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Riboflavin binding proteins as chiral selectors in HPLC and CE
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PSTT Vol. 2, No. 9 September 1999
Riboflavin binding proteins as chiral selectors in HPLC and CE Ersilia De Lorenzi and Gabriella Massolini The term riboflavin binding proteins (RfBPs) is applied to several molecular species that play the important role of vitamin delivery to the developing embryo, thus becoming essential for the survival of the fetus. In addition to this physiological significance, these proteins have recently been found to be successful chiral selectors. In this review, the authors address the use of such proteins, both as columns for high performance liquid chromatography (HPLC) and as additives in capillary electrophoresis (CE), for the enantioseparation of several racemic drugs.
Ersilia De Lorenzi* and Gabriella Massolini University of Pavia Department of Pharmaceutical Chemistry Via Taramelli 12 I-27100 Pavia Italy *tel: 139 0382 507383 fax: 139 0382 422975 e-mail: [email protected]
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▼ Several chemical compounds used in pharmaceutical formulations feature one or more chiral centres, responsible for optical activity, that can strongly influence their pharmacological and pharmacokinetic properties. In fact, optical isomers are often distinguished by biological systems in the human body, and may therefore have different pharmacokinetic profiles and qualitatively and quantitatively different pharmacological or toxicological effects. For these reasons the development of new approaches for the separation of chiral compounds is a source of global research efforts and innovative advances. Furthermore, the impetus for this vibrant activity lies with the increased pressure and concern from regulatory agencies such as the FDA. Enantioselective high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) are successfully used for analytical purposes; namely the direct separation of drug racemates, the monitoring of stereoselective metabolism and for optical purity tests in pharmaceutical dosage forms. Several chiral selectors have hitherto been employed as either chiral columns for HPLC or as additives to the background electrolyte in CE, and thus in this respect proteins play an important role.
Proteins feature several unique qualities that make them versatile, fascinating and successful chiral selectors. Because of their chiral nature, they often interact differently with stereoisomers of either chiral macromolecules or small molecules by reversible binding. As a result of the variety of functional groups present at their surface, proteins can interact with chiral entities by forming not only relatively weak and non-specific bonds, but also stronger and more specific interactions, which involve a well-defined site of the protein itself. Electrostatic and hydrophobic bonds, as well as hydrogen bonds, p–p bonds and inclusion phenomena, may participate in the interaction between a protein and a chiral drug. The broad applicability of proteins as chiral selectors is evident in the large number of racemates separated so far, and is extended by the possibility of operating in an aqueous buffered system, which is compatible with many biological samples. The versatility of proteins is further expressed by the potential of simple modifications to the mobile phase (or background electrolyte) parameters for inducing a reversible change of the selector conformation, thus obtaining different enantioselective properties of the same protein. Different pH values, type and concentration of organic modifier can, in fact, alter the interactions between the selector and the analyte. For these reasons, the past 15 years have seen a growing development of protein-based stationary phases for HPLC, which have been extensively used and successfully applied for the direct separation of enantiomers1–5.The first chiral columns to be developed and marketed were those made of bovine serum albumin6, human serum albumin7, a1-acid glycoprotein8 (AGP) and ovomucoid9 (OVM), later followed by cellobiohydrolase10 and avidin11. Also, the use of conalbumin12, pepsin13 and b-lactoglobulin14 has been reported by academic research groups. The physiological
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PSTT Vol. 2, No. 9 September 1999
role of human serum albumin and a1-acid glycoprotein has led to additional investigations15–18, with the aim of elucidating the stereoselective binding mechanisms and to obtain a more in-depth knowledge of the characterization of the binding site. In the early 1990s, proteins began to be used as chiral selectors in CE19,20, and since then increasing numbers of papers have appeared, as well as review articles21,22. As purified proteins are expensive and, in some cases, are not even commercially available, the CE approach is of particular advantage over HPLC. Most authors simply dissolve the selector in the background electrolyte (complete filling technique), and thus a few milligrams of protein are required and immobilization on a solid support is unnecessary. All of the proteins already mentioned as HPLC chiral stationary phases have been evaluated as chiral selectors in CE19,20,23–35, and, in addition, human serum transferrin36,37 and casein38 have been investigated. Riboflavin binding proteins In our laboratories we have undertaken the evaluation of riboflavin binding proteins (RfBPs) as potential chiral selectors for the enantioseparation of drug racemates. Several factors contributed to this choice, namely the physiological role of these proteins, the presence of a specific binding region, the possibility of extraction from different matrices and the awareness that, at the beginning of our investigation, the threedimensional structure characterization of chicken egg-white RfBP was about to be accomplished by X-ray diffraction39. The term riboflavin binding proteins is applied to several molecular species that are involved in the transport and storage of the vitamin, with particular emphasis on the role of supplying this micronutrient to the developing embryo40. Pregnancyspecific RfBPs have been found in mammalian species, including humans41–43, and they all appear to share similarities with the well known chicken riboflavin binding proteins. Chicken egg-white and -yolk RfBP share the same aminoacid sequence, but have undergone different post-translational modifications; that is, their carbohydrate chains are different and the latter lacks the last 11–13 aminoacids that are proteolytically cleaved. Peculiar characteristics of RfBPs extracted from chicken egg and chicken plasma are a highly phosphorylated region and a high degree of crosslinking by nine disulfide bridges, the former being essential for vitamin uptake and the latter being responsible for the high protein stability. Crystallographic data39 shed light on the ligand binding-site characterization and on the aminoacids involved in the binding with riboflavin. Interestingly, an additional succinate-binding cleft was described. RfBPs extracted and purified from the eggs of avian species other than chicken have also been investigated, including quail44, goose45 and duck46 RfBPs.
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Common features and differences among the RfBPs from these species and their chicken counterpart have been reported. For example, the RfBPs from domestic fowl and quail eggs have been compared44 by circular dichroism and fluorescence and peptide mapping, and the outcome of this study revealed small but significant differences in their tertiary structure that must reflect differences in the aminoacid sequence. It is interesting that the goose egg-yolk protein45 appears to have a significantly higher molecular weight and a much higher proportion of b-sheet than either chicken or quail RfBP. In addition, goose egg-yolk RfBP presents an unusually high carbohydrate content as well as glucose residues that are not believed to be present in any other RfBP.All these characteristics, together with differences in the far ultra violet circular dichroism spectra of goose egg-yolk and chicken egg-white RfBPs are important in accounting for differences in the secondary structures, despite the similar aminoacid composition. Furthermore, goose egg-yolk RfBP appears to have a larger number of phosphate residues than that reported for chicken egg-yolk RfBP, and its large amount of carbohydrates is in marked contrast to that of duck egg white, which lacks covalently bound oligosaccharides46. The ready availability of the matrix and the idea of obtaining a deeper insight into the subtle structural differences of these proteins through a chromatographic investigation, prompted us to set up extraction, purification and immobilization protocols to exploit chicken- and quail-egg RfBPs as chiral stationary phases for HPLC47,48. The large number of data produced by analysing drug racemates with different structures would offer additional knowledge for the comparison of RfBPs from avian species. Parallel CE experiments have also been planned on the basis of findings and ideas originated in HPLC in order to evaluate whether CE could be used as a rapid scouting technique for screening the enantioselectivity of novel proteins, and to verify to what extent the protein performance is affected by the immobilization process48. RfBPs have also been purified from the plasma of pregnant cows41, although the characterization of this protein is yet to be fully investigated. RfBP plays an important role in mammals in terms of fetal vitamin nutrition through the transplacental transport of riboflavin across the placental barrier49. Preliminary data are also available on the isolation and characterization of RfBP from human amniotic fluid50 and immunological similarities with the chicken counterpart have been demonstrated, although further studies are to be performed. It is important to mention that immunoneutralization of the endogenous RfBP in pregnant mammals leads to embryonic death due to acute vitamin deficiency51. Because binding of drugs is a part of the physiological role of proteins, a high performance liquid affinity chromatographic approach with stationary phases based on bovine plasma RfBP could be envisaged, in order to investigate the effect of drug binding on vitamin transport during pregnancy. Extraction and purification protocols of RfBPs from the plasma of pregnant cows 353
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Table 1. Best enantioseparations obtained in HPLC using RfBP-based chiral stationary phases Analyte
Column
k’
a
Mobile phase composition
Ref.
Ibuprofen
qWRfBP cWRfBP cYRfBP qYRfBP cWRfBP qWRfBP cYRfBP qYRfBP qWRfBP cYRfBP cYRfBP cWRfBP cYRfBP cYRfBP cWRfBP qYRfBP qWRfBP cYRfBP cWRfBP qYRfBP qWRfBP qYRfBP cYRfBP qWRfBP cYRfBP qYRfBP qWRfBP cYRfBP qYRfBP qWRfBP cYRfBP qYRfBP qWRfBP cYRfBP qYRfBP qWRfBP cYRfBP qYRfBP qWRfBP cYRfBP qYRfBP qWRfBP qWRfBP qYRfBP qWRfBP cYRfBP qWRfBP qYRfBP cWRfBP cWRfBP qYRfBP qWRfBP qWRfBP cWRfBP cWRfBP cWRfBP cWRfBP cWRfBP
3.92 8.52 1.38 1.2 23.94 9.71 2.56 19.2 13.83 9.84 6.10 17.99 8.27 30.42 38.25 7.18 11.60 4.67 16.41 2.72 4.01 3.43 1.56 5.78 4.91 5.31 11.34 4.87 7.06 14.00 8.57 4.7 3.57 0.72 37.7 20.08 10.61 29.8 17.00 232.94 17.3 31.32 7.8 6.17 4.42 11.96 3.09 24.1 0.96 0.79 2.89 5.93 1.21 13.13 3.46 1.48 5.61 1.01
1.69 1.07 1.13 1.07 1.29 1.07 1.22 1.32 1.24 1.16 1.13 1.06 1.08 2.20 1.50 2.36 2.59 1.39 2.16 1.16 1.28 2.14 1.20 2.91 1.87 6.92 8.42 6.68 1.21 1.15 1.28 1.43 1.95 1.10 1.58 1.53 1.32 1.43 1.38 1.32 1.17 1.25 1.07 1.14 1.11 1.21 2.19 2.57 1.09 1.27 1.28 1.37 8.77 1.14 1.51 1.72 3.79 1.52
50 mM NaH2PO4 (pH 3.5)-CH3OH (95:5 v/v) 50 mM CH3COONa (pH 5.0)- CH3OH (95:5 v/v) 50 mM KH2PO4 (pH 5.5)-CH3OH (95:5 v/v) 50 mM KH2PO4 (pH 6.5)-CH3OH (95:5 v/v) 50 mM KH2PO4 (pH 4.6)-CH3CH2OH (96:4 v/v) 50 mM NaH2PO4 (pH 4.5)-CH3OH (95:5 v/v) 50 mM KH2PO4 (pH 5.5) 50 mM KH2PO4 (pH 5.5)-CH3OH (95:5 v/v) 50 mM NaH2PO4 (pH 4.5)-CH3CN (95:5 v/v) 50 mM KH2PO4 (pH 6.5)-CH3CH2OH (90:10 v/v) 50 mM KH2PO4 (pH 5.5) 50 mM CH3COONa (pH 5.0)-CH3OH (95:5 v/v) 50 mM KH2PO4 (pH 5.5) 50 mM KH2PO4 (pH 5.5)-CH3OH (95:5 v/v) 50 mM CH3COONa (pH 5.0)-CH3OH (95:5 v/v) 50 mM KH2PO4 (pH 5.5)-CH3OH (95:5 v/v) 50 mM NaH2PO4 (pH 4.5)-CH3OH (95:5 v/v) 50 mM KH2PO4 (pH 4.6)-CH3CH2OH (90:10 v/v) 50 mM KH2PO4 (pH 4.6)-CH3CH2OH (96:4 v/v) 50 mM KH2PO4 (pH 3.5)-CH3OH (95:5 v/v) 50 mM NaH2PO4 (pH 3.5)-CH3OH (95:5 v/v) 50 mM KH2PO4 (pH 6.5)-CH3OH (95:5 v/v) 50 mM KH2PO4 (pH 4.5)-CH3OH (95:5 v/v) 50 mM NaH2PO4 (pH 6.5)-CH3OH (95:5 v/v) 50 mM KH2PO4 (pH 5.5)-CH3OH (95:5 v/v) 50 mM KH2PO4 (pH 6.5)-CH3OH (95:5 v/v) 50 mM NaH2PO4 (pH 6.5)-CH3OH (95:5 v/v) 50 mM KH2PO4 (pH 5.5)-CH3OH (95:5 v/v) 50 mM KH2PO4 (pH 6.5)-CH3OH (95:5 v/v) 50 mM NaH2PO4 (pH 4.5)-CH3OH (95:5 v/v) 50 mM KH2PO4 (pH 5.5) 50 mM KH2PO4 (pH 4.5)-CH3OH (95:5 v/v) 50 mM NaH2PO4 (pH 4.5)-CH3(CH2)2OH (95:5 v/v) 50 mM KH2PO4 (pH 5.5) 50 mM KH2PO4 (pH 6.5)-CH3OH (95:5 v/v) 50 mM NaH2PO4 (pH 5.5)-CH3OH (95:5 v/v) 50 mM KH2PO4 (pH 5.5)-CH3CH2OH (95:5 v/v) 50 mM KH2PO4 (pH 4.5)-CH3OH (95:5 v/v) 50 mM NaH2PO4 (pH 6.5)-CH3CN (95:5 v/v) 50 mM KH2PO4 (pH 5.5)-CH3CH2OH (95:5 v/v) 50 mM KH2PO4 (pH 5.5)-CH3OH (95:5 v/v) 50 mM NaH2PO4 (pH 5.5)-CH3OH (95:5 v/v) 50 mM NaH2PO4 (pH 6.5)-CH3OH (95:5 v/v) 50 mM KH2PO4 (pH 6.5)-CH3OH (95:5 v/v) 50 mM NaH2PO4 (pH 5.5)-CH3OH (95:5 v/v) 50 mM KH2PO4 (pH 5.5)-CH3CH2OH (95:5 v/v) 50 mM NaH2PO4 (pH 4.5)-CH3(CH2)2OH (95:5 v/v) 50 mM KH2PO4 (pH 6.5)-CH3OH (95:5 v/v) 20 mM KH2PO4 (pH 4.0)-CH3OH (90:10 v/v) 50 mM CH3COONa (pH 5.0)-CH3OH (95:5 v/v) 50 mM KH2PO4 (pH 6.5)-CH3OH (95:5 v/v) 50 mM NaH2PO4 (pH 6.5)-CH3OH (95:5 v/v) 50 mM NaH2PO4 (pH 5.5)-CH3OH (95:5 v/v) 50 mM KH2PO4 (pH 4.6)-CH3CH2OH (96:4 v/v) 50 mM KH2PO4 (pH 4.6)-(CH3)3COH (96:4 v/v) 20 mM KH2PO4 (pH 6.0)-CH3OH (98:2 v/v) 50 mM CH3COONa (pH 5.0)-CH3OH (95:5 v/v) 50 mM CH3COONa (pH 5.0)-CH3OH (95:5 v/v)
48 67 47 52 54 48 47 52 48 47 47 67 47 47 67 52 48 47 54 52 48 52 47 48 47 52 48 47 52 48 47 52 48 47 52 48 47 52 48 47 52 48 48 52 48 47 48 52 53 67 52 48 48 54 54 53 67 67
Ketoprofen
Indoprofen
Flurbiprofen Suprofen Carprofen Pranoprofen Warfarin
Lormetazepam Lorazepam
Oxazepam
Isradipine
Amlodipine Nimodipine Nicardipine
Manidipine
Lercanidipine Gallopamil Verapamil Bepridil
Propranolol Oxprenolol Fenfluoramine Bupivacaine a,e Dibenzoyllysine Benzoin Chlormezanone Proglumide Aminogluthetimide
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and from human amniotic fluid, together with identification with chicken plasma antibodies, are currently at a stage in our laboratories where their use in affinity CE or affinity HPLC will soon become feasible. RfBP-based HPLC columns RfBP is not very abundant in either the egg yolk or in the egg white of avian species40, and as the amount of protein required to produce an HPLC column is relatively large, an optimization of the purification process becomes necessary in order to obtain approximately 300– 400 mg per batch. So far, four stationary phases have been described as chiral selectors; namely those based on chicken egg yolk (cYRfBP)47, quail egg yolk (qYRfBP)52, quail egg white (qWRfBP)48 and chicken egg white (cWRfBP)53,54.
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Preparation of chicken- and quail-egg RfBP columns (e) (f) Purification. RfBPs from different avian species and different matrices were purified by suitable modification47,48 of widely used methods55,56. Monitoring of the purification process and analyses of the final preparation for homogeneity were performed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate and by isoelectric focusing with carrier ampholytes (pH 3.5–9.5). Immobilization. Immobilization was per0 10 20 30 40 50 0 5 10 15 20 25 30 formed as follows: 5 NH2 Nucleosil was Min Min slurried in HPLC-grade acetonitrile and Pharmaceutical Science & Technology Today N,N-disuccinylimidyl carbonate was added. Figure 1. Chromatograms of some of the compounds tested on chicken egg-yolk RfBP. After stirring, filtering and washing, this (a) nicardipine; (b) isradipine; (c) carprofen; (d) warfarin; (e) oxazepam; and (f) lorazepam. activated silica was added to the protein Chromatographic conditions were mobile phase 50 mM KH2PO4 (pH 5.5)–ethanol (95:5 v/v) in (a), previously suspended in buffer. The ob(c), (e) and (f); the mobile phase was 50 mM KH2PO4 (pH 5.5) in (b), and in (d) the mobile phase tained stationary phase was gently mixed was 50 mM KH2PO4 (pH 4.6)–ethanol (90:10 v/v). The flow rate was 0.8 ml min21. Column dimensions were 100 3 4.6 mm I.D. Reproduced, with permission, from Ref. 47. using the rotor evaporator and then 47,54 packed in a stainless steel column . Applicability. The applicability of the developed RfBP columns as chiral selectors for liquid chromatograare shown in Figs 1 and 2: high enantioselectivity and favourphy was tested by analysing a large number of racemic drugs with able chromatographic performance were obtained. Our interest different chemical properties (basic, acidic and neutral) and which was then focused on controlling the enantioselective retention belonged to different pharmaceutical classes. Chromatographic by varying the pH of the mobile phase and by adding different results are presented in Table 1 and representative chromatograms kinds and amounts of organic modifiers. 355
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Figure 2. Chromatograms of some of the compounds tested on quail egg-white RfBP. (a) amlodipine; (b) warfarin; (c) bupivacaine; (d) indoprofen; (e) fenfluramine; (f) bepridil; (g) oxazepam; and (h) lorazepam. Chromatographic conditions were mobile phase 50 mM phosphate buffer (pH 4.5)–methanol (90:10 v/v) in (a), (f) and (g); the mobile phase was 50 mM phosphate buffer (pH 4.5)–methanol (95:5 v/v) in (h); (d) and (b) featured a mobile phase of 50 mM phosphate buffer (pH 4.5)–acetonitrile (95:5 v/v); and the mobile phase was 50 mM phosphate buffer (pH 5.5)–methanol (95:5 v/v) in (c) and (e). The flow rate was 0.8 ml min21 and the column dimensions were 100 3 4.6 mm I.D. Reproduced, with permission, from Ref. 48.
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Influence of pH and organic modifiers on retention and chiral resolution Retention (k9) and enantioselectivity (Rs or a) of RfBP columns are essentially influenced by the pH of the mobile phase and by the percentage of organic solvent. Depending on the charge of the solute, a change in pH in the mobile phase, such as the one considered in our studies (3.5–6.5) (Refs 47,48), may cause either an increase or a decrease of the k9 values. In general, the effect of pH on retention is markedly larger for charged than for uncharged solutes, and the affinity for the stationary phase increases as the compounds become less ionized. This behaviour was observed for all of the RfBP columns and it provides evidence of the contribution of hydrophobic interactions on the binding between drugs and protein.
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RfBPs have an isoelectric point of approximately 4.0 and therefore they bear a net negative charge above this pH value. For the acidic compounds, whose pKa values are between 4.0 and 5.0, the maximum in retention and enantioselectivity was obtained at pH 4.5, where both protein and analytes are uncharged: this effect is caused by an increase in the non-polar interactions between solutes and RfBP. The k’s of basic compounds, whose pKa values are between 8.0 and 9.0, rose on increasing the pH, and this behaviour is related to the increase in the net negative charge of the protein that leads to a rise in the number of coulombic interactions with the cationic compounds. In the case of basic compounds, the enantioselectivity was not influenced to a great extent by pH variations. The retention time of uncharged solutes was almost independent of the pH of the mobile phase, although the enantioselectivity for benzodiaze12.5 pines, uncharged at the considered pH 10 values, rises upon increasing the pH from 7.5 5 3.5 to 6.5, and this trend is because 2.5 of the remarkable increase in the re0 tention time of the second eluted en-2.5 antiomer. The high selectivity shown by -5 oxazepam at pH 6.5 (Fig. 3) on the -7.5 RfBP–CSPs47,48,52 suggested that the two 80 100 0 20 40 60 Min enantiomers bind at different loci and, in Pharmaceutical Science & Technology Today particular, that the first eluted enantiomer interacts non-specifically with the proFigure 3. Chromatographic profile of oxazepam. Chromatographic conditions were 50 mM KH2PO4 tein, whereas the second eluted enan21 (pH 6.5)–methanol (95:5 v/v), and the flow rate was 0.8 ml min . The column used was qWRfBP 125 3 4.0 mm I.D. tiomer interacts with the riboflavin binding site.
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Figure 4. Influence of pH on k9 for some compounds tested on qYRfBP (a) and qWRfBP (b) columns. Chromatographic conditions were 50 mM KH2PO4–methanol (95:5 v/v). The sample was indoprofen (black diamond), carprofen (black circle), verapamil (black triangle), and bepridil (black square). Column dimensions were 100 3 4.6 mm I.D.
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25 20
k´
15 10 5 0 Warfarin
Oxazepam
Nicardipine
Amlodipine
Warfarin
Oxazepam
Nicardipine
Amlodipine
Warfarin
Oxazepam
Nicardipine
Amlodipine
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8 6 4 2 0 Pharmaceutical Science & Technology Today
Figure 5. Istograms of k9, a and Rs of selected drugs, analysed on chicken (black) and quail (white) egg-yolk RfBP. The selected drugs were warfarin, oxazepam, nicardipine and amlodipine. Chromatographic conditions were 50 mM KH2PO4 (pH 5.5)–methanol (95:5 v/v), the flow rate was 0.8 ml min21, and column dimensions were 100 3 4.6 mm I.D.
Different organic modifiers, such as methanol, ethanol, npropanol, 2-propanol and acetonitrile, have been used at different percentages to modulate retention and selectivity. By increasing their concentration in the mobile phase, capacity factors and Rs decrease, and this effect was more pronounced for the most hydrophobic compounds, such as carprofen and manidipine. These results indicate that the system works in a similar manner to a reversed-phase, and that hydrophobic interactions are important for the retention mechanism. In general, the addition of methanol to the mobile phase resulted in better resolution values than those obtained with other organic modifiers. 358
Species and matrices comparison The chromatographic performances and the enantioselective properties of the qYRfBP and qWRfBP columns were compared in order to display possible different behaviours connected to the different matrix origin52. The two columns gave a similar k9 pattern for all analytes, as shown in Fig. 4 for four selected drugs. In terms of the acidic compounds, an increase in the pH results in k9 values with a maximum at pH 4.5, where both the RfBPs and analytes are uncharged and ionic attraction phenomena do not occur. The k9 values of basic analytes were highly influenced by pH variations, whereas the capacity factors of neutrals (not reported in Fig. 4) were almost unaffected. RfBPs from both quail egg white and egg yolk immobilized on silica are good chiral selectors, as they could resolve a large number of the racemic compounds tested (Table 1), with a slightly better performance given by the former. In particular, antiinflammatory drugs were poorly resolved, whereas dihydropyridines and benzodiazepines gave the best enantioseparations. As the trend observed for the two columns was found to be similar, in terms of both retention and enantioselectivity, it is therefore reasonable to assume that the stereoselective regions of RfBPs extracted from different matrices (egg yolk and egg white) of the same species (quail) do not show considerable differences. Stationary phases prepared with egg-yolk RfBP from different species were used to investigate whether small differences in the aminoacid sequence, in both the tertiary structure and in the carbohydrate content, could influence the retention and, in particular, the selectivity properties of RfBP-based columns. In order to compare the chromatographic results of chicken47 and quail52 egg-yolk RfBP columns, we selected four model compounds: warfarin, oxazepam, amlodipine and nicardipine. Figure 5 shows the comparison of capacity factors (k9), selectivity (a) and resolution (Rs), measured under the same chromatographic conditions using the two chiral stationary phases.The retention of all compounds was higher for the chicken YRfBP column, except for the acidic compound warfarin. In terms of selectivity and resolution, improved a and Rs values were obtained for warfarin and nicardipine when analysed on the quail YRfBP column, whereas for oxazepam the best results were obtained with chicken YRfBP–CSP, and amlodipine enantiomers were successfully resolved only by the qYRfBP stationary phase. In general, the addition of increasing percentages of an organic modifier to the mobile phase meant that retention of the analytes on all YRfBP–CSPs decreased, while the enantiomeric separation was either reduced or remained substantially unchanged. The effect of the buffer pH on retention is markedly more evident for charged than for uncharged analytes, although it is similar for the two YRfBP columns. Generally, basic compounds are more strongly retained at higher pH values, whereas the opposite is true for acidic drugs.
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Table 2. Comparison of qWRfBP-based stationary phases Analyte k’
Column A qWRfBP a
Rs
k’
Column B qWRfBP a
Rs
k’
Column C qWRfBP a
Rs
Amlodipine
7.55
1.87
2.61
3.09
1.78
3.03
3.47
2.11
3.02
Bepridil
5.49
1.85
3.53
2.56
1.90
4.88
3.27
2.23
3.47
Ketoprofen
9.71
1.07
0.41
9.83
1.06
0.5
7.73
1.10
0.47
Lorazepam
4.45
1.66
3.04
2.34
1.78
3.41
2.73
1.86
3.09
Nicardipine
8.79
1.18
0.73
4.54
1.17
1
3.47
2.11
3.02
Warfarin
11.60
2.59
5.48
8.25
2.51
4.41
12.92
3.09
6.8
Indoprofen
53.77
1.31
2.00
31.72
1.39
2.61
49.15
1.48
3.04
Mobile phase: 50 mM KH2PO4 (pH 4.5)–methanol (95:5 v/v); flow rate 0.8 ml min21 Column dimensions: 100 x 4.6 mm I.D. (from Refs 48 and 52)
The enantioselective capability of the developed CSPs might be related to the interaction with the stereoselective riboflavinbinding site. Further systematic work on RfBP columns from different species with several model chiral drugs will be important, for a better understanding of both the RfBP structures and the stereoselective role of the binding site. Reproducibility and stability The measurement of capacity factors and resolution factors can be exploited for the study of column reproducibility, providing that the temperature and solvent composition are controlled.The enantioselective performance of three different qWRfBP stationary phases is presented in Table 2.A decrease in retention was observed between columns A and B, which were obtained from different protein purification batches; an explanation for the retention variation might be the different amount of immobilized protein, namely 104.26 mg g21 silica for column A and 54.41 mg g21 silica for column B, as calculated by elemental analysis. Good reproducibility of the enantioselectivity was obtained for columns packed with the same protein purification batch (columns B and C) and also from different batches (column A and B).The small differences in the Rs values observed between the three columns are related to the column hardware reproducibility and to the consistency of the column packing technique. Column stability was investigated by evaluating the chromatographic performance for warfarin at the start of the column lifetime and after 250 injections, under the required conditions
in method development studies, such as broad changes in mobile phase composition and pH. The chromatographic parameters, such as Rs, efficiency and retention, were almost unchanged (Table 3), thus proving that the column is robust as well as reliable for routine analysis. The same compound has since been used as a probe to ensure that adequate stereoselective performance was validated at regular intervals. RfBPs as additives in CE During the past ten years, CE has penetrated a wide range of areas of separation science and has been recognized as a powerful, viable, reliable, successful and versatile technique. CE features unique advantages, such as speed, minimum solvent Table 3. Stability of cYRfBP-based column using warfarin as test analytea Parameter
1st analysis
After 250 analyses
k’1 k’2 a Rs Plates/m
4.9 8.5 1.73 2.81 15,020
4.64 7.92 1.70 2.49 11,980
aMobile
phase: 50 mM KH2PO4 (pH 5.5)–ethanol (95:5 v/v); flow rate 0.8 ml min21
Column dimensions: 100 x 4.6 mm I.D. Reproduced, with permission, from Ref. 47.
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Box 1. Issues to be considered in method transfer (HPLC®CE) when using proteins as chiral selectors • • • • •
pH value Protein concentration Mobility match Protein batch Protein stability
consumption, small sample volumes, high selectivity and ease of automation. The peculiar characteristic of enabling simple method development is particularly favourable in chiral analysis, as the requested amount of chiral selector is low and the change of separation conditions in the capillary column is a fast procedure. CE is deemed to be complementary to the traditional analytical techniques, such as HPLC, and is suitable for the confirmation of results. Similarities between liquid chromatography and CE have been demonstrated for systems using cyclodextrins57,58, which are smaller and more conformationally stable than proteins, and it seems reasonable to assume that information on retention and selectivity obtained by using one technique could be used on a mutual basis in predicting separations achieved through the use of the other.With protein selectors it would be interesting to take a unified approach and compare the data obtained by using the same protein in both HPLC and CE, in order to demonstrate the potential of CE to compete with well established LC chiral-bonded columns, and to assess the role of the immobilization process on the protein performance as chiral selector, because the CE system works instead in solution. Once
results are found to be comparable, it becomes possible to make a rational choice regarding the proteins to prepare in large quantities for use as the immobilized phase in HPLC. Preliminary qualitative approaches in this respect were occasionally made by some authors14,19,20,23, and more precise quantitative relationships have been investigated on human serum albumins59,60 and, in our laboratory, with quail egg-white RfBP (Ref. 48). Following the HPLC data obtained through using this selector, we singled out a mobile phase composition and transferred it as a BGE composition in the CE system.This procedure implies a few constraints and issues that must be evaluated (Box 1). First, BGE pH values that are close or below the protein isoelectric point may result in protein precipitation or protein adsorption to the fused silica capillary wall, respectively. In addition, only pH values within the range of good buffering capacity for phosphate buffer were allowed, as phosphate was the buffer used for the chromatographic experiments.The amount of protein immobilized on the column and that dissolved in the capillary differed considerably, the latter being approximately 150 times lower.This parameter is important, as when comparing enantioseparations performed in LC and CE, retention at least has been shown to be directly proportional to the selector concentration59,60. Furthermore, a drawback of the CE method occurs when the net mobility of the drug sample is similar to that of the selector added to the BGE, as in order to achieve chiral separations the mobilities of the free and protein-bound analyte must be significantly different.
0.36 0.27
HPLC a k’1
Analyte a
CE b k’1
30¡C 0.18 AU
Table 4. Comparison of k’ and a values obtained in HPLC and CE for the same compounds
25¡C
0.09
a
18¡C
0.00
67.97 45.45 31.18 11.64 19.53 11.34 5.78 17.52 2.53 aMobile bBGE:
1.71 2.35 1.38 1.16 1.25 8.41 2.91 1.31 2.78
Nicardipine Bepridil Amlopidine Verapamil Nimodipine Oxazepam Lorazepam Indoprofen Warfarin
phase: 50 mM sodium phosphate buffer (pH 6.5)–methanol (95:5 v/v)
50 mM sodium phosphate buffer (pH 6.5)–methanol (99:1 v/v)
Reproduced, with permission, from Ref. 48.
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2.10 0.44 0.29 0.17 0.19 0.20 0.14
1.22 2.05
-0.09 4.50
1.15 2.61
5.40
6.30 Min
7.20
8.10
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Figure 6. Capillary electrophoresis: influence of temperature on the efficiency and resolution of prilocaine. Conditions were fused silica capillary 50 mm 3 61.5 cm (46.5 cm to the detector), background electrolyte of 30 mM qYRfBP in 50 mM phosphate buffer at pH 6.0. Analyte concentration was 200 mM. Voltage was 20 kV with UV detection at 225 nm.
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and this may be ascribable to the difference in protein concentration in the two systems. The table shows that as long as a -0.20 compound showed high a values in HPLC it could also be suc200 mM cessfully enantioseparated in CE, and this observation might be -0.40 a useful rule for prediction purposes. -0.60 It must be stressed that in such studies it is extremely important 100 mM to employ the same batch of protein, as the variability of the pro-0.80 teinaceous matrix could be responsible for false differences in the 50 mM performance of the chiral selector in HPLC and in CE. Another -1.00 factor to be considered when working with quail egg-white RfBP in solution and on a general basis with proteins in solution, is to 7.00 8.00 4.00 5.00 6.00 Min investigate the stability of the protein from either the end of purification to utilization or during the CE analyses. Quail egg-white Figure 7. Capillary electrophoresis: influence of analyte concentration RfBP was found to give reproducible results for up to ten analyses, on resolution of oxprenolol. Conditions were fused silica capillary 50 mm 3 61.5 cm (46.5 cm to the detector). Background electrolyte providing that a suitable capillary rinsing procedure is performed was 30 mM qYRfBP in 50 mM phosphate buffer at pH 7.0. Analyte between runs61. Purified holo RfBP must be kept in the freezer (at concentration was 200 mM. Voltage was 20 kV, the temperature was 2708C) until one week before use and then extensively dialysed 188C and UV detection was at 225 nm. at pH 3.0 in order to eliminate the riboflavin bound to the protein. Thawed aliquots in phosphate buffer must be kept in the fridge For these reasons, we had to select suitable cationic, un(at 48C) and used within seven days without compromising one charged and anionic analytes from the HPLC pool of drugs, and of the main advantages of the technique; that is, minimum selecthe study was performed in the conditions shown in Table 4. tor consumption. Aliquot concentration was spectrophotometriThe lower concentration of protein in CE accounts for the lower cally checked every day and a CE analysis of a sample of qRfBP was percentage of methanol used. A difference of two orders of magalso found to be useful on a daily basis in order to verify the repronitude is observed for k9 values between the two techniques, ducibility of the retention times and of the electrophoretic pattern. A wider and more systematic study on the enantioselective performance of quail egg-white and quail egg-yolk RfBPs was 0.40 later performed in our laboratory on 20 compounds62; other drugs were added to Initial capillary those listed in Table 4, including calcium lifetime 0.20 400 mM antagonists, b-blockers and local anaesthetics. In order to optimize enantioseparations, the operative pH (6.0, 7.0 and 0.00 200 mM 8.0), temperature (188C, 258C and 308C) and analyte concentration (50 mM, 100 mM 50 mM and 200 mM) were varied.The outcome of -0.20 this screening revealed that no significant After 41 difference is observed in the enantioanalyses selective performance of qRfBP extracted 400 mM -0.40 from egg white and egg yolk, as confirmed 200 mM by the HPLC data, despite the described structural difference of the two proteins -0.60 for the chicken counterpart. As a general 15.00 18.00 6.00 9.00 12.00 Min rule, the higher the k9 values, the better the enantioseparation. Higher temperature Figure 8. Capillary electrophoresis: effect of protein adsorption to the capillary wall (building up) on values improved the efficiency but resulted the enantioseparation of warfarin. Conditions were fused silica capillary 50 mm 3 61.1 cm (46.1 cm in a deterioration in the resolution (Fig. 6): to the detector). Background electrolyte was 30 mM qYRfBP in 50 mM phosphate buffer at pH 6.0. Voltage was 20 kV, temperature was 308C and UV detection was at 247 nm. this effect might be due to faster interaction kinetics between ligand and protein. Pharmaceutical Science & Technology Today
Pharmaceutical Science & Technology Today
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AU
0.00
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(a)
0,00055
0.45
AU
m EOF(cm2/Vs)
0,0005 0,00045 0,0004
0.40 0.35
0,00035 0,0003
5.6
4.8
0,00025
6.4
7.2
8.0
Min
0,0002 0
5
10 15 Number of analyses
20
25
(b)
Figure 9. Capillary electrophoresis: influence of protein building up on EOF. Conditions were fused silica capillary 50 mm 3 61.5 cm (46.5 cm to the detector). Background electrolyte was 30 mM qYRfBP in 50 mM phosphate buffer at pH 8.0. Analyte concentration was 200 mM, voltage was 20 kV and temperature was 258C.
mAU
Pharmaceutical Science & Technology Today
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7.5
8.5
9.5
Min
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(c)
AU
0.4
0.2
4.5
5.4
6.3
7.2
Min
(d) 60 40 mAU
As previously reported25,63, the optimum protein concentration that gives the maximum enantioselectivity is in the range 10–1000 mM. Because RfBP concentrations above 50 mM elicit a strong detection response when using the complete filling technique, the selector concentration was always kept at 30 mM, whereas the analyte concentration was varied, as reported above. When approaching a 1:1 stoichiometry between the individual enantiomer and the selector concentration, the a values increased (Fig. 7). Despite a suitable between-run rinsing procedure and favourable operative pH values, it was sometimes impossible to control protein adhesion to the fused silica capillary wall, which occurred progressively during analyses. This phenomenon, already described for human serum albumin64, was responsible for the enantioseparations of warfarin at concentrations of 400 and 200 mM, which were not achieved at the beginning of the capillary lifetime (Fig. 8). Indeed, this process of progressive sticking of the protein to the capillary walls randomly affects the EOF (Fig. 9), and is responsible for fluctuating and therefore irreproducible results. In general the low k9 and poor a values observed suggested that higher protein concentrations and better efficiencies had to be achieved in order to obtain improved separations. These observations, together with the need to lower the analyte concentration without detection problems, led us to undertake CE experiments65 by using the partial filling technique, which is known to overcome all these issues in an elegant manner19,25. In brief, the capillary is partially filled with the protein solution by applying a given pressure for a given time, in order to obtain a separation zone (plug) that does not reach the detection window. Conditions are chosen so that the protein has a net negative charge and, by applying the voltage, migrates towards
20 0 2
4
6 Min
8
10
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Figure 10. Capillary electrophoresis: comparison of electropherograms obtained for oxprenolol (a) and (b) and prilocaine (c) and (d) by using the complete filling technique (a) and (c) and the partial filling technique (b) and (d), with qWRfBP as chiral selector. Conditions: the complete filling technique used fused silica capillary 50 mm 3 61.5 cm (46.5 cm to the detector), the background electrolyte was 30 mM qWRfBP in 50 mM phosphate buffer at pH 6.0. Voltage was 20 kV, with UV detection at 225 nm. Analyte concentration was 200 mM. The partial filling technique used polyvinylalcohol-coated capillary 50 mm 3 49.5 cm (41 cm to the detector), with a background electrolyte of 100 mM phosphate buffer at pH 6.0. Voltage was 20 kV, with UV detection at 200 nm. Analyte concentration was 200 mM, qWRfBP plug concentration was 300 mM and the protein plug length was 10 cm in (b) and 20 cm in (d). Reproduced, with permission, from Ref. 66.
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Table 5. Best enantioseparations obtained in CE using quail egg-white RfBP as chiral selector Analyte (mM)
Technique
qWRfBP selector
Plug length (cm)
a
Ref.
Oxprenolol 100 Prilocaine 200 Bupivacaine 50 Bepridil 200 Benfluorex 200 Mefloquine 200 Amlodipine 50 Nicardipine 200 Oxazepam 200 Lorazepam 200
Partial filling Partial filling Partial filling Complete filling Complete filling Complete filling Complete filling Complete filling Complete filling Complete filling
300 mM in 100 mM phosphate buffer pH 6.0 300 mM in 100 mM phosphate buffer pH 6.0 900 mM in 100 mM phosphate buffer pH 6.0 30 mM in 50 mM phosphate buffer pH 7.0 30 mM in 50 mM phosphate buffer pH 6.0 30 mM in 50 mM phosphate buffer pH 6.0 30 mM in 50 mM phosphate buffer pH 7.0 30 mM in 50 mM phosphate buffer pH 6.0 30 mM in 50 mM phosphate buffer pH 6.5-methanol (99:1 v/v) 30 mM in 50 mM phosphate buffer pH 6.5-methanol (99:1 v/v)
10 20 30
1.02 1.04 1.03 1.69 1.37 3.15 1.52 1.44 1.15 2.61
65 65 65 48 62 62 48 48 48 48
Conditions: Temperature: 25°C. Voltage: 20 kV. Detection: UV at 200 nm (oxprenolol, prilocaine, bupivacaine); 247 nm (bepridil, benfluorex, mefloquine, amlodipine, nicardipine, oxazepam, lorazepam and warfarin).
the anode, whereas the analytes migrate towards the detector at the cathodic end.The use of a polyvinylalcohol-coated capillary was necessary for these experiments. Indeed, we modified the classical format of the technique65 with the aim of obtaining reproducible plug lengths and consequently reproducible results. The relative standard deviations for the retention times and peak areas of prilocaine were below 1% and 2%, respectively. By using this technique it was possible to increase the concentration of quail egg-white RfBP up to 900 mM, to achieve efficiencies up to 230,000 theoretical plates per meter and to detect concentrations as low as 80 ng ml21. Figure 10 contains the electropherograms obtained for oxprenolol and prilocaine by using both the complete filling technique and the partial filling technique66.Table 5 shows the overall results obtained in CE with quail egg-white RfBP as chiral selector62,65. Conclusions The amount of important information obtained thus far on RfBPs can be exploited for further studies.There will be a deeper investigation into the chromatographic chiral performance, through either the evaluation of other species or a more precise assessment of column stability, reproducibility and lifetime, in order to compete with commercially available protein-based columns, such as the well known Chiral AGP (ChromTech AB, Hägersten, Sweden) and Ultron ES-OVM (Shinwa Chemical Industries, Kyoto, Japan). In addition, the accomplishment of HPLC affinity studies with RfBP extracted from bovine plasma will enable a closer approach to human plasma RfBP and will shed light on the role of drug intake from vitamin transport during pregnancy. In both cases, CE would offer a reliable tool for a preliminary screening and for confirmation of HPLC results. Also, through affinity CE experiments, CE would avoid waste of precious purified protein amounts and would help in the calculation of the association constants between protein and drugs.
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In the September issue of Drug Discovery Today… Update – latest news and views Imaging systems in assay screening Peter Ramm Microdispensing technologies in drug discovery Don Rose RNA as a small-molecule drug target: doubling the value of genomics David J. Ecker and Richard H. Griffey Cell-based assays and instrumentation for screening ion-channel targets Jesús E. González, Kahuku Oades, Yan Leychkis, Alec Harootunian and Paul A. Negulescu
Monitor – new bioactive molecules, combinatorial chemistry, invited profile Products
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Riboflavin binding proteins as chiral selectors in HPLC and CE Ersilia De Lorenzi and Gabriella Massolini The term riboflavin binding proteins (RfBPs) is applied to several molecular species that play the important role of vitamin delivery to the developing embryo, thus becoming essential for the survival of the fetus. In addition to this physiological significance, these proteins have recently been found to be successful chiral selectors. In this review, the authors address the use of such proteins, both as columns for high performance liquid chromatography (HPLC) and as additives in capillary electrophoresis (CE), for the enantioseparation of several racemic drugs.
Ersilia De Lorenzi* and Gabriella Massolini University of Pavia Department of Pharmaceutical Chemistry Via Taramelli 12 I-27100 Pavia Italy *tel: 139 0382 507383 fax: 139 0382 422975 e-mail: [email protected]
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▼ Several chemical compounds used in pharmaceutical formulations feature one or more chiral centres, responsible for optical activity, that can strongly influence their pharmacological and pharmacokinetic properties. In fact, optical isomers are often distinguished by biological systems in the human body, and may therefore have different pharmacokinetic profiles and qualitatively and quantitatively different pharmacological or toxicological effects. For these reasons the development of new approaches for the separation of chiral compounds is a source of global research efforts and innovative advances. Furthermore, the impetus for this vibrant activity lies with the increased pressure and concern from regulatory agencies such as the FDA. Enantioselective high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) are successfully used for analytical purposes; namely the direct separation of drug racemates, the monitoring of stereoselective metabolism and for optical purity tests in pharmaceutical dosage forms. Several chiral selectors have hitherto been employed as either chiral columns for HPLC or as additives to the background electrolyte in CE, and thus in this respect proteins play an important role.
Proteins feature several unique qualities that make them versatile, fascinating and successful chiral selectors. Because of their chiral nature, they often interact differently with stereoisomers of either chiral macromolecules or small molecules by reversible binding. As a result of the variety of functional groups present at their surface, proteins can interact with chiral entities by forming not only relatively weak and non-specific bonds, but also stronger and more specific interactions, which involve a well-defined site of the protein itself. Electrostatic and hydrophobic bonds, as well as hydrogen bonds, p–p bonds and inclusion phenomena, may participate in the interaction between a protein and a chiral drug. The broad applicability of proteins as chiral selectors is evident in the large number of racemates separated so far, and is extended by the possibility of operating in an aqueous buffered system, which is compatible with many biological samples. The versatility of proteins is further expressed by the potential of simple modifications to the mobile phase (or background electrolyte) parameters for inducing a reversible change of the selector conformation, thus obtaining different enantioselective properties of the same protein. Different pH values, type and concentration of organic modifier can, in fact, alter the interactions between the selector and the analyte. For these reasons, the past 15 years have seen a growing development of protein-based stationary phases for HPLC, which have been extensively used and successfully applied for the direct separation of enantiomers1–5.The first chiral columns to be developed and marketed were those made of bovine serum albumin6, human serum albumin7, a1-acid glycoprotein8 (AGP) and ovomucoid9 (OVM), later followed by cellobiohydrolase10 and avidin11. Also, the use of conalbumin12, pepsin13 and b-lactoglobulin14 has been reported by academic research groups. The physiological
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PSTT Vol. 2, No. 9 September 1999
role of human serum albumin and a1-acid glycoprotein has led to additional investigations15–18, with the aim of elucidating the stereoselective binding mechanisms and to obtain a more in-depth knowledge of the characterization of the binding site. In the early 1990s, proteins began to be used as chiral selectors in CE19,20, and since then increasing numbers of papers have appeared, as well as review articles21,22. As purified proteins are expensive and, in some cases, are not even commercially available, the CE approach is of particular advantage over HPLC. Most authors simply dissolve the selector in the background electrolyte (complete filling technique), and thus a few milligrams of protein are required and immobilization on a solid support is unnecessary. All of the proteins already mentioned as HPLC chiral stationary phases have been evaluated as chiral selectors in CE19,20,23–35, and, in addition, human serum transferrin36,37 and casein38 have been investigated. Riboflavin binding proteins In our laboratories we have undertaken the evaluation of riboflavin binding proteins (RfBPs) as potential chiral selectors for the enantioseparation of drug racemates. Several factors contributed to this choice, namely the physiological role of these proteins, the presence of a specific binding region, the possibility of extraction from different matrices and the awareness that, at the beginning of our investigation, the threedimensional structure characterization of chicken egg-white RfBP was about to be accomplished by X-ray diffraction39. The term riboflavin binding proteins is applied to several molecular species that are involved in the transport and storage of the vitamin, with particular emphasis on the role of supplying this micronutrient to the developing embryo40. Pregnancyspecific RfBPs have been found in mammalian species, including humans41–43, and they all appear to share similarities with the well known chicken riboflavin binding proteins. Chicken egg-white and -yolk RfBP share the same aminoacid sequence, but have undergone different post-translational modifications; that is, their carbohydrate chains are different and the latter lacks the last 11–13 aminoacids that are proteolytically cleaved. Peculiar characteristics of RfBPs extracted from chicken egg and chicken plasma are a highly phosphorylated region and a high degree of crosslinking by nine disulfide bridges, the former being essential for vitamin uptake and the latter being responsible for the high protein stability. Crystallographic data39 shed light on the ligand binding-site characterization and on the aminoacids involved in the binding with riboflavin. Interestingly, an additional succinate-binding cleft was described. RfBPs extracted and purified from the eggs of avian species other than chicken have also been investigated, including quail44, goose45 and duck46 RfBPs.
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Common features and differences among the RfBPs from these species and their chicken counterpart have been reported. For example, the RfBPs from domestic fowl and quail eggs have been compared44 by circular dichroism and fluorescence and peptide mapping, and the outcome of this study revealed small but significant differences in their tertiary structure that must reflect differences in the aminoacid sequence. It is interesting that the goose egg-yolk protein45 appears to have a significantly higher molecular weight and a much higher proportion of b-sheet than either chicken or quail RfBP. In addition, goose egg-yolk RfBP presents an unusually high carbohydrate content as well as glucose residues that are not believed to be present in any other RfBP.All these characteristics, together with differences in the far ultra violet circular dichroism spectra of goose egg-yolk and chicken egg-white RfBPs are important in accounting for differences in the secondary structures, despite the similar aminoacid composition. Furthermore, goose egg-yolk RfBP appears to have a larger number of phosphate residues than that reported for chicken egg-yolk RfBP, and its large amount of carbohydrates is in marked contrast to that of duck egg white, which lacks covalently bound oligosaccharides46. The ready availability of the matrix and the idea of obtaining a deeper insight into the subtle structural differences of these proteins through a chromatographic investigation, prompted us to set up extraction, purification and immobilization protocols to exploit chicken- and quail-egg RfBPs as chiral stationary phases for HPLC47,48. The large number of data produced by analysing drug racemates with different structures would offer additional knowledge for the comparison of RfBPs from avian species. Parallel CE experiments have also been planned on the basis of findings and ideas originated in HPLC in order to evaluate whether CE could be used as a rapid scouting technique for screening the enantioselectivity of novel proteins, and to verify to what extent the protein performance is affected by the immobilization process48. RfBPs have also been purified from the plasma of pregnant cows41, although the characterization of this protein is yet to be fully investigated. RfBP plays an important role in mammals in terms of fetal vitamin nutrition through the transplacental transport of riboflavin across the placental barrier49. Preliminary data are also available on the isolation and characterization of RfBP from human amniotic fluid50 and immunological similarities with the chicken counterpart have been demonstrated, although further studies are to be performed. It is important to mention that immunoneutralization of the endogenous RfBP in pregnant mammals leads to embryonic death due to acute vitamin deficiency51. Because binding of drugs is a part of the physiological role of proteins, a high performance liquid affinity chromatographic approach with stationary phases based on bovine plasma RfBP could be envisaged, in order to investigate the effect of drug binding on vitamin transport during pregnancy. Extraction and purification protocols of RfBPs from the plasma of pregnant cows 353
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Table 1. Best enantioseparations obtained in HPLC using RfBP-based chiral stationary phases Analyte
Column
k’
a
Mobile phase composition
Ref.
Ibuprofen
qWRfBP cWRfBP cYRfBP qYRfBP cWRfBP qWRfBP cYRfBP qYRfBP qWRfBP cYRfBP cYRfBP cWRfBP cYRfBP cYRfBP cWRfBP qYRfBP qWRfBP cYRfBP cWRfBP qYRfBP qWRfBP qYRfBP cYRfBP qWRfBP cYRfBP qYRfBP qWRfBP cYRfBP qYRfBP qWRfBP cYRfBP qYRfBP qWRfBP cYRfBP qYRfBP qWRfBP cYRfBP qYRfBP qWRfBP cYRfBP qYRfBP qWRfBP qWRfBP qYRfBP qWRfBP cYRfBP qWRfBP qYRfBP cWRfBP cWRfBP qYRfBP qWRfBP qWRfBP cWRfBP cWRfBP cWRfBP cWRfBP cWRfBP
3.92 8.52 1.38 1.2 23.94 9.71 2.56 19.2 13.83 9.84 6.10 17.99 8.27 30.42 38.25 7.18 11.60 4.67 16.41 2.72 4.01 3.43 1.56 5.78 4.91 5.31 11.34 4.87 7.06 14.00 8.57 4.7 3.57 0.72 37.7 20.08 10.61 29.8 17.00 232.94 17.3 31.32 7.8 6.17 4.42 11.96 3.09 24.1 0.96 0.79 2.89 5.93 1.21 13.13 3.46 1.48 5.61 1.01
1.69 1.07 1.13 1.07 1.29 1.07 1.22 1.32 1.24 1.16 1.13 1.06 1.08 2.20 1.50 2.36 2.59 1.39 2.16 1.16 1.28 2.14 1.20 2.91 1.87 6.92 8.42 6.68 1.21 1.15 1.28 1.43 1.95 1.10 1.58 1.53 1.32 1.43 1.38 1.32 1.17 1.25 1.07 1.14 1.11 1.21 2.19 2.57 1.09 1.27 1.28 1.37 8.77 1.14 1.51 1.72 3.79 1.52
50 mM NaH2PO4 (pH 3.5)-CH3OH (95:5 v/v) 50 mM CH3COONa (pH 5.0)- CH3OH (95:5 v/v) 50 mM KH2PO4 (pH 5.5)-CH3OH (95:5 v/v) 50 mM KH2PO4 (pH 6.5)-CH3OH (95:5 v/v) 50 mM KH2PO4 (pH 4.6)-CH3CH2OH (96:4 v/v) 50 mM NaH2PO4 (pH 4.5)-CH3OH (95:5 v/v) 50 mM KH2PO4 (pH 5.5) 50 mM KH2PO4 (pH 5.5)-CH3OH (95:5 v/v) 50 mM NaH2PO4 (pH 4.5)-CH3CN (95:5 v/v) 50 mM KH2PO4 (pH 6.5)-CH3CH2OH (90:10 v/v) 50 mM KH2PO4 (pH 5.5) 50 mM CH3COONa (pH 5.0)-CH3OH (95:5 v/v) 50 mM KH2PO4 (pH 5.5) 50 mM KH2PO4 (pH 5.5)-CH3OH (95:5 v/v) 50 mM CH3COONa (pH 5.0)-CH3OH (95:5 v/v) 50 mM KH2PO4 (pH 5.5)-CH3OH (95:5 v/v) 50 mM NaH2PO4 (pH 4.5)-CH3OH (95:5 v/v) 50 mM KH2PO4 (pH 4.6)-CH3CH2OH (90:10 v/v) 50 mM KH2PO4 (pH 4.6)-CH3CH2OH (96:4 v/v) 50 mM KH2PO4 (pH 3.5)-CH3OH (95:5 v/v) 50 mM NaH2PO4 (pH 3.5)-CH3OH (95:5 v/v) 50 mM KH2PO4 (pH 6.5)-CH3OH (95:5 v/v) 50 mM KH2PO4 (pH 4.5)-CH3OH (95:5 v/v) 50 mM NaH2PO4 (pH 6.5)-CH3OH (95:5 v/v) 50 mM KH2PO4 (pH 5.5)-CH3OH (95:5 v/v) 50 mM KH2PO4 (pH 6.5)-CH3OH (95:5 v/v) 50 mM NaH2PO4 (pH 6.5)-CH3OH (95:5 v/v) 50 mM KH2PO4 (pH 5.5)-CH3OH (95:5 v/v) 50 mM KH2PO4 (pH 6.5)-CH3OH (95:5 v/v) 50 mM NaH2PO4 (pH 4.5)-CH3OH (95:5 v/v) 50 mM KH2PO4 (pH 5.5) 50 mM KH2PO4 (pH 4.5)-CH3OH (95:5 v/v) 50 mM NaH2PO4 (pH 4.5)-CH3(CH2)2OH (95:5 v/v) 50 mM KH2PO4 (pH 5.5) 50 mM KH2PO4 (pH 6.5)-CH3OH (95:5 v/v) 50 mM NaH2PO4 (pH 5.5)-CH3OH (95:5 v/v) 50 mM KH2PO4 (pH 5.5)-CH3CH2OH (95:5 v/v) 50 mM KH2PO4 (pH 4.5)-CH3OH (95:5 v/v) 50 mM NaH2PO4 (pH 6.5)-CH3CN (95:5 v/v) 50 mM KH2PO4 (pH 5.5)-CH3CH2OH (95:5 v/v) 50 mM KH2PO4 (pH 5.5)-CH3OH (95:5 v/v) 50 mM NaH2PO4 (pH 5.5)-CH3OH (95:5 v/v) 50 mM NaH2PO4 (pH 6.5)-CH3OH (95:5 v/v) 50 mM KH2PO4 (pH 6.5)-CH3OH (95:5 v/v) 50 mM NaH2PO4 (pH 5.5)-CH3OH (95:5 v/v) 50 mM KH2PO4 (pH 5.5)-CH3CH2OH (95:5 v/v) 50 mM NaH2PO4 (pH 4.5)-CH3(CH2)2OH (95:5 v/v) 50 mM KH2PO4 (pH 6.5)-CH3OH (95:5 v/v) 20 mM KH2PO4 (pH 4.0)-CH3OH (90:10 v/v) 50 mM CH3COONa (pH 5.0)-CH3OH (95:5 v/v) 50 mM KH2PO4 (pH 6.5)-CH3OH (95:5 v/v) 50 mM NaH2PO4 (pH 6.5)-CH3OH (95:5 v/v) 50 mM NaH2PO4 (pH 5.5)-CH3OH (95:5 v/v) 50 mM KH2PO4 (pH 4.6)-CH3CH2OH (96:4 v/v) 50 mM KH2PO4 (pH 4.6)-(CH3)3COH (96:4 v/v) 20 mM KH2PO4 (pH 6.0)-CH3OH (98:2 v/v) 50 mM CH3COONa (pH 5.0)-CH3OH (95:5 v/v) 50 mM CH3COONa (pH 5.0)-CH3OH (95:5 v/v)
48 67 47 52 54 48 47 52 48 47 47 67 47 47 67 52 48 47 54 52 48 52 47 48 47 52 48 47 52 48 47 52 48 47 52 48 47 52 48 47 52 48 48 52 48 47 48 52 53 67 52 48 48 54 54 53 67 67
Ketoprofen
Indoprofen
Flurbiprofen Suprofen Carprofen Pranoprofen Warfarin
Lormetazepam Lorazepam
Oxazepam
Isradipine
Amlodipine Nimodipine Nicardipine
Manidipine
Lercanidipine Gallopamil Verapamil Bepridil
Propranolol Oxprenolol Fenfluoramine Bupivacaine a,e Dibenzoyllysine Benzoin Chlormezanone Proglumide Aminogluthetimide
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and from human amniotic fluid, together with identification with chicken plasma antibodies, are currently at a stage in our laboratories where their use in affinity CE or affinity HPLC will soon become feasible. RfBP-based HPLC columns RfBP is not very abundant in either the egg yolk or in the egg white of avian species40, and as the amount of protein required to produce an HPLC column is relatively large, an optimization of the purification process becomes necessary in order to obtain approximately 300– 400 mg per batch. So far, four stationary phases have been described as chiral selectors; namely those based on chicken egg yolk (cYRfBP)47, quail egg yolk (qYRfBP)52, quail egg white (qWRfBP)48 and chicken egg white (cWRfBP)53,54.
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Preparation of chicken- and quail-egg RfBP columns (e) (f) Purification. RfBPs from different avian species and different matrices were purified by suitable modification47,48 of widely used methods55,56. Monitoring of the purification process and analyses of the final preparation for homogeneity were performed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate and by isoelectric focusing with carrier ampholytes (pH 3.5–9.5). Immobilization. Immobilization was per0 10 20 30 40 50 0 5 10 15 20 25 30 formed as follows: 5 NH2 Nucleosil was Min Min slurried in HPLC-grade acetonitrile and Pharmaceutical Science & Technology Today N,N-disuccinylimidyl carbonate was added. Figure 1. Chromatograms of some of the compounds tested on chicken egg-yolk RfBP. After stirring, filtering and washing, this (a) nicardipine; (b) isradipine; (c) carprofen; (d) warfarin; (e) oxazepam; and (f) lorazepam. activated silica was added to the protein Chromatographic conditions were mobile phase 50 mM KH2PO4 (pH 5.5)–ethanol (95:5 v/v) in (a), previously suspended in buffer. The ob(c), (e) and (f); the mobile phase was 50 mM KH2PO4 (pH 5.5) in (b), and in (d) the mobile phase tained stationary phase was gently mixed was 50 mM KH2PO4 (pH 4.6)–ethanol (90:10 v/v). The flow rate was 0.8 ml min21. Column dimensions were 100 3 4.6 mm I.D. Reproduced, with permission, from Ref. 47. using the rotor evaporator and then 47,54 packed in a stainless steel column . Applicability. The applicability of the developed RfBP columns as chiral selectors for liquid chromatograare shown in Figs 1 and 2: high enantioselectivity and favourphy was tested by analysing a large number of racemic drugs with able chromatographic performance were obtained. Our interest different chemical properties (basic, acidic and neutral) and which was then focused on controlling the enantioselective retention belonged to different pharmaceutical classes. Chromatographic by varying the pH of the mobile phase and by adding different results are presented in Table 1 and representative chromatograms kinds and amounts of organic modifiers. 355
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Figure 2. Chromatograms of some of the compounds tested on quail egg-white RfBP. (a) amlodipine; (b) warfarin; (c) bupivacaine; (d) indoprofen; (e) fenfluramine; (f) bepridil; (g) oxazepam; and (h) lorazepam. Chromatographic conditions were mobile phase 50 mM phosphate buffer (pH 4.5)–methanol (90:10 v/v) in (a), (f) and (g); the mobile phase was 50 mM phosphate buffer (pH 4.5)–methanol (95:5 v/v) in (h); (d) and (b) featured a mobile phase of 50 mM phosphate buffer (pH 4.5)–acetonitrile (95:5 v/v); and the mobile phase was 50 mM phosphate buffer (pH 5.5)–methanol (95:5 v/v) in (c) and (e). The flow rate was 0.8 ml min21 and the column dimensions were 100 3 4.6 mm I.D. Reproduced, with permission, from Ref. 48.
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Influence of pH and organic modifiers on retention and chiral resolution Retention (k9) and enantioselectivity (Rs or a) of RfBP columns are essentially influenced by the pH of the mobile phase and by the percentage of organic solvent. Depending on the charge of the solute, a change in pH in the mobile phase, such as the one considered in our studies (3.5–6.5) (Refs 47,48), may cause either an increase or a decrease of the k9 values. In general, the effect of pH on retention is markedly larger for charged than for uncharged solutes, and the affinity for the stationary phase increases as the compounds become less ionized. This behaviour was observed for all of the RfBP columns and it provides evidence of the contribution of hydrophobic interactions on the binding between drugs and protein.
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RfBPs have an isoelectric point of approximately 4.0 and therefore they bear a net negative charge above this pH value. For the acidic compounds, whose pKa values are between 4.0 and 5.0, the maximum in retention and enantioselectivity was obtained at pH 4.5, where both protein and analytes are uncharged: this effect is caused by an increase in the non-polar interactions between solutes and RfBP. The k’s of basic compounds, whose pKa values are between 8.0 and 9.0, rose on increasing the pH, and this behaviour is related to the increase in the net negative charge of the protein that leads to a rise in the number of coulombic interactions with the cationic compounds. In the case of basic compounds, the enantioselectivity was not influenced to a great extent by pH variations. The retention time of uncharged solutes was almost independent of the pH of the mobile phase, although the enantioselectivity for benzodiaze12.5 pines, uncharged at the considered pH 10 values, rises upon increasing the pH from 7.5 5 3.5 to 6.5, and this trend is because 2.5 of the remarkable increase in the re0 tention time of the second eluted en-2.5 antiomer. The high selectivity shown by -5 oxazepam at pH 6.5 (Fig. 3) on the -7.5 RfBP–CSPs47,48,52 suggested that the two 80 100 0 20 40 60 Min enantiomers bind at different loci and, in Pharmaceutical Science & Technology Today particular, that the first eluted enantiomer interacts non-specifically with the proFigure 3. Chromatographic profile of oxazepam. Chromatographic conditions were 50 mM KH2PO4 tein, whereas the second eluted enan21 (pH 6.5)–methanol (95:5 v/v), and the flow rate was 0.8 ml min . The column used was qWRfBP 125 3 4.0 mm I.D. tiomer interacts with the riboflavin binding site.
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Figure 4. Influence of pH on k9 for some compounds tested on qYRfBP (a) and qWRfBP (b) columns. Chromatographic conditions were 50 mM KH2PO4–methanol (95:5 v/v). The sample was indoprofen (black diamond), carprofen (black circle), verapamil (black triangle), and bepridil (black square). Column dimensions were 100 3 4.6 mm I.D.
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25 20
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Oxazepam
Nicardipine
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Warfarin
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Nicardipine
Amlodipine
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Figure 5. Istograms of k9, a and Rs of selected drugs, analysed on chicken (black) and quail (white) egg-yolk RfBP. The selected drugs were warfarin, oxazepam, nicardipine and amlodipine. Chromatographic conditions were 50 mM KH2PO4 (pH 5.5)–methanol (95:5 v/v), the flow rate was 0.8 ml min21, and column dimensions were 100 3 4.6 mm I.D.
Different organic modifiers, such as methanol, ethanol, npropanol, 2-propanol and acetonitrile, have been used at different percentages to modulate retention and selectivity. By increasing their concentration in the mobile phase, capacity factors and Rs decrease, and this effect was more pronounced for the most hydrophobic compounds, such as carprofen and manidipine. These results indicate that the system works in a similar manner to a reversed-phase, and that hydrophobic interactions are important for the retention mechanism. In general, the addition of methanol to the mobile phase resulted in better resolution values than those obtained with other organic modifiers. 358
Species and matrices comparison The chromatographic performances and the enantioselective properties of the qYRfBP and qWRfBP columns were compared in order to display possible different behaviours connected to the different matrix origin52. The two columns gave a similar k9 pattern for all analytes, as shown in Fig. 4 for four selected drugs. In terms of the acidic compounds, an increase in the pH results in k9 values with a maximum at pH 4.5, where both the RfBPs and analytes are uncharged and ionic attraction phenomena do not occur. The k9 values of basic analytes were highly influenced by pH variations, whereas the capacity factors of neutrals (not reported in Fig. 4) were almost unaffected. RfBPs from both quail egg white and egg yolk immobilized on silica are good chiral selectors, as they could resolve a large number of the racemic compounds tested (Table 1), with a slightly better performance given by the former. In particular, antiinflammatory drugs were poorly resolved, whereas dihydropyridines and benzodiazepines gave the best enantioseparations. As the trend observed for the two columns was found to be similar, in terms of both retention and enantioselectivity, it is therefore reasonable to assume that the stereoselective regions of RfBPs extracted from different matrices (egg yolk and egg white) of the same species (quail) do not show considerable differences. Stationary phases prepared with egg-yolk RfBP from different species were used to investigate whether small differences in the aminoacid sequence, in both the tertiary structure and in the carbohydrate content, could influence the retention and, in particular, the selectivity properties of RfBP-based columns. In order to compare the chromatographic results of chicken47 and quail52 egg-yolk RfBP columns, we selected four model compounds: warfarin, oxazepam, amlodipine and nicardipine. Figure 5 shows the comparison of capacity factors (k9), selectivity (a) and resolution (Rs), measured under the same chromatographic conditions using the two chiral stationary phases.The retention of all compounds was higher for the chicken YRfBP column, except for the acidic compound warfarin. In terms of selectivity and resolution, improved a and Rs values were obtained for warfarin and nicardipine when analysed on the quail YRfBP column, whereas for oxazepam the best results were obtained with chicken YRfBP–CSP, and amlodipine enantiomers were successfully resolved only by the qYRfBP stationary phase. In general, the addition of increasing percentages of an organic modifier to the mobile phase meant that retention of the analytes on all YRfBP–CSPs decreased, while the enantiomeric separation was either reduced or remained substantially unchanged. The effect of the buffer pH on retention is markedly more evident for charged than for uncharged analytes, although it is similar for the two YRfBP columns. Generally, basic compounds are more strongly retained at higher pH values, whereas the opposite is true for acidic drugs.
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Table 2. Comparison of qWRfBP-based stationary phases Analyte k’
Column A qWRfBP a
Rs
k’
Column B qWRfBP a
Rs
k’
Column C qWRfBP a
Rs
Amlodipine
7.55
1.87
2.61
3.09
1.78
3.03
3.47
2.11
3.02
Bepridil
5.49
1.85
3.53
2.56
1.90
4.88
3.27
2.23
3.47
Ketoprofen
9.71
1.07
0.41
9.83
1.06
0.5
7.73
1.10
0.47
Lorazepam
4.45
1.66
3.04
2.34
1.78
3.41
2.73
1.86
3.09
Nicardipine
8.79
1.18
0.73
4.54
1.17
1
3.47
2.11
3.02
Warfarin
11.60
2.59
5.48
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2.51
4.41
12.92
3.09
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Indoprofen
53.77
1.31
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31.72
1.39
2.61
49.15
1.48
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Mobile phase: 50 mM KH2PO4 (pH 4.5)–methanol (95:5 v/v); flow rate 0.8 ml min21 Column dimensions: 100 x 4.6 mm I.D. (from Refs 48 and 52)
The enantioselective capability of the developed CSPs might be related to the interaction with the stereoselective riboflavinbinding site. Further systematic work on RfBP columns from different species with several model chiral drugs will be important, for a better understanding of both the RfBP structures and the stereoselective role of the binding site. Reproducibility and stability The measurement of capacity factors and resolution factors can be exploited for the study of column reproducibility, providing that the temperature and solvent composition are controlled.The enantioselective performance of three different qWRfBP stationary phases is presented in Table 2.A decrease in retention was observed between columns A and B, which were obtained from different protein purification batches; an explanation for the retention variation might be the different amount of immobilized protein, namely 104.26 mg g21 silica for column A and 54.41 mg g21 silica for column B, as calculated by elemental analysis. Good reproducibility of the enantioselectivity was obtained for columns packed with the same protein purification batch (columns B and C) and also from different batches (column A and B).The small differences in the Rs values observed between the three columns are related to the column hardware reproducibility and to the consistency of the column packing technique. Column stability was investigated by evaluating the chromatographic performance for warfarin at the start of the column lifetime and after 250 injections, under the required conditions
in method development studies, such as broad changes in mobile phase composition and pH. The chromatographic parameters, such as Rs, efficiency and retention, were almost unchanged (Table 3), thus proving that the column is robust as well as reliable for routine analysis. The same compound has since been used as a probe to ensure that adequate stereoselective performance was validated at regular intervals. RfBPs as additives in CE During the past ten years, CE has penetrated a wide range of areas of separation science and has been recognized as a powerful, viable, reliable, successful and versatile technique. CE features unique advantages, such as speed, minimum solvent Table 3. Stability of cYRfBP-based column using warfarin as test analytea Parameter
1st analysis
After 250 analyses
k’1 k’2 a Rs Plates/m
4.9 8.5 1.73 2.81 15,020
4.64 7.92 1.70 2.49 11,980
aMobile
phase: 50 mM KH2PO4 (pH 5.5)–ethanol (95:5 v/v); flow rate 0.8 ml min21
Column dimensions: 100 x 4.6 mm I.D. Reproduced, with permission, from Ref. 47.
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Box 1. Issues to be considered in method transfer (HPLC®CE) when using proteins as chiral selectors • • • • •
pH value Protein concentration Mobility match Protein batch Protein stability
consumption, small sample volumes, high selectivity and ease of automation. The peculiar characteristic of enabling simple method development is particularly favourable in chiral analysis, as the requested amount of chiral selector is low and the change of separation conditions in the capillary column is a fast procedure. CE is deemed to be complementary to the traditional analytical techniques, such as HPLC, and is suitable for the confirmation of results. Similarities between liquid chromatography and CE have been demonstrated for systems using cyclodextrins57,58, which are smaller and more conformationally stable than proteins, and it seems reasonable to assume that information on retention and selectivity obtained by using one technique could be used on a mutual basis in predicting separations achieved through the use of the other.With protein selectors it would be interesting to take a unified approach and compare the data obtained by using the same protein in both HPLC and CE, in order to demonstrate the potential of CE to compete with well established LC chiral-bonded columns, and to assess the role of the immobilization process on the protein performance as chiral selector, because the CE system works instead in solution. Once
results are found to be comparable, it becomes possible to make a rational choice regarding the proteins to prepare in large quantities for use as the immobilized phase in HPLC. Preliminary qualitative approaches in this respect were occasionally made by some authors14,19,20,23, and more precise quantitative relationships have been investigated on human serum albumins59,60 and, in our laboratory, with quail egg-white RfBP (Ref. 48). Following the HPLC data obtained through using this selector, we singled out a mobile phase composition and transferred it as a BGE composition in the CE system.This procedure implies a few constraints and issues that must be evaluated (Box 1). First, BGE pH values that are close or below the protein isoelectric point may result in protein precipitation or protein adsorption to the fused silica capillary wall, respectively. In addition, only pH values within the range of good buffering capacity for phosphate buffer were allowed, as phosphate was the buffer used for the chromatographic experiments.The amount of protein immobilized on the column and that dissolved in the capillary differed considerably, the latter being approximately 150 times lower.This parameter is important, as when comparing enantioseparations performed in LC and CE, retention at least has been shown to be directly proportional to the selector concentration59,60. Furthermore, a drawback of the CE method occurs when the net mobility of the drug sample is similar to that of the selector added to the BGE, as in order to achieve chiral separations the mobilities of the free and protein-bound analyte must be significantly different.
0.36 0.27
HPLC a k’1
Analyte a
CE b k’1
30¡C 0.18 AU
Table 4. Comparison of k’ and a values obtained in HPLC and CE for the same compounds
25¡C
0.09
a
18¡C
0.00
67.97 45.45 31.18 11.64 19.53 11.34 5.78 17.52 2.53 aMobile bBGE:
1.71 2.35 1.38 1.16 1.25 8.41 2.91 1.31 2.78
Nicardipine Bepridil Amlopidine Verapamil Nimodipine Oxazepam Lorazepam Indoprofen Warfarin
phase: 50 mM sodium phosphate buffer (pH 6.5)–methanol (95:5 v/v)
50 mM sodium phosphate buffer (pH 6.5)–methanol (99:1 v/v)
Reproduced, with permission, from Ref. 48.
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2.10 0.44 0.29 0.17 0.19 0.20 0.14
1.22 2.05
-0.09 4.50
1.15 2.61
5.40
6.30 Min
7.20
8.10
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Figure 6. Capillary electrophoresis: influence of temperature on the efficiency and resolution of prilocaine. Conditions were fused silica capillary 50 mm 3 61.5 cm (46.5 cm to the detector), background electrolyte of 30 mM qYRfBP in 50 mM phosphate buffer at pH 6.0. Analyte concentration was 200 mM. Voltage was 20 kV with UV detection at 225 nm.
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and this may be ascribable to the difference in protein concentration in the two systems. The table shows that as long as a -0.20 compound showed high a values in HPLC it could also be suc200 mM cessfully enantioseparated in CE, and this observation might be -0.40 a useful rule for prediction purposes. -0.60 It must be stressed that in such studies it is extremely important 100 mM to employ the same batch of protein, as the variability of the pro-0.80 teinaceous matrix could be responsible for false differences in the 50 mM performance of the chiral selector in HPLC and in CE. Another -1.00 factor to be considered when working with quail egg-white RfBP in solution and on a general basis with proteins in solution, is to 7.00 8.00 4.00 5.00 6.00 Min investigate the stability of the protein from either the end of purification to utilization or during the CE analyses. Quail egg-white Figure 7. Capillary electrophoresis: influence of analyte concentration RfBP was found to give reproducible results for up to ten analyses, on resolution of oxprenolol. Conditions were fused silica capillary 50 mm 3 61.5 cm (46.5 cm to the detector). Background electrolyte providing that a suitable capillary rinsing procedure is performed was 30 mM qYRfBP in 50 mM phosphate buffer at pH 7.0. Analyte between runs61. Purified holo RfBP must be kept in the freezer (at concentration was 200 mM. Voltage was 20 kV, the temperature was 2708C) until one week before use and then extensively dialysed 188C and UV detection was at 225 nm. at pH 3.0 in order to eliminate the riboflavin bound to the protein. Thawed aliquots in phosphate buffer must be kept in the fridge For these reasons, we had to select suitable cationic, un(at 48C) and used within seven days without compromising one charged and anionic analytes from the HPLC pool of drugs, and of the main advantages of the technique; that is, minimum selecthe study was performed in the conditions shown in Table 4. tor consumption. Aliquot concentration was spectrophotometriThe lower concentration of protein in CE accounts for the lower cally checked every day and a CE analysis of a sample of qRfBP was percentage of methanol used. A difference of two orders of magalso found to be useful on a daily basis in order to verify the repronitude is observed for k9 values between the two techniques, ducibility of the retention times and of the electrophoretic pattern. A wider and more systematic study on the enantioselective performance of quail egg-white and quail egg-yolk RfBPs was 0.40 later performed in our laboratory on 20 compounds62; other drugs were added to Initial capillary those listed in Table 4, including calcium lifetime 0.20 400 mM antagonists, b-blockers and local anaesthetics. In order to optimize enantioseparations, the operative pH (6.0, 7.0 and 0.00 200 mM 8.0), temperature (188C, 258C and 308C) and analyte concentration (50 mM, 100 mM 50 mM and 200 mM) were varied.The outcome of -0.20 this screening revealed that no significant After 41 difference is observed in the enantioanalyses selective performance of qRfBP extracted 400 mM -0.40 from egg white and egg yolk, as confirmed 200 mM by the HPLC data, despite the described structural difference of the two proteins -0.60 for the chicken counterpart. As a general 15.00 18.00 6.00 9.00 12.00 Min rule, the higher the k9 values, the better the enantioseparation. Higher temperature Figure 8. Capillary electrophoresis: effect of protein adsorption to the capillary wall (building up) on values improved the efficiency but resulted the enantioseparation of warfarin. Conditions were fused silica capillary 50 mm 3 61.1 cm (46.1 cm in a deterioration in the resolution (Fig. 6): to the detector). Background electrolyte was 30 mM qYRfBP in 50 mM phosphate buffer at pH 6.0. Voltage was 20 kV, temperature was 308C and UV detection was at 247 nm. this effect might be due to faster interaction kinetics between ligand and protein. Pharmaceutical Science & Technology Today
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(a)
0,00055
0.45
AU
m EOF(cm2/Vs)
0,0005 0,00045 0,0004
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0,00035 0,0003
5.6
4.8
0,00025
6.4
7.2
8.0
Min
0,0002 0
5
10 15 Number of analyses
20
25
(b)
Figure 9. Capillary electrophoresis: influence of protein building up on EOF. Conditions were fused silica capillary 50 mm 3 61.5 cm (46.5 cm to the detector). Background electrolyte was 30 mM qYRfBP in 50 mM phosphate buffer at pH 8.0. Analyte concentration was 200 mM, voltage was 20 kV and temperature was 258C.
mAU
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0.2
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As previously reported25,63, the optimum protein concentration that gives the maximum enantioselectivity is in the range 10–1000 mM. Because RfBP concentrations above 50 mM elicit a strong detection response when using the complete filling technique, the selector concentration was always kept at 30 mM, whereas the analyte concentration was varied, as reported above. When approaching a 1:1 stoichiometry between the individual enantiomer and the selector concentration, the a values increased (Fig. 7). Despite a suitable between-run rinsing procedure and favourable operative pH values, it was sometimes impossible to control protein adhesion to the fused silica capillary wall, which occurred progressively during analyses. This phenomenon, already described for human serum albumin64, was responsible for the enantioseparations of warfarin at concentrations of 400 and 200 mM, which were not achieved at the beginning of the capillary lifetime (Fig. 8). Indeed, this process of progressive sticking of the protein to the capillary walls randomly affects the EOF (Fig. 9), and is responsible for fluctuating and therefore irreproducible results. In general the low k9 and poor a values observed suggested that higher protein concentrations and better efficiencies had to be achieved in order to obtain improved separations. These observations, together with the need to lower the analyte concentration without detection problems, led us to undertake CE experiments65 by using the partial filling technique, which is known to overcome all these issues in an elegant manner19,25. In brief, the capillary is partially filled with the protein solution by applying a given pressure for a given time, in order to obtain a separation zone (plug) that does not reach the detection window. Conditions are chosen so that the protein has a net negative charge and, by applying the voltage, migrates towards
20 0 2
4
6 Min
8
10
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Figure 10. Capillary electrophoresis: comparison of electropherograms obtained for oxprenolol (a) and (b) and prilocaine (c) and (d) by using the complete filling technique (a) and (c) and the partial filling technique (b) and (d), with qWRfBP as chiral selector. Conditions: the complete filling technique used fused silica capillary 50 mm 3 61.5 cm (46.5 cm to the detector), the background electrolyte was 30 mM qWRfBP in 50 mM phosphate buffer at pH 6.0. Voltage was 20 kV, with UV detection at 225 nm. Analyte concentration was 200 mM. The partial filling technique used polyvinylalcohol-coated capillary 50 mm 3 49.5 cm (41 cm to the detector), with a background electrolyte of 100 mM phosphate buffer at pH 6.0. Voltage was 20 kV, with UV detection at 200 nm. Analyte concentration was 200 mM, qWRfBP plug concentration was 300 mM and the protein plug length was 10 cm in (b) and 20 cm in (d). Reproduced, with permission, from Ref. 66.
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Table 5. Best enantioseparations obtained in CE using quail egg-white RfBP as chiral selector Analyte (mM)
Technique
qWRfBP selector
Plug length (cm)
a
Ref.
Oxprenolol 100 Prilocaine 200 Bupivacaine 50 Bepridil 200 Benfluorex 200 Mefloquine 200 Amlodipine 50 Nicardipine 200 Oxazepam 200 Lorazepam 200
Partial filling Partial filling Partial filling Complete filling Complete filling Complete filling Complete filling Complete filling Complete filling Complete filling
300 mM in 100 mM phosphate buffer pH 6.0 300 mM in 100 mM phosphate buffer pH 6.0 900 mM in 100 mM phosphate buffer pH 6.0 30 mM in 50 mM phosphate buffer pH 7.0 30 mM in 50 mM phosphate buffer pH 6.0 30 mM in 50 mM phosphate buffer pH 6.0 30 mM in 50 mM phosphate buffer pH 7.0 30 mM in 50 mM phosphate buffer pH 6.0 30 mM in 50 mM phosphate buffer pH 6.5-methanol (99:1 v/v) 30 mM in 50 mM phosphate buffer pH 6.5-methanol (99:1 v/v)
10 20 30
1.02 1.04 1.03 1.69 1.37 3.15 1.52 1.44 1.15 2.61
65 65 65 48 62 62 48 48 48 48
Conditions: Temperature: 25°C. Voltage: 20 kV. Detection: UV at 200 nm (oxprenolol, prilocaine, bupivacaine); 247 nm (bepridil, benfluorex, mefloquine, amlodipine, nicardipine, oxazepam, lorazepam and warfarin).
the anode, whereas the analytes migrate towards the detector at the cathodic end.The use of a polyvinylalcohol-coated capillary was necessary for these experiments. Indeed, we modified the classical format of the technique65 with the aim of obtaining reproducible plug lengths and consequently reproducible results. The relative standard deviations for the retention times and peak areas of prilocaine were below 1% and 2%, respectively. By using this technique it was possible to increase the concentration of quail egg-white RfBP up to 900 mM, to achieve efficiencies up to 230,000 theoretical plates per meter and to detect concentrations as low as 80 ng ml21. Figure 10 contains the electropherograms obtained for oxprenolol and prilocaine by using both the complete filling technique and the partial filling technique66.Table 5 shows the overall results obtained in CE with quail egg-white RfBP as chiral selector62,65. Conclusions The amount of important information obtained thus far on RfBPs can be exploited for further studies.There will be a deeper investigation into the chromatographic chiral performance, through either the evaluation of other species or a more precise assessment of column stability, reproducibility and lifetime, in order to compete with commercially available protein-based columns, such as the well known Chiral AGP (ChromTech AB, Hägersten, Sweden) and Ultron ES-OVM (Shinwa Chemical Industries, Kyoto, Japan). In addition, the accomplishment of HPLC affinity studies with RfBP extracted from bovine plasma will enable a closer approach to human plasma RfBP and will shed light on the role of drug intake from vitamin transport during pregnancy. In both cases, CE would offer a reliable tool for a preliminary screening and for confirmation of HPLC results. Also, through affinity CE experiments, CE would avoid waste of precious purified protein amounts and would help in the calculation of the association constants between protein and drugs.
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