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Enantiorecognition of profens by capillary electrophoresis using a novel chiral selector eremomycin
Journal of Chromatography A, 1216 (2009) 3674–3677
Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Short communication
Enantiorecognition of profens by capillary electrophoresis using a novel chiral selector eremomycin Aleksandra F. Prokhorova a , Elena N. Shapovalova a,∗ , Aleksei V. Shpak a , Sergei M. Staroverov b , Oleg A. Shpigun a a b
Division of Analytical Chemistry, Chemistry Department, Lomonosov Moscow State University, Leninskie Gory, 119992, Moscow, Russian Federation JSC BioChemMack S&T, Leninskie Gory, 119992, Moscow, Russian Federation
a r t i c l e
i n f o
Article history: Available online 11 February 2009 Keywords: Capillary electrophoresis Enantioseparation Eremomycin Profens
a b s t r a c t The evaluation of a macrocyclic glycopeptide antibiotic, eremomycin, as a chiral selector in capillary electrophoresis (CE) has been performed. The stability of eremomycin in solution and capillary electrolyte, as well as its optical and electrophoretic properties have been discussed. The effect of experimental parameters influencing the enantioseparation of several profens has been studied. Excellent enantioseparation of profens has been achieved and migration order has been validated. Comparison of enantioseparations of profens in CE by using eremomycin-mediated electrolytes and in HPLC with eremomycin immobilized on silica has revealed similar trends for both methods. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Separation of optical isomers is an important topic, especially in pharmaceutical and environmental analysis, since many drugs and agrochemicals are racemic compounds. The majority of chiral separations are carried out by liquid chromatography. The preparation of a chiral stationary phase (CSP) sometimes presents difficulties; moreover, it is hardly possible to predict the success of enantioseparation. Thus, it is necessary to evaluate enantioselective properties of chiral selectors without their immobilization on silica. In the past decade, capillary electrophoresis has advanced as an effective and versatile tool for enantiomeric separations [1]. Its feasibility, efficiency and rapidity, combined with low costs and low consumption of reagents and samples make CE a perfect method to separate optical isomers as well as evaluate enantioselectivity of novel chiral selectors [2–4]. The majority of CE enantioseparations have been performed using cyclodextrins and macrocyclic antibiotics as chiral selectors. Specifically, the antibiotics may be used in HPLC [5–8], CE [9–16], and CE-MS [17] to separate a wide range of compounds, particularly negatively charged analytes. Eremomycin, a macrocyclic antibiotic, is a recent addition to the family of chiral selectors. It is a structural analog of vancomycin [18]. Contrary to the latter, an eremomycin-based CSP has demonstrated high enantioselectivity in the separation of native amino acids, especially aromatic, by HPLC [8]. To the best of our knowledge, no published accounts on enantiomeric CE separations using eremomycin as a chiral additive can be found in literature.
∗ Corresponding author. Tel.: +7 495 939 5464; fax: +7 495 939 4675. E-mail address: [email protected] (E.N. Shapovalova). 0021-9673/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2009.02.017
To fill this gap, the present work is aimed on the evaluation of eremomycin as a chiral selector. The stability of eremomycin in electrolyte solution and its optical and electrophoretic properties have been investigated. The effects of experimental parameters influencing the enantioseparations of flurbiprofen, fenoprofen, ibuprofen, indoprofen, and ketoprofen have been tested. The migration order of profens has been determined. 2. Experimental 2.1. Chemicals and reagents Flurbiprofen, fenoprofen, ibuprofen, indoprofen, ketoprofen, S-(+)-ketoprofen, and S-(+)-ibuprofen were obtained from Sigma–Aldrich (St. Louis, MO, USA). Potassium dihydrogenphosphate, sodium hydrogenphosphate, sodium hydroxide, and sodium tetraborate decahydrate were purchased from Reachem (Moscow, Russia). Acetonitrile, methanol, and acetone were HPLC grade (Cryochrom, St. Petersburg, Russia). Eremomycin was supplied by the JSC BioChemMack S&T (Moscow, Russia). The purity of the antibiotic was about 95%. 2.2. Instrumentation CE separations were performed on a Capel-105 (Lumex, St. Petersburg, Russia) equipped with a UV detector. Fused-silica capillaries (58 cm (50 cm to the detector window) × 75 mm I.D., 35 cm (27 cm to the detector window) × 75 mm I.D., 35 cm (27 cm to the detector window) × 50 mm I.D.) were purchased from Polymicro Technologies (Phoenix, AZ, USA). All chiral compounds were detected at 230 nm and the cartridge coolant temperature was
A.F. Prokhorova et al. / J. Chromatogr. A 1216 (2009) 3674–3677
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Table 1 Effect of electrolyte pH on migration time and enantioresolution. Compound
pH 5.3
Ibuprofen Indoprofen Fenoprofen Flurbiprofen Ketoprofen
6.2
6.5
7.1
8.2
t1 (min)a
Rs
t1 (min)a
Rs
t1 (min)a
Rs
t1 (min)a
Rs
t1 (min)a
Rs
39.1 27.1 39.5 25.8 32.8
2.71 3.14 0.56 1.03 2.77
66.8 34.3 61.0 43.5 46.4
4.36 5.74 3.27 6.32 6.30
48.5 34.0 48.7 40.4 38.3
3.91 6.40 2.81 7.11 5.50
45.3 52.2 50.4 48.6 51.4
2.69 6.50 2.07 5.15 5.24
52.6 63.3 55.0 47.6 46.7
1.79 1.83 1.56 2.27 1.03
CE condition: fused-silica capillary, 75 m I.D. × 58.5/50 cm, 2.5 mM eremomycin, 50 mM phosphate buffer, +10 kV, external pressure 1 kPa. a Migration time of the first eluting enantiomer.
set at 20 ◦ C. All samples were introduced using pressure injection (12.5 kPa × s). To shorten the analysis time and increase its throughput, some external pressure was applied as specified below. 2.3. Procedure 25–100 mM phosphate buffers and 50 mM borate buffer were prepared by dissolving accurately weighed amounts of potassium dihydrogenphosphate and sodium hydrogenphosphate, and sodium tetraborate decahydrate, respectively, and adjusted to the desired pH using 0.1 M sodium hydroxide. Buffer solutions were filtered with 0.22 m membrane filter. The eremomycin stock solutions were prepared by dissolving eremomycin in a buffer solution followed by ultrasonic degasation. The eremomycin solutions were stored at +4 ◦ C when not in use. All chiral analytes (0.5 mg/ml) were dissolved in distilled water. Post-run washing of the capillary was required because of a strong adsorption of glycopeptide antibiotics on its walls [10,20,21]. The capillaries were purged daily with 0.1 M sodium hydroxide, followed by water and run buffer for 15 min each. Between runs, the capillary was rinsed at a high pressure with water and run buffer (for 5 min each). Acetone was used as an electroosmotic flow (EOF) marker. 3. Results and discussion
weeks before significant changes would occur (appearance of light pink coloring or precipitation). To study the stability of chiral selector in more detail, its 2.5 mM solution was placed into the capillary and absorption spectra (190–350 nm) were recorded at different temperatures after different periods of time. The examination of the absorption spectra obtained at ambient temperature showed that eremomycin solutions were stable during ∼170 h. Glycopeptide antibiotics are thermally labile. Therefore, increasing the temperature from 25 to 30 ◦ C or higher accelerated the decomposition of eremomycin. Changes caused by the decomposition of antibiotics were observed in the spectra (Fig. 1). At pH 9.2 the buffer solution containing eremomycin appears to decompose much faster: in 5–6 h at ambient temperature or within 2 days at −4 ◦ C. Thus, when the run buffer containing eremomycin is used, pH extremes (pH < 4.0 and >8.5), elevated temperatures, and extended storage (>2 weeks) should be avoided. It should be noted that eremomycin solutions are somewhat more stable than vancomycin solutions since the visible change of color was observed in ∼1.5 weeks (at 4 ◦ C) compared to <1 week for vancomycin. This observation agrees with the Armstrong theory consisting of the stability trend of glycopeptide antibiotics to increase with an increase in the number of attached saccharide moieties. Based on the existing data [13,22] and the data obtained in our work, the order of stability (5 < pH < 7) is the following: vancomycin < eremomycin < teicoplanin < ristocetin A < avoparcin.
3.1. Relevant properties of eremomycin 3.3. Effect of pH and electrolyte buffer composition Similarly to other macrocyclic antibiotics, eremomycin (MW 1558) does not significantly absorb in visible region [9,11,13,20], having weak absorbance bands between 260 and 350 nm with a small maximum around 280 nm. The absorption band varies with pH. In neutral solutions (pH 6.2–7.1) the absorbance at > 200 nm is lower than in basic solutions. At pH 6.2 a sag at approximately 240 nm forms. Taking into account that relatively dilute solutions of eremomycin (2.5 mM) were used in these measurements, direct UV detection was deemed feasible in the range from 225 to 265 nm. Eremomycin contains several ionizable groups that make its electrophoretic mobility dependent on pH of electrolyte. The reported pKa values are 3.1, 6.9, 7.9, 9.0, 9.7, 10.4, and 11.4 [21]. Also, pI = 8.3 has been reported (conditions are not specified) [18]. The pI was determined in 50 mM phosphate buffer; the value obtained was 7.6. This indicates that at pH < 7.6 eremomycin is positively charged and migrates in the same direction as the electroosmotic flow. In such a case, eremomycin would tend to interact with anionic compounds, which appears favorable for their enantioseparation.
As pH governs the charge and mobility of the chiral selector and analytes, considerable effect of pH of run buffer on the separation was observed. In order to examine the influence of pH and buffer composition on enantioseparation, CE experiments were carried out using phosphate buffers with pH ranged from 5.3 to 8.2 and a 50 mM borate buffer at pH 9.2. Higher concentration buffers (50
3.2. Stability in solution In aqueous solutions glycopeptide antibiotics are known to decompose with time; they are particularly unstable at elevated temperatures and in basic solutions [9,10,13,19]. A freshly prepared eremomycin solution can be stored at +4 ◦ C for approximately 2
Fig. 1. UV-spectra of 2.5 mM eremomycin solution in 100 mM phosphate buffer at different temperatures.
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A.F. Prokhorova et al. / J. Chromatogr. A 1216 (2009) 3674–3677
Fig. 2. Effect of eremomycin concentration on the separation of enantiomers. Electrolyte: 50 mM phosphate buffer (pH 6.5) containing variable selector concentration, other conditions as in Table 1.
and 100 mM) reduce chiral selector – capillary wall interaction and migration times and thereby improve the separation efficiency. The dilution of the buffer to 25 mM resulted in the significant decrease of the migration times of enantiomers. However, when 100 mM phosphate buffers were used, the baseline resolution of some profens (e.g. ibuprofen or ketoprofen) was not possible. Moreover, a significant increase in the current resulted in baseline noise. The results presented in Table 1 show the effect of increasing pH on the resolution and migration time. At pH 5.3, many system peaks appeared that were not identified, and the repeatability of migration time was unsatisfactory. An increase in pH from 7.1 to 8.2 worsened the resolution. None of the profens studied was resolved using borate buffer. As eremomycin is negatively charged at pH > 8.2, these pH values are unfavorable for achieving enantioseparation of the anionic analytes. The best enantioseparations were obtained at pH 6.5. 3.4. Effect of eremomycin concentration The concentration of chiral selector in the run buffer is an important variable controlling the chiral recognition in CE. Baseline resolution was achieved for all tested compounds at a 2.5 mM eremomycin concentration. In general, the resolution improved with increasing the eremomycin concentration from 1 to 7.5 mM, as shown in Fig. 2. The migration time increased as well. Therefore, at 7.5 mM of eremomycin, only enantiomers of flurbiprofen as well as R-(−)-enantiomers of indoprofen and ketoprofen were detected within 90 min. The calculated efficiencies (N/m) were not very high, probably due to the effect of external pressure, which resulted in less flat flow profile. The concentration of 2.5 mM was selected as optimum for attaining satisfactory resolution, peak shape, and baseline noise (Fig. 3). In comparison with vancomycin, eremomycin allows using more diluted solutions to achieve baseline resolution of all the selected profens [19]. The separation selectivity is similar to that of vancomycin, but better than in the case of teicoplanin or avoparcin. It is worthwhile to say that eremomycin-based separation of indoprofen and flurbiprofen was highly selective (resolution factor 1.35), while ristocetin A and vancomycin are more selective toward ketoprofen [19,24]. 3.5. Effect of the separation conditions The attempt to improve separation was made by varying capillary length, inner diameter, and applied voltage. The enantioseparations of all the profens could be achieved within 15 min
Fig. 3. Electrophoregram showing separation of selected profens (a: indoprofen; b: ketoprofen; c: flurbiprofen). Electrolyte: 50 mM phosphate buffer (pH 6.5), other conditions as in Table 1.
using a shorter capillary. As expected, the resulting efficiency of the separation, selectivity, and resolution factor decreased. For instance, the resolution of fenoprofen enantiomers lowered from 2.81 to 1.40. When 50 mM phosphate buffer was used in the capillary of 75 m I.D., an increase in applied voltage from +5 to +10 kV brought about a slight decrease (about 3 min) in migration times, and a higher efficiency (13,000 and 16,000 N/m, respectively) was obtained. A higher voltage (+15 kV) gives rise to excessive heat generation and strong baseline noise, and none of the analytes studied was detected. It is suggested that eremomycin could decompose in the capillary during the analysis at high voltage. A similar behavior has been observed for other antibiotics [19,23]. Experiments were carried out using a 50 m I.D. capillary using 25 mM phosphate buffer. In this case, the maximum voltage was +15 kV. Better efficiencies were achieved at the expense of the longer separation that took >100 min for each pair of enantiomers. 3.6. Effect of organic modifier An organic solvent may affect the effective charge of the enantiomers and chiral selector, EOF, interactions involved in the enantioseparation mechanism, etc. The addition of acetonitrile (<18%) led to insignificant changes in migration time, resolution, and separation selectivity. When the run buffer containing 18% (v/v) acetonitrile was used, a slight improvement in selectivity and peak shape was observed. The following addition of acetonitrile deteriorated the separation. In our opinion, such an effect is possibly due to reorganization of the adsorption layer related to solvation of eremomycin and analytes. When methanol was used as organic modifier (at 10% and 20% (v/v)), a decrease in analyte resolution and selectivity was observed. To achieve reproducible results, the capillary pre-treatment with the run buffer containing organic modifiers was needed over a longer period of time.
A.F. Prokhorova et al. / J. Chromatogr. A 1216 (2009) 3674–3677 Table 2 Comparison data on the separation of profens by HPLC and CE. Compound
Ibuprofen Indoprofen Fenoprofen Flurbiprofen Ketoprofen a
HPLCa
CE ˛
Rs
˛
Rs
1.12 1.35 1.10 1.35 1.26
3.91 6.40 2.81 7.11 5.50
1.70 1.99 1.32 – 1.55
2.18 2.08 1.42 – 2.47
From Ref. [23].
3.7. Migration order of enantiomers The migration order of ketoprofen and ibuprofen enantiomers was determined by injecting a racemate, a sample mixture of S-(+)/R-(−)-enantiomers at a ratio 2:1, and S-(+)-enantiomer. It was found that the R-(−)-enantiomer migrates faster than S-(+)enantiomer. The eremomycin migrates at almost the same rate as the EOF and analytes migrate in the opposite direction. The more stable complex between eremomycin and S-(+)-enantiomer thus passes along the detector first. 3.8. Comparison of HPLC and CE enantioseparations The selectivity study of different substances as chiral selectors by capillary electrophoresis is simpler and faster that than by HPLC. In Table 2, an attempt was made to compare the enantiomer-resolving potential of CE and HPLC using eremomycin. HPLC separations were performed with the eremomycin immobilized on silica in the reverse-phase mode (eluent was 40% methanol/60% 100 mM phosphate buffer, pH 6.5) [20]. Enantioseparation tendencies are commonly the same but in general HPLC allows better separation selectivity. The selectivity in CE was calculated as ˛ = e2 /e1 (e1 and e2 are the electrophoretic mobilities of the first and second migrating enantiomers, respectively). The selectivity factor ˛ for fenoprofen is the lowest in both HPLC and CE modes. The highest ˛ values were obtained for indoprofen (both methods) and flurbiprofen (CE). It is likely due to the presence of O and N atoms (indoprofen) and F atoms (flurbiprofen), capable of forming hydrogen bonds. As hydrogen bonding improves the stability of the enantiomer–chiral selector complex, its migration time decreases. The fastest enantioseparation (34.0 min) was achieved for indoprofen (Table 1). On the contrary, the resolution factors are greater in CE than in HPLC. This is a result of a higher efficiency of CE (N/m was about 11,000–15,000). The separation efficiency of HPLC did not exceed 6000 theoretical plates.
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It should be emphasized that the complexation between eremomycin and the individual profens exhibited similar trends in HPLC and CE. The results of enantiomeric separations indicate a rather similar interaction mechanism occurring in the two separation systems. 4. Conclusions This study has revealed that eremomycin is an effective chiral selector for the CE separation of enantiomeric compounds containing carboxylic groups. The separations of a range of profens have been optimized by adjusting the pH of the run buffer and the chiral selector concentration, which can be kept as low as 2.5 mM with no loss of resolution. CE allows relatively an easy adjustment of separation conditions. The comparison of data on electrophoretic and chromatographic separations showed that screening of chiral selectors is more effective by CE. References [1] B. Chankvetadze, Capillary Electrophoresis in Chiral Analysis, Wiley, Chichester, 1997. [2] P.D. Ferguson, D.M. Goodall, J.C. Loran, J. Chromatogr. A 745 (1996) 25. [3] E. Tesaˇrová, Z. Bosáková, I. Zusková, J. Chromatogr. A 879 (2000) 147. [4] B. Chankvetadze, J. Chromatogr. A 1168 (2007) 45. [5] A. Berthod, U.B. Nair, C. Bagwill, D.W. Armstrong, Talanta 43 (1996) 1767. [6] I. Ilisz, R. Berkecz, A. Peter, J. Sep. Sci. 29 (2006) 1305. [7] B. Kafková, Z. Bosáková, E. Tesaˇrová, P. Coufal, J. Chromatogr. A 1088 (2005) 82. [8] S.M. Staroverov, M.A. Kuznetsov, P.N. Nesterenko, G.G. Vasiyarov, G.S. Katrukha, G.B. Fedorova, J. Chromatogr. A 1108 (2006) 263. [9] D.W. Armstrong, K.L. Rundlett, J.-R. Chen, Chirality 6 (1994) 496. [10] D.W. Armstrong, M.P. Gasper, K.L. Rundlett, J. Chromatogr. A 689 (1995) 285. [11] D.W. Armstrong, K.L. Rundlett, J. Liq. Chromatogr. 18 (1995) 3659. [12] R. Vespalec, H. Corstjens, H.A.H. Billiet, J. Frank, K.C.A.M. Luyben, Anal. Chem. 67 (1995) 3223. [13] V.S. Sharp, D.S. Risley, A. McCarthy, B.E. Huff, M.A. Strege, J. Liq. Chromatogr. Rel. Technol. 20 (1997) 887. [14] K.H. Ekborg-Ott, G.A. Zientara, J.M. Schneiderheinze, K. Gahm, D.W. Armstrong, Electrophoresis 20 (1999) 2438. [15] P. Bednar, Z. Aturki, Z. Stransky, F. Fanali, Electrophoresis 22 (2001) 2129. [16] Z. Wang, J. Wang, Z. Hu, J. Kang, Electrophoresis 28 (2007) 938. [17] F. Fanali, C. Desiderio, G. Schulte, S. Heitmeier, D. Strickmann, B. Chankvetadze, G. Blaschke, J. Chromatogr. A 800 (1998) 69. [18] M.A. Kuznetsov, P.N. Nesterenko, G.G. Vasiyarov, S.M. Staroverov, Appl. Biochem. Microbiol. 42 (2006) 536. [19] G.F. Gause, M.G. Brazhnikova, N.N. Lomakina, T.F. Berdnikova, G.B. Fedorova, N.L. Tokareva, V.N. Borisova, G.Y. Batta, J. Antibiot. XLII (1989) 1790. [20] K.L. Rundlett, M.P. Gasper, E.Y. Zhou, D.W. Armstrong, Chirality 8 (1996) 88. [21] G. Gubitz, M.G. Schmid, Electrophoresis 21 (2000) 4112. [22] I.V. Polyakova, V.M. Kolikov, O.A. Pisarev, J. Chromatogr. A 1006 (2003) 251. [23] D.W. Armstrong, U.B. Nair, Electrophoresis 18 (1997) 2331. [24] M.P. Gasper, A. Berthod, U.B. Nair, D.W. Armstrong, Anal. Chem. 68 (1996) 2501.
Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Short communication
Enantiorecognition of profens by capillary electrophoresis using a novel chiral selector eremomycin Aleksandra F. Prokhorova a , Elena N. Shapovalova a,∗ , Aleksei V. Shpak a , Sergei M. Staroverov b , Oleg A. Shpigun a a b
Division of Analytical Chemistry, Chemistry Department, Lomonosov Moscow State University, Leninskie Gory, 119992, Moscow, Russian Federation JSC BioChemMack S&T, Leninskie Gory, 119992, Moscow, Russian Federation
a r t i c l e
i n f o
Article history: Available online 11 February 2009 Keywords: Capillary electrophoresis Enantioseparation Eremomycin Profens
a b s t r a c t The evaluation of a macrocyclic glycopeptide antibiotic, eremomycin, as a chiral selector in capillary electrophoresis (CE) has been performed. The stability of eremomycin in solution and capillary electrolyte, as well as its optical and electrophoretic properties have been discussed. The effect of experimental parameters influencing the enantioseparation of several profens has been studied. Excellent enantioseparation of profens has been achieved and migration order has been validated. Comparison of enantioseparations of profens in CE by using eremomycin-mediated electrolytes and in HPLC with eremomycin immobilized on silica has revealed similar trends for both methods. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Separation of optical isomers is an important topic, especially in pharmaceutical and environmental analysis, since many drugs and agrochemicals are racemic compounds. The majority of chiral separations are carried out by liquid chromatography. The preparation of a chiral stationary phase (CSP) sometimes presents difficulties; moreover, it is hardly possible to predict the success of enantioseparation. Thus, it is necessary to evaluate enantioselective properties of chiral selectors without their immobilization on silica. In the past decade, capillary electrophoresis has advanced as an effective and versatile tool for enantiomeric separations [1]. Its feasibility, efficiency and rapidity, combined with low costs and low consumption of reagents and samples make CE a perfect method to separate optical isomers as well as evaluate enantioselectivity of novel chiral selectors [2–4]. The majority of CE enantioseparations have been performed using cyclodextrins and macrocyclic antibiotics as chiral selectors. Specifically, the antibiotics may be used in HPLC [5–8], CE [9–16], and CE-MS [17] to separate a wide range of compounds, particularly negatively charged analytes. Eremomycin, a macrocyclic antibiotic, is a recent addition to the family of chiral selectors. It is a structural analog of vancomycin [18]. Contrary to the latter, an eremomycin-based CSP has demonstrated high enantioselectivity in the separation of native amino acids, especially aromatic, by HPLC [8]. To the best of our knowledge, no published accounts on enantiomeric CE separations using eremomycin as a chiral additive can be found in literature.
∗ Corresponding author. Tel.: +7 495 939 5464; fax: +7 495 939 4675. E-mail address: [email protected] (E.N. Shapovalova). 0021-9673/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2009.02.017
To fill this gap, the present work is aimed on the evaluation of eremomycin as a chiral selector. The stability of eremomycin in electrolyte solution and its optical and electrophoretic properties have been investigated. The effects of experimental parameters influencing the enantioseparations of flurbiprofen, fenoprofen, ibuprofen, indoprofen, and ketoprofen have been tested. The migration order of profens has been determined. 2. Experimental 2.1. Chemicals and reagents Flurbiprofen, fenoprofen, ibuprofen, indoprofen, ketoprofen, S-(+)-ketoprofen, and S-(+)-ibuprofen were obtained from Sigma–Aldrich (St. Louis, MO, USA). Potassium dihydrogenphosphate, sodium hydrogenphosphate, sodium hydroxide, and sodium tetraborate decahydrate were purchased from Reachem (Moscow, Russia). Acetonitrile, methanol, and acetone were HPLC grade (Cryochrom, St. Petersburg, Russia). Eremomycin was supplied by the JSC BioChemMack S&T (Moscow, Russia). The purity of the antibiotic was about 95%. 2.2. Instrumentation CE separations were performed on a Capel-105 (Lumex, St. Petersburg, Russia) equipped with a UV detector. Fused-silica capillaries (58 cm (50 cm to the detector window) × 75 mm I.D., 35 cm (27 cm to the detector window) × 75 mm I.D., 35 cm (27 cm to the detector window) × 50 mm I.D.) were purchased from Polymicro Technologies (Phoenix, AZ, USA). All chiral compounds were detected at 230 nm and the cartridge coolant temperature was
A.F. Prokhorova et al. / J. Chromatogr. A 1216 (2009) 3674–3677
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Table 1 Effect of electrolyte pH on migration time and enantioresolution. Compound
pH 5.3
Ibuprofen Indoprofen Fenoprofen Flurbiprofen Ketoprofen
6.2
6.5
7.1
8.2
t1 (min)a
Rs
t1 (min)a
Rs
t1 (min)a
Rs
t1 (min)a
Rs
t1 (min)a
Rs
39.1 27.1 39.5 25.8 32.8
2.71 3.14 0.56 1.03 2.77
66.8 34.3 61.0 43.5 46.4
4.36 5.74 3.27 6.32 6.30
48.5 34.0 48.7 40.4 38.3
3.91 6.40 2.81 7.11 5.50
45.3 52.2 50.4 48.6 51.4
2.69 6.50 2.07 5.15 5.24
52.6 63.3 55.0 47.6 46.7
1.79 1.83 1.56 2.27 1.03
CE condition: fused-silica capillary, 75 m I.D. × 58.5/50 cm, 2.5 mM eremomycin, 50 mM phosphate buffer, +10 kV, external pressure 1 kPa. a Migration time of the first eluting enantiomer.
set at 20 ◦ C. All samples were introduced using pressure injection (12.5 kPa × s). To shorten the analysis time and increase its throughput, some external pressure was applied as specified below. 2.3. Procedure 25–100 mM phosphate buffers and 50 mM borate buffer were prepared by dissolving accurately weighed amounts of potassium dihydrogenphosphate and sodium hydrogenphosphate, and sodium tetraborate decahydrate, respectively, and adjusted to the desired pH using 0.1 M sodium hydroxide. Buffer solutions were filtered with 0.22 m membrane filter. The eremomycin stock solutions were prepared by dissolving eremomycin in a buffer solution followed by ultrasonic degasation. The eremomycin solutions were stored at +4 ◦ C when not in use. All chiral analytes (0.5 mg/ml) were dissolved in distilled water. Post-run washing of the capillary was required because of a strong adsorption of glycopeptide antibiotics on its walls [10,20,21]. The capillaries were purged daily with 0.1 M sodium hydroxide, followed by water and run buffer for 15 min each. Between runs, the capillary was rinsed at a high pressure with water and run buffer (for 5 min each). Acetone was used as an electroosmotic flow (EOF) marker. 3. Results and discussion
weeks before significant changes would occur (appearance of light pink coloring or precipitation). To study the stability of chiral selector in more detail, its 2.5 mM solution was placed into the capillary and absorption spectra (190–350 nm) were recorded at different temperatures after different periods of time. The examination of the absorption spectra obtained at ambient temperature showed that eremomycin solutions were stable during ∼170 h. Glycopeptide antibiotics are thermally labile. Therefore, increasing the temperature from 25 to 30 ◦ C or higher accelerated the decomposition of eremomycin. Changes caused by the decomposition of antibiotics were observed in the spectra (Fig. 1). At pH 9.2 the buffer solution containing eremomycin appears to decompose much faster: in 5–6 h at ambient temperature or within 2 days at −4 ◦ C. Thus, when the run buffer containing eremomycin is used, pH extremes (pH < 4.0 and >8.5), elevated temperatures, and extended storage (>2 weeks) should be avoided. It should be noted that eremomycin solutions are somewhat more stable than vancomycin solutions since the visible change of color was observed in ∼1.5 weeks (at 4 ◦ C) compared to <1 week for vancomycin. This observation agrees with the Armstrong theory consisting of the stability trend of glycopeptide antibiotics to increase with an increase in the number of attached saccharide moieties. Based on the existing data [13,22] and the data obtained in our work, the order of stability (5 < pH < 7) is the following: vancomycin < eremomycin < teicoplanin < ristocetin A < avoparcin.
3.1. Relevant properties of eremomycin 3.3. Effect of pH and electrolyte buffer composition Similarly to other macrocyclic antibiotics, eremomycin (MW 1558) does not significantly absorb in visible region [9,11,13,20], having weak absorbance bands between 260 and 350 nm with a small maximum around 280 nm. The absorption band varies with pH. In neutral solutions (pH 6.2–7.1) the absorbance at > 200 nm is lower than in basic solutions. At pH 6.2 a sag at approximately 240 nm forms. Taking into account that relatively dilute solutions of eremomycin (2.5 mM) were used in these measurements, direct UV detection was deemed feasible in the range from 225 to 265 nm. Eremomycin contains several ionizable groups that make its electrophoretic mobility dependent on pH of electrolyte. The reported pKa values are 3.1, 6.9, 7.9, 9.0, 9.7, 10.4, and 11.4 [21]. Also, pI = 8.3 has been reported (conditions are not specified) [18]. The pI was determined in 50 mM phosphate buffer; the value obtained was 7.6. This indicates that at pH < 7.6 eremomycin is positively charged and migrates in the same direction as the electroosmotic flow. In such a case, eremomycin would tend to interact with anionic compounds, which appears favorable for their enantioseparation.
As pH governs the charge and mobility of the chiral selector and analytes, considerable effect of pH of run buffer on the separation was observed. In order to examine the influence of pH and buffer composition on enantioseparation, CE experiments were carried out using phosphate buffers with pH ranged from 5.3 to 8.2 and a 50 mM borate buffer at pH 9.2. Higher concentration buffers (50
3.2. Stability in solution In aqueous solutions glycopeptide antibiotics are known to decompose with time; they are particularly unstable at elevated temperatures and in basic solutions [9,10,13,19]. A freshly prepared eremomycin solution can be stored at +4 ◦ C for approximately 2
Fig. 1. UV-spectra of 2.5 mM eremomycin solution in 100 mM phosphate buffer at different temperatures.
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Fig. 2. Effect of eremomycin concentration on the separation of enantiomers. Electrolyte: 50 mM phosphate buffer (pH 6.5) containing variable selector concentration, other conditions as in Table 1.
and 100 mM) reduce chiral selector – capillary wall interaction and migration times and thereby improve the separation efficiency. The dilution of the buffer to 25 mM resulted in the significant decrease of the migration times of enantiomers. However, when 100 mM phosphate buffers were used, the baseline resolution of some profens (e.g. ibuprofen or ketoprofen) was not possible. Moreover, a significant increase in the current resulted in baseline noise. The results presented in Table 1 show the effect of increasing pH on the resolution and migration time. At pH 5.3, many system peaks appeared that were not identified, and the repeatability of migration time was unsatisfactory. An increase in pH from 7.1 to 8.2 worsened the resolution. None of the profens studied was resolved using borate buffer. As eremomycin is negatively charged at pH > 8.2, these pH values are unfavorable for achieving enantioseparation of the anionic analytes. The best enantioseparations were obtained at pH 6.5. 3.4. Effect of eremomycin concentration The concentration of chiral selector in the run buffer is an important variable controlling the chiral recognition in CE. Baseline resolution was achieved for all tested compounds at a 2.5 mM eremomycin concentration. In general, the resolution improved with increasing the eremomycin concentration from 1 to 7.5 mM, as shown in Fig. 2. The migration time increased as well. Therefore, at 7.5 mM of eremomycin, only enantiomers of flurbiprofen as well as R-(−)-enantiomers of indoprofen and ketoprofen were detected within 90 min. The calculated efficiencies (N/m) were not very high, probably due to the effect of external pressure, which resulted in less flat flow profile. The concentration of 2.5 mM was selected as optimum for attaining satisfactory resolution, peak shape, and baseline noise (Fig. 3). In comparison with vancomycin, eremomycin allows using more diluted solutions to achieve baseline resolution of all the selected profens [19]. The separation selectivity is similar to that of vancomycin, but better than in the case of teicoplanin or avoparcin. It is worthwhile to say that eremomycin-based separation of indoprofen and flurbiprofen was highly selective (resolution factor 1.35), while ristocetin A and vancomycin are more selective toward ketoprofen [19,24]. 3.5. Effect of the separation conditions The attempt to improve separation was made by varying capillary length, inner diameter, and applied voltage. The enantioseparations of all the profens could be achieved within 15 min
Fig. 3. Electrophoregram showing separation of selected profens (a: indoprofen; b: ketoprofen; c: flurbiprofen). Electrolyte: 50 mM phosphate buffer (pH 6.5), other conditions as in Table 1.
using a shorter capillary. As expected, the resulting efficiency of the separation, selectivity, and resolution factor decreased. For instance, the resolution of fenoprofen enantiomers lowered from 2.81 to 1.40. When 50 mM phosphate buffer was used in the capillary of 75 m I.D., an increase in applied voltage from +5 to +10 kV brought about a slight decrease (about 3 min) in migration times, and a higher efficiency (13,000 and 16,000 N/m, respectively) was obtained. A higher voltage (+15 kV) gives rise to excessive heat generation and strong baseline noise, and none of the analytes studied was detected. It is suggested that eremomycin could decompose in the capillary during the analysis at high voltage. A similar behavior has been observed for other antibiotics [19,23]. Experiments were carried out using a 50 m I.D. capillary using 25 mM phosphate buffer. In this case, the maximum voltage was +15 kV. Better efficiencies were achieved at the expense of the longer separation that took >100 min for each pair of enantiomers. 3.6. Effect of organic modifier An organic solvent may affect the effective charge of the enantiomers and chiral selector, EOF, interactions involved in the enantioseparation mechanism, etc. The addition of acetonitrile (<18%) led to insignificant changes in migration time, resolution, and separation selectivity. When the run buffer containing 18% (v/v) acetonitrile was used, a slight improvement in selectivity and peak shape was observed. The following addition of acetonitrile deteriorated the separation. In our opinion, such an effect is possibly due to reorganization of the adsorption layer related to solvation of eremomycin and analytes. When methanol was used as organic modifier (at 10% and 20% (v/v)), a decrease in analyte resolution and selectivity was observed. To achieve reproducible results, the capillary pre-treatment with the run buffer containing organic modifiers was needed over a longer period of time.
A.F. Prokhorova et al. / J. Chromatogr. A 1216 (2009) 3674–3677 Table 2 Comparison data on the separation of profens by HPLC and CE. Compound
Ibuprofen Indoprofen Fenoprofen Flurbiprofen Ketoprofen a
HPLCa
CE ˛
Rs
˛
Rs
1.12 1.35 1.10 1.35 1.26
3.91 6.40 2.81 7.11 5.50
1.70 1.99 1.32 – 1.55
2.18 2.08 1.42 – 2.47
From Ref. [23].
3.7. Migration order of enantiomers The migration order of ketoprofen and ibuprofen enantiomers was determined by injecting a racemate, a sample mixture of S-(+)/R-(−)-enantiomers at a ratio 2:1, and S-(+)-enantiomer. It was found that the R-(−)-enantiomer migrates faster than S-(+)enantiomer. The eremomycin migrates at almost the same rate as the EOF and analytes migrate in the opposite direction. The more stable complex between eremomycin and S-(+)-enantiomer thus passes along the detector first. 3.8. Comparison of HPLC and CE enantioseparations The selectivity study of different substances as chiral selectors by capillary electrophoresis is simpler and faster that than by HPLC. In Table 2, an attempt was made to compare the enantiomer-resolving potential of CE and HPLC using eremomycin. HPLC separations were performed with the eremomycin immobilized on silica in the reverse-phase mode (eluent was 40% methanol/60% 100 mM phosphate buffer, pH 6.5) [20]. Enantioseparation tendencies are commonly the same but in general HPLC allows better separation selectivity. The selectivity in CE was calculated as ˛ = e2 /e1 (e1 and e2 are the electrophoretic mobilities of the first and second migrating enantiomers, respectively). The selectivity factor ˛ for fenoprofen is the lowest in both HPLC and CE modes. The highest ˛ values were obtained for indoprofen (both methods) and flurbiprofen (CE). It is likely due to the presence of O and N atoms (indoprofen) and F atoms (flurbiprofen), capable of forming hydrogen bonds. As hydrogen bonding improves the stability of the enantiomer–chiral selector complex, its migration time decreases. The fastest enantioseparation (34.0 min) was achieved for indoprofen (Table 1). On the contrary, the resolution factors are greater in CE than in HPLC. This is a result of a higher efficiency of CE (N/m was about 11,000–15,000). The separation efficiency of HPLC did not exceed 6000 theoretical plates.
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It should be emphasized that the complexation between eremomycin and the individual profens exhibited similar trends in HPLC and CE. The results of enantiomeric separations indicate a rather similar interaction mechanism occurring in the two separation systems. 4. Conclusions This study has revealed that eremomycin is an effective chiral selector for the CE separation of enantiomeric compounds containing carboxylic groups. The separations of a range of profens have been optimized by adjusting the pH of the run buffer and the chiral selector concentration, which can be kept as low as 2.5 mM with no loss of resolution. CE allows relatively an easy adjustment of separation conditions. The comparison of data on electrophoretic and chromatographic separations showed that screening of chiral selectors is more effective by CE. References [1] B. Chankvetadze, Capillary Electrophoresis in Chiral Analysis, Wiley, Chichester, 1997. [2] P.D. Ferguson, D.M. Goodall, J.C. Loran, J. Chromatogr. A 745 (1996) 25. [3] E. Tesaˇrová, Z. Bosáková, I. Zusková, J. Chromatogr. A 879 (2000) 147. [4] B. Chankvetadze, J. Chromatogr. A 1168 (2007) 45. [5] A. Berthod, U.B. Nair, C. Bagwill, D.W. Armstrong, Talanta 43 (1996) 1767. [6] I. Ilisz, R. Berkecz, A. Peter, J. Sep. Sci. 29 (2006) 1305. [7] B. Kafková, Z. Bosáková, E. Tesaˇrová, P. Coufal, J. Chromatogr. A 1088 (2005) 82. [8] S.M. Staroverov, M.A. Kuznetsov, P.N. Nesterenko, G.G. Vasiyarov, G.S. Katrukha, G.B. Fedorova, J. Chromatogr. A 1108 (2006) 263. [9] D.W. Armstrong, K.L. Rundlett, J.-R. Chen, Chirality 6 (1994) 496. [10] D.W. Armstrong, M.P. Gasper, K.L. Rundlett, J. Chromatogr. A 689 (1995) 285. [11] D.W. Armstrong, K.L. Rundlett, J. Liq. Chromatogr. 18 (1995) 3659. [12] R. Vespalec, H. Corstjens, H.A.H. Billiet, J. Frank, K.C.A.M. Luyben, Anal. Chem. 67 (1995) 3223. [13] V.S. Sharp, D.S. Risley, A. McCarthy, B.E. Huff, M.A. Strege, J. Liq. Chromatogr. Rel. Technol. 20 (1997) 887. [14] K.H. Ekborg-Ott, G.A. Zientara, J.M. Schneiderheinze, K. Gahm, D.W. Armstrong, Electrophoresis 20 (1999) 2438. [15] P. Bednar, Z. Aturki, Z. Stransky, F. Fanali, Electrophoresis 22 (2001) 2129. [16] Z. Wang, J. Wang, Z. Hu, J. Kang, Electrophoresis 28 (2007) 938. [17] F. Fanali, C. Desiderio, G. Schulte, S. Heitmeier, D. Strickmann, B. Chankvetadze, G. Blaschke, J. Chromatogr. A 800 (1998) 69. [18] M.A. Kuznetsov, P.N. Nesterenko, G.G. Vasiyarov, S.M. Staroverov, Appl. Biochem. Microbiol. 42 (2006) 536. [19] G.F. Gause, M.G. Brazhnikova, N.N. Lomakina, T.F. Berdnikova, G.B. Fedorova, N.L. Tokareva, V.N. Borisova, G.Y. Batta, J. Antibiot. XLII (1989) 1790. [20] K.L. Rundlett, M.P. Gasper, E.Y. Zhou, D.W. Armstrong, Chirality 8 (1996) 88. [21] G. Gubitz, M.G. Schmid, Electrophoresis 21 (2000) 4112. [22] I.V. Polyakova, V.M. Kolikov, O.A. Pisarev, J. Chromatogr. A 1006 (2003) 251. [23] D.W. Armstrong, U.B. Nair, Electrophoresis 18 (1997) 2331. [24] M.P. Gasper, A. Berthod, U.B. Nair, D.W. Armstrong, Anal. Chem. 68 (1996) 2501.