Phase-modulation uorescence lifetime resolution of quinine and quinidine by ion-pair diastereomeric complexation

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1 Analytica Chimica Acta 381 (1999) 11±16 Phase-modulation uorescence lifetime resolution of quinine and quinidine by ion-pair diastereomeric complexation A. Navas DõÂaz *, F. GarcõÂa SaÂnchez, M.C. Torijas Departamento de QuõÂmica AnalõÂtica, Facultad de Ciencias, Universidad de MaÂlaga, MaÂlaga, Spain Received 23 July 1998; received in revised form 22 September 1998; accepted 26 September 1998 Abstract Quinine (QN) and quinidine (QD) ion-pair diastereomeric complex resolution based on phase-modulation resolved uorescence has been developed. ( )-10-Camphorsulfonic acid used as a counter-ion gives diastereomeric complexes with QN and QD which have different stabilities. They show different lifetimes (QN complex, ˆ21.79 ns and QD complex, ˆ22.89 ns). On the basis of this difference both complexes have been determined, obtaining a detection limit of 1.8 mm for the QN complex and a 0.97 mm for the QD complex. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Phase-modulation; Lifetime; Quinine; Quinidine; Ion-pair diastereomeric complex 1. Introduction Many uorophores exhibit very similar spectra and are not always amenable to the techniques which rely upon differences in the excitation or emission spectra. This is the case of enantiomeric compounds because they have identical uorophores. Time domain data offer a powerful alternative or supplemental means for examining and quantitating heterogeneous emitting systems. Phase-modulation resolved uorescence spectroscopy (PMRFS) provides a means by which uorescence lifetime selectivity can be implemented [1]. Chiral phase liquid chromatography with photometric or uorimetric detection is widely used for enantiomer separation. Alternatively, enantiomers' *Corresponding author. Fax: ; a_navas@uma.es uorescence lifetime differences can be used to resolve them without separation. Modulation and phase uorescence spectroscopy can be used for the determination of quinine (QN) and quinidine (QD). QN and QD show small lifetime differences with emission and excitation wavelengths, ph and solvents [2±4]. But these differences in lifetimes are not suitable for QN and QD resolution. However, luminescence characteristics of organic molecules may be altered by the environment for a variety of reasons. The formation of diastereomeric complexes (ion pairs) may alter the photophysical properties of the enantiomers on the basis of their different interactions with the environment. Thus, we have used an indirect resolution of enantiomers as diastereomeric derivatives. A chiral counter ion (( )-10-camphorsulfonic acid) is added to the QN and QD solution and two diastereomeric complexes are formed (formation of diastereomeric ion pairs) [5] /99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S (98)

2 12 A. Navas DõÂaz et al. / Analytica Chimica Acta 381 (1999) 11±16 This present work describes the uorescence lifetime resolution of QN and QD which involves several factors that in uence the system, lifetime determinations and data manipulations. The theory and instrumentation of phase-modulation uorescence spectroscopy (PMFS), described elsewhere [6,7], are based on the phase-modulation technique for the determination of uorescence lifetimes. A technique for recording the individual component spectra of two and three-component mixtures which uses phase or modulation measurements has been described [8]. For two component mixtures, information about either the phase angle or modulation ratio (together with the total intensity) is suf cient to calculate the relative contributions when the lifetimes of the two components are known. Two explicit expressions for these fractional contributions (f 1 and f 2 ) calculated from either phase or demodulation data have been previously reported [9]: R p ˆ 2 p 1!2 1 2 p 1 1! ; (1) s, 1! R m ˆ! ! ! 2 m 2 1 s 1! ! ! 2 m 2 1! 2 ; (2) Fig. 1. Structure and stereochemistry of the ``pseudo-enantiomeric'' (A) quinine and (B) quinidine, (C) structure of ( )-10- camphorsulfonic acid. where Rˆf 1 /f 2 (the subscripts p and m refer to data obtained using either phase (p) or modulation (m) values), and p and m are the phase and modulation lifetimes of the mixture, respectively. Phase-modulation resolved uorescence spectroscopy uses the lifetimes determined from modulation measurements to calculate the relative fractional contributions of the two components (in this case, QN and QD) of the samples (Eq. (2)). QN and QD have received much attention as a cheap natural source for stereodiscriminating auxiliaries. Structurally, QN and QD consist of a planar quinoline and a rigid quinuclidine ring, which are connected by a secondary methanol bridge. Conformational exibility is available only for the torsions of the C 8 ±C 9 and C 9 ±C 4 bonds. The arrangement of the ve stereogenic centers within the rigid molecules QN and QD may provide an excellent basis for effective chiral recognition. Although they are diastereomers, QN and QD often display quasi-enantiomeric behavior. Therefore, they are sometimes called ``pseudoenantiomers'' (Fig. 1). This term can be addressed to the fact that in most cases stereoselectivity is under C 8 /C 9 control and also to the opposite con gurations at these chiral centers. 2. Experimental 2.1. Chemicals Quinine hemisulfate was provided by Sigma (>93% of purity; Q-1250, St. Louis, MO) and quinidine sulfate salt dihydrate also by Sigma (Q-0875). Both compounds were recrystallized twice from ethanol. We prepared stock solutions of QN and QD (110 4 M) dissolving the appropriate amount in ethanol. (1S)-( )-10-Camphorsulfonic acid was from Sigma (C-1395) and was prepared as a stock solution (110 2 M) in n-hexane. 9-Aminoacridine hydrochloride hydrate (98%) was obtained from Aldrich (A3,840-1, Steinhein, Germany) and a stock solution (110 3 M) was prepared in ethanol. n-hexane was provided by Merck (Darmstadt, Germany) Apparatus Fluorescence measurements were made with an Aminco SLM 48000S spectro uorimeter equipped with a 450 W xenon lamp source, a Hamamatsu R928 photomultiplier tube detector, and a Pockel cell

3 A. Navas DõÂaz et al. / Analytica Chimica Acta 381 (1999) 11±16 13 electro-optic modulator. An IBM PC AT microcomputer was used for on-line data acquisition and data processing. Fluorescence lifetime determinations were made using multifrequency modulated excitation beams and a 9-aminoacridine solution was the reference (ˆ12.0 ns). Lifetime values based on modulation measurements were obtained at a xed modulation frequency of 15 MHz at the ``200 average'' mode in which each measurement is the average of 200 samplings, carried out automatically by the instrument circuitry during a period of approximately 50 s. The excitation monochromator was set at 358 nm, and the slits were set at 16 nm with a 16 nm bandpass entrance. To select the emission wavelength, instead of a monochromator, a cut-off lter (470 nm) was placed in the sample emission receiving channel and the start channel received the solution reference. The measurements (sample and reference) were made in quartz cuvettes. The temperature was controlled by a Frigiterm (Selecta) and xed at 208C. 3. Results and discussion In aqueous solution, the uorescence lifetimes of QN and QD increase with the wavelength of emission studied at different ph values and these QN lifetimes present small differences with regard to QD lifetimes [3]. However, these differences are not suitable for the resolution of both compounds. So, the formation of diastereomeric ion-pairs were studied to obtain greater differences in their lifetimes. The ion-pairs were obtained by adding ( )-10- camphorsulfonic acid as the counter ion. The ( )- 10-camphorsulfonic acid structure is shown in Fig. 1. This counter ion has been previously used to separate enantiomeric b-aminoalcohols [10,11]. QN and QD form diastereomeric complexes with ( )-10-camphorsulfonic acid with different stabilities [12]. QN -10-Camphorsulfonic acid! QN : -10-Camph: QD -10-Camphorsulfonic acid! QD : -10-Camph: The chiral counter-ion is used with organic solvents of low polarity to promote a high degree of ion-pair formation. In this case, n-hexane has been selected as the solvent because it has a very low polarity ET N ˆ 0:009 and is miscible with ethanol (sample solvent). The QN and QD lifetimes were measured under two different conditions: (a) QN or QD in n-hexane and (b) QN or QD plus ( )-10-camphorsulfonic acid in n-hexane. To obtain uorescence lifetimes, a multifrequency data acquisition was applied and the values of lifetime were obtained using multi-frequency heterogeneity analysis. The lifetimes obtained without counter-ion were 1.82 ns ( 2ˆ6.2) for QN and 1.84 ns ( 2ˆ5.4) for QD. When ( )-10-camphorsulfonic acid was added, dramatic changes in the lifetimes were obtained, as a consequence of complex formation, i.e., to ns ( 2ˆ5.8) for the QN complex and ns ( 2ˆ7.5) for the QD complex. Also, the excitation and emission spectra of both compounds under conditions (a) and (b) were obtained and the spectra are shown in Fig. 2. As a consequence of QN and QD complex formation, there is a large shift of excitation and emission spectra in the presence of the counter-ion ( excˆ 358 nm and emˆ444 nm), besides a great change in their lifetimes, as shown above (Fig. 3). As well, we can observe 1 ns difference in their lifetimes (although the excitation and emission spectra have identical maxima). This lifetime difference gives us a suitable difference to apply the above mentioned technique Choice of modulation frequency To calculate the QN and QD complex lifetime multi-frequency data acquisition was applied. The lifetime obtained at each frequency and the AC and DC intensity values allow us to choose the best modulation frequency. At 15 MHz a 1.2 ns lifetime difference was obtained. Besides, at this frequency high AC and DC intensities were obtained which give good reproducibility in the measurements. The lifetime modulation values at 15 MHz were m (QN complex)ˆ ns and m (QD complex)- ˆ ns Reference solution Errors in phase and modulation lifetime measurements could be reduced by using single lifetime

4 14 A. Navas DõÂaz et al. / Analytica Chimica Acta 381 (1999) 11±16 Fig. 2. Excitation and emission spectra of QN (dotted line) and QD (solid line) in n-hexane (A) without counter ion, and (B) with counter ion. RFIˆrelative fluorescence intensity. Table 1 Measured modulation lifetime values of QN and QD complexes at several counter ion concentrations ( )-10-Camph. acid (M) (QN a ) (ns) (QD b ) (ns) c c c c c c d d Fig. 3. Excitation and emission spectra of QN without counter ion (solid line) and with counter ion (dotted line). a [QN]ˆ110 5 M. b [QD]ˆ610 6 M. c excˆ358 nm; em cut-off filter at 470 nm, 9-aminoacridine as reference solution, 15 MHz. d excˆ332 nm; em cut-off filter at 370 nm, glycogen as reference solution, 40 MHz. (homogeneous) reference standards whose uorescence lifetime approximated to that of the unknown sample (isochronal standards) [13]. Glycogen is commonly used as a scatter reference and its lifetime is 0 ns. QN and QD complexes have a long lifetime and considerable errors in lifetimes have been observed using glycogen as reference. The use of isochronal uorescence standards minimise errors. Thus, 9-aminoacridine (ˆ12.0 ns) solution was used as a reference standard for all the lifetime measurements Counter-ion concentration A series of measurements were made at several counter-ion concentrations, in which the QN and QD concentrations were kept constant and variable volumes of ( )-10-camphorsulfonic acid stock solution were added. The results obtained are shown in Table 1. The last measurement in Table 1 has been obtained with counter-ion concentrations lower than the QN and QD concentrations, so the complexes are not formed and the instrumental parameters and lifetimes change. Thus, the ( )-10-camphorsulfonic acid concentration must be higher than the QN plus QD concentrations. If the counter-ion concentration is not high enough, the complexes are not formed and lifetimes decrease. To assure complex formation the counter-ion concentration used is M Temperature and cut-off emission filter selection A temperature study has been made and the results are collected in Table 2. The QN and QD lifetimes increase as the temperature drops and the viscosity increases. However, the lifetime difference

5 A. Navas DõÂaz et al. / Analytica Chimica Acta 381 (1999) 11±16 15 Table 2 Comparative study of QN and QD complex modulation lifetimes against temperature change T a (8C) (n-hexane), cp. (QN) a (ns) (QD) a (ns) 10 ± a excˆ358 nm; em cut-off filter at 470 nm, 9-aminoacridine as reference solution, 15 MHz. between QN and QD complexes are constant and room temperature has been selected to make measurements. To obtain good AC and DC readings a high power excitation beam must be used. With two monochromators (excitation and emission) the power impinging on the photomultiplier is low, and an enhancement can be obtained by replacing the emission monochromator with a cut-off lter. Two different cut-off lters were used. The rst is a cut-off lter at 418 nm and the second at 470 nm. In the rst case, the maximum emission wavelength ( emˆ444 nm) was used to calculate the uorescence lifetimes. In the second case, the maximum emission wavelength cannot go through the cut-off lter and a range of longer wavelengths was used. The lifetimes obtained were: (A) m (QN complex)ˆ20.99 ns, m (QD complex)ˆ21.58 ns with a cut-off lter at 418 nm. (B) m (QN complex)ˆ ns, m (QD complex)ˆ21.80 ns with a cut-off lter at 470 nm. The cut-off lter at 470 nm produces a greater lifetime difference ( mˆ1 ns) than the cutoff lter at 418 nm ( mˆ0.6 ns). This small lifetime change using different emission lters is produced by Fig. 4. Calibration plots for QN (*) and QD (^). the QN and QD lifetimes dependence on the emission wavelengths [2]. This previous study re ects a lifetime increment with the wavelength increment, besides a greater lifetime increase for QD Calibration The instrumental parameters were xed at the previously optimized conditions: excˆ358 nm, em cutoff lter at 470 nm, 9-aminoacridine (in ethanol solution) as reference solution (ˆ12.0 ns), modulation frequency 15 MHz and 208C. Five mixtures of QN and QD were selected in order to give the same DC intensity. The appropriate volumes of QN, QD, coun- Table 3 Analytical parameters of the photometric/polarimetric liquid chromatographic (LC) detection and phase-modulation resolved fluorescence intensity for QN and QD resolution QN QD PMRFI PLC PMRFI PLC Linear range 2±10.6 mm 3±30.8 mm 1±6 mm 3±30.8 mm Regression coefficient a DL b 1.8 mm 2.5 mm 0.97 mm 2.6 mm RSD c 9.5% 5.9% 10.5% 4.0% a nˆ6. b 3SD of blank/m (slope of calibration graph). c Four measurements at nˆ6.

6 16 A. Navas DõÂaz et al. / Analytica Chimica Acta 381 (1999) 11±16 Table 4 Obtained results from QN and QD recovery assay Compound Taken (mm) Found (mm) Recovery (%) QN QD ter-ion and n-hexane were put into the quartz cuvette (total volume of 3 ml) and the cuvette was shaken. The individual fractional contribution of the QN and QD complexes (f 1 and f 2 ) were plotted against concentration to obtain calibration graphs (Fig. 4). The analytical parameters are shown in Table 3. Also, in Table 3 is a comparison of results obtained by this method and liquid chromatography with photometric and polarimetric detection for QN and QD resolution. The precision of the method was assessed by measuring three replicates of each sample. A recovery assay was realised and the results are collected in Table Conclusions Diastereomeric ion-pair formation between QN/QD and counter-ion produce lifetime shifts high enough to resolve the enantiomeric pair by applying modulation uorescence lifetime measurements. The obtained results allowing us to compare favorably with photometric/polarimetric liquid chromatography detection in terms of precision and sensitivity. References [1] L.B. McGown, F.V. Bright, Anal. Chem. 56 (1984) [2] S.G. Schulman, R.M. Threatte, A.C. Capomacchia, W.L. Poul, J. Pharm. Sci. 63 (1974) 876. [3] D. Pant, H.B. Tripathi, D.D. Pant, J. Lumin. 51 (1992) 223. [4] S. Pant, H.B. Tripathi, D.D. Pant, J. Photochem. Photobiol. A 85 (1995) 33. [5] C. Pettersson, K. No, J. Chromatogr. 282 (1983) 671. [6] T.V. Vaseleva, A.S. Cherkasov, V.I. Shirokov, Opt. Spectrosc. 29 (1970) 617. [7] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, Plenum Press, New York, [8] E. Gratton, D.M. Jameson, Anal. Chem. 57 (1985) [9] D.M. Jameson, E. Gratton, R.D. Hall, Appl. Spectra Rev. 20 (1984) 55. [10] C. Pettersson, G. Schill, J. Chromatogr. 204 (1981) 179. [11] C. Pettersson, G. Schill, Chromatographia 16 (1982) 192. [12] C. Petterson, in: A. Krstulovic (Ed.), Chiral Separations by HPLC, Horwood, Chichester, 1989, p [13] D.A. Barrow, B.R. Lentz, J. Biochem. Biophys. Methods 7 (1983) 217.