Quenched Phosphorescence as a Detection Method in Capillary

Anal. Chem. 2004, 76, 399-403

Quenched Phosphorescence as a Detection Method in Capillary Electrophoretic Chiral Separations. Monitoring the Stereoselective Biodegradation of Camphorquinone by Yeast Carmen Garcı´a-Ruiz,† Marco Siderius,‡ Freek Ariese,† and Cees Gooijer*,†

Department of Analytical Chemistry and Applied Spectroscopy, Laser Centre, and Department of Biochemistry and Molecular Biology, IMBW, Biocentrum Amsterdam, Vrije Universiteit, De Boelelaan 1083, NL-1081 HV Amsterdam, The Netherlands

Quenched phosphorescence detection of camphorquinone in cyclodextrin-based electrokinetic chromatography provides very favorable detection limits, i.e., 7 × 10-7 M, 3 orders of magnitude lower than conventional UV absorption detection at 200 nm. The detection is based on the dynamic quenching by the analyte of the strong phosphorescence emission of brominated naphthalenesulfonate, under deoxygenated buffer solution conditions. This approach has been used to detect (1S)-(+)- and (1R)(-)-camphorquinone after enantiomeric separation by CE. Although the use of the negatively charged carboxymethyl β-cyclodextrin (CM-β-CD) alone was not successful, the addition of a second, neutral cyclodextrin, r-CD, provided an adequate enantiomeric separation of camphorquinone. Using 25 mM borate buffer (pH 8.5) with 10 mM CM-βCD and 20 mM r-CD (applied voltage 20 kV, ambient temperature), the enantiomeric separation was performed in ∼14 min. The chiral method was applied to monitor the stereoselectivity of the biotransformation of a racemic mixture of camphorquinone by yeast. It was found that the enantiomeric ratio calculated from the peak areas in the electropherogram (RSD ) 5%) after 24 h of incubation decreased from 0.92 for the control solution (culture medium without yeast) to 0.24 for the culture medium; a similar ratio of 0.25 was observed for cell extract solutions. Therefore, racemic camphorquinone is enantioselectively degraded by yeast, the biodegradation of (1S)-(+)-camphorquinone being faster than that of the (1R)-(-)-enantiomer. Chirality is very important in biochemistry and pharmacochemistry because the enantiomers of a chiral molecule often have totally different effects on cellular processes. As a consequence, the development of chiral analytical methods is essential for various purposes, for example, to control stereoselective processes * To whom correspondence should be addressed. Fax: 31 (0) 20 4447543. E-mail: [email protected]. † Department of Analytical Chemistry and Applied Spectroscopy. ‡ Department of Biochemistry and Molecular Biology. 10.1021/ac034949q CCC: $27.50 Published on Web 12/09/2003

© 2004 American Chemical Society

of chemical or biological nature, to determine the enantiomeric purity of commercial pharmaceutical products, or to detect chiral pollutants in biological or environmental matrixes.1 Capillary electrophoresis (CE) has shown to be very useful for chiral separations, as is reflected by the exponential number of papers published since the first paper on chiral separations by CE2 and its increasing use in industry, mainly in the pharmaceutical sector. CE is characterized by high-efficiency separation, large flexibility, and low reagent consumption. This explains why it is attractive for the separation of stereoisomeric compounds, which often requires the use of (expensive) chiral selectors.3 A wide variety of chiral selectors has been employed, not only native cyclodextrins and neutral or charged cyclodextrin derivatives but also bile salts, monomeric and polymeric chiral surfactants, crown ethers, calixarenes, proteins, macrocyclic antibiotics, and polysaccharides or alkaloids.4 Nonetheless, cyclodextrins are among the most frequently used and the best studied chiral selectors; they can form cyclodextrin-analyte inclusion complexes with various types of compounds.5-9 This CE working mode using cyclodextrins is denoted as cyclodextrin-based electrokinetic chromatography (CDEKC).3 Unfortunately, in many cases, the application of CD-EKC combined with conventional UV absorption detection is hampered by poor concentration detection limits. To solve this problem, either analyte preconcentration procedures have to be involved or alternative detection systems have to be developed. In the present paper, it is demonstrated that quenched phosphorescence detection fulfills the sensitivity requirement for camphorquinone. (1) Kallenborn, R.; Hu ¨ hnerfuss, H. Chiral Environmental Pollutants. Trace Analysis and Ecotoxicology; Springer; Berlin, 2001. (2) Fanali, S. J. Chromatogr., A 1989, 474, 441-446. (3) Chankvetadze, B.; Blaschke, G. J. Chromatogr., A 2001, 906, 309-363. (4) Chankvetadze, B. Capillary Electrophoresis in Chiral Analysis; John Wiley & Sons: Chichester, U.K., 1997. (5) Garcı´a-Ruiz, C.; Martı´n Biosca, Y.; Crego, A. L.; Marina, M. L. J. Chromatogr., A 2001, 910, 157-164. (6) Martı´n Biosca, Y.; Garcı´a-Ruiz, C.; Marina, M. L. Electrophoresis 2000, 21, 3240-3248. (7) Martı´n Biosca, Y.; Garcı´a-Ruiz, C.; Marina, M. L. Electrophoresis 2001, 22, 3216-3225. (8) Garcı´a-Ruiz, C.; Marina, M. L. Electrophoresis 2000, 21, 1565-1573. (9) Garcı´a-Ruiz, C.; Marina, M. L. Electrophoresis 2001, 22, 3191-3197.

Analytical Chemistry, Vol. 76, No. 2, January 15, 2004 399

Phosphorescence detection in CE as an alternative for UV absorption has recently been introduced.10-14 Although initially the focus was only on capillary zone electrophoresis and thus on charged analytes, the use of quenched phosphorescence detection for neutral nitroaromatic compounds in CD-EKC has also been reported, providing limits of detection down to the 10-8 M range.14 Apparently, the dynamic phosphorescence quenching reaction between the analytes and the phosphorophore, i.e., brominated naphthalenesulfonate, is not severely hampered by the formation of the inclusion complex with cyclodextrin. Here, it will be shown that the same holds for the detection of the enantiomers of chiral compounds with emphasis on camphorquinone. Within this context it should be noted that the applicability range of quenched phosphorescence detection for neutral molecules is fairly wide. This was already illustrated some twenty years ago in a study dealing with column liquid chromatography using biacetyl as the phosphorophore.15 Camphorquinone is a photochemically reactive compound, which upon irradiation at wavelengths in the 400-500-nm range (its maximum absorption is ∼450 nm) generates radicals.16 For this reason, it is frequently used as a photosensitizer for dental resin composities17 and, furthermore, as a photoinitiator for lightcured resin compositions.18,19 Since camphorquinone shows low UV-visible absorptivity, an alternative, more sensitive detection system would be welcome. Camphorquinone is a chiral bicyclic 2,3-dione-type monoterpene which according to the literature can be reduced stereoselectively to the corresponding R-keto alcohols by yeast,20,21 various other fungi,22 or plant cultured cells.23 These studies20-23 focused on the stereochemistry of the generated products, while only scarce information was provided about the stereoselectivity of the biodegradation process as such. In the present work, we intend to monitor the degradation of camphorquinone by yeast in order to establish whether this biodegradation process is enantioselective and to test the chiral detection system in practice. EXPERIMENTAL SECTION Chemicals and Samples. Boric acid was obtained from J. T. Baker (Deventer, The Netherlands). Carboxymethyl β-cyclodex(10) Kuijt, J.; Brinkman, U. A. Th.; Gooijer, C. Anal. Chem. 1999, 71, 13841390. (11) Kuijt, J.; Brinkman, U. A. Th.; Gooijer, C. Electrophoresis 2000, 21, 13051311. (12) Kuijt, J.; de Rijke, E.; Brinkman, U. A. Th.; Gooijer, C. Anal. Chim. Acta 2000, 417, 15-17. (13) Kuijt, J.; van Teylingen, R.; Nijbacker, T.; Ariese, F.; Brinkman, U. A. Th., Gooijer, C. Anal. Chem., 2001, 73, 5026-5029. (14) Kuijt, J.; Arraez Roman, D.; Ariese, F.; Brinkman, U. A. Th.; Gooijer, C. Anal. Chem. 2002, 78, 5139-5145. (15) Donkenbroek, J. J.; Veltkamp, A. C.; Gooijer, C.; Velthorst, N. H.; Frei, R. W. Anal. Chem. 1983, 55, 1886-1893. (16) Atsumi, T.; Iwakura, I.; Fujisawa, S.; Ueha, T. Arch. Oral Biol. 2001, 46, 391-401. (17) Park, Y. J.; Chae, K. H.; Rawls, H. R. Dent. Mater. 1999, 15, 120-127. (18) Ziani-Cherif, H.; Abe, Y.; Imachi, K.; Matsuda, T. J. Biomed. Mater. Res. 2002, 59, 386-389. (19) Kawada, T.; Nakayama, Y.; Zheng, C.; Ohya, S.; Okuda, K.; Sunagawa, K. Biomaterials 2002, 23, 3169-3174. (20) Rebolledo, F.; Roberts, S. M.; Willetts, A. J. Biotechnol. Lett. 1991, 13, 245248. (21) Chenevert, R.; Thiboutot, S. Chem. Lett. 1988, 1191-1192. (22) Miyazawa, M.; Nobata, M.; Hyakumachi, M.; Kamboka, H. Phytochemistry 1995, 39, 569-573. (23) Chai, W.; Hamada, H.; Suhara, J.; Akira Horiuchi, C. Phytochemistry 2001, 57, 669-673.

400 Analytical Chemistry, Vol. 76, No. 2, January 15, 2004

Figure 1. Structures of the enantiomers of camphorquinone.

trin (CM-β-CD, degree of substitution ∼3), R-cyclodextrin (R-CD), and (()-camphorquinone were purchased from Fluka (Steinheim, Germany). β-Cyclodextrin (β-CD), (1R)-(-)-camphorquinone, and (1S)-(+)-camphorquinone were from Aldrich (Steinheim, Germany). The structures of the enantiomers of camphorquinone are shown in Figure 1. 1-Bromo-4-naphthalenesulfonic (BrNS) acid was synthesized in-house.13 Water used to prepare solutions was purified through a Milli-Q system from Millipore (Bedford, MA). Buffer solutions were filtered through 45-µm syringe filters (Schleicher & Schuell, Dassel, Germany). Instrumentation. The setup developed by Kuijt el al.14 was further optimized and modified. A CE injection/high-voltage system from PrinCE (Lauerlabs, Emmen, The Netherlands) and an LS-40 luminescence LC detector (Perkin-Elmer, Beaconsfield, U.K.) adapted for CE10 were used. Because thorough deoxygenation of the buffer is required to observe room-temperature phosphorescence, a modified vial was used for the buffer solution.14 This vial contained a hole connected to Teflon HPLC tubing (0.50 mm) for a continuous flow of nitrogen through the buffer solution. Since this flow should be very low and constant to observe stable baselines, a flow meter (Porter, Hatfield, PA) was used between the vial and the nitrogen cylinder. This setup, which is very simple, has enabled us to work automatically in the normal polarity mode; the creation of an overpressure in the CE system is avoided, since during the analysis (when voltage is applied) the dynamic injection system of the PrinCE is open to the ambient air. Buffer solutions were deoxygenated for 10 min with a nitrogen flow of 18 mL/min14 and after that the capillary was filled by pressure, observing in this case a low overpressure, which was adjusted by the dynamic injection system. With the LS-40 luminescence LC detector an uncoated fused-silica capillary from BGB Analytik (Anwil, Switzerland) with 50-µm inner diameter (i.d.), 375-µm outer diameter (o.d.) and with an effective length of 60 cm (100 cm total length) was employed. The measurements were made at room temperature. For detection, a delay time of 0.05 ms and a gating time of 5.00 ms were chosen. Excitation of BrNS, the phosphorophore present in the buffer at a concentration of 1 mM, was performed at 294 nm and emission was collected by means of the total emission mirror and a 390-nm cutoff filter provided with the instrument.13,14 An HP3D CE system (Hewlett-Packard, Waldbronn, Germany) equipped with an on-column diode array detector and HP 3D-CE Chemstation software was also used for comparison. In this case, a shorter capillary was used (68.5 cm total length); the other characteristics were the same. The capillary temperature was set to 25 °C, and UV detection was performed at 200 and 450 nm. Analytical Procedure. Electrolytic solutions were prepared by weighing and dissolving the appropriate amount of buffer in Milli-Q water to obtain the required concentration, adjusting the

pH to the desired value with 0.1 and 1 M sodium hydroxide solution and adding solvent to the required volume. Stock solutions of racemic camphorquinone were prepared by dissolving it in Milli-Q water up to a final concentration of 5 × 10-6 M for phosphorescence detection and 5 × 10-3 M for UV detection. Buffers and stock solutions were stored in the refrigerator at 4 °C. At the beginning of the day, the capillaries were rinsed for 15 min (1000 mbar) with 0.1 M NaOH. Injections were made by pressure using 50 mbar for 6 s (1.4 nL). In all cases the applied voltage was 20 kV. Biochemical Procedure. The yeast strain used in the experiments performed in this work was the TM141 strain (MATa leu2 ura3 trp1 his3).24 Cell were cultured at 30 °C in 150 mL of YPD medium (2% glucose, 2% bactopeptone, and 1% yeast extract) using Erlenmeyer flasks on a rotary shaker (200 rpm). Racemic camphorquinone (12 × 10-3 M) was added when the culture reached an OD660 of 0.2. At the same time, the same concentration (12 × 10-3 M) of racemic camphorquinone was added to the YPD medium at 30 °C as control. Aliquots (20 mL) were taken at 0 h (directly after adding the camphorquinone to the yeast culture) and after 1, 2, 3, and 24 h. Cell were pelleted by centrifugation (5 min, 4000 rpm, Heraeus Megafuge 1.0); the supernatant was frozen in liquid nitrogen and stored at -80 °C. After thawing, the supernatant was injected in the CE system after a 100 times dilution with methanol. The cell fraction was frozen by directly pipetting drops of cells into liquid nitrogen. Frozen cells were stored at -80 °C. Frozen cells were broken using a MikroDismembranator (Braun Biotech) for 2 min at 2000 rpm, after which the resulting cell powder was extracted with 1 mL of methanol and centrifugated (2 min, 14 000 rpm, Eppendorf 5417C microcentrifuge) in order to separate the solid particles from the extract. The extracts were 100 times diluted with methanol prior to injection in the CE system, yielding solutions that were ∼20 times more concentrated than the control and culture solutions. Data Treatment. The experimental data were processed using Microsoft Excel 97 from Microsoft Office software and Origin 6.1 from OriginLab Corp.

Figure 2. Signal of racemic camphorquinone employing UV detection at 200 nm (a, right axis) and quenched phosphorescence detection using BrNS as phosphorophore (b and c, left axis) for high and low camphorquinone concentrations, respectively. Buffer, 25 mM borate, 10 mM CM-β-CD, 1 mM BrNS (pH 9.0); applied voltage, 20 kV; injection by pressure, 50 mbar for 6-s sample. Note: differences in migration times are due to the different total lengths of the capillaries used in the two CE systems. EOF, electroosmotic flow; (()CQ, (()camphorquinone.

RESULTS AND DISCUSSION The photophysical properties of camphorquinone show some similarities to those of the well-known phosphorophore biacetyl,25 which can be readily conceived since both molecules contain an R-diketone chromophoric group. They both show low absorptivities in the UV-visible region, which makes their absorption detection in CE rather cumbersome. However, they show an energetically low-lying T1 electronic state (corresponding to ∼550 nm for camphorquinone), which makes them appropriate energytransfer acceptors for the dynamic quenching of brominated naphthalenesulfonate phosphorescence with the T1 state at ∼490 nm.13 Contrary to biacetyl, the phosphorescence quantum yield of camphorquinone in aqueous solutions is negligible; therefore, sensitized phosphorescence as a result of energy transfer plays no role. Figure 2a shows the electropherogram obtained when a 5 × 10-3 M solution of racemic camphorquinone was injected in the

CE system. UV detection at 200 nm yielded only a small peak (at 450 nm, the maximum absorption wavelength in the visible range, no signal was observed under these conditions). Figure 2b shows that the signal obtained by quenched phosphorescence detection for the same concentration (5 × 10-3 M) is a strong and broad band due to complete quenching at that concentration. In fact, it was necessary to decrease the concentration 1000-fold (5 × 10-6 M) to see an acceptable peak in the electropherogram (Figure 2c). The associated limits of detection (calculated as three times the peak-to-peak noise measured over 4 min) were 2 × 10-3 M by UV detection and 7 × 10-7 M by phosphorescence detection. For the electropherograms in Figure 2, an anionic cyclodextrins 10 mM CM-β-CD in borate bufferswas used at a sufficiently high pH to ensure that it was negatively charged. Apparently, under these conditions, camphorquinone interacts with CM-β-CD, but the enantiomers are not separated. Since the use of dual cyclodextrin systems can provide unique selectivities that cannot be achieved using single cyclodextrins,5,26,27 two dual cyclodextrin systems were tested in order to achieve enantiomeric separation, i.e., β-CD/CM-β-CD and R-CD/ CM-β-CD. The use of 10 mM β-CD/10 mM CM-β-CD in borate buffer at pH 8.5 did not induce any chiral separation. However, by utilizing R-CD instead of β-CD, a good separation of the enantiomers of camphorquinone could be performed. Figure 3 shows the enantiomeric separation of camphorquinone at increasing concentrations of R-CD. From these results, the combination of 10 mM CM-β-CD and 20 mM R-CD with 1 mM BrNS in borate at pH 9 was chosen as the optimum composition, yielding the best enantiomeric separation in the shortest migration time. It should be noted that in this system the negatively charged cyclodextrin (CM-β-CD) acts as the carrier of the neutral camphorquinone while the neutral cyclodextrin R-CD acts as the chiral

(24) Alonso-Monge, R.; Real, E.; Wojda, I.; Bebelman, J. P.; Mager, W. H.; Siderius, M. Mol. Microbiol. 2001, 41, 717-730. (25) Donkerbroek, J. J.; Gooijer, C.; Velthorst, N. H.; Frei, R. W. Anal. Chem. 1982, 6, 891-895.

(26) Lurie, I. S.; Klein, R. F. X.; Dal Cason, T. A.; LeBelle, M. J.; Brenneisen, R.; Weinberger, R. E. Anal. Chem. 1994, 66, 4019-4026. (27) Lelie`vre, F.; Gareil, P.; Bahaddi, Y.; Galons, H. Anal. Chem. 1997, 69, 393401.

Analytical Chemistry, Vol. 76, No. 2, January 15, 2004

401

Table 1. ER Values Calculated for the Control and Medium Solutions and for the Extract of Cells at Different Incubation Times (0, 1, 2, 3, and 24 h)a incubation time (h)

ER cells

ER medium

ER control

0 1 2 3 24

0.93 0.92 0.98 0.92 0.25

1.01 0.99 0.94 0.95 0.24

1.01 1.01 0.92 1.02 0.92

a Reproducibility, calculated as the RSD of the five different control solutions, was 5%.

Figure 3. Variation of the enantiomeric resolution of racemic camphorquinone (5 × 10-6 M) in a 25 mM borate buffer (pH 8.5), 10 mM CM-β-CD as a function of the R-CD concentration added: (a) 0, (b) 5, (c) 10, (d) 15, and (e) 20 mM; other experimental conditions as in Figure 2.

selector discriminating between both enantiomers. Under the selected conditions, the pure enantiomers of camphorquinone were injected individually, enabling the identification of each enantiomer in the racemic mixture. The first migrating peak enantiomer corresponds to (1S)-(+)-camphorquinone and the second to (1R)-(-)-camphorquinone. This is in line with the fact that (1R)-(-)-camphorquinone forms less strong complexes with R-CD28 so that it is more efficiently transported by CM-β-CD. It should be noted that in the electropherograms in Figure 3 the relative peak height variations observed for the two enantiomers are mainly caused by differences in peak widths. In fact, it was found that the area for the first migrating peak was always slightly smaller than for the second one (within 12%). This indicates the presence of a slightly higher concentration of the latter enantiomer, (1R)-(-)-camphorquinone, in the aqueous phase where the dynamic quenching of the phosphorescence signal provided by the negatively charged phosphorophore BrNS takes place. Small differences in peak height should be expected since the concentrations of uncomplexed (1S)-(+)-camphorquinone and (1R)(-)-camphorquinone will not be identical due to the different association constants with R-CD.28 Therefore, separate calibration lines were determined for the two enantiomers. The above technique was used to investigate whether the degradation of racemic camphorquinone by yeast is enantioselective. For this purpose, the enantiomeric ratio (ER) was determined at different incubation times. The ER values were calculated as the ratio of the (1S)-(+)- and (1R)-(-)-camphorquinone concentrations, which were determined by means of the calibration lines obtained for each enantiomer under the selected separation conditions. To compensate for differences in peak width, especially notable in the yeast samples, peak areas instead of peak heights were determined. In a quenching experiment, peak areas do not increase linearly with the analyte concentration. Linear calibration curves were obtained by multiplying these areas with Io/I, according to the Stern-Volmer equation, where Io is (28) Bortolus, P.; Marconi, G.; Monti, S.; Mayer, B. J. Phys. Chem. A 2002, 106, 1686-1694.

402 Analytical Chemistry, Vol. 76, No. 2, January 15, 2004

Figure 4. Signal for the enantiomers of camphorquinone in the control (a), in the medium (b), and in the extract from the cells (c), obtained after 24 h, injected directly after 100 times dilution with methanol in the CE-phosphorescence detection system. Other experimental conditions as in Figure 2.

the phosphorescence signal in the absence of analyte and I is the signal in the presence of analyte (bottom of the negative peak). The linear dynamic range thus obtained was from 1 × 10-6 to 5 × 10-5 M and R2 values were better than 0.999 for both enantiomers. Migration times of the camphorquinone enantiomers were corrected by dividing them by the migration time of the electroosmotic flow; their precision, determined for the five independent control solutions analyzed, was ∼2%. The ER values determined for the medium and control solutions as well as for the extraction of cells are given in Table 1. It can be seen that during the first 3 h the change in ER values was not significant. However, after 24 h of incubation, the ER had decreased from 0.92 for the control solution, where yeast was not added, to 0.24 for the medium or to 0.25 for the camphorquinone extracted from the cells. These results show unambiguously that the first migrating enantiomer, which corresponds to (1S)-(+)camphorquinone, is reduced as much as 70% in the medium and also in the cells. The fact that a similar ER was found in the cells excludes the possibility that the selective disappearance from the medium was due to different membrane diffusion rates. We conclude that racemic camphorquinone is degraded enantioselectively by the yeast strain used in this work. These results can also be observed in Figure 4, showing the electropherograms obtained when the control and medium solution and the extract of cells obtained after 24 h were injected directly, after 100 times dilution with methanol, in the CE-phosphorescence detection system.

CONCLUSIONS The use of quenched phosphorescence detection provides sufficient sensitivity for camphorquinone in CD-EKC so that preconcentration techniques are not needed to study real samples. Camphorquinone enantiomers could be adequately separated in a charged dual cyclodextrin system (CM-β-CD/R-CD), where only R-CD offers chiral discrimination for camphorquinone. The stereoselectivity of the degradation of camphorquinone by yeast could

be directly demonstrated. Similar approaches will be followed for other chiral compounds. ACKNOWLEDGMENT C.G.-R. gratefully thanks the European Commission for a postdoctoral Marie Curie Individual fellowship (Contract HPMFCT-2002-01826). AC034949Q

Analytical Chemistry, Vol. 76, No. 2, January 15, 2004

403