Chiral selectors in CE: recent developments and applications (2012-mid 2014).

101

Electrophoresis 2015, 36, 101–123

Ioannis J. Stavrou∗ Maria C. Mavroudi∗ Constantina P. KapnissiChristodoulou Department of Chemistry, University of Cyprus, Nicosia, Cyprus

Received June 30, 2014 Revised September 25, 2014 Accepted September 26, 2014

Review

Chiral selectors in CE: Recent developments and applications (2012-mid 2014) There is a large number of chiral selectors (CSs) that have, over the years, been synthesized and used in electrophoretic enantioseparations. This report highlights the most important CSs applied in CE during the period 2012 to mid-2014. It is mainly focused on method developments and validations, along with pharmaceutical and biomedical applications. Even though numerous publications have, through the years, reported the utilization of CSs in enantioseparations, only the ones applied in electrophoretic techniques the last approximately three years are demonstrated in this review article. In particular, cyclodextrins, cyclofructants, linear and branched oligo- and polysaccharides, antibiotics, and polymeric surfactants are presented, and their advantages, their chiral recognition mechanisms, and their performance are discussed. Keywords: Capillary electrophoresis / Chiral selectors / Enantioseparations DOI 10.1002/elps.201400310

1 Introduction Correspondence: Dr. Constantina P. Kapnissi-Christodoulou, Department of Chemistry, University of Cyprus, 1678 Nicosia, Cyprus E-mail: [email protected] Fax: +357-22-892801

Abbreviations: AMBIMCD, 6A -ammonium-6C butylimidazolium-␤-CD chloride; BCD3AM, 3-amino-3deoxy-␤-CD; BIMCDCl, mono-6A -butylimidazolium-␤-CD; BNP, 1,1 -binaphthalene-2,2 -diyl hydrogen phosphate; BuAMCD, mono-6A -butylammonium-6A -deoxy-␤-CD tosylate; CDNH3 Cl, mono-6A -ammonium-␤-CD chloride; CE-␤-CD, carboxyethyl-␤-CD; CF, cyclofructan; CIL, chiral IL; CM-␣-CD, carboxymethyl-␣-CD; CM-␤-CD, carboxymethyl␤-CD; CM-␥-CD, carboxymethyl-␥-CD; CS, chiral selector; CSD, chondroitin sulfate D; CSE, chondroitin sulfate E; DM-␤-CD, dimethyl-␤-CD; DS, degree of substitution; DTAC, dodecyl trimethyl ammonium chloride; [EMIM][L-lactate], 1-ethyl-3-methylimidazolium-L-lactate; FITC-AAs, ␣-amino acids derivatized with FITC; GCD3AM, 3-amino-3-deoxy-␥-CD; HDAS-␤-CD, heptakis-(2,3-diacetyl6-sulfato)-CD; HDB, hexadimethrine bromide; HDMS-␤-CD, heptakis-(2,3-dimethyl-6-sulfo)-CDs; HEtAMCD, mono6A -(2-methoxypropan-1-ammonium)-6A -␤-CD chloride; HP-␣-CD, hydroxypropyl-␣-CD; HP-␤-CD, hydroxypropyl␤-CD; HP-␥-CD, hydroxypropyl-␥-CD; [HPTMA-␤-CD][BF4 ], 6-O-2-hydroxypropyltrimethylammonium-␤-CD tetrafluoroborate; HS-␥-CD, highly sulfated-␥-CD; IL, ionic liquid; IL-MWNT, IL dispersed multiwalled carbon nanotube; IPCF6, isopropyl-CF-6; L-AlaC4 NTf2 , L-alanine tert butyl ester bis(trifluoromethane)sulfonimide; L-ValC4 NTf2 , L-valine tert butyl ester bis(trifluoromethane)sulfonimide; M-␤-CD, methyl-␤-CD; M-␥-CD, methyl-␥-CD; MD, maltodextrin; MEtAMCD, mono-6A -(2-methoxyethyl-1-ammonium)-6A ␤-CD chloride; MPrAMCD, mono-6A -(3-methoxypropan1-ammonium)-6A -␤-CD chloride; NSAID, nonsteroidal anti-inflammatory drug; P-␥-CD, phosphate-␥-CD; PA-␤ C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

The separation of chiral compounds has been of great interest in many industries, and particularly the pharmaceutical industry. It is well established that enantiomers differ qualitatively or quantitatively in their pharmacological, toxicological, and biological activity [1]. Most biological molecules are present in only one chiral form; so, the enantiomers of a drug compound bind differently to target receptors. One of them is usually active, while the other may be less active, inactive, or has adverse effects. Thus, the Food and Drug Administration and the European Medicines Agency require enantiomer drug purity of pharmaceuticals in pharmacokinetic and metabolic studies [1, 2]. Therefore, a great variety of chromatographic methods have been developed and applied, through the years, for the enantioseparation of bioorganic compounds [3–16]. For more than two decades, there has been a growing interest in the separation of enantiomeric compounds in CE CD, 6-monodeoxy-6-mono(hydroxyl)propylamino-␤-CD hydrochloride; poly-LL-SULV, poly(sodium N-undecanoylLL-leucine-valinate); PrAMCD, mono-6A -propylammonium6A -␤-CD chloride; QA-␤-CD, quaternary ammonium ␤-CD; S-␣-CD, sulfated-␣-CD; S-␤-CDs, sulfated-␤-CDs; SBE-␤CD, sulfobutyl ether-␤-CD; SCF6, sodium sulfated CF 6; SCF7, sodium sulfated CF 7; SET-␤-CD, mono(6-deoxy6-sulfoethylthio)-␤-CD; SMHT-␤-CD, mono[-6-deoxy-6-(6sulfoxy-5,5-bis-sulfooxymethyl)hexylthio]-␤-CD; SP-␤-CD, sulfopropylated-␤-CD; THALAH, 6A,6D-dideoxy-6A,6D-N[6,6 -di-(␤-alanylamido)]-6,6 -dideoxy-␣,␣ -trehalose]-␤-CD; TM-␣-CD, trimethyl-␣-CD; TM-␤-CD, trimethyl-␤-CD; TM␥-CD, trimethyl-␥-CD; 18C6H4 , (+)-(18-crown-6)-2,3,11,12tetracarboxylic acid ∗

These authors contributed equally to this work.

Colour Online: See the article online to view Figs. 2 and 4 in colour.

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I. J. Stavrou et al.

due to its versatility, high separation efficiency, and low consumption of reagents and samples [17–19]. It can be used for achiral and chiral analysis of pharmaceutical, biomedical and food samples [20–24]. CE has various modes of operation depending on the composition of the BGE and the nature of the capillary. The most common CE modes are CZE, EKC, MEKC, MEEKC, and NACE. In all cases, one or more chiral selectors (CSs) are added into the BGE in order to perform chiral discrimination. The chiral separation in CE is achieved due to the electrophoretic mobility and the interactions between the CSs and the analytes. The stereoselective recognition of the enantiomers by the CS is based on the formation of transient diastereomeric complexes, which have different mobilities. The enantioseparation is therefore based on the different binding constants of the enantiomers with the CS and/or the different mobilities of the diastereomeric associates [25]. During the last decades, various CSs have been used in CE for enantioseparations due to their chiral recognition abilities [26–31] and numerous review articles have been published [32–39]. Thereby, herein, an overview of different kinds of CSs that were used in CE during the period 2012 to mid2014 is summarized. It is worth here to mention that this is a continuity of our previous review paper, which was focused on the literature before 2012 [32].

2 Chiral selectors The CSs that were utilized from 2012 until mid-2014, in EKC, MEKC, MEEKC, CD-EKC, CD-MEKC and NACE, for improved chiral separations, along with their applications, are reported and discussed in this review. In particular, CDs, cyclofructans (CFs), linear oligo- and polysaccharides, branched polysaccharides, polymeric surfactants, antibiotics, crown ethers and some other CS systems, are demonstrated.


tively and positively charged CD derivatives as CSs in CE are classified.

2.1.1 Native CDs ␤-CD is used more often in chiral CE applications than the other native CDs due to its cavity size, which enables tight inclusion complex formation with most of the analytes [40–45]. Song et al. [40] achieved the separation and determination of DL-penicillamine in the oral penicillamine tablets by using native ␤-CD as the CS in a well-grounded CD-CE method at a broad pH range (from pH 2 to over pH 10). Quantification of standard D- and L- penicillamine was performed by assaying the real samples of oral penicillamine tablets at three pH values (acidic, neutral and basic). LOD was estimated to be 2.58 ␮g/mL in acidic and neutral conditions and 1.41 ␮g/mL in basic conditions. Sánchez-Hernández et al. [41] used different neutral CDs to develop a CE-ESI-MS2 methodology for the identification of amino acid enantiomers that were derivatized with fluoresceine-5-isothiocyanate. Both the native ␤-CD and ␥ -CD achieved enantiomeric separation of 14 amino acids with resolution values of above 1.0 and LODs between 0.02 and 0.8 ␮M. They also developed enantiomeric purity control of hydrolyzed protein fertilizers. Results demonstrated that, depending on the type of hydrolysis of the analyzed sample, different percentages of enantiomeric impurities were produced and the enzymatic hydrolysis was the one with no or almost negligible presence of D-amino acids. Native ␣-CD, at a concentration of 10 mM (pH 2.0), was used as a CS for the enantioseparation of trimipramine enantiomers in plasma and urine samples [46]. As far as the native ␥ -CD is concerned, it has not been chosen as the optimum CS for the last three years, possibly due to its large cavity size, which does not allow effective stereoselective interactions with most of the analytes.

2.1.2 Neutral CD derivatives 2.1 Cyclodextrins CDs are cyclic oligosaccharides composed usually of 6–8 ␣-(1,4)-linked D-(+)-glucopyranose units forming a hollow truncated cone structure. The size of the CD cavity can be varied according to the number of the glycopyranose units. The three main CDs are ␣-CD (6 units), ␤-CD (7 units) and ␥ -CD (8-units). Their structure makes them a perfect CS due to the inclusion complex formation with several types of chiral analytes. Moreover, CDs have good solubility in aqueous solvents, low toxicity, low UV absorbance and widespread application range. Thus, CDs have been extensively used for chiral separations in CE. Despite the fact that native CDs have successfully separated a number of chiral compounds, different CD derivatives have been synthesized in order to improve and enhance their enantiorecognition capability. In this review, the application of native, neutral, and both nega C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Although native cyclodextrins demonstrated good enantioseparation abilities, their application range increased when different derivatives of the three native CDs (␣-, ␤-, ␥ -CD) were synthesized. In particular, several neutral and charged CDs have been synthesized, over the years, and have been widely used as CSs in CE. Various neutral CD derivatives have been applied in CE for the chiral separation of many compounds. Hydroxypropyl-␤-CD (HP-␤-CD) appears to be the most commonly used neutral CD derivative. Tabani et al. [47] used electrically assisted liquidphase microextraction in combination with CE for the quantification of propranalol enantiomers in human body fluids. Its enantioseparation was achieved by use of four types of CDs (␣-CD, hydroxypropyl-␣-CD (HP-␣-CD), ␤-CD and HP␤-CD) as CSs. Good resolution was obtained with HP-␤-CD, because this neutral CD has a suitable cavity that provides www.electrophoresis-journal.com


better interaction with the propranalol enantiomers. On the other hand, HP-␣-CD and ␣-CD did not provide a satisfactory resolution, due to the small size of their cavities, which did not allow effective stereoselective interaction with the analyte enantiomers. Similarly, El-Gizawy et al. [48] used 10 mM of HP-␤-CD for the chiral separation of perindopril erbumine enantiomers. The optimized method was applied for the determination of S-perindopril in bulk powder and commercial tablets containing perindopril erbumine racemate. In another study, the enantiomeric separation of the antidepressant basic (pKa = 9.7) drug duloxetine was investigated by using 15 neutral CDs as CSs at acidic pH in CE [49]. HP-␤-CD and methyl-␥ -CD (M-␥ -CD) provided the best enantioseparations with a resolution value of 1.6 in both cases, while acetyl-␥ -CD and 2-(hydroxybutenyl)-␤CD gave resolution values of 0.6 and 0.7, respectively. Although there were no significant differences in the analysis time when HP-␤-CD and M-␥ -CD were applied, the enantiomer migration order was reversed. When HP-␤-CD was used, the enantiomeric impurity of duloxetine (R-duloxetine) was the first migrating enantiomer, while in the case of M-␥ CD, R-duloxetine was the second-migrating enantiomer. This was originated by the different electrophoretic mobilities of the enantiomer-CS complexes. HP-␤-CD was also used by Theurillat et al. [50] for the analysis of threo-methylphenidate enantiomers in oral fluid by CE-based assay. The enantioseparation was achieved within 10 minutes. The performance of the assay with six point multilevel internal calibration was assessed with calibration and control samples and was applied to patient samples. A linear response for each enantiomer was found in the 10–200 ng/mL concentration range. Ibrahim et al. [51] separated all four stereoisomers of vinpocetine by using five neutral modified CDs, namely DM␤-CD, trimethyl-␤-CD (TM-␤-CD), HP-␣-CD, HP-␤-CD, and hydroxypropyl-␥ -CD (HP-␥ -CD), in CD-MEKC. Another important observation, in this study, involves the low resolution that was obtained when the HP-CDs were used. In particular, broad and unshaped peaks were observed when HP-␣-CD was added into the BGE at different concentrations. In the case of HP-␤-CD, no enantioseparations were achieved at 15, 25 and 35 mM, and only three peaks slightly appeared when the concentration increased to 40 mM. Partial separation (Rs = 0.4) was also observed when HP-␥ -CD was used at a concentration of 40 mM. Zhang et al. [52] investigated the synergistic effect of CD derivatives and amino acid-derived CILs (L-alanine tert butyl ester bis (trifluoromethane) sulfonamide, L-AlaC4 NTf2 , and L-valine tert butyl ester bis (trifluoromethane) sulfonimide, L-ValC4 NTf2 ). Methyl-␤-CD (M-␤-CD), HP-␤-CD and glucose-␤-CD were used as CSs for the enantioseparation of six anionic racemic drugs (naproxen, pranoprofen, warfarin, carprofen, ibuprofen and ketoprofen). The synergistic effect was significant for the first three analytes, since the resolution values were lower in the case where the CD derivative was used as the sole CS. It is worth though mentioning that, when the amino acid-derived CILs were used alone, no enantioseparations were observed. In a sim C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

CE and CEC

103

ilar study by Zhao et al. [53], a dual system of HP-␤-CD and three different types of ionic liquids (ILs) (1-ethyl-3methylimidazolium-L-lactate [EMIM][L-lactate], N-methyl-Nethylpyrrolidinium tetrafluoroborate, and dodecyl trimethyl ammonium chloride (DTAC)) for the simultaneous enantioseparation of four azole antifungals was employed. When HP-␤-CD was used as the sole CS, no enantioseparation was observed. On the other hand, when the ILs were individually added into the BGE, the enantioseparations were improved significantly. Among the three additives, DTAC proved to be the most effective in regard to resolution and effective electrophoretic selectivity. The interactions between DTAC and HP-␤-CD affected the formation of inclusion complex, which, in turn, influenced the enantioseparation (Fig. 1). Another type of neutral CD derivative that is frequently used in CE is the M-CD. Lee et al. [54] developed and validated two methods (CE and NMR) for the separation and quantitation of sibutamine enantiomers by using M-␤-CD as the CS. In the CE study, the method was fully validated and successfully applied to determine S-sibutamine in a commercial sibutamine hydrochloride capsule. The results obtained from the CE method optimization demonstrated that, at 10 mg/mL of M-␤-CD, the enantioseparation was influenced by the formation of the diastereomeric inclusion complex between the neutral CD derivative and the analyte, while at higher concentrations of M-␤-CD, the enantioseparation was affected by the mobility difference between the temporary diastereomeric associates. Li et al. [56] developed an easy, cost-effective and rapid CE method for the separation of repaglidine enantiomers by using ␤-CD and its derivatives as CSs. It was observed that ␤-CD and M-␤-CD could not separate the analyte enantiomers, while sulfobutyl ether-␤-CD (SBE-␤-CD) and DM-␤-CD provided baseline enantioseparation. In addition, DM-␤-CD demonstrated better enantioselective ability than SBE-␤-CD, possibly due to the cavity size as well as the role of the side-chain-related sites of repaglidine. The separation of ketoprofen enantiomers was achieved by use of three TM-CDs derivatives, namely trimethyl-␣-CD (TM-␣-CD), TM-␤-CD, and trimethyl-␥ -CD (TM-␥ -CD) in CE [57]. In this report, it was observed that the elution order of the enantiomers depended on the CD’s cavity size. Therefore, with TM-␣-CD and TM-␤-CD, the R enantiomer eluted first, while in the case of TM-␥ -CD the enantiomer migration order was reversed. The change in the structure of the complexes between the analyte enantiomers and the CSs was confirmed by NMR experiments. In particular, ketoprofen entered the cavity from the wider secondary side of TM-␣-CD with the benzoyl moiety located deep inside the cavity, while in the second case, the phenyl and alkyl moieties of ketoprofen entered the cavity from the narrower, primary side of TM-␤-CD. In the third and last case, the analyte entered the TM-␥ -CD cavity from the narrower side with the benzyl ring located outside (Fig. 2). Another recent study by Kodama et al. [58] reported the use of TM-␤-CD for the enantioseparation of the antioxidant lipoic acid in nine dietary www.electrophoresis-journal.com

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Figure 1. Electropherograms of four azole antifungal enantiomers. (1) Miconazole; (2) econazole; (3) ketoconazole; (4) itraconazole. CE conditions: pH 3.5, (A) 30 mM HP-␤-CD for 1,2, and 4, while 50 mM HP-␤-CD for 3, 50 mM NaH2 PO4 /H3 PO4 (B) 30 mM HP-␤-CD, 30 mM NaH2 PO4 /H3 PO4 , with 20 mM dodecyl trimethyl ammonium chloride added. Reprinted with permission from [53].

supplement samples. In this study though, the resolution of the racemic lipoic acid by using the optimized separation conditions was approximately 1.2. 2.1.3 Negatively charged CD derivatives Although native cyclodextrins demonstrated good enantioseparation abilities, their application range increased when different derivatives of the three native CDs (␣-, ␤-, ␥ -CD) were synthesized. In particular, several negatively and positively charged CDs have been synthesized, over the years, and have been widely used as CSs in CE. In the first case, the negative charge provides a suitable interaction site for the positively charged analytes. Thus, the negatively charged CD derivatives interact strongly with basic compounds. It is worth pointing out that the negatively charged CDs are more widely used than the positively charged CDs because a broad spectrum of analyzed chiral compounds are pharmaceuticals, which are mostly basic and positively charged. Table 1 summarizes the negatively charged CDs that have been used as CSs during the period 2012 to mid 2014, along with their applications in different modes of CE. The most commonly used negatively charged CDs are sulphur-contained CD derivatives. Deng et al. [59] developed a chiral electrophoretic method for the quantification of 0.1% of the enantiomeric purity (dextrocetirizine) in levocetirizine by using sulfated-␤-CDs (S-␤-CD) as the CS. In their study, different other CSs, such as cellulose, dextran and heptakis-(2,3-diacetyl-6-sulfato)-CD (HDAS-␤-CD), were used and their performance was compared. After validation, the method was applied to the quality control and enantiomeric purity control of levocetirizine dihydrochloride tablets. They were able to determine 0.1% (w/w)

C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

chiral impurity of the drug substance. The examined levocetirizine tablets were 99.2% assay of label claim (5 mg), which complies with the requirements for drug product content (95–105%) and 0.42% (w/w) of dextrocetirizine enantiomer impurity. In 2013, 13 amphetamine-like designer drugs were separated using neutral and negatively charged CD derivatives [60]. S-␤-CD proved to be the best chiral selector among the other used CDs (DM-CD and carboxymethyl-CD (CM-CD)). Briefly, it was observed that, when the negatively charged CD CM-CD was used as the CS, only three amphetamines were able to be separated, and the obtained RS values were not satisfactory. On the other hand, the neutral DM-CD was not able to separate any of the examined chiral drugs, because there were no electrostatic interactions between the positively charged analyte and the neutral CD. It is well known that the enantiomer that interacts strongly with the negatively charged CS has no net positive charge, while the enantiomer that has no interactions with the CS has net positive charge [60]. Thus, the separation resolution is much better when a charged CD is used as a CS, instead of a neutral one. Different amphetamine-like drugs were enantioseparated by using different S-␤-CD concentrations each time (1–10 mg/mL). It is worth pointing out that there are also more reports regarding the use of S-␤-CD and highly sulfated-␤-CD (HS-␤-CD) as CSs for the enantioseparation of several drugs and clinically important compounds, such as tolterodine and methoxytolterodine [61], escitalopram [62], verapamil, norverapamil [63], cathinone derivatives [64], and nicotine butyric acid [65]. Although, during the reported period 2012 – mid 2014, the use of S- and HS-␤-CDs as CSs in CE was significant, only a few studies reported the use of sulfated-␣-CD (S-␣-CD) and sulfated-␥ -CD (S-␥ -CD). Particularly, Iwata et al. [66] reported



Fucose Pipecolic acid Zopiclone, repaglinide, chlorphenamine maleate, brompheniramine maleate, dioxopromethazine hydrochloride, promethazine hydrochloride, liarozole, carvedilol, homatropine hydrobromide, homatropine methylbromide, venlafaxine, sibutramine hydrochloride 5 Acyclic nucleoside phosphonates

10 mM 30 mM

10 mM

Neutral derivatives

NACE

¨ 6 Troger’s base derivatives

100 mM

Duloxetine Threo-methylphenidate Vinpocetine Miconazole, econazole, ketonazole, itraconazole Naproxen, pranoprofen, warfarin

1% (w/v) 20 mg/mL

40 mM 30 mM or 50 mM

20 mM

10 mg/ml 1.25% (w/v)

M-␤-CD

M-␤-CD DM-␤-CD

EKC EKC

EKC

CD-MEKC EKC

30 mM sodium citrate/15 mM L-AlaC4 NTf2 or L-ValC4 NTf2 , 20 % (v/v) ethanol or acetonitrile 50 mM phosphate 20 mM sodium phosphate

100 mM phosphate/15 % methanol (v/v) 50 mM phosphate 50 mM phosphate/30 mM triethanolamine 50 mM phosphate/40 mM SDS 50 mM phosphate/20 mM DTAC

20 mM phosphate/10 % acetonitrile 80 mM ammonium acetate

50 mM ammonium carbonate

100 mM phosphate

50 mM sodium tetraborate

20 mM phosphate, 5-15 % acetonitrile (v/v) Formamide, 200 mM sodium citrate, 50 mM Tris 40 mM borax 40 mM borax/30 mM SDS/15% isopropanol/10 mM D-AlaC4 NTf2 30 mM Tris-H3 PO4 /20 mM [EMIM][L-lactate]

200 mM acetic acid or 30 mM sodium phosphate or 80 mM boric acid 50 mM ammonium carbonate

BGE

3 2.5

5

7 3.5

2 3

7

2.5 2.5

10

9.6 or 9.85 or 10.3 2

2.5

9.5 9.5

–

2.5

10

4.5 or 7.4 or 9.7

pH

– Pharmaceutical formulations

–

Plasma and urine samples Hydrolyzed protein fertilizers – Plasma and urine samples Bulk powder and commercial tablets – Patient samples Oral fluid Assay – –

–

Commercial tablets

– –

–

Oral penicillamine tablets Hydrolyzed protein fertilizers –

Applications

[54] [56]

[52]

[51] [53]

[49] [50]

[48]

[42] [47]

[41]

[46]

[85]

[45]

[44] [44]

[43]

[42]

[41]

[40]

Ref.

CE and CEC

Sibutamine Repaglinide

Perindopril erbumine

10 mM EKC EKC

EKC EKC

¨ 2 Troger’s base derivatives Propranolol

20 mM 8 mM

HP-␤-CD

EKC

13 Protein amino acids, ornithine

5 mM

␥-CD

EKC

10 mM

EKC

EKC CD-MEKC

␣-CD Trimipramine

EKC

¨ 5 Troger’s base derivatives

5 mM

20 mg/mL

EKC

13 Protein amino acids, ornithine

5 mM

EKC

Penicillamine

17 mM

␤-CD

CE mode

Native

Analyte

Concentration

CD

Type of CD

Table 1. Enantioseparation of chiral compounds by using CDs as CSs Electrophoresis 2015, 36, 101–123

105


Negatively charged CDs

Type of CD


Verapamil, norverapamil

2.5% (m/v)

Monosaccharide-NAIM enantiomers Tolterodine, methoxytolterodine Norephedrine Talinolol Talinolol

10 mg/mL 3% (w/v) 30 mg/mL 50 mg/mL 50 mg/mL

Pantoprazole Armodafinil Bupivacaine hydrochloride

20 mg/mL 60 mg/mL 50 mg/mL

20 mg/mL

20 mM 0.48 mM

SBE-␤-CD

Norephedrine Talinolol Talinolol

Tolterodine, methoxytolterodine Ornidazole

4% (w/v) 2% (w/v)

4 or 5% (w/v)

Nicotine butyric acid Amphetamine, ephedrine, pseudoephedrine,norephedrine, norpseudoephedrine 16 ␤-carboline derivatives

EKC EKC

EKC

EKC EKC NACE

EKC EKC EKC EKC NACE

EKC EKC

EKC (partial filling technique) EKC EKC (S-type modified capillary) EKC

EKC EKC EKC

EKC Sulfonated capillary EKC EKC EKC EKC EKC

CE mode

20 mM phosphate /20% methanol 4 mM Ammonium acetate/sodium acetate/acetic acid

300 mM phosphate 70 mM Tris/phosphate 200 mM Sodium Phosphate 100 mM H3 PO4 /triethanolamine 10 mM Ammonium formate/formic acid in methanol 200 mM Sodium Phosphate 100 mM H3 PO4 /triethanolamine 10 mM Ammonium formate/ Formic acid in methanol 50 mM borax/150 mM phosphate

60 mM phosphate (H3 PO4 /triethylamine) 70 mM Tris/phosphate 20 mM Tris/phosphate

75mM H3 PO4 /triethylamine 50 mM phosphate

50 mM phosphate

63.5 mM H3 PO4 /46.9 mM NaOH 70 mM Tris/phosphate 25mM phosphate

50 mM phosphate 70 mM acetate/20 % (v/v) methanol 60 mM acetic acid 60 mM acetic acid 50mM sodium tetraborate

60 mM acetic acid 100 mM phosphate

BGE

7.5 4.0

6.5

2.5 3.0 3.0

3.0 2.5 2.5 3.0 3.0

2.5 2.1

2.5

7 2.6

8.8

2.6 2.5 7.0

2 4.5 5 5 8.2

5 7

pH

S-(-)-Pantoprazole bulk samples Armodafinil bulk samples Rabbit serum and pharmaceutical injections

– – –

– Drug substance and injection solution of levornidazole – Commercial pills – –

-

– –

– – – Bulk samples and tablets of levocetirizine – – Bulk samples and tablets of escitalopram –

– Dietary supplements

Applications

[73] [74]

[72]

[70] [71] [71]

[68] [61] [70] [71] [71]

[61] [69]

[67]

[65] [66]

[63]

[60] [61] [62]

[49] [55] [57] [57] [59]

[57] [58]

Ref. I. J. Stavrou et al.

HDAS-␤-CD

P-␥-CD HDMS-␤-CD

S-␣-CD

HS-B-CD HS-␥-CD

10% (m/v) 10 mM

Amphetamine-like designer drugs Tolterodine, methoxytolterodine Escitalopram

1-10 mg/mL 4% (w/v) 1.6% (w/v)

TM-␥-CD TM-␣-CD S-B-CD

Duloxetine Dapoxetine Ketoprofen Ketoprofen Cetirizine

1% (w/v) 3 mM 50 mM 50 mM 1% (w/v)

M-␥-CD

Ketoprofen Lipoic acid

50 mM 8 mM

TM-␤-CD

Analyte

Concentration

CD

Table 1. Continued

106 Electrophoresis 2015, 36, 101–123



Positively derivatives

Type of CD

1.8 mM 2.5 mM 1.5 mM

2.5 mM

5 mM 0.25 mM-2 mM

MEtAMCD

HEtAMCD GCD3AM

0.25 mM-2 mM

BCD3AM

THALAH MPrAMCD AMBIMCD

60 mg/mL

QA-␤-CD

10 mM

2I -O-CM-␤-CD

Fenfluramine

40 mM

10 Dansyl amino acids 10 Acidic and ampholytic racemates 14 Acidic enantiomers and amino acids 24 Ampholytic, acidic and neutral racemates 22 Acidic and amino acids FITC-AAs

13 FITC-AAs

¨ ¨ Baclofen, Troger’s base 1, Troger’s base 2, primaquine, tryptophan ¨ ¨ Baclofen, Troger’s base 1, Troger’s base 2, primaquine, tryptophan 1 Acyclic nucleoside phosphonate

Pindolol

40 mM

10 mM

Amphetamine, methamphetamine Verapamil

3 mM 40 mM

2 mM

3I -O-CM-␣-CD

CM-␥-CD

CM-␤-CD

20 mM 0.25 mM 0.5 mM 1 mM 2.5 mM 10 mM

Indapamide Verapamil Clenbuterol, Pseudoephedrine, terbutaline Ephedrine Verpamil, propranolol Terbutaline, pseudoephedrine Clenbuterol Ephedrine Clenbuterol, bambuterol, tulobuterol, procaterol, salbutamol Meptazinol and its intermidiate

5 mM 1 mM 5 mM

SET-␤-CD

SMHT-␤-CD

Analyte

Concentration

CD

Table 1. Continued

EKC EKC

EKC

EKC EKC EKC

EKC

EKC

EKC

EKC

NACE

NACE

EKC NACE

EKC

EKC EKC EKC EKC EKC EKC

EKC EKC EKC

CE mode

50 mM phosphate 20 mM acetic acid/ammonium acetate

50 mM phosphate

20 mM acetic acid/ammonium acetate 20 mM ammonium acetate 50 mM phosphate 50 mM acetate

20 mM Tris/40 mM phosphate

20 mM sodium phosphate

6 6.4

6.5

5.1 6 6

6.4

2.2

2.5

2.5

–

–

2.8 –

6.0

20 mM H3 PO4 /5% acetonitrile 150 mM acetic acid/ 0.2% (w/v) HPMC 125 mM ammonium acetate/1 M acetic acid in DMSO-methanol 80:20 125 mM ammonium acetate/0.5 M acetic acid in DMSO-methanol 70:30 125 mM ammonium acetate/0.5 M acetic acid in DMSO 20 mM sodium phosphate

2.5 2.5 2.5 2.5 2.5 3.5

7 2.5 2.5

pH

60 mM H3 PO4 /triethanolamine 60 mM H3 PO4 /triethanolamine 60 mM H3 PO4 /triethanolamine 60 mM H3 PO4 /triethanolamine 60 mM H3 PO4 /triethanolamine 50 mM phosphate

25 mM Na2 HPO4 60 mM H3 PO4 /triethanolamine 60 mM H3 PO4 /triethanolamine

BGE

– –

–

– –

–

–

–

–

–

–

– – – – Pharmaceutical formulations Pharmaceutical formulations Human urine sample –

– –

Applications

[91] [86]

[90]

[87] [88] [89]

[86]

[85]

[84]

[83]

[82]

[82]

[133] [82]

[79]

[76] [76] [76] [76] [76] [78]

[75] [76] [76]

Ref.


CE and CEC 107


108



Figure 2. Structure of the ketoprofen/CD complexes as derived from 1D ROESY spectra. Ketoprofen/TM-␣-CD (A), ketoprofen/TM-␤-CD (B), and ketoprofen/TM-␥-CD (C). Reprinted with permission from [57].

the use of highly sulfated-␥ -CD (HS-␥ -CD) for methamphetamine profiling. Some amphetamine-type stimulants (amphetamine, ephedrine, pseudoephedrine, norephedrine and norpseudoephedrine) were simultaneously enantioseparated by using a modified capillary column (Type S, sulfated groups) and HS-␥ -CD as the CS. This method provided repeatable, rapid and good resolution results, and it was proposed for routine methamphetamine profiling. In another study, Lipka et al. [67] used HS-␥ -CD for the separation of 16 new synthesized chiral ␤-carboline derivatives. These are compounds that are structurally related to melatonin, and they are proposed as possible antioxidants inhibiting the low-density lipoprotein. As far as the use of S-␣-CD is concerned, Lehnert et al. [61] reported its use for the enantioseparations of tolteridine and methoxytolteridine. Despite the fact that S-␤-CD demonstrated the best chiral recognition ability, S-␣-CDs were able to baseline resolve the two chiral compounds (RS ⬎2). In the same study, they also utilized a rarely used negatively charged CD (phosphate-␥ -CD (P-␥ -CD)), which was able to successfully baseline separate tolterodine and methoxy tolterodine enantiomers [61]. In a different study, S-␣-CD was used as the CS for the chiral separation of monosaccharidenaphthimidazole derivatives (monosaccharide-NAIMs) [68]. Finally, the authors noted that, in comparison to other methods (e.g. GC-MS), their method was simpler due to the easy chemical modification of the monosaccharides to their NAIM derivatives in order to analyze them fast in CE and determine the composition and the absolute configuration in one shot. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

In another study, S-␣-CD was chosen as the optimum CS for the separation of levornidazole and ornidazole [69]. Among different neutral and negatively charged CDs, only the negatively charged CDs were able to resolve the two enantiomers in a reversed polarity mode. Briefly, the CSs S-␣-CD, S-␤-CD, sulfopropylated-␤-CD (SP-␤-CD) and SBE-␤-CD provided enantiomer separation, while the also negatively charged carboxymethyl-␤-CD (CM-␤-CD) and carboxyethyl-␤-CD (CE-␤-CD) did not. S-␣-CD, at a concentration of 2% (w/v), was selected as the optimum CS due to greater RS values (RS ⬎5) and short analysis times (around 7 min). Although S-CDs have been reported as excellent CSs, some drawbacks have limited their use. The degree of substitution of the commercially available S-CDs is not known and differs from one batch to another. Thus, the resolution obtained may differ each time. In addition, the concentrations required are relatively high, and thus, the current, passing through the capillary column, is increased [65]. To circumvent such problems, researchers sometimes use other CD derivatives as alternatives (HDAS-␤-CD, heptakis-(2,3-dimethyl-6-sulfo)-CD (HDMS-␤-CD), mono(6deoxy-6-sulfoethylthio)-␤-CD (SET-␤-CD), mono[-6-deoxy6-(6-sulfoxy-5,5-bis-sulfooxymethyl)hexylthio]-␤-CD (SMHT␤-CD), SP-␤-CD, CM-␤-CD, CE-␤-CD). Lomsadze et al. [70] studied the separation of norephedrine enantiomers using HDAS-␤-CD and HDMS-␤-CD as the CSs in CE. The results obtained were compared with those obtained by using the native ␣-CD, ␤-CD, ␥ -CD and some neutral CD-derivatives www.electrophoresis-journal.com


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Figure 3. CE separation of norephedrine enantiomers with native ␣-CD, ␤-CD and HDMS-␤-CD and HDAS-␤-CD. Reprinted with permission from [70].

(DM-␤-CD, TM-␤-CD and heptakis(2,3-di-O-acetyl)-␤-CD). All CDs, except from ␥ -CD, were able to separate the enantiomers of norephedrine. HDAS-␤-CD though, proved to be the best CS because it provided the best baseline separation with a selectivity factor, ␣, of 1.34. Comparing the two native CDs (␣-CD and ␤-CD) to the negatively charged HDAS-␤CD S and HDMS-␤-CD, the last two were able to baseline separate the enantiomers of norephedrine, while the formers provided partial separation of the enantiomers (Fig. 3). In addition, it was observed that the elution order was different. Briefly, when ␤-CD derivatives were used as CSs, the elution order of the norephedrine enantiomers was the same as in the case of the native ␣-CD, while it was reversed when the native ␤-CD was used. For better understanding, Lomsadze et al. [70] investigated the molecular mechanisms by performing 1 H NMR and ROESY experiments. It was demonstrated that, in the native ␣- and ␤-CD, the aromatic ring of norephedrine entered the cavity from the wider, secondary rim. In the case of ␤-CD, the complex seemed to be tighter and the inclusion deeper. In HDAS- and HDMS-␤-CD, in comparison to the native CDs, the interactions occurred in their primary, smaller openings. HDAS-␤-CD formed an inclusion complex with norephedrine, while only a superficial binding was observed for HDMS-␤-CD. In a similar work, the same research group [71] reported the use of the last two CDs (HDAS- and HDMS-␤-CD) for the enantioseparation of talinolol. Although both CD-derivatives were able to separate the analyte in aqueous BGE, HDAS-␤CD proved to be a more effective CS than the HDMS-␤-CD, which partially separated the talinolol enantiomers. In nonaqueous BGE, both CD-derivatives gave baseline separation of talinolol. Using NMR studies, the structures of CD-talinolol complexes were examined. The authors concluded the follow C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

ing: different enantiomer affinity pattern can be observed by using chemically different CD derivatives; chiral separations can be achieved even if the geometry of analyte-cyclodextrin complexes are not the same; the resolution and the chiral recognition ability that rely on the formation of external complexes between analyte and CDs can be the same as in the case where analyte/CDs inclusion complexes are formed. SBE-␤-CD is another sulphur-containing CD derivative, which has been widely used in CE during the last few years. Guan et al. [72] developed a method for the determination of pantoprazole enantiomers by use of SBE-␤-CD as the optimum CS. It was observed that, as the concentration of the SBE-␤-CD increased, both the migration time and resolution increased. In addition to the above-mentioned application, more studies have been reported on the use of SBE-␤-CD as a CS for the chiral separation of different types of analytes, such as armodafinil [73], bupivacaine hydrochloride [74] and indapamide [75]. In all cases, the resolution values were above 3. In 2013, Boonleang et al. [76] synthesized two new negatively charged sulphur-␤-CD derivatives. Particularly, two mono-substituted CD derivatives were synthesized (SET-␤CD and SMHT-␤-CD) in order to avoid the drawbacks that are caused by randomly substituted charged CD derivatives. Thus, SET-␤-CD and SMHT-␤-CD bearing one and three negative charges, respectively, were utilized and evaluated as CSs in CE. Both CD derivatives provided good separation abilities, and they were able to resolve several basic compounds, such as clenbuterol, ephedrine, phseudoephedrine, terbutaline, verapamil, and propranolol. However, the resolution values obtained by the use of SMHT-␤-CD were greater than the ones obtained with SET-␤-CD. According to the researchers, this is possibly due to the higher binding affinity of SMHT-␤-CD www.electrophoresis-journal.com

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to the compounds and the wider separation window, which, in turn, increased the countercurrent mobility of the selector. In addition, they calculated the apparent binding constants of the compounds to the two CD derivatives, and they found that the majority of the examined compounds had almost the same affinity to SET-␤-CD and SMHT-␤-CD. Thus, it was concluded that the enantioseparation of the compounds followed the predictions of the charged-resolving-agent migration model. In addition, the observed degree of enantioseparation difference was due to the magnitude of differences in both enantiomer-CS binding affinities and the mobilities of the complexed enantiomers [76, 77]. Another type of negatively charged CD derivatives that have been used as CSs in CE during the 2012-mid 2014 period are the CM-␤-, -␥ -, and -␣-CDs. CM-␤-CD was used for the chiral separation of different drug compounds, such as ␤-agonists and opioid-type analgesics (e.g. meptazinol) [78–81]. It was observed that, among ␤-CD, ␥ -CD, DM-␤CD, TM-␤-CD and HP-␤-CD, the third type separated all the enantiomers with higher Rs values. Meptazinol and its three intermediate enantiomers were also separated by using CM-␤-CD, and the method was validated for the application of quantitative determination of meptazinol in tablets [79]. CM-␥ -CD was used for the chiral separation of verapamil, pindolol and fenfluramine by using DMSO-based BGEs in NACE [82]. Verapamil and pindolol enantiomers were baseline separated, while fenfluramine enantiomers were partially separated, with Rs values of 1.5, 2.0 and 1.2, respectively. ˇ In 2012, Rezanka et al. [83] studied the effect of substituent position in monosubstituted CM-␣-CDs on enantioselectivity in CE, and their results were compared to those obtained using native ␣-CD. Monosubstituted CM-␣-CDs with the carboxymethyl group attached at position 3 (3I O-CM-␣-CD) provided better resolution values for the analytes baclofen, Tröger’s base 1, Tröger’s base 2, primaquine and tryptophan than the native ␣-CD, the two monosubstituted derivatives (2I -O-CM-␣-CD and 6I -O-CM-␣-CD) and their mixture. One year later, the same research group [84] examined the enantioselectivity of monosubstituted CM-␤-CD derivatives in CE. The results were similar to their previous work. They observed that different substitution locations on ␤-CD skeleton had an important influence on the enantioseparation of the investigated enantiomers. In contrast to the CM-␣-CD monosubstituted derivatives, 2I -O-CM-␤-CD demonstrated better enantioselectivity than the 3I -O-CM-␤CD and 6I -O-CM-␤-CD. It is worth here to mention that the enantioseparations with the last two CM-␤-CD derivatives were worse than in the case where the native ␤-CD was used as the CS in CE.

2.1.4 Positively charged CD derivatives Positively charged CD derivatives had limited number of applications in comparison to the negatively charged CDs, due to their absorbing character to the column wall and the C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim


more complicated synthesis procedure [32]. However, they demonstrate an advantage over the anionic CDs, and this includes the lower migration times of the analytes. The majority of the positively charged CD derivatives are derived from ␤-CD. ˇ ınová et al. [85] developed a method for the fast and Sol´ sensitive chiral analysis of new types of six acyclic nucleoside phosphonates (nucleotide analogs with potential biological activities). A variety of native and derivatized neutral and charged CDs (␣-, ␤-, and ␥ -CDs, HP-␤-CD, HP-␥ -CD, DM-␤-CD, and quaternary ammonium ␤-CD (QA-␤-CD)) were examined. Among them, ␤-CD, at a concentration of 20 mg/mL, proved to be the best CS for the baseline separation of the R and S enantiomers of five acyclic nucleoside phosphonates (Rs = 1.51–2.94). On the other hand, QA-␤-CD, at a concentration of 60 mg/mL, was determined as the most effective CS for the baseline separation of the enantiomers of the sixth acyclic nucleoside phosphonates. In 2012, Giuffrida et al. [86] reported the separation of thirteen enantiomeric pairs of ␣-amino acids derivatized with fluorescein isothiocyanate (FITC-AAs). The single isomer derivatives 3-amino-3-deoxy-␤-cyclodextrin (BCD3AM) and 3-amino-3-deoxy-␥ -cyclodextrin (GCD3AM) were added individually into the BGE, and their performance was evaluated and compared. The cavity size, along with the conformational steric and electrostatic effects, were the determining factors for their chiral selecting ability. Their results demonstrated that BCD3AM was the most effective CS, since GCD3AM was able to separate only eight of the thirteen enantiomeric pairs. The same group, in 2013, [87] separated ten different pairs of dansyl-derivatives of ␣-amino acids, with resolution values that ranged from 0.59 to 4.46 by the use of GCD3AM and 6A,6D-dideoxy-6A,6D-N-[6,6’di-(␤-alanylamido)]-6,6’-dideoxy-␣,␣’-trehalose]-␤-CD (THALAH) as CSs. Mono-6A -(3-methoxypropan-1-ammonium)-6A -␤-CD chloride (MPrAMCD), a novel cationic CD was designed, used and evaluated by Dai et al. [88]. MPrAMCD has three recognition sites: ␤-CD, ammonium cation and methoxy group in the sidearm. These sites contribute to three corresponding driving forces that involve inclusion complexation, electrostatic interaction and hydrogen bonding. This CD demonstrated excellent enantioselectivities in CE for a wide range of acidic and ampholytic racemates. An important observation, in this study, was the very low concentration that was required (2.5 mM) in order to achieve baseline enantioseparations of acidic racemates (Rs ⬎ 10). In addition, it was observed that the effective mobilities of ␣-hydroxy and carboxylic acids decreased upon increasing the CD concentration, partially due to the increased BGE viscosity and the interplay between increasing complexation and ionic-induced mobility depression. In another study by Dai et al. [89], 6A -ammonium-6C butylimidazolium-␤-CD chloride (AMBIMCD) was synthesized and used for the separation of acidic enantiomers and amino acids. This dicationic CD exhibited excellent enantioselectivities toward selected analytes in CE, at a concentration www.electrophoresis-journal.com

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Figure 4. Comparison of chiral resolutions of (A) 4 dansyl-amino acids and 3 hydroxy acids with 5 mM or 10 mM HEtAMCD and CD-NH3 Cl, (B) 5 carboxylic acids with 5 mM HEtAMCD, MPrAMCD and CD-NH3 Cl, respectively, in pH 6 BGEs. Reprinted with permission from [91].

as low as 0.5 mM. The enantioseparation capabilities toward both dansyl-amino acids and acidic racemates are comparable or even better than the monocationic counterpart CDs (mono-6A -butylimidazolium-␤-CD (BIMCDCl) and mono-6A -ammonium-␤-CD chloride (CDNH3 Cl)), at the cost of slightly longer analyte migration times. In this system, the chiral discrimination is affected by the degree of inclusion complexation, the electrostatic interactions, and/or the hydrogen bonding between the substituents on the CD rim and the moieties of the enantiomers. So, the effective mobilities of the enantiomers were much stronger for the dicationic CDs than for the monocationic CDs. In addition, they synthesized a new monocationic positively charged single-isomer ␤-CD, mono-6A -(2methoxyethyl-1-ammonium)-6A -␤-CD chloride (MEtAMCD) [90]. It was utilized as a CS for the enantioseparation of ampholytic, acidic and even neutral racemates. In comparison to the counterpart amine-substituted CDs (mono-6A -butylammonium-6A -deoxy-␤-cyclodextrin tosylate (BuAMCD), mono-6A -propylammonium-6A -␤-CD chloride (PrAMCD)), the use of MEtAMCD provided better enantioselectivities for dansyl-amino acids, hydroxyl acids and phenoxyalkanoicm acids due to enhanced hydrogen-bonding. In their latest study, another cationic CD, mono-6A (2-methoxypropan-1-ammonium)-6A -␤-CD chloride (HEtAMCD), was synthesized and applied as a CS in CE [91]. (Fig. 4). It was concluded that, the hydroxyethylammonium moiety of HEtAMCD increased the enantioselective capability significantly. This is possibly due to the extra hydrogen bonding formed between the hydroxyl moiety of CD and the hydroxyl or amine group of the analytes. In addition to this, the higher polarity of the hydroxyethylammonium moiety may enhance the interactions between the cationic CD and the analyte enantiomers, and this may, in turn, provide better chiral resolutions toward carboxylic acids. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2.2 Cyclofructans CFs (macrocyclic oligosaccharides) are novel components obtained from the enzymatic conversion of inulin. They consist of a crown ether skeleton and six or more ␤-(1→2) D-fructofuranose units, which contain four stereogenic centers each. These units are linked to the crown ether ring in a manner that leads to a spiral configuration, so that the shape of CFs is like a disc with central in dentation [92–95]. The high solubility of CFs into water is due to the three hydroxyl groups, which are attached in each fructofuranose unit. In addition, the hydroxyl groups provide multiple hydrogenbonding sites for derivatization with aliphatic or aromatic groups, and they affect the complexation and chiral recognition properties. They have been proposed as novel selective complex-forming agent for metal cations [96] and as CSPs and CSs in HPLC, GC and CE [97–100]. Native CFs did not demonstrate any significant enantioselectivity, while several cyclofructan derivatives have been successfully used as CSs in CE. Reijenga et al. [96] reported that, although the six chirally oriented D-fructose groups of native CFs should have given to the molecule cyclodextrin-like characteristics, the fructose groups clearly pointed away from the center of the crown ether ring, and they were oriented perpendicular to the plane of this ring. This is very different from CDs, since the entrance of the conical wall of a cavity is formed by the sugar molecules. Thus, it was concluded that the enantioselective behaviour of CFs is different from that of CDs. In CE, only a few studies regarding the use of CF derivatives as CSs have been reported. In 2009, Armstrong’s group [98] developed a derivatization/sulfation procedure for the synthesis of sodium sulfated cyclofructan 6 (SCF6) and sodium sulfated cyclofructan 7 (SCF7). These derivatives demonstrated exceptional selectivity toward many cationic analytes withamine moieties, including primary, secondary,


112


tertiary amines and amino acids. Since the first application of CF derivatives in CE as CSs, in 2009, only two research articles have been, so far published [101, 102]. Zhang et al. [101] introduced SCF-6 for the chiral separation of basic pharmaceuticals (tamsulosin, tiropramide, bupivacaine and norephedrine) in CE. The RS values for the racemic analytes tamsulosin, tiropramide, bupivacaine and norephedrine by using 0.7% SCF-6 were 5.60, 5.43, 6.87, 0.59, respectively. In comparison to S-CDs, SCF-6 demonstrated greater enantioselectivities. Thus, the latter was proposed as a promised CS with a possible wide range of chiral applications in the future. Recently, Maier et al. [102] used a neutral CF-6, called isopropyl-cyclofructan-6 (IP-CF6), in both HPLC and CE. The chiral separation of all five binaphthyl atropoisomers was successfully achieved in HPLC, when IP-CF6 was used as CSP bonded onto the silica particles, while in the case of CE, only 1,1’-binaphthalene-2,2’diyl hydrogen phosphate (BNP) was able to be baseline resolved, when IP-CF6 was utilized as a CS. It was observed that the addition of Ba2+ into the BGE improved the chiral separation of BNP, and as the Ba2+ amount increased, the EOF decreased. It was suggested that the incorporation of Ba2+ into the IP-CF6 core made the CS’s structure more rigid. In addition, it was reported that the presence of the positive charge in the IP-CF6 core, developed electrostatic interactions with the negatively charged analytes, which, in turn, provided better separations.

2.3 Surfactants Surfactants are molecules with hydrophobic and hydrophilic parts. At a low concentration, they are dispersed in solution, while at a concentration above the CMC, the surfactant molecules aggregate together to form micelles. The micelle is the most commonly used pseudostationary phase in MEKC. The separation mechanism, in this mode of CE, is based on the electrophoretic mobility of the charged species and the partitioning of the analytes between the mobile phase and the micellar pseudostationary phase via hydrogen bonding, dipole-dipole, and dispersive interactions. Even though a variety of natural and synthetic monomeric and polymeric chiral surfactants have been utilized as CSs over the years, only a few were reported the period 2012 to mid-2014. In 2012, Tsioupi et al. [103] developed a MEKC method for the enantioseparation of (±)-Huperzine. The baseline separation was achieved by using a BGE of 50 mM acetate (pH 5.0), supplemented with 0.2% w/v poly(sodium N-undecanoyl-LL-alanyl-valinate) as a CS. An important observation, in this study, was the significant increase in peak efficiency by adding into the BGE 10% v/v tert-butanol. The optimum conditions were applied successfully to a pharmaceutical formulation that contained (–)-Huperzine A. This demonstrated the ability of the method to control the purity of (–)-Huperzine A in commercially available capsules. Another polymeric surfactant, and particularly poly(sodium N-undecanoyl-LL-leucine-valinate) (poly-LLSULV), was used as a CS by Hadjistasi et al. [44] for C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim


the separation of the enantiomers of pipecolic acid. The optimum BGE, which consisted of 40 mM borax (pH 9.5) and 1% poly-LL-SULV, provided a resolution of 2.78. However, due to the fact that polymeric surfactants are not commercially available, an alternative method was also developed by using ␤-CD as a CS and a CIL as the additive.

2.4 Antibiotics Antibiotics are considered an important class of CSs, and their use in CE has been reported extensively. A number of review articles have, so far, been published regarding the significance of antibiotics as CSs in HPLC and CE [14, 104, 105]. Various classes of antibiotics have been reported, and these include ansamycins, macrolides, lincosamides, aminoglycosides and ␤-lactams (Table 2). Each class may have different physiochemical properties. Glycopeptides are composed of an aglycone portion, which has three or four macrocyclic rings forming a characteristic basket-like structure. Onto the aglycon part, some saccharides are attached, which are able to rotate free. Due to the substituted phenols, which are bonded to the basket-like moiety, this class of antibiotics absorb in UV. Unlike glycopeptides, macrolides do not have any aromatic ring attached to them. Instead, they consist of a macrocyclic lactone ring. Therefore, they exhibit low UV absorption. Another group of antibiotics is called ␤-lactam antibiotics. They are non-macrocyclic, negatively charged antibiotics, which consist of a ␤-lactam group and a carboxylic group that is attached to the ring. This negatively charged group makes the ␤-lactam antibiotics promising CSs because they can interact electrostatically with basic compounds [106, 107]. Glycopeptide antibiotic, which is the most commonly used class of antibiotics, has been extensively used as a CS in CE. Vancomycin, eremomycin and bromobalhimycin are the three glycopeptide antibiotics that were used during the period 2012 to mid-2014. The separation and determination of stereoisomeric impurity of folinic acid and diastereomers in CE was performed by using vancomycin as the CS [108]. Due to the presence of amino groups in the vancomycin structure, the adsorption onto the inner wall of the negatively charged capillary was possible. Thus, in this method, a solution of poly(dimethylacrylamide) (PDMA) was used to coat the capillary wall in order to minimize the electrostatic interactions between vancomycin and the silanol groups of the inner wall. After optimization, the method was applied to a commercial calcium folinate sample in order to determine and calculate the stereoisomeric impurity in the sample. It was found that 0.08% of stereoisomeric impurity was included in the (6S,2’S)-calcium folinate sample. In 2014, the chiral separation of FMOC-amino acids was achieved by using vancomycin in CE, which was coupled to ion-trap-MS [109]. In this method, a noncovalent polycationic coated capillary column (hexadimethrine bromide, HDB) was used in the CE-ESI-MS/MS2 mode. Vancomycin was initially added into the BGE, and it was partially filled in the coated capillary in order to avoid the introduction of vancomycin in www.electrophoresis-journal.com

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Table 2. Enantioseparation of chiral compounds by use of antibiotics as CSs Antibiotics

Concentration

Analytes

CE mode

BGE

pH

Applications

Ref.

Vancomycin

1 mM

Folinic acid diastereomers

7.0

Calcium folinate sample –

[108]

FMOC-ornithinephenylalanine-alanine 17 FMOC-amino acids

100 mM Tris/phosphate buffer/5% acetonitrile 50 mM ammonium formate/10% methanol 50 mM ammonium formate

6.0

0.5 mM

EKC(PDMA modified capillary) EKC

7.0

–

[109]

50 mM phosphate /20% methanol/ 15 mM L-AlaC4 NTf2 50 mM phosphate /20% methanol/ 15 mM L-ValC4 NTf2 50 mM phosphate /methanol (40:60 vol%) 25 mM phosphate

7.0

Bulk S-naproxen drug

[110]

7.0

Bulk S-naproxen drug

[110]

5.8

Bystrumgel gel

[111]

7.0

–

[112]

25 mM phosphate

7.0

–

[112]

75 mM Tris/50 mM borate in methanol

9.0

–

[113]

30 mM boric acid/75 mM tributylamine in acetonitrile/methanol (80:20) CS dissolved in water/methanol (90:10) 16.1 mM penicillin G/methanol (90:10)

–

–

[114]

6.41

–

[115]

6.36

–

[115]

10 mM

2 mM

EKC (partial filling technique, HDB modified capillary) EKC

Eremomycin

0.5 mM

Bromobalhimycin

4 mM

Naproxen, ibuprofen, carprofen, ketoprofen, pranoprofen Naproxen, ibuprofen, carprofen, ketoprofen, pranoprofen Flurbiprofen, ketoprofen, fenoprofen, indoprofen 16 Amino acid derivatives

15 mM

26 Amino acid derivatives

Boromycin

20 mM

Azithromycin

72 mM

Norepinephrine, octapamine, methylbenzyl amine, hydroxynorephedrine, 2-amino-1phenyloethanol Tetrahydrozoline

Penicillin G (potassium salt)

10.7 mM

Citalopram, darifenacin, propranolol, sertaline

EKC

16.1 mM

Metoprolol

EKC

2 mM

EKC

EKC EKC (partial filling technique, HDB modified capillary) EKC (partial filling technique, eCAP modified capillary) NACE

NACE

the ionization source (low MS sensitivity). A partial filling of the column was performed by introducing 10 mM vancomycin into the capillary for 150 s x 50 mbars. When the optimum electrophoretic and spectrometric conditions were applied, the simultaneous chiral separation of 17 amino acids was achieved (Fig. 5). In another study by Zhang et al. [110], vancomycin was used in combination with amino acid ester-based CILs as additives (L-AlaC4 NTf2 , and L-ValC4 NTf2 ) for the enantioseparation of non-steroidal anti-inflammatory drugs (NSAIDs). They demonstrated that the addition of L-AlaC4 NTf2 and LValC4 NTF2 into the BGE improved the chiral separation of NSAIDs drugs since resolution was better than that obtained when vancomycin was used as the sole CS. In this report, pH was considered an important factor for the enantioseparation, because it affected the dissociation of the carboxyl groups of the NSAIDs, the carboxyl and amino groups in vancomycin and the amino group in the CILs. The concentrations of CIL and vancomycin in the BGE were also evaluated. As the first concentration increased from 0 to 15 mM, the RS increased, while in the case of a CIL concentration of above15 mM, the RS decreased due to the gradually saturated complexation. As far as the vancomycin concentration is con C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

[109]

cerned, a concentration of 2–3 mM proved to be the optimum, since a higher concentration resulted in longer migration times due to the adsorption of vancomycin onto the capillary wall. Other glycopeptides that have recently been used as CSs in CE are eremomycin and bromobalhimycin. Eremomycin was added into the BGE and used as a CS for the enantioseparation of some NSAIDs (indoprofen, flurbiprofen, ketoprofen and fenoprofen) [111]. In this study, two different BGEs (aqueous BGE and water-methanol BGE) were used and their chiral separation performance was evaluated and compared. Eremomycin has amino groups; so, at low pHs, it is adsorbed onto the capillary wall. On the other hand, the use of an organic-aqueous BGE reduces its adsorption onto the walls, when reversed polarity CE mode is applied, and the analysis time is shorter. After method optimization and validation, Lebedeva et al. determined ketoprofen in a sample of Bystrumgel. It was found that a racemic mixture of ketoprofen was contained in the sample. Peng et al. [112] examined the enantioseparation of N-benzoylated amino acids by using bromobalhimycin, a vancomycin-like antibiotic, as a CS in CE. This antibiotic has a very strong UV absorbance, which interferes with the


114



Figure 5. CE-MS2 extracted ion electropherograms for the 17 FMOC-amino acids enantiomerically resolved. CE conditions: BGE, 50 mM ammonium formate buffer (pH 7.0); partial filling, 150 s x 50 mbar of 10 mM vancomycin in BGE; HDB coated capillary, 50 ␮m id x 100 cm; injection by pressure at 50 mbar x 15 s; applied voltage, -20 kV; temperature, 25°C; MS conditions: see Section 2.7 of reference. *Unknown peaks. Reprinted with permission from [109].

analyte detection. In addition, such antibiotics are positively charged at low pHs; so, in most cases, the use of coated capillaries is necessary in order to avoid the adsorption of the antibiotic onto the negatively charged walls. In this study, HDB and eCAP neutral-modified capillaries were used, and partial filling of bromobalhimycin BGE was applied in order to achieve chiral separation of some N-benzoylated amino acids. The researchers noted that, when the eCAP-modified column was used, the enantioseparation was better than in the case where the HDB-modified column was used. Briefly, 26 of the 28 examined amino acids were successfully separated by using the eCAP column, while with the HDB column, only 16 amino acids were able to be partially or baseline separated. Another class of antibiotics that were applied in CE are the macrolides. In 2012, Maier et al. [113] investigated the enantioselectivity ability of boromycin. Several analytes were examined, such as norepinephrine, octapamine, tryptophanol, methylbenzyl amine, hydroxynorephedrine, and 2-amino-1-phenyloethanol. It is worth here to note that boromycin is non soluble in water; so, an organic solvent (methanol) was necessary to be used in this study. Baseline chiral separation was achieved for all the analytes, except from tryptophanol, which was partially separated, C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

when a concentration of 20 mM boromycin was used. According to the authors, the main reason for this, is the structure of tryptophanol, which is slightly different from all the other examined analytes. It has a different aromatic moiety, and the chiral center is located in a different place, in relation to the other examined primary amine analytes. Azithromycin is another macrolide antibiotic that has recently been used as a CS in NACE for the chiral separation of tetrahydozoline [114]. Unlike other antibiotics, azithromycin did not demonstrate any adsorption onto the inner capillary walls. The chiral recognition ability of penicillin G potassium salt, a sub class of ␤-lactam antibiotics, was examined, for the first time, by Dixit et al. [115]. Basic drugs, such as darifenacin, citalopram hydrobromide, sertraline hydrochloride, metoprolol and propranolol were analyzed, and their chiral separation was performed by using penicillin G potassium salt dissolved in water/methanol. The analytes were determined via indirect detection, because penicillin G absorbs in UV. It is important here to note that penicillin G has three chiral centers, an aromatic ring, two amide functional groups and a carboxyl group. At the working pH (below pH 7), penicillin G is negatively charged (due to carboxyl group); so, it can interact with positively charged analytes, such as basic chiral drugs. According to the authors, the


115

– 3

[123]

–

[120] –

2.8-3.4

4.5-5.3 2.8–3.4

2.8-3.4

[118] [120]

[116] [117]

Commercial tablets Plasma and urine samples – – 4 4

pH

Application

Ref.

chiral separation was performed due to the ion-pair ability of the CS, which formed diastereomeric ion-pairs with the positively charged analyte enantiomers. As they suggested, the enantioseparation of the above-mentioned drugs was attributed to the difference in mobilities of the free and complexed analytes, and the equilibrium of constants of ion-pair formation for R- and S- enantiomers. In addition, they explained that, at the optimum CS concentration, the mobilities of the two enantiomers reached a maximum difference, and this, in turn, provided the optimum resolution for the enantioseparations.

[121]

CE and CEC



CZE

20 mM Tris/10% (v/v) H3 PO4 /2.4 ␮g/mL ILs-MWNTs 40 mM Tris/ H3 PO4 /60 mM tetramethylammonium-Larginine or tetramethylammonium-Laspartic acid

20 mM Tris/10% (v/v) H3 PO4 CZE

CZE

100 mM phosphate 20 mM Tris/10% (v/v) H3 PO4

2.5% Branched polysaccharides

Glycogen

2.5%

5% CSE

CSE

4% 5% Heparin CSD

Pheniramine, chloroquine Amlodipine, laudanosine, nefopam, sulconazole Amlodipine, laudanosine, nefopam, sulconazole, tryptophan methyl ester, citalopram, duloxetine, propranolol Amlodipine, laudanosine, citalopram, propranolol Nefopam, citalopram, duloxetine

100 mM phosphate 100 mM phosphate CZE CZE 10% (w/v) 20% (w/v) Maltodextrin Linear polysaccharides

Amlodipine Tolterodine

CE mode Analytes Concentration Polysaccharide Type of polysaccharide

Table 3. Enantioseparation of chiral compounds by use of polysaccharides as CSs

A big number of linear oligo- and polysaccharides have, over the years, been successfully employed in CE. In this report, anionic linear polysaccharides, such as heparin and chondroidin sulphates (D (CSD) and E (CSE)) and neutral polysaccharides, such as maltodextrin (MD) that have been used in CE during the period 2012 to mid-2014, are summarized (Table 3). The positively charged linear polysaccharides have not been used as CSs recently due to their low solubility and their adsorption to the inner surface of the capillary wall. Alternatively, the anionic linear polysaccharides are effective for the enantioseparation of neutral and charged analytes under acidic and neutral conditions. MDs are the first CSs, in the family of linear oligoand polysaccharides, that have been used for enantioseparations in CE. They consist of D-(+)-glucose units, which are connected through a Glu-(1–4)-␣-D-Glu linkage. In addition, they are complex malto-oligo and polysaccharide mixtures, obtained from partial acid and/or enzymatic hydrolysis of starch. They are characterized by their dextrose equivalent, which is the equivalent of the degree of polymerization of malto-oligosaccharides. MDs have shown highly efficient chiral selectivity in CE for a broad range of acidic and basic compounds. Nojavan et al. [116] examined MDs with different dextrose equivalent values as CSs in CE for the analysis of amlodipine enantiomers. Although all MDs were able to separate amlodipine enantiomers, the one with a dextrose equivalent value of 4–7 demonstrated the best chiral recognition ability. It was, therefore, used for the development and validation of a stereoselective CE method and for the determination of amlodipine enantiomers in commercial tablets. In the same study, the enantioseparation capability of heparin was also evaluated, and the results obtained demonstrated that heparin did not have any chiral selector activity for the particular analyte enantiomers. Fakhari et al. [117] combined, for the first time, the electromembrane extraction technique with the MD-modified CE for the separation, preconcentration and determination of tolterodine enantiomers in plasma and urine samples. The optimum extract was analyzed successfully by use of 20% (w/v) MD with dextrose equivalent of 4–7. The reproducibility was satisfactory with intra- and

BGE

2.5.1 Linear oligo- and polysaccharides

CZE CZE

2.5 Oligo- and polysaccharides


116


inter-day RSD values below 6.5%., and the LOQ and LOD for both enantiomers were 10 and 3 ng/mL, respectively. Heparin is a complex biopolymer and, due to its extensive sulfonation, is one of the most anionic biopolymers currently known. It cannot be synthesized directly, and it must be extracted from various animal tissues; so, the basic subunit of heparin can vary depending on the source. In addition, due to the way heparin is manufactured, a number of impurities occur naturally. The most common impurity is dermatan sulfate, which is present in pharmaceutical-grade heparins at the range of 1–7%. Szekely et al. [118] focused on heparin’s ability to serve as a CS for the qualitative and quantitative determination of heparin contamination. Specifically, the effect of contamination on the enantiomeric separation of pheniramine and chloroquine was explored by using chondroitin sulfate A as the contaminant. These two particular analytes were chosen in order to evaluate enantiomers that have stronger (chloroquine) and weaker (pheniramine) interactions with heparin. An interesting observation, in this study, was the improvement in resolution of chloroquine enantiomers with the addition of chondroitin sulfate A. In particular, the resolution increased from 1.3 at 0% contamination to 2.3 at 60% contamination. However, according to the authors, even though the method showed potential, further optimization is required. Currie et al. [119] demonstrated the potential of heparin to form a dynamic layer on the surface of the capillary at acidic pH. Zhang et al. [120] investigated the performance of the anionic linear polysaccharides CSD and CSE as potential CSs in CE. CSD and CSE, which belong to the group of glycosaminoglycans, are linear, sulfated polysaccharides with large mass. They are composed of unit structures of ␤(1,4)-linked disaccharide subunits within which hexosamine and uronic acid are connected through ␤-(1,3) linkages. The same group [121], in a subsequent study, used ionic liquid dispersed multi-walled carbon nanotubes (IL-MWNTs) as a modifier and CSE as a CS for the enantioseparation of different drug compounds. Satisfactory separations were achieved when 2.4 ␮g/mL IL-MWNTs were introduced into the 20 mM Tris/H3 PO4 buffer solution, which contained 2.5% CSE.

2.5.2 Branched polysaccharides Glycogen is an electrically neutral and branched polysaccharide of high molecular weight. It consists of ␣-(1,4)-linked glucose subunits and ␣-(1,6)-linked branching. It exhibits high solubility, low viscosity in water and weak UV absorbance. Although it demonstrated satisfactory enantiorecognition towards four basic and one acidic compound in a study performed in 2010 [122], its enantioseparation mechanism is still not well understood. In addition, only one study by Zhang et al. [123] was performed by use of glycogen as a CS in CE during the period 2012 to mid-2014. In particular, a glycogenbased synergistic system with two amino acid-based CILs as additives was used in CE in order to evaluate their poten C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim


tial enantioselectivity. In comparison to the single separation system, significantly improved enantioseparations of all the examined compounds were observed (Rs = 1.82 (nefopam), 2.59 (citalopram), and 4.39 (duloxetine)).

2.6 Other CSs In this reported period, additional CSs, which do not belong to the CS systems mentioned above, were utilized in CE. These include bacteria, chiral ILs, chiral IL functionalized ␤-CD, dialkyltartrate- and polyols-boric acid complexes (Table 4). Li et al. [124] used bacteria such as Escherichia coli, Pseudomonas aeruginosa (Gram-negative), and Staphylococcus aureus (Gram-positive) as CSs on the basis of highly stereoselective and affinity between bacteria and antibacterial enantiomers. Baseline enantioseparation of ofloxacin was achieved within 7 min by using E. coli and P. aeruginosa as CSs. These last two CSs demonstrated better chiral recognition ability for ofloxacin than S. aureus. This is in good agreement with the fact that ofloxacin has better antimicrobial activity on Gram-negative rather than Gram-positive bacteria. The results demonstrated that there is a difference in affinity between antimicrobial enantiomers and bacterias, and this may be attributed to the pharmacokinetic differentiation of these enantiomers. However, further work is required in order to understand the enantioselective interactions between the particular CSs and the antibacterial enantiomers. Yu et al. [125] synthesized and used a novel CIL functionalized ␤-CD, 6-O-2-hydroxypropyltrimethylammonium␤-CD tetrafluoroborate ([HPTMA-␤-CD][BF4 ]) as a CS in CE for the enantioseparation of eight drugs. An important parameter examined in this study was the concentration of [HPTMA-␤-CD][BF4 ]. An increase in concentration from 3 to 13 mg/mL resulted in a significant increase in both analysis time and resolution. When the concentration increased further, the elution times increased, while resolution was either reduced or unaffected. Moreover, the chiral recognition ability of [HPTMA-␤-CD][BF4 ] was demonstrated by comparing the performance of the CIL with that of the native ␤-CD. For [HPTMA-␤-CD][BF4 ], the functional moiety of the CD rim may change the dimensions of the CD cavity, and this, in turn, may increase the stability of the inclusion complex. In addition, in comparison to the native ␤-CD, charge localization creates a nonsymmetrical chemical environment in [HPTMA-␤-CD][BF4 ], which provides greater stereodifferentiation in the intermolecular interactions between the analyte and the CS. Stavrou et al. [126] investigated, for the first time, the applicability of a chiral IL as the sole CS in CE. Among different amino acid-based ILs, L-alanine tert butyl ester lactate proved to the optimum CS for the baseline separation of BNP enantiomers (Rs = 1.94). The run-to-run and batch-tobatch reproducibilities were very good, since all RSD values were below 1%. From the studies performed in this report, it was concluded that the mechanism of enantiorecognition www.electrophoresis-journal.com


Pipecolic acid Ofloxacin Ofloxacin

1% (w/v) 6×108 cells/mL 6×108 cells/mL 13 mg/mL

60 mM 80 mM dialkyl tartrate/100 mM boric acid 40 mM D-mannitol/150 mM boric acid 40 mM D-sorbitiol/150 mM boric acid 80 mM di-n-butyl L-tartrate/100 mM boric acid complexes

P. aeruginosa

[HPTMA-␤-CD][BF4 ]

L-Alanine tert butyl ester lactate

12 Dialkyltartrate/boric acid complexes

Others

Di-n-butyl L-tartrate/boric acid complexes

D-Sorbitiol/boric acid complexes

D-Mannitol /boric acid complexes

Huperzine A

0.2% (w/v)

Poly(sodium N-undecanoyl-LLalanyl-valinate) Poly(sodium N-undecanoyl-LLleucine-valinate) E. coli

Polymeric Surfactants

20 mM

IP-CF6

NACE

NACE

6 ␤-blockers, 5 ␤-agonists


NACE

NACE


Propranolol, sotalol, atenolol, bisoprolol, bambuterol

CZE

EKC (partial-filling technique) EKC (partial-filling technique) EKC

MEKC

MEKC

CZE

CZE

CE mode

Chlorpheniramine, brompheniramine, promethazine, liarozole, tropicamide, warfarin, pheniramine, bifonazole BNP

Tamsulosin, tiropramide, bupivacaine, norephedrine BNP

0.7% (w/v)

SCF-6

Analyte

Cyclofructans

Concentration

CS

Type of CS

Table 4. Enantioseparation of chiral compounds by using other CSs

–

–

–

–

8

4 or 8

30 mM phosphate

100 mM Tris/10 mM sodium tetraboratedecahydrate 80 mM dialkyl tartrate/100 mM boric acid/50 mM triethylamine in methanol 40 mM D-mannitol/150 mM boric acid/108 mM triethylamine in methanol 40 mM D-sorbitiol/150 mM boric acid/180 mM triethylamine in methanol 80 mM di-n-butyl L-tartrate/100 mM boric acid/45 mM NH4 OH in methanol

7.4

7.4

9.5

–

–

–

–

–

–

–

–

Pharmaceutical formulations –

–

10 5

-

Application

2.5

pH

10 mM phosphate

10 mM phosphate

100 mM sodium borate/ 8 mM Ba2+ 50 mM Sodium acetate/10% tert-butanol 40 mM borax

100 mM Tris/H3 PO4

BGE

[128]

[127]

[127]

[127]

[126]

[125]

[124]

[124]

[44]

[103]

[102]

[101]

Ref.


CE and CEC 117


118


process is based on: (i) steric hindrance (tert butyl group), (ii) electrostatic interactions (between the cation of the CIL and the negatively charged analyte) and (iii) hydrogen bonding (hydrogen-bonding capability of the phosphate group in BNP). A new type of CS has recently been proposed in NACE for the chiral separation of six ␤-blockers and five ␤-agonists. In particular, twelve dialkyltartrate-boric acid complexes and two polyols (D-mannitol and D-sorbitol)-boric acid complexes have been applied as chiral ion-pair selectors in NACE [127]. In this study, the chiral separation of propranolol was successfully achieved by using twelve dialkyltartrate-boric acid complexes. The resolution, in all cases, was excellent with values above 2.0. The active center for the enantioseparation in these chiral ion-pair selectors is the stereogenic center of dialkyltartrates because a change in their configuration reverses the elution order of the analytes. It is worth also to mention that the dialkyltartrate-boric acid complexes with different alkyl groups, but with the same number of carbons, provide the same enantioseparations. In addition, the D-mannitol and D-sorbitiol-boric acid complexes were able to separate all the examined chiral compounds. The same research group, in 2013, used the this ion-pair principle for the achievement of chiral separations in NACE [128]. They used methanolic BGEs and a di-n-butyl-L-tartrate/boric acid complex. It was concluded, by examining several basic BGEs, that triethylamine plays an important role in the apparent pH of the BGE. MS experiments were also performed, which suggested that triethylamine promoted the formation of a negatively charged di-n-butyl-L-tartrate/boric complex counter ion with a complex of 2:1 ratio.

2.7 Dual selector systems In some cases, the use of a single CS in the BGE cannot achieve partial or baseline separation. Therefore, the combination of two CSs may improve the enantioseparations. The most common dual selector systems include different CDs, usually ionic with neutral CD derivatives or with native CDs or two neutral CD derivatives, [129–134] (Table 5). Imperanene, a novel phenolic compound, was enantiomerically resolved by using different CD derivatives as single CSs in CE [129]. Among 27 neutral or charged CD derivatives, only SBE-␥ -CD (degree of substitution, DS 4) was able to separate the two enantiomers with a resolution value of 1.26. Although the particular CD proved to be the best CS, the imperanene enantiomers were not baseline separated. Therefore, the utilization of dual selector/cyclodextrin systems was investigated. In the dual cyclodextrin systems, a charged and a neutral or two charged CDs were combined. The optimum CS, SBE-␥ -CD, was chosen as the primary selector, while the second component involved several CD derivatives with different DS. The neutral CDs HP-␣-CD, HP-␤-CD, HP-␤CD, HP-␤-CD and the charged CDs SP-␤-CD, SBE-␤-CD, SBE-␣-CD, 6-monodeoxy-6-mono(hydroxyl)propylamino-␤cyclodextrin hydrochloride (PA-␤-CD) were examined at vari C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim


ous concentrations. Although PA-␤-CD could not resolve the enantiomers when it was used as a single CS, it enhanced the enantioseparation when it was added as a second component in the dual-cyclodextrin system. The optimum dual selector system was the combination of 12.5 mM SBE-␥ -CD and 10 mM PA-␤-CD, which provided a significant enantioseparation of imperanene (RS = 4.47) (Fig. 6). A CD-MECKC mode was developed for the separation of some imidazole enantiomers by using a dual cyclodextrin system [130]. Firstly, three CDs (HP-␤-CD, HP-␥ -CD and DM␤-CD) were evaluated as single CSs in CD-MEKC in order to simultaneously separate the enantiomers of three imidazole drugs. None of these three CDs were able to provide a baseline chiral separation of these drugs. After method validation, human urine samples and cream formulations were analysed in order to qualitatively and quantitatively determine tioconazole, isoconazole and fenticonazole. In another study by Petr et al. [131], a dual selector system (S-␤-CD and TM-␤-CD), in combination with SDS, was utilized for the chiral separation of ketoprofen enantiomers. The method was also applied for the determination of ketoprofen enantiomers in waste water samples by using simple filtration as a clean-up step. It was clearly demonstrated from this study that the addition of an extra modifier in a dual selector system is sometimes crucial for successful enantioseparations. Jin et al. [132] used an achiral IL (glycidyltrimethylammonium chloride) as a modifier in a BGE that contained both DM-␤-CD and TM-␤-CD for the chiral separation of three ␤-blockers. In this study, the ratio of 2:1 TM-␤-CD/DM-␤-CD was suggested as the optimum with resolution values above 0.8. According to the authors, the enantioseparation of pindolol and oxprenolol are mainly based on the interactions between the enantiomers and IL/DM-␤-CD complex, while the enantioseparation of propranolol is based on the interactions with the IL/TM-␤-CD complex. A different type of dual selector system was applied by Mantim et al. [133] for the enantioseparation of amphetamine, methamphetamine, ephedrine, pseudoephedrine, norephedrine and norpseudoephedrine. They particularly used DM-␤-CD, CM-␤-CD and the chiral crown ether (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid (18C6H4 ) as CSs. In another study, 5 mM ␤-CD and 4 mM of the chiral amino acid-based IL tetra-N-butylammonium L-aspartate [TBA] [L-Asp] were added into the BGE and proved to be the optimum for the chiral separation of phenylalanine and tryptophan [134]. The possible separation mechanism of this type of dual selector system was also reported. It was suggested that, the presence of ␤-CD and CIL in the BGE lead to a synergy effect, which affected the enantioseparation of the analytes. The analyte forms complexes with ␤-CD, while steric hindrances and hydrogen bonds are generated between the CIL and the analyte. Moreover, it was proposed that the structure of the anion of the IL (L-aspartic acid) might change the complexation between ␤-CD and analyte, increasing though the resolutions. www.electrophoresis-journal.com


30 mM TM-␤-CD/5 mM L-UCLB 30 mM TM-␤-CD/5 mM L-UCLB 30 mM TM-␤-CD/3 mM L-UCLB 30 mM TM-␤-CD/5 mM L-UCAB

40 mM HP-␤-CD/30 mM [EMIM][L-lactate]

50 mM sodium phosphate/H3 PO4

10 mM sodium acetate

EKC

EKC

CD-MEKC CD-MEKC CD-MEKC CD-MEKC

Phenylalanine, tryptophan

Ofloxacin, propranalolhydrochloride, dioxopromethazine hydrochloride, isoprenaline hydrochloride, chlorpheniramine maleate, liarozole, tropicamide, amlodipine benzenesulfonate, brompheniramine maleate, homatropine methylbromide 1,1’-bi-2-naphtol

2,2,2-trifluoro-1-(9-anthryl)ethanol

7,8,9,10-tetrahydrobenzo[a]pyren-7-ol

Transstilbene oxide

10 mM phosphate



15 mM sodium tetraborate

EKC

50 mM phosphate/30 mM glycidyltrimethylammonium chloride 150 mM acetic acid/0.2% (w/v) HPMC 500 mM acetic acid

Norephedrine, norpseudoephedrine

EKC

Pindolol, oxprenolol, propranolol

EKC

CD-MEKC

Ketoprofen

35 mM phosphate/50 mM SDS/15% (v/v) acetonitrile 50 mM phosphate/20 mM SDS

75 mM borate

BGE

Ephedrine, pseudoephedrine

CD-MEKC

Tioconazole, isoconazole, fenticonazole

3 mM CM-␤-CD/3 mM DM-␤-CD 5 mM DM-␤-CD/5 mM 18C6H4 5 mM ␤-CD/4 mM [TBA][L-Asp]

EKC

Imperanene

12.5 mM SBE-␥-CD/10 mM PA-␤-CD 35 mM HP-␥-CD/10 mM DM-␤-CD 4.0% (w/v) S-␤-CD/0.5% (w/v) TM-␤-CD 10 mM TM-␤-CD/5 mM DM-␤-CD

CE mode

Analyte

Dual selector system

Table 5. Enantioseparation of chiral compounds by using dual selector systems as CSs

7

5

5

5

2.75

9.5

2.5

2.8

4.4

2.5

7.0

9.0

pH

–

–

–

–

Compound amino acid injections (18AA-I, 18AA-II and 3AA) S-ofloxacin bulk samples

Human urine sample

Humar urine sample

Human urine sample

Waste water samples

Human urine sample

-

Application

[136]

[136]

[136]

[136]

[135]

[134]

[133]

[133]

[132]

[131]

[130]

[129]

Ref.


CE and CEC 119


120



Figure 6. Enantioseparation of imperanene enantiomers at various SB-␥-CD concentrations (A) and with a dual CD system of 12.5 mM SB-␥-CD and 10 mM PA-␤-CD (B). Reprinted with permission from [129].

In a similar work, Cui et al. [135], used HP-␤-CD and CIL ([EMIM][L-lactate]) as CSs for the chiral separation of several drugs (Rs: 1.43–5.45). It was observed that the chiral impurity (R-ofoxacin) in S-ofloxacin bulk samples was well below 0.2%. It is important here to note that, from the proposed recognition mechanisms reported in the last two studies, it is not clear if the CIL works as a second CS in a dual selector system or it is just a chiral modifier added into the BGE. In 2014, Liu et al. [136] utilized the combination of CIL surfactants and the neutral CD TM-␤-CD for the enantioseparation of neutral compounds in CE. In particular, the effect of the IL chiral head group and their complexation ability with TM-␤-CD was evaluated by comparing the resolution, resolution per unit time and selectivity of four neutral chiral compounds. It was clearly stated in this work that the addition of 1 mM CIL surfactant, not only demonstrated an increased retention of the analytes, but also an increase in resolution (up to 2.5). The concentration of each type of CIL surfactant was optimized for each neutral analyte.

technique that provides high sensitivities and selectivities. When EKC though is used, the CSs that are added into the BGE are usually incompatible with the ion sources in MS. This causes interferences with the detection of the analyte of interest. Therefore, further research is required to circumvent this problem. Overall though, considering the large number of applications reported, it is easy to conclude that CE enantioseparations show potential for routine industrial separations. The authors have declared no conflict of interest.

4 References [1] Scriba, G. K. E., J. Pharm. Biomed. Anal. 2011, 55, 688–701. [2] Waldeck, B., Chirality 1993, 5, 350–355. [3] Płotka, J. M., Biziuk, M., Morrison, C., TrAC Trend. Anal. Chem. 2011, 30, 1139–1158. [4] Li, L., Zhou, S., Jin, L., Zhang, C., Liu, W., J. Chromatogr. B 2010, 878, 1264–1276.

3 Concluding remarks

¨ [5] Konig, W. A., Ernst, K., J. Chromatogr. A 1983, 280, 135–141.

This review reported all the CSs that were applied in CE during the period 2012 to mid 2014. As demonstrated, numerous CSs have been successfully used for the chiral separation of a wide range of analytes in chemical, biochemical, pharmaceutical, and forensic sciences. It is clearly demonstrated, in this review paper, that, even though native CDs and CD derivatives are the most widely applied CSs in electrophoretic separations, the search for new CSs still continues. It is also easily concluded from the studies presented that the future of CF derivatives, CILs and antibiotics as effective and widely applicable CSs is very promising. Further research is required though to understand the chiral recognition mechanisms between the particular CSs and the enantiomers. Another parameter that needs further examination is the use of these CSs for the analysis of chiral compounds at trace levels. The most common detector in EKC is the UV detector due to its easy manageability and low cost. Its major drawback is the low sensitivity that results from the short optical path length. A mass spectrometer is considered a viable alternative to UV detection. The coupling of CE with MS is a well-established

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