Capillary Electrophoresis for the Analysis of Small-molecule Pharmaceuticals

Kevin Altria1

Alex Marsh1

Cari Sänger-van de Griend2

1GlaxoSmithKlineResearch & Development, Harlow,Essex, UK

2AstraZeneca Pharmaceuticaland Analytical R&D,Södertälje, Sweden

Received January 17, 2006Revised March 16, 2006Accepted March 17, 2006

Review

Capillary electrophoresis for the analysis ofsmall-molecule pharmaceuticals

This paper reviews the application of CE to the analysis of small-molecule pharma-ceuticals. The areas of pharmaceutical analysis covered are enantiomer separation,the analysis of small molecules such as amino acids or drug counter-ions, pharma-ceutical assay, determination of related substances and physicochemical measure-ments such as log P and pKa of compounds. The different electrophoretic modesavailable and their advantages for pharmaceutical analysis are described. Recentapplications of CE for each subject area are tabulated with electrolyte details.

Keywords: Capillary electrophoresis / Chiral separation / Log P determination / Phar-maceuticals / pKa determination DOI 10.1002/elps.200600030

1 Introduction

The use of CE methods for pharmaceutical analysis hasbecome increasingly popular in recent years. The widerange of applications for which its use has proved suc-cessful includes [1] assay of drugs, determination of drug-related impurities, physicochemical measurements ofdrug molecules, chiral separation and the analysis ofpharmaceutical excipients.

Pharmaceutical analysis is dominated by HPLC. Otherseparative techniques used include TLC and GC, but therange of applications for which they can be used and theirquantitative capabilities are not as widespread as HPLC.Other techniques include IR and UV spectroscopy, whichare often used for identity testing of pharmaceuticals, anda range of flask-based methods, e.g. titration, which areused for physicochemical parameter determinations.

The advantages of CE for pharmaceutical analysisinclude its speed and cost of analysis, reductions in sol-vent consumption and disposal, and the possibility ofrapid method development. CE also offers the possibilitythat a single set of separation conditions can be applica-

ble for a wide range of analyses, giving efficiency savings.CE instruments can be coupled to a variety of detectortypes, including mass spectrometers, for special appli-cations and more detailed analysis.

1.1 Electrophoretic modes

There are several electrophoretic modes that can be usedto analyse pharmaceuticals. Free-solution capillary elec-trophoresis (FSCE) is very popular and involves the use ofsimple buffered aqueous electrolytes. Separation of ionicdrugs is achieved in FSCE through pH control. The addi-tion of reagents to the electrolyte, such as organic sol-vents or ion-pair reagents, can also be used to achievethe required separation selectivity. For the separation ofdrug enantiomers, FSCE with chirally selective agents,such as CDs, added to the electrolyte to facilitate chiralresolution is the most common method used [2].

For the separation of water-insoluble or sparingly solublepharmaceuticals, NACE, which employs electrolytescomposed of organic solvents, has been used success-fully [3–7]. NACE is also useful for the resolution of water-soluble charged solutes as the selectivity obtained can bedifferent to aqueous-based separations.

MEKC and microemulsion electrokinetic chromato-graphy (MEEKC) employ electrolytes that contain surfac-tant molecules which form micelles (MEKC) and micro-emulsion droplets (MEEKC). These micelles or dropletsadd a chromatographic element to the separation thatenables separation of acidic, basic and neutral drugs.

Correspondence: Dr. Kevin Altria, GlaxoSmithKline R&D, BuildingH89, New Frontiers Science Park South, Harlow, Essex, CM19 5AW,UKE-mail: [email protected]: 144-1279-643860

Abbreviations: API, active pharmaceutical ingredients; FSCE, freesolution capillary electrophoresis; MEEKC, microemulsion electroki-netic chromatography

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2264 K. Altria et al. Electrophoresis 2006, 27, 2263–2282

MEEKC is the most flexible of all the separation modes,offering the greatest selectivity to the widest range ofcompounds and can be considered the separation meth-od of choice when performing CE analysis.

1.1.1 FSCE

The majority of drugs are basic and thus ionised at lowpH. FSCE using low-pH buffer systems has beenemployed to separate a range of basic drugs; separationsare based on analyte size and number of positive charges,but neutral compounds do not migrate through thedetector. This offers an advantage over HPLC analysis offormulated pharmaceutical products where the analytepeak can be masked by the co-elution of neutral excipientor flavouring compounds. A low-pH phosphate buffer hasbeen used to analyse 550 different basic drugs [8] and asimilar method has been validated [9] for analysis of avariety of basic drugs, excipients and raw materials.

To separate mixtures of acidic and basic drugs (or cati-onic and anionic species), FSCE at high pH can be utilised[10]. At pH 7 or greater, the EOF generated by the appliedcurrent is sufficiently strong to sweep anions to thedetector. Analyte migration time is dependent on solutecharge type and density; strongly cationic speciesmigrate first while small, highly charged anions attempt tomigrate against the EOF and are detected last. Neutralspecies migrate unresolved at the EOF solvent front.

1.1.2 NACE

Water-insoluble basic drugs can be difficult to separateusing FSCE methods, and NACE can be applied for suchanalyses. [3–6, 11]. NACE electrolytes do not containwater, with organic solvents such as methanol and ACNbeing used instead. Selectivities which are difficult to ob-tain using aqueous buffers, even when using surfactants orcomplexing agents, may be easily obtained with non-aqueous systems [12]. Separation selectivity can bealtered in NACE by changing the composition of organicsolvents in the electrolyte. The compatibility of MS cou-pling is increased due to the solvent volatility and the use oforganic buffers [3]. NACE is starting to be more frequentlyconsidered and its use for the analysis of basic drugs hasbeen reported. These reports include the separation of anumber of opium alkaloids [12], a mixture of cationic drugsubstances [13], a range of tropane alkaloids [6], a range ofb-blockers [14], tricyclic antidepressants [7] and differentbasic drugs [5, 15]. NACE has also been successfully usedto separate polar acidic and basic drugs [16], to performchiral separation of pharmaceutical amines [17] and tocalculate pKa* values of basic analytes in methanol [15].

1.1.3 MEKC and MEEKC

MEKC or MEEKC can be employed to achieve separa-tion of mixtures of acidic, basic and neutral pharma-ceutical compounds. Both techniques enhance electro-phoretic separations by providing a chromatographicpartitioning mechanism into the micelles (MEKC) ormicroemulsion droplets (MEEKC). This partitioningallows resolution of neutral compounds as well ascharged species. Neutral solutes are separated solelybased on their solubility. Charged solutes are separatedbased on their electrophoretic movement, solubility-based partitioning and potential ion-pair interaction withthe charged micelle or droplet. MEEKC has been foundto provide superior separation efficiency to MEKC,probably due to improved mass transfer between themicroemulsion droplet and aqueous phase [18]. Solutesalso penetrate the MEEKC droplet more easily than therigid MEKC micelle, which allows MEEKC to be appliedto a wider range of analytes. MEEKC also offers greaterseparation capability for water-insoluble compoundsthan 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 ofdrugs [21–29] and is an important pharmaceutical appli-cation of the technique.

Many reports have been published [22, 30–42] detailingthe use of MEEKC for pharmaceutical applications.Table 1 details selected pharmaceutical applicationsusing MEEKC and the composition of the microemulsionused.

Readers are referred to two recent reviews [43, 44] forfurther examples of MEEKC applications and descrip-tions of operating parameter effects.

1.2 CE-MS

There is an increased utility of CE-MS in pharmaceuticalanalysis as the methodology and equipment skills/relia-bility/robustness have all improved in recent years.

CE-MS methods can offer sufficient sensitivity to beoperational methods. For example, 0.05% galantamineimpurities were monitored [45] in Reminyl capsules usingCE-MS (ESI).

Standard CE-MS methods have been developed andapplied to a wide range of drug types. For example, Vas-sort et al. [46] reported the successful application ofstandard CE-MS method conditions to 22 drugs and theirprocess impurities.


Electrophoresis 2006, 27, 2263–2282 CE and CEC 2265

Table 1. Selected pharmaceutical applications of MEEKC

Application Microemulsion composition Ref.

Analysis of betamethasoneand derivatives

1.44% w/w SDS, 0.81% w/w octane, 6.61% w/w butan-1-ol, 91.14% 20 mMsodium phosphate pH 7.51.9% 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

[22]

Determination of nine preser-vatives in pharmaceuticaland cosmetic products

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

[30]

Analysis of 4-hydroxybenzoatepreservatives inpharmaceuticals

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

[31]

Analysis of formulated drugproducts

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

[32]

Insoluble ingredients ofpharmaceutical ointment

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

[33]

Levetiracetam from otherantiepileptic drugs

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

[34]

Separation of immunosup-pressive drugs

1.44% w/w SDS, 0.81% w/w octane, 6.61% w/w butan-1-ol, 91.14% 20 mMsodium phosphate pH 7.52.0% 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 7.52.0% 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

[35]

Analysis of ephedrine andpseudoephedrine

23.3 mM SDS, 16.4 mM n-heptane, 180.85 mM butan-1-ol, 8% ACN,20 mM borate

[36]

Amino acid derivatives usingLIFD detection

2.12% w/w SDS, 0.52% w/w heptane, 4.21% w/w butanol,84 mM borate pH 8.4

[37]

Nicotine-related alkaloids 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

[38]

Analysis of vitamins 6.0% w/w SDS, 0.8% octane, 6.6% butanol, 20.0% propan-2-ol,66.6% 25 mM phosphate pH 2.75

[39]

Analysis of water andfat-soluble vitamins

80 mM SDS, 1% w/w octane, 5% v/v butanol, 40 mM borate pH 8.5 [40]

Separation of fat-solublevitamins

6.0% w/w SDS, 0.8% octane, 6.6% butanol, 20.0% propan-2-ol,66.6% 25 mM phosphate pH 2.75

[41]

UV filters in sunscreen lotions 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

[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 studiedareas in CE. It has been shown that CE is a powerfulanalytical technique for separating chiral compounds andfast, efficient, sensitive and selective methods have beendeveloped. Extensive research has shown that chiralanalysis by CE can be superior compared to conventionaltechniques such as LC and GC. Advantages includereduced costs as the chiral selector is added to the BGE

instead of using expensive chiral columns. The possibilityof low-UV wavelength detection for CE also allows theseparation and detection of analytes with poor chromo-phores which are difficult to detect by LC. CE has becomethe technique of choice for enantiomeric separations [47–49] in some companies. Several companies [46, 50–54]have published generic approaches for chiral methoddevelopment. The importance of using orthogonal meth-ods and techniques in method evaluation and validationhas been stressed [46].



There are many types of chiral selectors that can beapplied to achieve enantioseparation, but the most com-mon are native and derivatised CDs. (Table 2 contains alisting of CD examples and abbreviations.) An illustrativeexample from AstraZeneca [55, 56] is presented in Fig. 1,which shows chiral resolution of a series of five localanaesthetics using heptakis(2,6-di-O-methyl)-b-CD in alow-pH buffer. Recently, the enantiomeric purity determi-nation of ropivacaine hydrochloride based on theseresults was published in the US Pharmacopeia [57]. Other

chiral selectors that have been applied to CE separationsinclude natural and synthetic chiral micelles, crownethers, chiral ligands, proteins, carbohydrates and mac-rocyclic antibiotics. Readers are referred to textbooks[58–60] and recently published reviews on the funda-mental aspects and applications of chiral CE [61–73].Scriba [61] specifically reviewed the use of chiral CEtechniques for pharmaceutical and bioanalysis. Otherreviews 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 Substance/product Electrolyte Ref.

R209130 Substance 5 mM a-CD, 2% S-b-CD, 10 mM phosphoricacid–triethanolamine buffer pH 3.0

[51]

BIX Substance 2.5% w/v CM-b-CD, 25 mM NaH2PO4 buffer pH 4.0 [53]

SLV307 (antipsychotic) Capsules 50 mM HP-a-CD, 100 mM Tris–phosphoric acid pH 2.5 [75]

Calcium levofolinate Substance 20 mg/mL DM-b-CD, 40 mM sodium tetraborate pH 9.9 [76]

Methotrexate Tablets, injectionsolutions

45 mM b-CD, 100 mM SDS, 50 mM borate bufferpH 9.30 containing 25% methanol

[77]

Ragaglitazar, arginine Substance, tablets 90% (2% SB-b-CD, 0.7% DM-b-CD, 25 mM phosphatebuffer pH 8.0) 10% ACN

[78]

Citalopram Tablets 0.15% CM-g-CD, 20 mM NaH2PO4 pH 7.0 (with NaOH) [80]

Moxiflaxin Substance, ophthalmic/otic solutions

5% HS-g-CD, 12.5 mM triethylammonium phosphatebuffer pH 2.5, 6% ACN

[81]

Nicardipine Substance-CDcomplexes

3% S-b-CD, 2% TM-b-CD, 20 mM triethanolaminephosphate pH 3.0

[83]

Propanolol Tablets, injection solutions 100 mM HSA, 67 mM phosphate buffer pH 7.4 [84]

Adrenaline Local anaestheticsolutions

40 mM DM-b-CD, 100 mM phosphoric acid,50 mM triethanolamine

[86]

Butorphanol, cycloamine Substance, intermediate 0.02% HS-b-CD, 250 mM Tris, 350 mM phosphoric acid [87]

L-DOPA Tablet 5 mM HP-b-CD, 100 mM borate pH 8.0, 120 mM SDS [88]

Lisuride Substance 20 mM g-CD, 100 mM Tris, 140 mM phosphoric acid [89]

Ketoconazole, terconazole Substance, tablets,syrup, gel

10 mM TM-b-CD, 50 mM phosphate buffer pH 2.5 [90]

3-(4-Methylbenzylidene)-camphor

Cream 15 mM CM-b-CD, 120 mM a-CD, 100 mM borate pH 9.2 [91]

Aziridine-type proteaseinhibitors

Substance 2 mM sulphated b-CD, 50 mM phosphate buffer pH 2.5 [92]

Etodolac Tablets 20 mM HP-b-CD, 100 mM phosphate buffer pH 7 [93]

Baclofen Substance 3% w/v highly S-b-CD, 25 mM phosphate buffer pH 2.5 [94]

Substituted imidazole p38MAP kinase inhibitor

Substance 2% S-b-CD, 25 mM phosphate buffer pH 2.2 [95]

Apomorphine Substance 14 mM HP-b-CD, 100 mM Tris-phosphate pH 3.0 [96]

Balaglitazone, pioglitazone,thiaglitazone

Substance, tablets 90% (2% SB-b-CD, 0.7% DM-b-CD, 25 mM phosphatebuffer pH 8.0) 10% ACN

[97]

Rivastigmine Substance 7.5 mM b-CD, 75 mM triethanolammonium phosphatepH 2.5

[98]



Table 2. Continued

Application Substance/product Electrolyte Ref.

Pheniramine Granulate 2.5 mg/mL CE-b-CD, 0.2% MHEC, 20 mM e-aminoca-proic acid pH 4.5 (with acetic acid)

[99]

Cefoperazone Substance, solutions 0.04 mM b-CD 75 mM NaH2PO4 pH 4.0 [100]

Simendan Substance 50% (12 mM b-CD, 20 mM borate pH 11.0),50% methanol

[101]

Organic disulphates Substance 0.5 mM QA-b-CD, 10 mM glycine pH 2.4 [102]

Sertraline Substance 20 mM HP-b-CD, 30 mM sodium deoxycholate,35 mM borate pH 11.5

[103]

Anticholinergic drugs Substance 10 mM HDMS-b-CD, 20 mM phosphoric acid,10 mM NaOH, in methanol

[104]

Omeprazole Capsules 30 mM M-b-CD, 5 mM sodium disulphide,40 mM phosphate buffer pH 2.2

[105]

Abbreviations:a-CD a-cyclodextrinb-CD b-cyclodextring-CD g-cyclodextrinCE-b-CD carboxyethyl-b-cyclodextrinCM-b-CD carboxymethyl-b-cyclodextrinCM-g-CD carboxymethyl-g-cyclodextrinDM-b-CD dimethyl-b-cyclodextrinHDMS-b-CD heptakis(2,3-di-O-methyl-6-O-sulphato)-b-cyclodextrinHP-a-CD hydroxypropyl-a-cyclodextrinHP-b-CD hydroxypropyl-b-cyclodextrinHS-b-CD highly sulphated b-cyclodextrinHS-g-CD highly sulphated g-cyclodextrinQA-b-CD quaternary ammonium b-cyclodextrinM-b-CD methyl-b-cyclodextrinS-b-CD sulphated-b-cyclodextrinSB-b-CD sulphobutylether-b-cyclodextrinTM-b-CD trimethyl-b-cyclodextrin

Lämmerhofer [69, 70] recently reviewed chiral NACE [69]and CEC [70]. In the chiral CE review the focus was onsolvent effects on the chiral separation, while the CECreview discussed potentials and peculiarities of chiralnonaqueous CEC. NACE considerably expands thepotential for chiral CE, since both analytes and chiralselectors that are poorly soluble in water can beemployed. But the number of publications is still modest,probably due to the dominant role of CDs as chiral selec-tor [69].

Mangelings et al. [71] have recently reviewed the use ofchiral CEC for pharmaceutical analysis, highlightingpotential practical pharmaceutical applications publishedin the past 10 years. Chiral stationary phases based onmacrocyclic antibiotics and polysaccharide selectors aremost frequently used, but more recently monolithic sta-tionary phases have gained interest.

The usefulness of chiral CEC has been proven manytimes. But in order to survive as a separation techniquewithin pharmaceutical industry, it is important that thebetween-column reproducibility problems and technicalissues (e.g. sample loop injections) reportedly encoun-tered [71] are resolved.

Mertzman and Foley [72] evaluated the use of CD-modifiedMEEKC in different modes, thereby opening possibilities forresolution of enantiomeric hydrophobic compounds.

A series of overviews sorted by chiral selectors andtechnique was recently published in Methods in Molecu-lar Biology [73].

Patel reviewed the use of CE techniques for the enantio-meric resolution of a group of NSAIDs, the 2-arylpropionicacids, with the application to real samples of pharma-ceutical and biomedical interest [67]. They concluded that



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. Reproducedwith permission from [55, 56].

the majority of the reports have been focused on themechanism of resolution and the evaluation of the effectof parameters on the separation, while only a limitedamount of reports describe fully validated methods thathave subsequently been applied to pharmaceutical orbioanalytical problems.

The various modes of chiral CE for the analysis of pollu-tants have been reviewed by Hernández-Borges et al.[68]. They concluded that CE is an impressive tool for thispurpose, but some effort needs to be put into on-line andoff-line concentration techniques [68].

2.1 Pharmaceutical applications of chiral CE:generic approaches

Several pharmaceutical companies [46, 49–54] haverecently published generic approaches for chiral methoddevelopment.

Jimidar [49] (Johnson & Johnson) described a widely ap-plicable strategy for screening through a number of CDsfor the separation of basic, acidic or neutral components.The type of CD, the CD concentration, buffer electrolyteconcentration and the amount of organic modifier in theBGE is evaluated in these screens. In the first instance,

the pH of the BGE is aligned to the nature of the com-pound. Fine-tuning of the experimental parameters isthen performed. An example was also described in asubsequent paper [51]. A Box–Behnken experimentaldesign was used [51] in which four factors were optimisedto obtain separation of the eight stereoisomers of thecompound of interest (three chiral centres). Finally, themethod development was continued with optimising themethod for QC performance, i.e. appropriate sensitivityand stability. The method was then validated according tothe demands during phase II clinical studies. Anotherillustration of their approach was presented [52].

Sokoliess [54] (Boehringer Ingelheim Pharma) publisheda similar generic approach for the initial screening,which was illustrated with the enantiomeric separationof an active pharmaceutical ingredient. Particular atten-tion was paid to the conditioning of the capillary whilstoptimising the method. The inlet and outlet buffer vialswere swapped between injections to diminish bufferdepletion and chiral selector concentration changes.This allowed the same 1-mL buffer vials to be used forup to 20 runs.

Vassort and Szücs et al. [46] (Pfizer) included an evalua-tion of CE vs. LC and LC vs. CE orthogonality, as well asthe compatibility to CE-ESI-MS [46] in their generic



screen. They conclude that their screen offers significanttime and resource savings and will speed up the processof pharmaceutical analysis. A logical further advancewould be to evaluate additional techniques such as GC,SFC, NACE and CEC to extend the versatility of theapproach.

Rocheleau [53] (Bristol-Myers Squibb) used only sulph-ated b-CDs in her screen, and additionally evaluated thedifferent suppliers and batches of CD. The approach hasbeen successfully applied to more than a dozen of thecompany’s compounds in different phases of develop-ment.

Matthijs [74] compared the use of HSCD/neutral CD dualsystems with single HSCD systems, in order to define asequential strategy for method development with dualsystems when resolutions obtained with single systemsdo not suffice.

2.2 Pharmaceutical applications of chiral CE:Applications

Muijselaar [75] described method development and vali-dation for a basic potential antipsychotic compound. Ahigh concentration of Tris was used as the cationic buffercomponent to prevent excessive electromigration dis-persion of the eutomer (main, desired enantiomer). Al-though the electrodispersion is limited by this approach,the triangular shape of the eutomer peak may still influ-ence the peak shape of the distomer (low level, undesiredenantiomer impurity). Therefore it is important to have thematrix components as well as the eutomer present in thesamples when evaluating the method performance whilemeasuring the distomer. This was illustrated by deter-mining the corrected peak area ratios and RSDs for thedistomer at five levels with and without the presence ofthe eutomer at nominal concentration. The method wasvalidated and found to give good analytical performancefor substance and capsule formulations.

Süss [76] developed and validated a method for calciumlevofolinate, taking other impurities besides the enantio-mer into account. The method was optimised with help of acentral composite experimental design and the impurities,enantiomeric as well as others, could be quantified downto 0.05%, well below the European Pharmacopoeia (EP)demands. Thus, this one CE method could replace twoseparate LC methods currently required in the EP.

A CD-MEKC method to simultaneously determine thechiral and achiral purity of methotrexate was published byGotti [77]. The method was validated and proved to besufficiently robust and sensitive for the analysis of com-mercially available tablets and injection solutions. Jamali

and Lehmann [78] also published a method that coveredseveral specification tests. A single chiral CE methodachieved [78] the assay and identification of the activepharmaceutical ingredient ragaglitazar and its counter-ion arginine, and the achiral and the chiral purity of raga-glitazar, all in API and in low-dose tablets.

A method was developed for the determination of thediastereomeric stability of S-adenosylmethionine inaqueous solutions and was applied for a short-term sta-bility assessment (freeze–thaw cycles, 14 days incuba-tion at 387C) [79]. No chiral selector was needed for theseparation of the diastereoisomers as the high resolutionpower of CE was sufficient. Compared to LC, the CEmethod could simultaneously quantify the diastereoi-somers and the degradation products.

A rapid method to assay citalopram racemate or escita-lopram (S-citalopram) in tablets was reported [80]. Thevalidation included an experimentally designed robust-ness test using the Plackett–Burman model.

A chiral CE method was developed to evaluate whetherracemization is a potential degradation pathway for mox-ifloxacin drug substance or ophthalmic/otic solution [81].The study demonstrated that racemization into the RR-enantiomer or RS- or SR-diastereoisomers is unlikely.Therefore stereoselective testing may not be required forthis drug substance or its ophthalmic/otic solution.

Van Eeckhaut et al. [82] compared different chiral MEKCmethods for the enantiomeric separation of the neutralcalcium channel-blocking dihydropyridines. Tests com-prised chiral MEKC with bile salts, CD-MEKC withuncharged CD and CD-EKC with charged CD. Best resultswere obtained with CM-b-CD in a pH 5.0 MES buffer.

The photostability of nicardipine–CD complexes wasstudied by combining chiral CE analysis with DSC (differ-ential scanning calorimetry) and XRPD (X-ray powder dif-fraction) [83]. Some CDs protected nicardipine from pho-todegradation, some had no effect, and one even pro-moted photodegradation.

Martínez-Pla [84] reported the use of HSA as chiralselector in a quality control method for propranolol tabletsand injection solutions. The capillary is filled with the HSAsolution during preconditioning, while the inlet and outletvials during the run contain only buffer, resulting in verylow costs per analysis.

The enantiomeric purity of a series of 2- and 3- sub-stituted 2,3-dihydro[1,4]dioxino[2,3-b]pyridine deriva-tives, synthesized for early-phase drug discovery, wasdetermined with chiral CE using a-CD and/or S-b-CD aschiral selectors in a formic acid/ammonia buffer pH 4.0[85].



The enantiomeric separation of low concentrations ofadrenaline in high-concentrated local anaesthetic (LA)injection solutions was presented in a recent publication[86]. Adrenaline is added to LA solutions in amounts ofaround 0.1% of the LA. Racemization as low as 2–3%, i.e.0.002–0.003% of the LA and corresponding to ca. 0.1 mg/mL of d-adrenaline could be determined with sufficientprecision in a simple and robust chiral CE system. LA–adrenaline solutions were injected without sample prepa-ration.

Further applications presented during the last two yearsare listed in Table 2 [51, 53, 75–105].

3 Analysis of small molecules and ions

The separation and detection of small organic and inor-ganic ions is an important activity in the pharmaceuticalindustry. Most drugs molecules are charged and as suchare manufactured with a counterion; commonly a metalcation, e.g., K1 for acidic drugs, and an ionic salt, e.g., Cl2

or small organic acid (e.g., acetate) for basic drugs. It isimportant to analytically characterise the drug stoichio-metry (ratio of drug:counterion) to ensure that the potencyof the batch of drug substance is known. Because suchcounterions usually have little or no chromophore, popu-lar techniques for the analysis of small ions include ionexchange chromatography (IEC) and flame atomicabsorption spectrometry; but CE is becoming increas-ingly popular for such applications due to its simplicity.Commercial kits are available for ion analysis and are fre-quently used. Consequently methods are simple to oper-ate with analysis times of 2–10 min, which comparesfavourably with IEC. Additionally, IEC columns areexpensive and require regeneration, so CE offers bothreduced analysis times and costs. Table 3 contains aselection of CE methods that have been used to analysesimple ions for pharmaceutical applications.

Excipients such as SDS [106] or arginic acid [107] can beanalysed as raw materials or when present within for-mulations. For example, SDS levels were determined[106] within various tablet batches.

Ions with a poor UV response can be analysed by indirectUV detection, using a UV absorbing BGE, and metal ionscan be detected directly through on-capillary complexa-tion to form UV active metal chelates.

Some simple organic acids are sufficiently UV active to bedetected directly [108]. The separation of amino acids canbe quite complicated by HPLC as they need to first bederivatised to provide a chromophore for detection.However, CE can be used at lower operating wave-lengths. Amino acids, usually zwitterionic, become

cations at low pH, and a pH 2.8 electrolyte of 50 mMethanesulphonic acid was used to resolve a number ofamino acids which were detected directly at 185 nmwithout requiring sample pretreatment, as shown in Fig. 2[109]. A BGE of 100 mM sodium tetraborate pH 10 and20 mM sodium deoxycholate and 15 mM b-CD was usedto separate all major and minor components of gentami-cin amino sugar antibiotic and its impurities by derivati-sation with o-phthaldialdehyde [110].

The presence of small-ion contaminant impurities in drugsubstance or formulations can also be measured by CE.For instance, a NACE method using an electrolyte con-taining imidazole as the UV absorber has been used [111]to monitor ammonium ion contaminant. Ammonium wasmonitored with an LOD of 50 ppb.

4 Determination of pharmaceutical content

Assay of drug substance and formulated products is avery important and regulated activity. Analytical methodsmust be validated to strict standards to show that they arerobust, accurate, repeatable and suitable for their pur-pose. Most routine analytical assay determinations in thepharmaceutical industry are performed by HPLC. One ofthe major advantages of implementing CE methods inplace of HPLC is its relatively small level of solvent con-sumption, millilitres compared to the litres of mobilephase used in an HPLC run. An additional advantage isthat sample pre-treatment requirements are oftenreduced compared to HPLC as the CE capillary can bewashed with NaOH between injections and many inter-fering components do not migrate because they are neu-tral. The ability to quantify a range of sample types using asingle set of CE conditions is another strong feature sincethis can make considerable savings in analysis and sys-tem set-up times [9, 10, 128–131].

The electrophoretic conditions inside the capillary varyslightly between injections, which leads to greater varia-bility in peak migration times and area than seen in HPLC.A number of approaches can be taken to overcome thisproblem. Migration times and peak areas can be calcu-lated relative to those of an internal standard peak whichresults in great improvement in method repeatability[132]. Greater migration time reproducibility can also beachieved by applying the separation voltage across thecapillary for a very short time prior to injection andseparation [133]. A commercial capillary treatment sys-tem of buffers and rinse solutions has been shown toimprove CE repeatability as the capillary is coated with abilayer of surfactants ensuring that the surface coverageand EOF is consistent between injections and betweencapillaries. The paper highlighted the consistency of EOF



Table 3. Small molecules and ions, pharmaceutical analysis application table

Application Electrolyte Ref.

Anions – indirect detection

SDS levels in tablets Barbital buffer [106]

Determination of Br, Cl and SO4 as impurities in calciumacamprosate

Chromate 1 1 mM borate pH 9.15 [112]

TFA counterion of an opioid peptide analgesic Phthalate, CTAB [113]

Acetate content in acetate drug salt Phthalate, OFM [114]

Acetate residues in drug substance Phthalate, OFM [115]

Drug inorganic counterion determination Chromate, TTAB [116]

Drug organic acid counterion determination Phthalate, MES, TTAB [117]

Inorganic anion contaminant in drug substance Chromate, OFM [118]

Anions – direct detection

Arginic acid (an excipient level) Borate/boric acid [107]

Determination of residual Br in excess of chloride for localanaesthetic analysis

60:40 MeCN: methanesulphonic acid buffer pH 1.3 [119]

Drug organic acid counterion determination,e.g. benzoate, hydroxynapthoate

Borate pH 9.5 [120]

Arginine counterion determination for ragaglitazone [121]

Cations indirect detection

Ammonium ion impurities NACE method, imidazole [111]

Ca, Li, K and Na counterions of glycosaminoglycans 4-Aminopyridine buffer pH 9 [122]

Ca in calcium acamprosate drug substance Imidazole, sulphuric acid [123]

Cationic counter ion content in drug substance Imidazole, formic acidImidazole, low pH

[124][116]

K in drug substance Imidazole, formic acid [125]

Quarternary amine residues in drug substance Quinine, THF [126]

Cations direct detection

K counterion and inorganic cationic impurities of acidicdrugs by conductivity detection

Creatine, acetic acid, 18-crown-6 [129]

CTAB = cetyltrimethylammonium bromide, MES = 4-morpholineethanesulphonic acid, OFM = proprietary Waters chemical,TTAB = tetradecyltrimethylammonium bromide

when using the buffer coating system compared to astandard phosphate buffer using a capillary composedof 19 separate channels. Using the phosphate buffer,the peaks in the 19 individual channels had differentspeeds and the separation was poor giving a split peak.The EOF was consistent in each channel using thecoating system and the peaks moved at the samespeed, resulting in a single peak [134]. For a morecomprehensive guide to improving the precision of CEmethods, readers are referred to a review on the subject[135].

An FSCE method with CDs in the electrolyte has beendeveloped [136] for analysis of the diabetic therapy raga-glitazar and its counterion arginine in active pharmaceu-tical ingredients (API) and low-dose tablets. The methodis suitable for 12 different analyses of the API and tablets– active ingredient assay and identification of ragaglitazarand arginine, chiral purity of ragaglitazar and purity ofragaglitazar. The accuracy of the method (% recovery) wasfound to be 101–106% for ragaglitazar and 101–125% forarginine, while precision for the detection of peaks(% RSD) was found to be 0.63% for ragaglitazar and



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 withpermission from [109].

3.50% for arginine. An MEEKC method was used for thequantitative determination of folic acid in tablets [137]giving a precision of ,1.2% RSD and recovery of99.8 6 1.8% at three concentration levels.

CE methods have been reported that monitor bothdrugs and polymers released from drug delivery sys-tems in a single run (HPLC methods allow the monitor-ing of either the drug or the polymer alone). FSCE wasused for monitoring of the controlled delivery of recom-binant growth hormone from a system based in VP-HEMA copolymer. The CE method used a running bufferof 100 mM sodium tetraborate pH 9 and allowed thesimultaneous monitoring of both the liberation of rHGHand the polymer’s degradation by solubilisation [138]. ACE method has been reported to monitor R- and S-ibuprofen released from hydrophilic copolymer systemsusing dextrin 10 as a chiral selector. The methodallowed detection at concentrations of 1 mg mL21 and0.5% of the R-ibuprofen enantiomer [139]. An assay

method has been reported [142] for quantitative deter-mination of the weakly acidic antibiotic benzylpenicillinand procaine, benzathine and clemizole, the basicforms of benzylpenicillin found in pharmaceutical prep-arations.

Table 4 contains a selection of CE methods that havebeen validated for the quantitative assay of various phar-maceutical substances and dosage forms.

CE can also be applied (simultaneously or separately tothe drug content measurement) to monitor excipientlevels within formulations. For example [141], a single setof CE method conditions have been developed andapplied to the determination of the most commonlyemployed formulation preservatives (including parabens,cresols, etc.). The 10-min method employed pH 9.2borate buffer containing 20 mM SDS. The method wasvalidated and applied to a range of samples and obtainedgood agreement with reference methods.



Table 4. Validated CE methods for pharmaceutical assay

Application Method details Ref.

FSCE methods

Assay and identification of ragaglitazar, a diabetic therapy,and its counterion arginine, chiral purity of ragaglitazarand purity of ragaglitazar in API and low-dose tablets

90% v/v 25 mM phosphate buffer pH 8.0 1 10% v/vACN 1 2% w/v sulphobutyl ether b-CD 1 0.7% w/vdimethyl b-CD

[136]

Acetaminophenol, phenylephedrine, chlorepheniraminecontent in combination tablets

Phosphate pH 6.2 [142]

Simultaneous determination of procaine, dihydro-streptomycin and penicillin G in multiantibiotic veterinarypreparations

0.08 M borate pH 8 [143]

Determination of indinavir sulphate, a protease inhibitorused in HIV therapy in capsule formulations

20 mM phosphate pH 2.52 [144]

Method for assay of raloxifene, an oestrogen agonistin bone

20 mM acetate pH 4.5 [145]

Determination of pravastatin, a cholesterol-reducing agent,in tablet formulation

10 mM borate pH 8.5 1 10% MeCN [146]

Determination of the anti-fungal ketoconazole in tabletsand creams

10 mM phosphate buffer pH 2.3 [147]

Stability indicating, validated method for cyclizine hydro-chloride tablets and suppositories (motion sickness)


Determination and separation of antipsychotics –clothiapine, clozapine, olanzapine, quetiapine


Simultaneous determination of six angiotensin-II-receptorantagonists – candesartan, eprosartan, irbesartan,losartan potassium, telmisartan and valsartan

FSCE: 60 mM phosphate pH 2.5MEKC: 55 mM phosphate pH 6.5, 15 mM SDS

[150][151]

Dynamic capillary coating system methodsAnalysis of benzodiazepines CEofix™a) buffer system [152]

Characterisation and quantification of heroin and its basicimpurities and adulterants

CElixir™b) buffer system [153]

NACE methodsAnalysis of quinolizidine alkaloids in Chinese herbs,

which are difficult to separate in aqueous media1% acetic acid, 50 mM ammonium acetate,

20% ACN in methanol[154]

Tricyclic antidepressant content in tablets 1 M acetic acid, 50 mM ammonium acetate in ACN [155]

MEKC methods

Benzylpenicillin salts Phosphate-borate buffer pH 8.7 1 14.4 gL21 SDS [140]Determination of acyclovir in Zovirax cream 20 mM borate buffer pH 10 1 10 mM SDS [156]

Determination of preservatives in formulations Borate pH 9.20 1 20 mM SDS [157]Determination of piribedil content in tablets Borate 1 SDS [158]

MEEKC methodsQuantitative determination of the fat-soluble vitamin E

acetate (suppressed EOF environment enabled timelymigration of the analyte peak)

0.8% w/w n-octane, 6.6% w/w butan-1-ol, 6% w/wSDS, 20% v/v propan-2-ol, 66.6% w/w 25 mMphosphate pH 2.5

[41]

Quantitative determination of folic acid (a water-solublevitamin) in tablets

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/w10 mM tetraborate pH 9.2

[137]

a) Analis proprietary chemicalb) MicroSolv proprietary chemical



The simplicity and low cost of CE methods affords the appli-cation of CE to the analysis of dissolution test samples wherehigh throughput testing is required. For example [144], apH 6.2 phosphate buffer was used to separate acet-aminophenol (paracetamol), phenylephedrine and chloro-pheniramine in a combination formulation within 4 min.

5 Determination of drug-related impurities

CE can be used for the separation of structural-relatedimpurities and degradants from the main drug peak.Impurity profiling is generally carried out by HPLC andcross-correlated with other chromatographic methodssuch as TLC or another HPLC method. CE offers a com-pletely different selectivity process to chromatographyand thus is a complementary technique to HPLC.

CE has been proven as an alternative to TLC or HPLC forquantitation of compounds and the determination ofdrug-related impurities [157–159].

The structural impurities of a drug will possess similarstructural properties to the main component, whichmakes achieving their resolution challenging. The highseparation efficiencies possible for CE often allow asmall degree of selectivity to provide an acceptable res-olution. Standard requirements of an impurity methodare that all likely synthetic and degradative impurities areresolved from one another, and from the main drug peak,and that impurities can be monitored at 0.1% area/arealevel or below. Table 5 contains a selection of successfulCE methods for impurity determination for pharmaceu-ticals.

Table 5. Drug-related impurity pharmaceutical analysis application table

Application Electrolyte Ref.

22 drug substances and their process impurities by CE-MS Various NACE methods [52]

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

[153]

Ranitidine hydrochloride and related substances 190 mM trisodium citrate pH 2.6. [160]

3,4-Diaminopyridine and 4-aminopyridine potassiumchannel blockers

50 mM phosphate buffer pH 2.5 [161]

Homotaurine as an impurity in calcium acamprosate 40 mM borate pH 9.2 [162]

New substance [LAS 35917) 60 mM tetraborate pH 9.2 [163]

5-Aminosalicylic acid and its major impurities 120 mM CAPS buffer pH 10.2, 65 mM SDS,55 mM TBAB, 5% MeOH

[164]

Ketorolac tromethamine and its known related impurities 13 mM boric acid-phosphoric acid pH 9.1 [165]

Vancomycin and related impurities 120 mM Tris-phosphate buffer pH 5.2 containing50 mM CTAC

[166]

Ximelagatran thrombin inhibitor and related substancesin drug substance and tablet formulation

Phosphate buffer pH 1.9, 22% v/v MeCN,11 mM hydroxypropyl b-CD

[167]

Penicillin and related impurities 20 mM ammonium acetate pH 6.5 [168]

N-Acetylcysteine and its impurities 100 mM borate pH 8.40 [169]

Ciprofloxacin and its impurities Phosphate buffer pH 6.0 0.075 M pentane-1-sulphonic acid sodium salt

[170]

Metacycline and its related substances 160 mM sodium carbonate 1 1 mM EDTA pH 10.35,13% v/v MeOH

[171]

Loratadine and related impurities 100 mM H3PO4 pH 2.5 10% ACN [172]

Rofecoxib and photodegradation impurities 25 mM borate, 15 mM SDS, 10% ACN [173]

Galantamine impurities by CE-MS 100 mM ammonium acetate/ACN/methanol50:25:25 v/v/v

[45]

a) MicroSolv proprietary chemicalCAPS = 3-(cyclohexylamino)-1-propanesulphonic acid, CTAC = cetyltrimethylammonium chloride, TBAB = tetra-butylammonium bromide



5.1 Low pH

Basic drug impurities can be separated at pH 2–4 and alow pH electrolyte is often employed in analysis of basicdrugs and their related impurities. A CE assay was devel-oped and validated for ranitidine (Zantac) and potentialrelated substances in bulk drug and pharmaceutical pre-parations [160], with the ionic strength and pH of theelectrolyte shown to be the most critical parametersaffecting selectivity. The CE assay gave detection limitsof: diamine (0.03% area/area, a/a), oxime (0.04%, a/a),Bis (0.1% a/a), nitroacetamide (0.24% a/a) and a numberof unknown peaks, which were not resolved by either TLCor HPLC.

All known impurities of 3,4-diaminopyridine, a potassiumchannel blocker, were separated and quantified at a levelof 0.05% [161].

5.2 High-pH

High-pH buffers such as phosphate and borate areemployed in the analysis of acidic components. At highpH the acidic components migrate against the EOF,maximising mobility differences.

High-pH borate buffer at pH 9.2 has been used in CE forthe determination of homotaurine, a doubly chargedanion at this pH, as an impurity in calcium acamprosateby CZE [162]. The method was validated and detectionlimits of between 0.01 and 0.15% homotaurine withrespect to drug substance were reported.

A high pH CE method was successfully developed toquantitatively profile the chloromethylated, mono-methylated and hydroxylated impurities of a new sub-stance (LAS 35917) [163]. These impurities co-elutedwhen analysed by HPLC; the CE method allowed detec-tion and quantitation of impurities present at levels of0.04–0.08% of the parent drug.

5.3 MEKC

Due to its chromatographic-style separation mechanism,MEKC is often employed to separate mixtures ofcharged and neutral components. An MEKC methodwas developed for the quantification of mesalazine 5–aminosalicylic and its major impurities [164]. A fast,selective MEKC method was used for the simultaneousassay of ketorolac tromethamine and its known relatedimpurities [165], and quantitative analysis of vancomycinantibiotic and related impurities has also been carriedout by MEKC [166].

An SDS-based MEKC method has been used [158] todetermine peribedil content in formulations. Difunisal wasused as an internal standard and good performance wasobtained with assay results in agreement with a validatedspectrophotometric method.

The separation and determination of drug-related impu-rities using CE have been extensively studied and themethod performance and validation data obtained clearlyshow that CE methods can be successfully applied to thisarea and give equal or superior performance to HPLCmethods.

6 Physicochemical measurements

In the early stages of drug discovery, it is important todetermine the pharmacokinetic properties of compoundsin order to predict their bioavailability and blood–brainbarrier distribution and to develop appropriate formula-tions. There are a large number of compounds requiringsuch screening, mainly because of the high volume ofsyntheses which can be carried out by combinatorialchemistry. Consequently there is a need for rapid and re-liable methods of physicochemical determination in orderto maintain high throughput and efficiency. An extensivereview of the use of CE in this area has recently beenpublished [174]. CE has been applied successfully forphysicochemical analysis of many pharmaceutical com-pounds and has many advantages over the traditionalmethods of log P and pKa determination. An integratedautomated process of measuring the log P, pKa andchemical stability of compounds has been reported [175].

6.1 Log P

Extremes of pH, i.e. pH 1.19 and pH 12, can be used inCE to measure the log P of acids and bases respectivelyin their uncharged forms [176]. This is an advantagecompared to HPLC where stationary-phase stability mustbe considered.

Early work in this area was performed [177–179] usingMEKC with solute partitioning with the micelle beingrelated to its log P (solubility).

The wider range of solutes that are separated in MEEKChas enabled its use [21–23, 26, 27, 30, 179–181] forcompound solubility measurement techniques. TheMEEKC method of octanol–water partition coefficientdetermination has been demonstrated for pharmaceu-ticals [21–29, 176]. The compound solubility is assessedby bracketing it with neutral ‘marker’ compounds ofknown log P, which are used to create a calibration graphof log P against migration time, as shown in Fig. 3. The



Figure 3. Use of MEEKC to determine partition coeffi-cients: plot of MEEKC migration times versus log P datafor a range of phenones. Separation conditions: 0.81%w/w octane, 6.61% w/w butan-1-ol, 3.31% w/w SDS and89.27% w/w 10 mM sodium tetraborate buffer, 15 kV,30 cm650 mm id capillary (detection window at 22 cm),407C, 200 nm.

log P of the analyte of interest can then be calculated byits migration time using the graph. The higher the com-pound’s log P value, the more it partitions into the micro-emulsion droplet and the longer it takes to migrate [23,182–183].

A rapid screening assay for the determination of log P hasbeen developed [28] which uses pressure-assistedMEEKC. This technique applies pressure to the capillary,driving its contents through the capillary at a faster ratethan offered by the EOF.

The use of MEEKC for log P determinations has also beendemonstrated with a 96-capillary array instrument for thehigh throughput screening of compound solubility [21,184]. Table 6 contains a selection of published applica-tions of CE for log P determination.

6.2 pKa

The dissociation constants (pKa) of pharmaceuticals canbe determined from migration time data obtained by run-ning the compound with FSCE electrolytes of a range of

Table 6. Log P application table


Carbonate esters and small organic molecules 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 bufferpH 7.41.44–2.88% w/w SDS, 0.82% w/w n-heptane,6.49% w/w butan-1-ol, 0.05 M acetate bufferpH 4.752.16% w/w SDS, 0.82% w/w n-heptane,6.49% w/w butan-1-ol, 0.1 M borate-0.05 M HClpH 1.4

[22]

Multiplexed 96-capillary method for neutral and basiccompounds

3.3% w/w SDS, 0.8% w/w n-heptane, 6.6% w/wbutan-1-ol, 92% w/w 68 mM (cyclohexylamino)-1-propanesulphonic acid pH 10.3

[22]

24 acidic and basic pharmaceuticals Heptane-butanol-SDS borate pH 12 [23]

45 weakly acidic, weakly basic and neutralpharmaceuticals

50 mM SDS, 82 mM n-heptane, 50 mM borate-phosphate buffer pH 10, 0.87 M butan-1-ol

[28]

Neutral and weakly acidic compounds using dynamicallycoated capillary columns

1.4% w/v SDS, 1.2% v/v n-heptane, 8% v/vbutan-1-ol, 85% v/v 50 mM sodium phosphatebuffer pH 3

[29]

Neutral pharmaceuticals 25 mM borate buffer pH 8.5 – 30–150 mM SDS(MEKC)

[177]

Neutral pharmaceuticals and steroids 0.05 M phosphate buffer pH 7 (pH 9 for steroids) –80 mM SC, 100 mM SDS or 100 mM CTABsurfactant (MEKC)

[178]

Anionic pharmaceuticals Heptane-butanol-SDS borate pH 7 [182]

SC = sodium cholate, CTAB = cetyltrimethylammonium bromide



Table 7. pKa application table


pKa* values of bases pH* range 4.9–9.7Methanol and sodium acetate buffer 1 acetic acid

[15]

Medium throughput pKa of 48 pharmaceuticalcompounds – acidic, basic and multivalent

pH range 2.5–11.0.Electrolytes of 0.1 M ionic strength composedof phosphate, acetate and borate buffers adjustedto required pH with phosphoric, acetic or boric acidor NaOH.Pressure-assisted CE (PACE) at 2 psi

[188]

Rapid pKa screening of 26 acidic, basic and multivalentpharmaceuticals

pH range 2.5–11.0Electrolytes of 0.05 M ionic strength composedof 0.5 M phosphate and 1 M acetate buffers mixedto obtain required pH.PACE at 25 mbar“Short-end” injection

[189]

pKa determination of labile drug compounds pH range 2.0–12.0Electrolytes of 0.05 M ionic strength composedof 1 M phosphate, 0.1 M borate and 1 M acetatebuffers mixed and adjusted with phosphoric acid,acetic acid and NaOH to obtain required pH“Short-end” injection

[190]

2-Amino-2-oxazolines (anti-hypertensive agents) pH 4.77–9.69 [195]

Cephalosporins (antibiotics) pH 2.0–9.0 [196]

pKa determination of 99mTechnetium radiopharmaceuticals pH range 1.3–6.650 mbar PACECitric acid adjusted with NaOH

[200]

Anthrocyclines (antibiotics) pH 4.20–8.20 [202]

Cytokinins (phytohormones) pH 1.5–6.0 [203]

Dihydrofolate reductase inhibitors pH 2.1–4.550 mM phosphate buffer adjusted with phosphoricacid/NaOH

[204]

Quinolones (antibacterials) pH 2.0–11.0 [205]

Ropinirole and impurities (Parkinson’s treatment) pH 2.20–11.42 [206]

pKa of organic bases in aqueous methanol (0–70% v/v) pH range 4.76–9.5Tris, ethanolamine and acetate buffers

[207]

pKa of N-imidazole derivative aromatase inhibitors pH range 3.88–9.1625 mM phosphate buffer adjusted withtriethylamine

[208]

pH values [185–197]. Table 7 details a selection ofreported applications. The mobility of the solute at eachpH can be calculated from its migration time and the EOF(measured using a neutral marker such as DMSO), and aplot of mobility against pH can be constructed. Figure 4shows the mobility of the antibiotic cephalexin plottedover a range of electrolyte pH values. The pKa value of acompound is calculated using the electrophoretic mobili-ties, and cephalexin is calculated to have an acidic and abasic pKa of 2.49 and 7.08, respectively, compared to itsliterature values of 2.53 and 7.14 [198].

The dissociation constants of water-insoluble and sparinglysoluble compounds have been measured by CE throughadding solvents such as methanol to the electrolyte [187].

A medium-throughput method of pKa screening by pres-sure-assisted CE has been developed and demonstratedsuccessfully for 48 acidic, basic and multivalent pharma-ceutical compounds of known pKa [188]. The pressureassistance can greatly reduce the measurement time,especially for the low-pH analyses which can be very slowdue to the low EOF level.



Figure 4. Plot of cephalexinelectrophoretic mobility againstelectrolyte pH, used to calculatecompound pKa. Analysis per-formed using CombiSep 96-capillary array instrument,CombiSep pKa determinationbuffers and CombiSep pKa

determination software. (Unpub-lished material provided by theauthor).

By combining the application of pressure assistancewith the use of “short-end injection” technique and ashort capillary, pKa determinations can be performedeven more rapidly, and this method was also found togive greater repeatability of mobility measurements[189].

CE compares very favourably to other methods of pKa

analysis [199]. CE instruments are highly automated andcan be used for high-throughput applications. Unliketitration methods, precise information of sample con-centration is not required, as only analyte mobilities areused to calculate pKa by CE. Sparingly soluble com-pounds are easily analysed and only very small amountsof material are required, which is especially useful forthe screening of newly synthesised pharmaceuticalentities where only small quantities exist [195] or forapplications where the molecule of interest is presentonly in very small quantities such as radio-pharmaceuticals. A CE system equipped with a radio-activity detector has been used to determine the dis-sociation constants of 99mTechnetium radio-pharmaceuticals [200]. By using the “stacking”technique of sample introduction, concentrations ofpharmaceutical compounds as low as 2 mM haveresulted in successful pKa determinations [189]. Tech-niques commonly used for pKa determination such as

potentiometric titration or UV-Vis spectroscopy do notdifferentiate in analytical response between the analyteof interest and any analogue impurities, which causesproblems when attempting to measure compounds thatare not highly pure or are unstable in solution. BecauseCE is a separative technique, it is appropriate for pKa

determination of impure or unstable samples, and alsoelectrolyte purity is not essential. The CE method of pKa

determination has been demonstrated successfully forsets of labile drug compounds which are unstable in arange of acidic, basic and neutral solutions [190].

The standard pH scale for aqueous acids and bases haslimited applicability to organic solvents where pH* is usedinstead. Correspondingly, the pKa* values of compoundsin non-aqueous solvents have been calculated by CE[201]. The pKa* values in methanol of a series of baseshave been determined [15].

7 Conclusions

The range of pharmaceutical analysis applications for CEis extensive, possibly eclipsing those of HPLC. CE alsooffers a number of advantages over HPLC and otheranalytical techniques: analysis and method developmentspeed, reduced consumable and solvent expenses, sim-



plicity of operations and a greater possibility of imple-mentation of a single set of method conditions for theanalysis of several different samples, giving the large effi-ciency savings which are desirable in today’s busy labo-ratory.

Low-wavelength detection (190–200 nm) can beemployed in CE due to the short path-length of the capil-lary employed for detection. This can allow direct detec-tion of analytes that would require derivatisation (or analternative detection system to UV absorbance) to be dedetected by HPLC. CE therefore has a niche for the effi-cient and effective analysis of small organic and inorganicions such as amino acids, metal ions, simple organicacids and inorganic ions such as chloride and nitrate.These analytes are often drug (or excipient) counterions.These analyses are generally conducted using commer-cially available reagent kits or standard method condi-tions, and they are generally simpler and faster than ion-chromatography and are conducted on standard CEequipment, which eliminates the need for specialist ion-chromatography equipment.

For some pharmaceutical applications, such as chiralanalysis or the measurement of physicochemical proper-ties, CE is a superior technique to the conventionalmethods in terms of cost, ease of use and automationpossibilities. CE does have its disadvantages, but withthe considered method development, such as the incor-poration of internal standards to overcome poor injectionprecision, its performance can match that of HPLC. Thishas been shown by the number of reported validated CEmethods and also by several CE methods which havebeen accepted by regulatory authorities in new drugproduct submissions. CE also suffers from not being aswidely experienced as other techniques by pharmaceu-tical analysts, but with its routine applications increasing,this is improving.

CE systems are flexible and can be automatically pre-programmed to vary parameters such as voltage, wave-length, temperature, and operate using vials containingvarious electrolyte compositions. This flexibility allowsexperimental designs such as Plackett–Burman [209] tobe effectively used to optimise CE methods or to performrobustness testing.

In many analytical laboratories, CE is regularly used toprovide complementary information to HPLC or othermethods as well as being the technique of choice forcertain applications. As the instrumentation furtherimproves, automation possibilities expand and the list ofapplications increases, the use of CE for pharmaceuticalanalysis is certain to continue to be popular and becomemore widespread.

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Capillary Electrophoresis for the Analysis of Small-molecule Pharmaceuticals