Immobilized β-cyclodextrin-based silica vs polymer monoliths for chiral nano liquid chromatographic separation of racemates

Talanta 132 (2015) 301–314

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Immobilized β-cyclodextrin-based silica vs polymer monoliths for chiral nano liquid chromatographic separation of racemates Ashraf Ghanem a,n, Marwa Ahmed a, Hideaki Ishii b, Tohru Ikegami b a b

Chirality Program, Biomedical Science, University of Canberra, Canberra, ACT, Australia Department of Biomolecular Engineering, Graduate School of Science and Technology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto, Japan

art ic l e i nf o

a b s t r a c t

Article history: Received 23 June 2014 Received in revised form 2 September 2014 Accepted 3 September 2014 Available online 16 September 2014

The enantioselectivity of immobilized β-cyclodextrin phenyl carbamate-based silica monolithic capillary columns was compared to our previously described polymer counterpart. 2,3,6-Tris(phenylcarbamoyl)β-cyclodextrin-6-methacrylate was used as a functional monomer for the preparation of β-cyclodextrin (β-CD)-based silica and polymer monoliths. The silica monoliths were prepared via the sol–gel technique in fused silica capillary followed by modification of the bare silica monoliths with an anchor group prior to polymerization with β-CD methacrylate using either 2,20 -azobis(isobutyronitrile) or benzoylperoxide as radical initiators. On the other hand, the polymer monoliths were prepared via the copolymerization of β-CD methacrylate and ethylene glycol dimethacrylate in different ratios in situ in fused silica capillary. The prepared silica/polymer monoliths were investigated for the chiral separation of different classes of pharmaceuticals namely; α- and β-blockers, anti-inflammatory drugs, antifungal drugs, dopamine antagonists, norepinephrine-dopamine reuptake inhibitors, catecholamines, sedative hypnotics, diuretics, antihistaminics, anticancer drugs and antiarrhythmic drugs. Baseline separation was achieved for alprenolol, bufuralol, carbuterol, cizolertine, desmethylcizolertine, eticlopride, ifosfamide, 1-indanol, propranolol, tebuconazole, tertatolol and o-methoxymandelic acid under reversed phase conditions using mobile phase composed of methanol and water. The silica-based monoliths showed a comparative enantioselectivity to the polymer monoliths. Crown Copyright & 2014 Published by Elsevier B.V. All rights reserved.

Keywords: β-Cyclodextrin methacrylate Silica monolith Polymer monolith Chiral separation Nano-LC Reversed phase chromatography

1. Introduction The interest in chirality and its consequences has risen significantly in the last two decades due to scientific and economic reasons. Chiral drugs constitute approximately one-third of all drug sales worldwide and it is expected that nearly 95% of the pharmaceutical drugs will be chiral by 2020 [1]. This imposed strict regulations in approving new chiral entities that include full documentation of the separate pharmacological, toxicological and pharmacokinetic profiles for the individual enantiomers as well as their racemates (mixture of enantiomers) [2]. Accordingly, pharmaceutical companies are yet shifting toward the development of single pure enantiomer drugs via preparative chiral separation techniques rather than the time-consuming chiral syntheses [3]. This raises the need for a high throughput, robust, reliable, environmentally benign and relatively cheaper separation technique of chiral pharmaceuticals to allow their commercialization [4,5].

n Correspondence to: Professor of Organic Chemistry, President of the Royal Australian Chemical Institute (RACI) ACT, Biomedical Science Program, University of Canberra, ACT 2601 Australia. Tel.: þ61 2 6201 2089. E-mail address: [email protected] (A. Ghanem). URL: http://www.chiralitygroup.com (A. Ghanem).

http://dx.doi.org/10.1016/j.talanta.2014.09.006 0039-9140/Crown Copyright & 2014 Published by Elsevier B.V. All rights reserved.

Miniaturization of the analytical techniques reduces the total analysis time, reagents consumption and is able to handle small samples. Nano-LC is one of the microfluidic techniques where reduction in both column inner diameter (10–300 μm) and flow rate (200–1000 nL/min) are combined [6]. The advantages of miniaturization also include the reduced cost of the packing stationary phase; this is of particular interest when costly chiral stationary phases (CSPs) are used [7]. However, packing the capillaries with small particles (usually 3–5 mm diameter) require high level of experience because of the need of retaining frits [8]. This problem can be overcome using monolithic materials [9–15]. Silica-based monoliths are prepared by sol–gel process which involves the hydrolysis of a mixture of silane oxides followed by condensation and polycondensation reactions [16]. They have a characteristic bi-continuous structure providing mesopores with high surface area (several hundred square meters per gram) which makes it highly efficient for the separation of small molecules [17]. The polar surface of silica-based monolith renders it useful for normal phase liquid chromatography (NPLC) and hydrophilic interaction liquid chromatography (HILIC). Alternatively, silica skeleton needs to be derivatized to be suitable for reversed phase liquid chromatography (RPLC) [16]. On the other hand, polymerbased monoliths are prepared via the in situ copolymerization of

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one or more monovinyl monomer with a divinyl cross linker in suitable porogenic solvents (binary or trinary) in a single step process [9]. Free radical polymerization is the common mechanism and the polymer is formed within the capillary to form a continuous porous bed. Polymer monoliths are not only considered for the separation of macromolecules (proteins and peptides) but also for small organic molecules. Remarkable amount of research has been done to increase the surface area of the polymer monolith to ensure adequate separation of small organic molecules [18,19]. The ease and speed of preparation of polymer-based monoliths compared to the technically challenging procedures for silicamonoliths preparation render polymer monoliths an attractive tool in the field of separation science. Nonetheless, polymer monoliths tend to swell or shrink upon exposure to organic solvents compared to silica monoliths, while silica monoliths show lower stability at temperature higher than 60 1C and pH higher than 8.5 compared to the higher temperature and chemical stability of the polymer monoliths. Therefore, both polymer and silica monoliths have their own merits and drawbacks and each of the stationary phases could be tailored to obtain the optimum separation according to the sample nature. To introduce chirality in silica-based monolith, the CS is immobilized on the activated silica skeleton [20] or encapsulated during the sol–gel procedure [21]. On the other hand, to introduce chirality in polymer-based monoliths, the chiral selector (CS) is copolymerized as a functional monomer [22] or immobilized after the monolith preparation [23], [24] in a one pot polymerization procedure or post-polymerization surface modification of reactive groups, respectively [25]. Various classes of CSs are available for enantioselective separation of racemic mixtures, such as, molecularly imprinted polymers, ligand exchange, brush-type, macrocyclic antiobiotics-based, protein/glycoprotein- based, cellulose/amylose derivatives and cyclodextrin (s) derivatives [26–35]. Bonding of β-CD into monoliths has been previously achieved via the copolymerization or post modification approaches. The former is considered a simple and less time consuming compared to the latter. However, due to diversity of monomers precursors, it is time and laborious consuming to prepare different kinds of monolithic columns to establish a robust monolithic backbone. Thus, post modification of raw monoliths with high robustness, tailorability and column efficiency becomes a good alternative [36,37]. The purpose of this study is to compare the enantioselectivity of β-CD-based silica monoliths prepared via the post modification approach to that of β-CD-based polymer monoliths previously prepared via the one pot copolymerization approach [38]. Whether

the two approaches would display similar, broader or complimentary chiral discrimination ability is another raised question.

2. Experimental 2.1. Reagents and materials Benzoyl peroxide (BOP, 497%), ethylene glycol dimethacrylate (EDMA, 98%), 3-(trimethoxysilyl)propyl methacrylate (98%), 1-propanol (99%), 1,4-butanediol (99%), methacryloyl chloride (97%), phenyl isocyanate (498%), trifluoroacetic acid (TFA, Z99.5%), sodium hydroxide, toluene, tetramethoxysilane (TMOS), methyltrimethoxysilane (MTMS), poly(ethylene glycol) with molecular weight of 10,000, urea, hydrochloric acid and acetic acid were purchased from Aldrich (Milwaukee, WI, USA). β-Cyclodextrin was purchased from TCA (Tokyo, Japan). Acetone (AR grade), ethanol (HPLC grade) were purchased from BDH (Kilsyth, Vic., Australia). Acetonitrile (HPLC grade) was purchased from Merck (Darmstadt, Germany). Methanol (HPLC grade) was purchased from Scharlau (Sentmenat, Spain). All other reagents were of the highest available grade and used as received. The fusedsilica capillaries (100 mm and 150 μm inner diameter) were purchased from Polymicro Technologies (Phoenix, AZ, USA). 2,20 -azobis(isobutyronitrile) (AIBN) was obtained from Wako (Osaka, Japan). (3-Aminopropyl)triethoxysilane, tetrahydrofuran, pyridine and uracil were purchased from Sigma Aldrich (St. Louis, MO, USA). Pyridine and toluene were dried before use over molecular sieves (4 Å) also from Sigma Aldrich (St. Louis, MO, USA), toluene was distilled from calcium hydride (Sigma Aldrich, USA). Tetrahydrofuran was distilled prior to use. Water used for dilutions and experiments was purified by Nanopure Infinity water system (NJ, USA). Racemic analytes were mostly purchased from sigma Aldrich. 2.2. Preparation of the monolithic columns β-CD functional monomer (Fig. 1) was prepared according to the previously described method [38]. The hybrid-type monolithic silica columns were prepared from a mixture (9 mL) of TMOS and MTMS in a 3:1 ratio, poly(ethylene glycol) (0.9 g), and urea (2.025 g) in 0.01 M acetic acid (20 mL) as described previously [39] resulting in a monolithic silica having through-pore size of 2.0 μm and skeleton size of 1.5 μm. Briefly, TMOS (9 mL) was added to a solution of poly(ethylene glycol) (PEG, 0.9 g, Mwt ¼10,000) and urea (2.025 g) in 0.01 M acetic acid (20 mL) and stirred at 0 1C for 45 min. The resultant homogeneous solution was charged into a fused–silica capillary tube (100 mm ID, Polymicro, AZ), which had

Fig. 1. Schematic diagram showing the preparation of 2,3,6-tris(phenylcarbamoyl)-β-CD-6-methacrylate [38].

A. Ghanem et al. / Talanta 132 (2015) 301–314

been treated in advance with 1 M NaOH solution at 40 1C for 2 h, and allowed to react at 40 1C. Gelation occurred within 2 h, and the gel was subsequently aged in the capillary overnight at the same temperature. Then, temperature was raised, and the monolithic silica column was treated for 4 h at 120 1C to complete the formation of the mesopores with ammonia generated by the hydrolysis of urea, followed by methanol wash. After drying, heat treatment was carried out at 330 1C for 24 h, resulting in the decomposition of organic moieties in the capillary [40]. Usually, 4-m capillary columns were prepared from the same reaction mixture. After preparation, both ends (10–15 cm) of the capillary were trimmed, and several 50-cm long columns were obtained. Before modification of the surface silanol groups, the monolithic silica capillary columns were evaluated using methanol/ water: 80/20 (v/v) as a mobile phase and uracil as the test analyte. The columns exhibited the plate heights in the range of 18–27 μm and a theoretical plate numbers in the range of 16,000–76,000. After the evaluation, the monolithic silica columns were used for modification with 3-methacrylamidopropyltriethoxysilane (MAS) (Fig. 2) followed by polymerization with 2,3,6-tris(phenylcarbamoyl) β-cyclodextrin-6-methacrylate. The anchor group MAS was prepared via previously reported method [41]. In situ polymerization of the CS onto the MAS-modified silica surface Columns were prepared in situ as illustrated in Fig. 3; 0.40 μL (0.38 mmol) methacryloyl chloride was added to 0.42 g (0.37 mmol) β-CD dissolved in 9 mL pyridine. The solution was stirred for 6 h at room temperature then 1.9 mL phenyl isocyanate was added and the solution was refluxed for 18 h. 20 mg/mL AIBN or BOP in dioxan was then prepared separately and added to the reaction mixture at a concentration of 0.1 g/mL. The mixture was then filtered through Sartorius Minisart RC 15 0.2 μm pore size filters (Goettingen, Germany), the MAS modified silica columns were then filled with the mixture using a syringe pump or via N2 gas, the capillaries were placed into 80 1C oven for 24 h to complete the polymerization reaction. Finally the modified columns were washed with dioxan for 24 h and conditioned with the mobile phase prior to use. The surface modification in fused silica capillaries (150 mm ID) was done by using the same procedure of Schaller et al. [42]. As previously described, only the two permeable polymer-based monolithic capillary columns were prepared via in situ copolymerization of binary monomer mixtures consisted of β-CD derived functional monomer and EDMA as a crosslinker along with three porogens namely; 1-propanol, 1,4-butanediol and water in the presence of 1 wt% AIBN (with respect to monomers) [38]. The filled capillaries were then sealed with a septum, placed in 70 1C water bath for 18 h for the polymerization reaction to take place. The unreacted monomers were removed from the monolithic columns by pumping with methanol at a flow rate of 100 μL/h for 4 h before being conditioned with water and mobile phase, both for 1 h at 30 μL/h. Polymer composition in the prepared columns is shown in Table 1.

2.3. Instrumentation The nano liquid chromatographic system was from Dionex Corporation (UltiMates 3000 capillary LC system) which featured an

303

integrated flowsplitter connected to a continuously monitored flow meter and control valve to maintain a constant flow rate in the range from 0.1 to 1.0 μL/min. It also comprised a binary pump, a vacuum degasser, an autosampler and a UV detector. The Chromeleons Chromatography Management System version 7.00 was used for the data processing and control of the HPLC system. 2.4. Standard solutions and sample preparation Stock solutions of the racemic analytes at concentrations of 1 mg/mL in filtered HPLC grade methanol were prepared. Prior to injection, the stock solutions were further diluted 10  and filtered through Sartorius Minisart RC 15 0.2 μm pore size filters (Goettingen, Germany). Tested compounds—β-blockers: alprenolol, celiprolol, metoprolol, pindlol, propranolol, acebutolol, carbuterol, bufuralol, tertatolol, atenolol. α-blockers: naftopidil. Antiinflammatory drugs: ketoprofen, ibuprofen, naproxen, flurbiprofen, indoprofen, cizolirtine, cizolirtine carbinol, carprofen, etodolac, desmethylcizolirtine, glafenine. Antifungal drugs: hexaconazole, cyproconazole, tebuconazole, miconazole, diniconazole, sulconazole. Dopamine antagonists: eticlopride. Norepinephrine–dopamine reuptake inhibitor: nomifensine. Catecholamines: arterenol, normetanephrine. Sedative hypnotics: aminoglutethimide, phenylglutethimide, N-acetylaminoglutethimide, 4-bromogluthethimide, pentobarbital, phydroxyphenobarbital. Diuretics: etozoline. Antihistaminics: chlorpheneramine. Anticancer drugs: ifosfamide. Antiarrhythmic drugs: tocainide. Miscellaneous: 1-acenaphthenol, o-methoxy mandelic acid, 4-hydroxy-3-methoxymandelic acid, 1-indanol, 1-(2-chlorophenyl)ethanol, 1-phenyl-2-propanol. Chemical structures of the investigated racemates and their suppliers are listed in Table 2. 2.5. HPLC conditions The mobile phase A and B consisted of 0.1% TFA in water (v/v) and methanol (v/v) or 0.1% TFA in water (v/v) and acetonitrile. For all samples, the injected volume was 0.2 μL. Preliminary UV analyses were performed at several different wavelengths (219– 270 nm) for each compound, in order to select the optimum wavelength for all the analytes and best utilise a single wavelength UV detector.

3. Results and discussion 3.1. Silica monoliths: Preparation and characterization In order to attach the β-CD derivative on silica monolith via the copolymerization approach, surface activation of the bare silica is required. Hence, at the first step of modification, an anchor, N-(3trimethoxysilylpropyl)methacrylamide, was chemically bonded to the silica skeleton at 80 1C. At that temperature, no thermal polymerization of the anchor was detectable by a 1H-NMR measurement of the residue [41]. Surface coverage of the anchor group was evaluated by injecting toluene/benzanilide mixture into MAS-modified capillaries to compare the dead volume (toluene retention) to benzanilide retention time. Columns showed retention

Fig. 2. Schematic preparation of the anchor group (MAS).

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Fig. 3. Schematic diagram for the preparation of β-cyclodextrin-modified silica monolith.

Table 1 Polymer composition in the prepared monoliths [38]. Entry β-CD monomer (wt%)

EDMA (wt%)

1-Propanol (wt%)

1,4-Butanediol (wt%)

Water (wt%)

A1 A2

30 20

48 48

6 6

6 6

10 20

factors in the range of 1.5–2 were used for the β-CD copolymerization to ensure optimum coverage of the chiral selector. SEM was used to examine anchor-modified silica monoliths for agglomeration of the silane solution during modification. Fig. 4 shows SEM of the silica monolith before (left) and after (right) MAS modification. The capillaries showed homogenous porous structure (Fig. 4). 3.2. Polymer monoliths: Preparation and characterization For comparison purposes, the previously described polymer monoliths [38] were prepared via in situ copolymerization of 2,3,6-tris(phenylcarbamoyl)-β-CD-6-methacrylate as a functional monomer and EDMA as a cross linker in the presence of ternary porogenic system composed of 1-propanol (48%), 1,4-butanediol

(6%) and water (6%) (Fig. 5). While the porogen composition was kept constant, the ratio of the functional monomer to the cross linker was varied in attempt to study the effect of CS concentration and column porosity on the enantioselective separation of rcaemates (Table 1). SEM for A1 and A2 showed homogenous porous structures with interconnecting channels allowing the flow of mobile phase under low column backpressure (Fig. 6). 3.3. Enantioseparation of different classes of pharmaceutical racemates using silica- and polymer-based monoliths Four columns; AIBN, BOP, A1 and A2, were investigated for the enantioselective nano liquid chromatographic separation of a set of different classes of racemic pharmaceuticals namely: β-blockers, α-blockers, antiinflammatory drugs, antifungal drugs, dopamine antagonists, norepinephrine-dopamine reuptake inhibitors, catecholamines, sedative hypnotics, diuretics, antihistaminics, anticancer drugs and antiarrhythmic drugs. The choice of compounds was arbitrary and guided by our previous investigations [25,38]. A mobile phase composed of acetonitrile and water mixture ranged from 10 to 90% (v/v) was initially tested for the enantioselective separation. No enantioselective separation was observed under

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Table 2 Chemical structures and suppliers of the investigated racemates. Class

Compound name

β-Blockers

Alprenolol and L-alpenolol

3B Scientific Corporation, USA

Celiprolol and S-celiprolol, carbuterol, tertatolol

American Custom Chemicals Corp., USA

Atenolol, metoprolol, propranolol, acebutolol, bufuralol, pindolol

Sigma (St. Louis, MO, USA)

β-Blockers

Structure

Supplier

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Table 2 (continued ) Class α-Blockers

Compound name Naftopidil

Structure

Supplier Sigma (St. Louis, MO, USA)

AntiKetoprofen, ibuprofen, inflammatories naproxen, carprofen, etodolac, glafenine, flurbiprofen, indoprofen

Sigma (St. Louis, MO, USA

AntiDesmethylcizolirtine, inflammatories cizolirtine, S-cizolirtine, cizolirtine carbinol

American Custom Chemicals Corp., USA

Antifungals

3B Scientific Corporation, USA

Hexaconazole, cyproconazole, tebuconazole, miconazole, diniconazole, sulconaole

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307

Table 2 (continued ) Class

Compound name

Structure

Supplier

Dopamine antagonists

Eticlopride

American Custom Chemicals Corp., USA

Norepinephrinedopamine reuptake inhibitors

Nomifensine

Sigma (St. Louis, MO, USA

Sedative hypnotics

Aminoglutithimide, phenylglutethimide, 4-bromoglutethimide, N-acetylglutethimide, pentobarbital, phydroxyphenobarbital

American Custom Chemicals Corp., USA

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Table 2 (continued ) Class

Compound name

Structure

Supplier

Diuretics

Etozoline

American Custom Chemicals Corp., USA

Antihistaminics

Chlorpheniramine

Sigma (St. Louis, MO, USA

Anticancers

Ifosfamide

Sigma (St. Louis, MO, USA

Antiarrhythmics

Tocainide

Sigma (St. Louis, MO, USA

Catecholamines

Arterenol

Sigma (St. Louis, MO, USA

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309

Table 2 (continued ) Class

Compound name

Structure

Supplier

Flavonoids

Flavanone, 6-hydroxyflavanone

3B Scientific Corporation, USA

Miscellaneous

1-Acenaphthenol, omethoxy mandelic acid, 1-indanol, 4-hydroxy3-methoxymandelic acid, 1-(2chlorophenyl)ethane, 1-phenyl-2-propanol

Sigma (St. Louis, MO, USA

Fig. 4. Scanning electron micrograph of bare silica monolithic column (left) vs MAS-modified monolithic column (right) showing homogenous porous monolithic structure in both without any apparent agglomeration.

this condition. However, when aqueous methanol-based mobile phase was used, baseline separation (RsZ 1.5) was achieved for propranolol 7, ifosfamide 39, alprenolol 1, tertatolol 4, 1-indanol 47, tebuconazole 25, o-methoxymandelic acid 46, celiprolol 2, cizolirtine 21 and eticlopride 29 while acceptable resolution (Rs 1–1.5) was achieved for diniconazole 27, ketoprofen 12, metoprolol 6, desmethylcizolertine 20, carbuterol 3, normetanephrine 42 and 6-hydroxyflavanone 44. Separation (α) and resolution (Rs)

factors for the baseline/acceptable resolved compounds are listed in Table 3 (cf. Table 3). Comparative chromatograms showing the enantioselective separation on polymer monoliths (A1 and A2 columns) vs silica monoliths (AIBN and BOP columns) are also shown in Figs. 7–10. It is worth pointing out that some analytes were resolved on either of the polymer (A1/A2) or silica (AIBN/ BOP) monoliths. It was also observed that the silica-based monoliths showed broader enantioselctivities toward diverse chemical

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Fig. 5. Schematic diagram showing the preparation of β-CD functionalized polymer monolith [38].

Fig. 6. Scanning electron micrograph of A1 (left) and A2 (right) at 500  showing homogenous porous monolithic structure [38].

classes of pharmaceutical racemates for example polyhalogenated aromatics such as miconazole 26 and aromatic amides such as eticlopride 29 (Figs. 11 and 12, respectively). Interestingly, the sympathomimetic arternol 41 and its metabolite normetanephrine 42 were acceptably resolved using the silica-based β-CD columns (Figs. 13 and 14) which was not achievable using the polymer monoliths. It is worth mentioning that the enantioselective separation was achieved under reversed phase conditions which allow the use of less costly solvents and provide easier sample preparation from serum or plasma [43]. Moreover, it eliminates the use of health hazardous solvents such as n-hexane. The capillary columns prepared in this study are 10,000 less in internal diameter and operate with one million time less solvent volume than the conventional columns. Consequently, they consume less solvent and produce faster and reproducible separations.

3.4. Insights into the chiral recognition mechanism The prediction of the chiral recognition mechanisms of CSPs has mainly been based on empirical rules or speculations due to the complexity of the enantioselective separation [44]. Racemates of homologous series of closely related chemical structures tend to interact differently with the chiral selectors resulting in large differences in both enantioselectivities and elution order [45]. It is well established that CDs form transient diastereomeric inclusion complexes with enantiomers by means of the cavity under reversed phase chromatographic conditions. Additionally, derivatization of the external rim hydroxyl groups affords CD derivatives with higher solubility and variable cavity depth with multiple interaction sites [8]. Phenyl carbamate derivatives have been widely explored for the enantioselective separation, their enantioselectivity can be further modulated by introduction of groups

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Table 3 Chromatographic parameters, separation and resolution factors for the baseline resolved compounds. Column

Separated compound

Separation factor (α)

Resolution (Rs)

A1

Propranolol Ifosfamide Alprenolol Diniconazole Ketoprofen Tertalol 1-Indanol

1.39 1.44 1.89 1.65 1.46 1.67 1.76

2.22 1.65 2.2 1.26 1.32 2.56 2.46

A2

Propranolol Metoprolol Tebuconazole 6-Hydroxyflavanone o-Methoxymandelic acid Cizolertine Celiprolol Tertalol

1.52 1.54 1.45 1.67 1.71 1.72 1.52 1.65

2.53 1.23 2.5 1.18 1.58 2.62 1.93 2.56

AIBN

Arterenol Alprenolol Celiprolol Cizolirtine Eticlopride Metoprolol

1.65 2.1 2.12 1.68 2.28 1.62

1.36 1.79 2 2.04 2.44 1.67

BOP

Alprenolol Miconazole Bufuralol Carbuterol Celiprolol Cizolirtine Desmethyl cizolirtine Methoxymandelic acid Metoprolol Normetanephrine

1.67 1.92 1.7 2.44 2.61 2.28 2.03 2.1 1.75 2.43

1.4 1.26 1.46 1.3 1.42 1.68 1.45 1.41 1.39 1.28

100

Bufuralol on polymer

80

10

Absorbance (mAu)

Absorbance (mAu)

Bufuralol on silica

12

60

40

20

8 6 4 2 0

0

-2 0

5

10

15

20

25

30

Time (min)

0

5

10

15

20

25

30

Time (min)

Fig. 7. Enantioselective nano-LC separation of bufuralol: Left: on A1 capillary column (150 μm ID, 25 cm length). Mobile phase: methanol/water (0.1% TFA) 10:90 v/v, UV: 270 nm, flow rate: 0.3 μL/min, Right: BOP capillary column (100 mm, 15 cm), mobile phase: methanol/water (0.1% TFA) 80:20 v/v.

with different electronic effects on the phenyl ring. This suggests that the most important adsorbing sites for the chiral discrimination of on phenyl carbamate derivatives is the polar carbamate group [46]. As demonstrated from testing 50 racemates from different chemical and pharmaceutical classes on four 2,3,6-tris (phenylcarbamoyl)-β-CD-based CSPs, 7 out of the 11 baseline resolved racemates were for chiral centers with secondary alcohols with adjacent electronegative nitrogen bearing alkyl side chain; this underlines the importance of these groups in providing three points of interactions which is required for the chiral recognition as the side chain on the chiral carbon is also thought

to be concerned in the chiral recognition mechanism [47]. We postulate that the chiral separation was achieved via the formation of hydrogen bond-stabilized inclusion complexes within the β-CD cavity and the carbamate moiety. It was also observed that the enantioselective separation was mostly achieved at high water content mobile phase; this indicates that water improved the magnitude of interaction between the racemates and the CSP. Aqueous mobile phases allow the inclusion of the whole molecule or just its lipophilic part into the hydrophobic cavity of CD, it has been previously reported that the resolution of this kind of CSP is improved by increase in water content [47]. Interestingly, mobile

312

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120

6

(R)

Cizolertine on polymer

Absorbance (mAu)

Absorbance (mAu)

(S)

N

60

N

O

N

40

Cizolertine on silica

5

100 80

(R)

4

N

3

N

O

N

(S)

2 1

20 0 0 -1 0

5

10

15

20

25

0

30

5

10

15

20

25

30

Time (min)

Time (min)

Fig. 8. Enantioselective nano-LC separation of cizolertine: Left: on A2 capillary column (150 μm ID, 25 cm length). Mobile phase: methanol/water (0.1% TFA) 10:90 v/v, UV: 240 nm, flow rate: 0.3 μL/min, Right: BOP capillary column (100 mm, 15 cm), mobile phase: methanol/water (0.1% TFA) 55:45 v/v.

(S)

140

(R)

(S)

2.5

Celiprolol on silica

100

Absorbance (mAu)

Absorbance (mAu)

120

(R)

3.0

Celiprolol polymer

80 60 40

2.0 1.5 1.0

20

0.5

0

0.0

-20

-0.5 0

5

10

15

20

25

0

30

10

20

30

40

50

60

Time (min)

Time (min)

Fig. 9. Enantioselective nano-LC separation of celiprolol: Left: on A2 capillary column (150 μm ID, 25 cm length). Mobile phase: methanol/water (0.1% TFA) 10:90 v/v, UV: 254 nm, flow rate: 0.3 μL/min, Right: AIBN capillary column (100 mm, 25 cm), mobile phase: methanol/water (0.1% TFA) 55:45 v/v.

(R)(S)

100

Alprenolol on polymer

(R)

2.0

ALprenolol on silica

Absorbance (mAu)

Absorbance (mAu)

80

60

40

1.5

(S)

1.0

0.5

20 0.0 0 -0.5 10

12

14

16

18

20

Time (min)

0

10

20

30

40

50

60

Time (min)

Fig. 10. Enantioselective nano-LC separation of alprenolol: Left: on A2 capillary column (150 μm ID, 25 cm length). Mobile phase: methanol/water (0.1% TFA) 10:90 v/v, UV: 254 nm, flow rate: 0.3 μL/min, Right: AIBN capillary column (100 mm, 25 cm), mobile phase: methanol/water (0.1% TFA) 55:45 v/v.

phase composed of mixture of acetonitrile and water in the range of 10–90% v/v did not furnish any enantioselective separation. On the other hand, when methanol-based mobile phase was used, enantioselective separation was observed; this underlines the importance of solvent polarity in determining retention and

chiral separation mechanism in terms of the inclusion complex stability. It was also demonstrated that the monolith backbone did not play a major role in the chiral separation as mostly the same racemates were resolved on silica or polymer backbone. It is worth

A. Ghanem et al. / Talanta 132 (2015) 301–314

313

3.5 20

3.0

Absorbance (mAu)

Absorbance (mAu)

2.5 2.0 1.5 1.0

15

10

5

0.5 0

0.0

0

-0.5 0

5

10

15

20

25

5

Time (min) Fig. 11. Enantioselective nano-LC separation of miconazole on BOP capillary column (100 mm, 15 cm), mobile phase: methanol/water (0.1% TFA) 80:20 v/v, UV: 240 nm, flow rate: 0.3 μL/min.

10

15

20

25

30

Time (min)

30

Fig. 14. Enantioselective nano-LC separation of noremetanephrine on BOP capillary column (100 mm, 15 cm), mobile phase: methanol/water (0.1% TFA) 85:15 v/v, UV: 219 nm, flow rate: 0.3 μL/min.

mentioning that polymer monoliths are of smaller surface area and bigger pore size compared to the silica monolith and this had affected the retention times of the racemates but not the enantioselectivity.

3.0

Absorbance (mAu)

2.5

O

2.0

O

Cl

4. Conclusions

N H OH

1.5

N

1.0 0.5 0.0 -0.5 0

10

20

30

40

50

60

Time (min) Fig. 12. Enantioselective nano-LC separation of eticloprode on AIBN capillary column (100 mm, 25 cm), mobile phase: methanol/water (0.1% TFA) 55:45 v/v, UV: 270 nm, flow rate: 0.3 μL/min.

Acknowledgements

12

10

Absorbance (mAu)

New β-CD-based CSPs were prepared via the copolymerization of β-CD methacrylate either in one pot or post modification approaches. The prepared columns were investigated for the enantioselective separation of racemates from twelve pharmaceutical classes. β-CD-based silica monolithic capillary columns displayed broader enantioselectivities, nonetheless, the polymer monolith is less time consuming and can be easily reproduced compared with the technically challenging silica monolith preparation. With further option of variation of the porogens/functional monomers ratio composition, tailoring of polymer skeleton will be possible for each separation problem which is still a time consuming process. The enantioselective separation was conducted under reversed phase conditions which augments the environmentally benign nano-liquid chromatographic separations.

OH HO

8

6

NH2

HO

4

The authors gratefully acknowledge The Endeavour Program/ Australia Research Award (Project no. ERF_PDR_3853_2014) and the Japan Society for promotion of Science (JSPS) for sponsoring Dr Ghanem’s stay at KIT, Kyoto, Japan. Dr. Karsten Gömann, Central Science Laboratory, University of Tasmania (UTAS) for the SEM imaging and the W.J. Weeden scholarship program at the University of Canberra, Australia, for the PhD scholarship offered to Ms. Marwa Ahmed. References

2

0 0

10

20

30

40

Time (min) Fig. 13. Enantioselective nano-LC separation of arterenol on AIBN capillary column (100 mm, 25 cm), mobile phase: methanol/water (0.1% TFA) 15:85 v/v, UV: 219 nm, flow rate: 0.5 μL/min.

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