use of HSCD/neutral CD dual systems with single HSCD systems, in order to define a sequential strategy for method development with dual systems when resolutions obtained with single systems do not suffice.
and Lehmann [78] also published a method that covered several specification tests. A single chiral CE method achieved [78] the assay and identification of the active pharmaceutical ingredient ragaglitazar and its counterion arginine, and the achiral and the chiral purity of ragaglitazar, all in API and in low-dose tablets. A method was developed for the determination of the diastereomeric stability of S-adenosylmethionine in aqueous solutions and was applied for a short-term stability assessment (freezethaw cycles, 14 days incubation at 387C) [79]. No chiral selector was needed for the separation of the diastereoisomers as the high resolution power of CE was sufficient. Compared to LC, the CE method could simultaneously quantify the diastereoisomers and the degradation products. A rapid method to assay citalopram racemate or escitalopram (S-citalopram) in tablets was reported [80]. The validation included an experimentally designed robustness test using the PlackettBurman model. A chiral CE method was developed to evaluate whether racemization is a potential degradation pathway for moxifloxacin drug substance or ophthalmic/otic solution [81]. The study demonstrated that racemization into the RRenantiomer or RS- or SR-diastereoisomers is unlikely. Therefore stereoselective testing may not be required for this drug substance or its ophthalmic/otic solution. Van Eeckhaut et al. [82] compared different chiral MEKC methods for the enantiomeric separation of the neutral calcium channel-blocking dihydropyridines. Tests comprised chiral MEKC with bile salts, CD-MEKC with uncharged CD and CD-EKC with charged CD. Best results were obtained with CM-b-CD in a pH 5.0 MES buffer. The photostability of nicardipineCD complexes was studied by combining chiral CE analysis with DSC (differential scanning calorimetry) and XRPD (X-ray powder diffraction) [83]. Some CDs protected nicardipine from photodegradation, some had no effect, and one even promoted photodegradation. Martnez-Pla [84] reported the use of HSA as chiral selector in a quality control method for propranolol tablets and injection solutions. The capillary is filled with the HSA solution during preconditioning, while the inlet and outlet vials during the run contain only buffer, resulting in very low costs per analysis. The enantiomeric purity of a series of 2- and 3- substituted 2,3-dihydro[1,4]dioxino[2,3-b]pyridine derivatives, synthesized for early-phase drug discovery, was determined with chiral CE using a-CD and/or S-b-CD as chiral selectors in a formic acid/ammonia buffer pH 4.0 [85].
2.2 Pharmaceutical applications of chiral CE: Applications

Muijselaar [75] described method development and validation for a basic potential antipsychotic compound. A high concentration of Tris was used as the cationic buffer component to prevent excessive electromigration dispersion of the eutomer (main, desired enantiomer). Although the electrodispersion is limited by this approach, the triangular shape of the eutomer peak may still influence the peak shape of the distomer (low level, undesired enantiomer impurity). Therefore it is important to have the matrix components as well as the eutomer present in the samples when evaluating the method performance while measuring the distomer. This was illustrated by determining the corrected peak area ratios and RSDs for the distomer at five levels with and without the presence of the eutomer at nominal concentration. The method was validated and found to give good analytical performance for substance and capsule formulations. Sss [76] developed and validated a method for calcium levofolinate, taking other impurities besides the enantiomer into account. The method was optimised with help of a central composite experimental design and the impurities, enantiomeric as well as others, could be quantified down to 0.05%, well below the European Pharmacopoeia (EP) demands. Thus, this one CE method could replace two separate LC methods currently required in the EP. A CD-MEKC method to simultaneously determine the chiral and achiral purity of methotrexate was published by Gotti [77]. The method was validated and proved to be sufficiently robust and sensitive for the analysis of commercially available tablets and injection solutions. Jamali
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The enantiomeric separation of low concentrations of adrenaline in high-concentrated local anaesthetic (LA) injection solutions was presented in a recent publication [86]. Adrenaline is added to LA solutions in amounts of around 0.1% of the LA. Racemization as low as 23%, i.e. 0.0020.003% of the LA and corresponding to ca. 0.1 mg/ mL of d-adrenaline could be determined with sufficient precision in a simple and robust chiral CE system. LA adrenaline solutions were injected without sample preparation. Further applications presented during the last two years are listed in Table 2 [51, 53, 75105].
cations at low pH, and a pH 2.8 electrolyte of 50 mM ethanesulphonic acid was used to resolve a number of amino acids which were detected directly at 185 nm without requiring sample pretreatment, as shown in Fig. 2 [109]. A BGE of 100 mM sodium tetraborate pH 10 and 20 mM sodium deoxycholate and 15 mM b-CD was used to separate all major and minor components of gentamicin amino sugar antibiotic and its impurities by derivatisation with o-phthaldialdehyde [110]. The presence of small-ion contaminant impurities in drug substance or formulations can also be measured by CE. For instance, a NACE method using an electrolyte containing imidazole as the UV absorber has been used [111] to monitor ammonium ion contaminant. Ammonium was monitored with an LOD of 50 ppb.
3 Analysis of small molecules and ions

The separation and detection of small organic and inorganic ions is an important activity in the pharmaceutical industry. Most drugs molecules are charged and as such are manufactured with a counterion; commonly a metal cation, e.g., K1 for acidic drugs, and an ionic salt, e.g., Cl2 or small organic acid (e.g., acetate) for basic drugs. It is important to analytically characterise the drug stoichiometry (ratio of drug:counterion) to ensure that the potency of the batch of drug substance is known. Because such counterions usually have little or no chromophore, popular techniques for the analysis of small ions include ion exchange chromatography (IEC) and flame atomic absorption spectrometry; but CE is becoming increasingly popular for such applications due to its simplicity. Commercial kits are available for ion analysis and are frequently used. Consequently methods are simple to operate with analysis times of 210 min, which compares favourably with IEC. Additionally, IEC columns are expensive and require regeneration, so CE offers both reduced analysis times and costs. Table 3 contains a selection of CE methods that have been used to analyse simple ions for pharmaceutical applications. Excipients such as SDS [106] or arginic acid [107] can be analysed as raw materials or when present within formulations. For example, SDS levels were determined [106] within various tablet batches. Ions with a poor UV response can be analysed by indirect UV detection, using a UV absorbing BGE, and metal ions can be detected directly through on-capillary complexation to form UV active metal chelates. Some simple organic acids are sufficiently UV active to be detected directly [108]. The separation of amino acids can be quite complicated by HPLC as they need to first be derivatised to provide a chromophore for detection. However, CE can be used at lower operating wavelengths. Amino acids, usually zwitterionic, become
4 Determination of pharmaceutical content

Assay of drug substance and formulated products is a very important and regulated activity. Analytical methods must be validated to strict standards to show that they are robust, accurate, repeatable and suitable for their purpose. Most routine analytical assay determinations in the pharmaceutical industry are performed by HPLC. One of the major advantages of implementing CE methods in place of HPLC is its relatively small level of solvent consumption, millilitres compared to the litres of mobile phase used in an HPLC run. An additional advantage is that sample pre-treatment requirements are often reduced compared to HPLC as the CE capillary can be washed with NaOH between injections and many interfering components do not migrate because they are neutral. The ability to quantify a range of sample types using a single set of CE conditions is another strong feature since this can make considerable savings in analysis and system set-up times [9, 10, 128131]. The electrophoretic conditions inside the capillary vary slightly between injections, which leads to greater variability in peak migration times and area than seen in HPLC. A number of approaches can be taken to overcome this problem. Migration times and peak areas can be calculated relative to those of an internal standard peak which results in great improvement in method repeatability [132]. Greater migration time reproducibility can also be achieved by applying the separation voltage across the capillary for a very short time prior to injection and separation [133]. A commercial capillary treatment system of buffers and rinse solutions has been shown to improve CE repeatability as the capillary is coated with a bilayer of surfactants ensuring that the surface coverage and EOF is consistent between injections and between capillaries. The paper highlighted the consistency of EOF
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Table 3. Small molecules and ions, pharmaceutical analysis application table Application Anions indirect detection SDS levels in tablets Determination of Br, Cl and SO4 as impurities in calcium acamprosate TFA counterion of an opioid peptide analgesic Acetate content in acetate drug salt Acetate residues in drug substance Drug inorganic counterion determination Drug organic acid counterion determination Inorganic anion contaminant in drug substance Anions direct detection Arginic acid (an excipient level) Determination of residual Br in excess of chloride for local anaesthetic analysis Drug organic acid counterion determination, e.g. benzoate, hydroxynapthoate Arginine counterion determination for ragaglitazone Cations indirect detection Ammonium ion impurities Ca, Li, K and Na counterions of glycosaminoglycans Ca in calcium acamprosate drug substance Cationic counter ion content in drug substance K in drug substance Quarternary amine residues in drug substance Cations direct detection K counterion and inorganic cationic impurities of acidic drugs by conductivity detection Creatine, acetic acid, 18-crown-6 [129] NACE method, imidazole 4-Aminopyridine buffer pH 9 Imidazole, sulphuric acid Imidazole, formic acid Imidazole, low pH Imidazole, formic acid Quinine, THF [111] [122] [123] [124] [116] [125] [126] Borate/boric acid 60:40 MeCN: methanesulphonic acid buffer pH 1.3 Borate pH 9.5 [107] [119] [120] [121] Barbital buffer Chromate 1 1 mM borate pH 9.15 Phthalate, CTAB Phthalate, OFM Phthalate, OFM Chromate, TTAB Phthalate, MES, TTAB Chromate, OFM [106] [112] [113] [114] [115] [116] [117] [118] Electrolyte Ref.
CTAB = cetyltrimethylammonium bromide, MES = 4-morpholineethanesulphonic acid, OFM = proprietary Waters chemical, TTAB = tetradecyltrimethylammonium bromide
when using the buffer coating system compared to a standard phosphate buffer using a capillary composed of 19 separate channels. Using the phosphate buffer, the peaks in the 19 individual channels had different speeds and the separation was poor giving a split peak. The EOF was consistent in each channel using the coating system and the peaks moved at the same speed, resulting in a single peak [134]. For a more comprehensive guide to improving the precision of CE methods, readers are referred to a review on the subject [135].
An FSCE method with CDs in the electrolyte has been developed [136] for analysis of the diabetic therapy ragaglitazar and its counterion arginine in active pharmaceutical ingredients (API) and low-dose tablets. The method is suitable for 12 different analyses of the API and tablets active ingredient assay and identification of ragaglitazar and arginine, chiral purity of ragaglitazar and purity of ragaglitazar. The accuracy of the method (% recovery) was found to be 101106% for ragaglitazar and 101125% for arginine, while precision for the detection of peaks (% RSD) was found to be 0.63% for ragaglitazar and
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Figure 2. Electropherogram of 20 common amino acids. 50 mM ethanesulphonic acid, pH 2.8; applied voltage, 30 kV; injection time, 10 s. Peaks: 1 = Lys; 2 = Arg; 3 = His; 4 = Gly; 5 = Ala; 6 = Val; 7 = Ser; 8 = Ile; 9 = Leu; 10 = Thr; 11 = Asn; 12 = Met; 13 = Gln; 14 = Trp; 15 = Glu; 16 = Phe; 17 = Pro; 18 = Tyr; 19 = Cys, UV detection at 185 nm. Reproduced with permission from [109].
3.50% for arginine. An MEEKC method was used for the quantitative determination of folic acid in tablets [137] giving a precision of ,1.2% RSD and recovery of 99.8 6 1.8% at three concentration levels. CE methods have been reported that monitor both drugs and polymers released from drug delivery systems in a single run (HPLC methods allow the monitoring of either the drug or the polymer alone). FSCE was used for monitoring of the controlled delivery of recombinant growth hormone from a system based in VPHEMA copolymer. The CE method used a running buffer of 100 mM sodium tetraborate pH 9 and allowed the simultaneous monitoring of both the liberation of rHGH and the polymers degradation by solubilisation [138]. A CE method has been reported to monitor R- and Sibuprofen released from hydrophilic copolymer systems using dextrin 10 as a chiral selector. The method allowed detection at concentrations of 1 mg mL21 and 0.5% of the R-ibuprofen enantiomer [139]. An assay
method has been reported [142] for quantitative determination of the weakly acidic antibiotic benzylpenicillin and procaine, benzathine and clemizole, the basic forms of benzylpenicillin found in pharmaceutical preparations. Table 4 contains a selection of CE methods that have been validated for the quantitative assay of various pharmaceutical substances and dosage forms. CE can also be applied (simultaneously or separately to the drug content measurement) to monitor excipient levels within formulations. For example [141], a single set of CE method conditions have been developed and applied to the determination of the most commonly employed formulation preservatives (including parabens, cresols, etc.). The 10-min method employed pH 9.2 borate buffer containing 20 mM SDS. The method was validated and applied to a range of samples and obtained good agreement with reference methods.
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Table 4. Validated CE methods for pharmaceutical assay Application FSCE methods Assay and identification of ragaglitazar, a diabetic therapy, and its counterion arginine, chiral purity of ragaglitazar and purity of ragaglitazar in API and low-dose tablets Acetaminophenol, phenylephedrine, chlorepheniramine content in combination tablets Simultaneous determination of procaine, dihydrostreptomycin and penicillin G in multiantibiotic veterinary preparations Determination of indinavir sulphate, a protease inhibitor used in HIV therapy in capsule formulations Method for assay of raloxifene, an oestrogen agonist in bone Determination of pravastatin, a cholesterol-reducing agent, in tablet formulation Determination of the anti-fungal ketoconazole in tablets and creams Stability indicating, validated method for cyclizine hydrochloride tablets and suppositories (motion sickness) Determination and separation of antipsychotics clothiapine, clozapine, olanzapine, quetiapine Simultaneous determination of six angiotensin-II-receptor antagonists candesartan, eprosartan, irbesartan, losartan potassium, telmisartan and valsartan Dynamic capillary coating system methods Analysis of benzodiazepines Characterisation and quantification of heroin and its basic impurities and adulterants NACE methods Analysis of quinolizidine alkaloids in Chinese herbs, which are difficult to separate in aqueous media Tricyclic antidepressant content in tablets MEKC methods Benzylpenicillin salts Determination of acyclovir in Zovirax cream Determination of preservatives in formulations Determination of piribedil content in tablets MEEKC methods Quantitative determination of the fat-soluble vitamin E acetate (suppressed EOF environment enabled timely migration of the analyte peak) Quantitative determination of folic acid (a water-soluble vitamin) in tablets Method details Ref.
90% v/v 25 mM phosphate buffer pH 8.0 1 10% v/v [136] ACN 1 2% w/v sulphobutyl ether b-CD 1 0.7% w/v dimethyl b-CD Phosphate pH 6.2 [142] 0.08 M borate pH 8 [143]
20 mM phosphate pH 2.52 20 mM acetate pH 4.5 10 mM borate pH 8.5 1 10% MeCN 10 mM phosphate buffer pH 2.3 50 mM phosphate pH 2.3 80 mM phosphate pH 3.5 FSCE: 60 mM phosphate pH 2.5 MEKC: 55 mM phosphate pH 6.5, 15 mM SDS
[144] [145] [146] [147] [148] [149] [150] [151]
CEofixa) buffer system CElixirb) buffer system
[152] [153]
1% acetic acid, 50 mM ammonium acetate, 20% ACN in methanol 1 M acetic acid, 50 mM ammonium acetate in ACN
[154] [155]
Phosphate-borate buffer pH 8.7 1 14.4 gL21 SDS 20 mM borate buffer pH 10 1 10 mM SDS Borate pH 9.20 1 20 mM SDS Borate 1 SDS
[140] [156] [157] [158]
0.8% w/w n-octane, 6.6% w/w butan-1-ol, 6% w/w SDS, 20% v/v propan-2-ol, 66.6% w/w 25 mM phosphate pH 2.5 0.5% w/w ethyl acetate, 1.2% w/w butan-1-ol, 0.6% w/w SDS, 15% v/v propan-2-ol, 82.7% w/w 10 mM tetraborate pH 9.2
[41]
[137]
a) Analis proprietary chemical b) MicroSolv proprietary chemical
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The simplicity and low cost of CE methods affords the application of CE to the analysis of dissolution test samples where high throughput testing is required. For example [144], a pH 6.2 phosphate buffer was used to separate acetaminophenol (paracetamol), phenylephedrine and chloropheniramine in a combination formulation within 4 min.
CE has been proven as an alternative to TLC or HPLC for quantitation of compounds and the determination of drug-related impurities [157159].
5 Determination of drug-related impurities

CE can be used for the separation of structural-related impurities and degradants from the main drug peak. Impurity profiling is generally carried out by HPLC and cross-correlated with other chromatographic methods such as TLC or another HPLC method. CE offers a completely different selectivity process to chromatography and thus is a complementary technique to HPLC.
The structural impurities of a drug will possess similar structural properties to the main component, which makes achieving their resolution challenging. The high separation efficiencies possible for CE often allow a small degree of selectivity to provide an acceptable resolution. Standard requirements of an impurity method are that all likely synthetic and degradative impurities are resolved from one another, and from the main drug peak, and that impurities can be monitored at 0.1% area/area level or below. Table 5 contains a selection of successful CE methods for impurity determination for pharmaceuticals.
Table 5. Drug-related impurity pharmaceutical analysis application table Application Electrolyte Ref. [52] [153]
22 drug substances and their process impurities by CE-MS Various NACE methods Heroin and its basic impurities 100 mM DM-b-CD in Celixira) reagent B pH 2.5, 100 mM HP-b-CD in Celixir a) reagent B pH 2.5, 103.2 mM SDS, 50 mM phosphate-borate pH 6.5 190 mM trisodium citrate pH 2.6. 50 mM phosphate buffer pH 2.5 40 mM borate pH 9.2 60 mM tetraborate pH 9.2 120 mM CAPS buffer pH 10.2, 65 mM SDS, 55 mM TBAB, 5% MeOH 13 mM boric acid-phosphoric acid pH 9.1 120 mM Tris-phosphate buffer pH 5.2 containing 50 mM CTAC Phosphate buffer pH 1.9, 22% v/v MeCN, 11 mM hydroxypropyl b-CD 20 mM ammonium acetate pH 6.5 100 mM borate pH 8.40 Phosphate buffer pH 6.0 0.075 M pentane-1sulphonic acid sodium salt 160 mM sodium carbonate 1 1 mM EDTA pH 10.35, 13% v/v MeOH 100 mM H3PO4 pH 2.5 10% ACN 25 mM borate, 15 mM SDS, 10% ACN 100 mM ammonium acetate/ACN/methanol 50:25:25 v/v/v
Ranitidine hydrochloride and related substances 3,4-Diaminopyridine and 4-aminopyridine potassium channel blockers Homotaurine as an impurity in calcium acamprosate New substance [LAS 35917) 5-Aminosalicylic acid and its major impurities Ketorolac tromethamine and its known related impurities Vancomycin and related impurities Ximelagatran thrombin inhibitor and related substances in drug substance and tablet formulation Penicillin and related impurities N-Acetylcysteine and its impurities Ciprofloxacin and its impurities Metacycline and its related substances Loratadine and related impurities Rofecoxib and photodegradation impurities Galantamine impurities by CE-MS
[160] [161] [162] [163] [164] [165] [166] [167] [168] [169] [170] [171] [172] [173] [45]
a) MicroSolv proprietary chemical CAPS = 3-(cyclohexylamino)-1-propanesulphonic acid, CTAC = cetyltrimethylammonium chloride, TBAB = tetrabutylammonium bromide
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5.1 Low pH
Basic drug impurities can be separated at pH 24 and a low pH electrolyte is often employed in analysis of basic drugs and their related impurities. A CE assay was developed and validated for ranitidine (Zantac) and potential related substances in bulk drug and pharmaceutical preparations [160], with the ionic strength and pH of the electrolyte shown to be the most critical parameters affecting selectivity. The CE assay gave detection limits of: diamine (0.03% area/area, a/a), oxime (0.04%, a/a), Bis (0.1% a/a), nitroacetamide (0.24% a/a) and a number of unknown peaks, which were not resolved by either TLC or HPLC. All known impurities of 3,4-diaminopyridine, a potassium channel blocker, were separated and quantified at a level of 0.05% [161].
An SDS-based MEKC method has been used [158] to determine peribedil content in formulations. Difunisal was used as an internal standard and good performance was obtained with assay results in agreement with a validated spectrophotometric method. The separation and determination of drug-related impurities using CE have been extensively studied and the method performance and validation data obtained clearly show that CE methods can be successfully applied to this area and give equal or superior performance to HPLC methods.
6 Physicochemical measurements
In the early stages of drug discovery, it is important to determine the pharmacokinetic properties of compounds in order to predict their bioavailability and bloodbrain barrier distribution and to develop appropriate formulations. There are a large number of compounds requiring such screening, mainly because of the high volume of syntheses which can be carried out by combinatorial chemistry. Consequently there is a need for rapid and reliable methods of physicochemical determination in order to maintain high throughput and efficiency. An extensive review of the use of CE in this area has recently been published [174]. CE has been applied successfully for physicochemical analysis of many pharmaceutical compounds and has many advantages over the traditional methods of log P and pKa determination. An integrated automated process of measuring the log P, pKa and chemical stability of compounds has been reported [175].
5.2 High-pH
High-pH buffers such as phosphate and borate are employed in the analysis of acidic components. At high pH the acidic components migrate against the EOF, maximising mobility differences. High-pH borate buffer at pH 9.2 has been used in CE for the determination of homotaurine, a doubly charged anion at this pH, as an impurity in calcium acamprosate by CZE [162]. The method was validated and detection limits of between 0.01 and 0.15% homotaurine with respect to drug substance were reported. A high pH CE method was successfully developed to quantitatively profile the chloromethylated, monomethylated and hydroxylated impurities of a new substance (LAS 35917) [163]. These impurities co-eluted when analysed by HPLC; the CE method allowed detection and quantitation of impurities present at levels of 0.040.08% of the parent drug.
6.1 Log P
Extremes of pH, i.e. pH 1.19 and pH 12, can be used in CE to measure the log P of acids and bases respectively in their uncharged forms [176]. This is an advantage compared to HPLC where stationary-phase stability must be considered. Early work in this area was performed [177179] using MEKC with solute partitioning with the micelle being related to its log P (solubility). The wider range of solutes that are separated in MEEKC has enabled its use [2123, 26, 27, 30, 179181] for compound solubility measurement techniques. The MEEKC method of octanolwater partition coefficient determination has been demonstrated for pharmaceuticals [2129, 176]. The compound solubility is assessed by bracketing it with neutral marker compounds of known log P, which are used to create a calibration graph of log P against migration time, as shown in Fig. 3. The
5.3 MEKC
Due to its chromatographic-style separation mechanism, MEKC is often employed to separate mixtures of charged and neutral components. An MEKC method was developed for the quantification of mesalazine 5 aminosalicylic and its major impurities [164]. A fast, selective MEKC method was used for the simultaneous assay of ketorolac tromethamine and its known related impurities [165], and quantitative analysis of vancomycin antibiotic and related impurities has also been carried out by MEKC [166].
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log P of the analyte of interest can then be calculated by its migration time using the graph. The higher the compounds log P value, the more it partitions into the microemulsion droplet and the longer it takes to migrate [23, 182183]. A rapid screening assay for the determination of log P has been developed [28] which uses pressure-assisted MEEKC. This technique applies pressure to the capillary, driving its contents through the capillary at a faster rate than offered by the EOF. The use of MEEKC for log P determinations has also been demonstrated with a 96-capillary array instrument for the high throughput screening of compound solubility [21, 184]. Table 6 contains a selection of published applications of CE for log P determination. Figure 3. Use of MEEKC to determine partition coefficients: plot of MEEKC migration times versus log P data for a range of phenones. Separation conditions: 0.81% w/w octane, 6.61% w/w butan-1-ol, 3.31% w/w SDS and 89.27% w/w 10 mM sodium tetraborate buffer, 15 kV, 30 cm650 mm id capillary (detection window at 22 cm), 407C, 200 nm.
6.2 pKa
The dissociation constants (pKa) of pharmaceuticals can be determined from migration time data obtained by running the compound with FSCE electrolytes of a range of
Table 6. Log P application table Application Carbonate esters and small organic molecules Method details 1.80% w/w SDS, 0.82 w/w% n-heptane, 6.49 w/w% butan-1-ol, 0.1 M borate-0.5 M phosphate buffer pH 7.4 1.442.88% w/w SDS, 0.82% w/w n-heptane, 6.49% w/w butan-1-ol, 0.05 M acetate buffer pH 4.75 2.16% w/w SDS, 0.82% w/w n-heptane, 6.49% w/w butan-1-ol, 0.1 M borate-0.05 M HCl pH 1.4 3.3% w/w SDS, 0.8% w/w n-heptane, 6.6% w/w butan-1-ol, 92% w/w 68 mM (cyclohexylamino)1-propanesulphonic acid pH 10.3 Heptane-butanol-SDS borate pH 12 50 mM SDS, 82 mM n-heptane, 50 mM boratephosphate buffer pH 10, 0.87 M butan-1-ol 1.4% w/v SDS, 1.2% v/v n-heptane, 8% v/v butan-1-ol, 85% v/v 50 mM sodium phosphate buffer pH 3 25 mM borate buffer pH 8.5 30150 mM SDS (MEKC) 0.05 M phosphate buffer pH 7 (pH 9 for steroids) 80 mM SC, 100 mM SDS or 100 mM CTAB surfactant (MEKC) Heptane-butanol-SDS borate pH 7 Ref. [22]
Multiplexed 96-capillary method for neutral and basic compounds 24 acidic and basic pharmaceuticals 45 weakly acidic, weakly basic and neutral pharmaceuticals Neutral and weakly acidic compounds using dynamically coated capillary columns Neutral pharmaceuticals Neutral pharmaceuticals and steroids
[22]
[23] [28] [29]
[177] [178]
Anionic pharmaceuticals
[182]
SC = sodium cholate, CTAB = cetyltrimethylammonium bromide

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Table 7. pKa application table Application pKa* values of bases Medium throughput pKa of 48 pharmaceutical compounds acidic, basic and multivalent Method details pH* range 4.99.7 Methanol and sodium acetate buffer 1 acetic acid Ref. [15]
pH range 2.511.0. [188] Electrolytes of 0.1 M ionic strength composed of phosphate, acetate and borate buffers adjusted to required pH with phosphoric, acetic or boric acid or NaOH. Pressure-assisted CE (PACE) at 2 psi pH range 2.511.0 [189] Electrolytes of 0.05 M ionic strength composed of 0.5 M phosphate and 1 M acetate buffers mixed to obtain required pH. PACE at 25 mbar Short-end injection pH range 2.012.0 Electrolytes of 0.05 M ionic strength composed of 1 M phosphate, 0.1 M borate and 1 M acetate buffers mixed and adjusted with phosphoric acid, acetic acid and NaOH to obtain required pH Short-end injection pH 4.779.69 pH 2.09.0 pH range 1.36.6 50 mbar PACE Citric acid adjusted with NaOH pH 4.208.20 pH 1.56.0 [190]
Rapid pKa screening of 26 acidic, basic and multivalent pharmaceuticals
pKa determination of labile drug compounds
2-Amino-2-oxazolines (anti-hypertensive agents) Cephalosporins (antibiotics) pKa determination of 99mTechnetium radiopharmaceuticals
[195] [196] [200]
Anthrocyclines (antibiotics) Cytokinins (phytohormones) Dihydrofolate reductase inhibitors
[202] [203]
pH 2.14.5 [204] 50 mM phosphate buffer adjusted with phosphoric acid/NaOH pH 2.011.0 pH 2.2011.42 pH range 4.769.5 Tris, ethanolamine and acetate buffers pH range 3.889.16 25 mM phosphate buffer adjusted with triethylamine [205] [206] [207] [208]
Quinolones (antibacterials) Ropinirole and impurities (Parkinsons treatment) pKa of organic bases in aqueous methanol (070% v/v) pKa of N-imidazole derivative aromatase inhibitors
pH values [185197]. Table 7 details a selection of reported applications. The mobility of the solute at each pH can be calculated from its migration time and the EOF (measured using a neutral marker such as DMSO), and a plot of mobility against pH can be constructed. Figure 4 shows the mobility of the antibiotic cephalexin plotted over a range of electrolyte pH values. The pKa value of a compound is calculated using the electrophoretic mobilities, and cephalexin is calculated to have an acidic and a basic pKa of 2.49 and 7.08, respectively, compared to its literature values of 2.53 and 7.14 [198].
The dissociation constants of water-insoluble and sparingly soluble compounds have been measured by CE through adding solvents such as methanol to the electrolyte [187]. A medium-throughput method of pKa screening by pressure-assisted CE has been developed and demonstrated successfully for 48 acidic, basic and multivalent pharmaceutical compounds of known pKa [188]. The pressure assistance can greatly reduce the measurement time, especially for the low-pH analyses which can be very slow due to the low EOF level.
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Figure 4. Plot of cephalexin electrophoretic mobility against electrolyte pH, used to calculate compound pKa. Analysis performed using CombiSep 96capillary array instrument, CombiSep pKa determination buffers and CombiSep pKa determination software. (Unpublished material provided by the author).
By combining the application of pressure assistance with the use of short-end injection technique and a short capillary, pKa determinations can be performed even more rapidly, and this method was also found to give greater repeatability of mobility measurements [189]. CE compares very favourably to other methods of pKa analysis [199]. CE instruments are highly automated and can be used for high-throughput applications. Unlike titration methods, precise information of sample concentration is not required, as only analyte mobilities are used to calculate pKa by CE. Sparingly soluble compounds are easily analysed and only very small amounts of material are required, which is especially useful for the screening of newly synthesised pharmaceutical entities where only small quantities exist [195] or for applications where the molecule of interest is present only in very small quantities such as radiopharmaceuticals. A CE system equipped with a radioactivity detector has been used to determine the dis99m sociation constants of Technetium radiopharmaceuticals [200]. By using the stacking technique of sample introduction, concentrations of pharmaceutical compounds as low as 2 mM have resulted in successful pKa determinations [189]. Techniques commonly used for pKa determination such as
potentiometric titration or UV-Vis spectroscopy do not differentiate in analytical response between the analyte of interest and any analogue impurities, which causes problems when attempting to measure compounds that are not highly pure or are unstable in solution. Because CE is a separative technique, it is appropriate for pKa determination of impure or unstable samples, and also electrolyte purity is not essential. The CE method of pKa determination has been demonstrated successfully for sets of labile drug compounds which are unstable in a range of acidic, basic and neutral solutions [190]. The standard pH scale for aqueous acids and bases has limited applicability to organic solvents where pH* is used instead. Correspondingly, the pKa* values of compounds in non-aqueous solvents have been calculated by CE [201]. The pKa* values in methanol of a series of bases have been determined [15].
7 Conclusions
The range of pharmaceutical analysis applications for CE is extensive, possibly eclipsing those of HPLC. CE also offers a number of advantages over HPLC and other analytical techniques: analysis and method development speed, reduced consumable and solvent expenses, simwww.electrophoresis-journal.com
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plicity of operations and a greater possibility of implementation of a single set of method conditions for the analysis of several different samples, giving the large efficiency savings which are desirable in todays busy laboratory. Low-wavelength detection (190200 nm) can be employed in CE due to the short path-length of the capillary employed for detection. This can allow direct detection of analytes that would require derivatisation (or an alternative detection system to UV absorbance) to be de detected by HPLC. CE therefore has a niche for the efficient and effective analysis of small organic and inorganic ions such as amino acids, metal ions, simple organic acids and inorganic ions such as chloride and nitrate. These analytes are often drug (or excipient) counterions. These analyses are generally conducted using commercially available reagent kits or standard method conditions, and they are generally simpler and faster than ionchromatography and are conducted on standard CE equipment, which eliminates the need for specialist ionchromatography equipment. For some pharmaceutical applications, such as chiral analysis or the measurement of physicochemical properties, CE is a superior technique to the conventional methods in terms of cost, ease of use and automation possibilities. CE does have its disadvantages, but with the considered method development, such as the incorporation of internal standards to overcome poor injection precision, its performance can match that of HPLC. This has been shown by the number of reported validated CE methods and also by several CE methods which have been accepted by regulatory authorities in new drug product submissions. CE also suffers from not being as widely experienced as other techniques by pharmaceutical analysts, but with its routine applications increasing, this is improving. CE systems are flexible and can be automatically preprogrammed to vary parameters such as voltage, wavelength, temperature, and operate using vials containing various electrolyte compositions. This flexibility allows experimental designs such as PlackettBurman [209] to be effectively used to optimise CE methods or to perform robustness testing. In many analytical laboratories, CE is regularly used to provide complementary information to HPLC or other methods as well as being the technique of choice for certain applications. As the instrumentation further improves, automation possibilities expand and the list of applications increases, the use of CE for pharmaceutical analysis is certain to continue to be popular and become more widespread.
8 References
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Electrophoresis 2006, 27, 22632282

Kevin Altria1 Alex Marsh1 Cari Snger-van de Griend2