Anal. Chem. 1996, 68, 2826-2831
Mechanism of Enantioseparation of Salsolinols, Endogenous Neurotoxins in Human Brain, with Ion-Pair Chromatography Using β-Cyclodextrin as a Mobile Phase Additive Yulin Deng,† Wakako Maruyama,§ Hatsuo Yamamura,† Masao Kawai,† Philippe Dostert,⊥ and Makoto Naoi*,‡
Departments of Applied Chemistry and Biosciences, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya, Japan, Laboratory of Biochemistry and Metabolism, Department of Basic Gerontorlogy, National Institute for Longevity Sciences, Obu, Japan, and Research and Development, Pharmacia-Upjohn, Milan, Italy
A novel method for direct separation of the enantiomers of salsolinol and N-methylsalsolinol was devised. The enantiomers were completely separated with ion-pair chromatography on a reversed-phase column with β-cyclodextrin as a chiral mobile phase additive and sodium 1-heptanesulfonate as a counterion. The mechanism for enantioseparation with this ion-pair system containing β-cyclodextrin was discussed. The effects of β-cyclodextrin, counterions, pH, ionic strength, and organic solvent on retention were investigated, and a retention model was proposed and proved to be consistent with the experimental data. A preliminary study of the enantiomeric composition of salsolinol and N-methylsalsolinol in banana and in human brain was made as an example of the application of this assay. There is currently intense interest in the development of chiral chromatographic methods and the application of such methods, particularly to biological materials. 1-Methyl-6,7-dihydroxy-1,2,3,4tetrahydroisoquinoline (salsolinol, Sal) was found to occur in human brain1 and to be an endogenous neurotoxin.2 The N-methylation of Sal into N-methylsalsolinol (NMSal) was demonstrated by in vivo microdialysis in rat brain.3 NMSal was identified in human brain further by GC/MS.4 As shown in Figure 1, Sal and NMSal have an asymmetric center at C1 and exist as (R)- and (S)-enantiomers. Several reports demonstrated that the chiral properties of Sal and NMSal play an important role in their cytotoxicity, selective to dopamine neurons.5-8 Therefore, it is quite important to determine the enantiomeric composition of Sal †
Department of Applied Chemistry, Nagoya Institute of Technology. Department of Biosciences, Nagoya Institute of Technology. § National Institute for Longevity Sciences. ⊥ Pharmacia-Upjohn. (1) Sjoequist, B.; Eriksson, A; Winblad, B. Prog. Clin. Biol. Res. 1982, 90, 57. (2) Naoi, M.; Maruyama, W.; Dostert, P.; Hashizume, Y.; Nakahara, D.; Takahashi, T.; Ota, M. Brain Res. 1996, 709, 285. (3) Maruyama, W.; Nakahara, D.; Ota, M.; Takahashi, T.; Takahashi, A.; Nagatsu, T.; Naoi, M. J. Neurochem. 1992, 59, 395. (4) Niwa, T.; Takeda, N.; Yoshizumi, H.; Tatematsu, A.; Yoshida, M.; Dostert, P.; Naoi, M.; Nagatsu, T. Biochem. Biophys. Res. Commun. 1991, 177, 603. (5) Cohen, G.; Heikkila, R. E.; Dembiec, D.; Sang, D.; Teitel, S.; Brossi, A. Eur. J. Pharmacol. 1974, 29, 292. (6) Minami, M.; Takahashi, T.; Maruyama, W.; Takahashi, A.; Dostert, P.; Nagatsu, T.; Naoi, M. J. Neurochem. 1992, 58, 2097. (7) Minami, M.; Maruyama, W.; Dostert, P.; Nagatsu, T.; Naoi, M. J. Neural Transm.: Gen. Sect. 1993, 92, 125. ‡
2826 Analytical Chemistry, Vol. 68, No. 17, September 1, 1996
Figure 1. Structures of enantiomers of salsolinol and N-methylsalsolinol.
and NMSal in biological samples, such as brain tissues. The separation of Sal enantiomers with GC or HPLC has been reported.9-11 In all the previous methods, the enantiomers had to first be derivatized to the diasteroisomers, and it is difficult to use these methods to analyze human brain samples because they are time-consuming and require careful control of experimental conditions. Recently, we developed a method to determine the enantiomers of Sal and NMSal without derivatization using a cyclodextrin-bonded column (ChiraDex, E. Merck, Darmstadt, Germany).12 This method is sensitive enough to determine Sal and NMSal in human brain samples, but the column was too fragile to be used for the crude biological samples. Applications of cyclodextrins in LC have been intensively studied for their utility as mobile phase additives.13 There are several advantages to using chiral selectors in the mobile phase. Less expensive conventional packed columns (such as reversedphase columns) can be used. The type and concentration of the cyclodextrins can be easily changed, and the selectivity is sometimes different from that of the respective chiral stationary phase. Thus, this technique can offer unique selectivity for the chiral separation of complex biological samples. The enantioseparation mechanism for reversed-phase liquid chromatography (RPLC) in the presence of cyclodextrin as a chiral mobile phase (8) Takahashi, T.; Deng, Y.; Maruyama, W.; Dostert, P; Kawai, M.; Naoi, M. J. Neural Transm.: Gen. Sect. 1994, 98, 107. (9) Dostert, P.; Strolin Benedetti, M.; Dedieu, M. Pharmacol. Toxicol. 1987, 60 (Suppl. 1), 12. (10) Strolin Benedetti, M.; Bellotti, V.; Pianezzola, E.; Moro, E.; Carminati, P.; Dostert, P. J. Neural Transm. 1989, 77, 47. (11) Pianezzola, E.; Bellotti, V.; Fontana, E.; Moro, E.; Dal, G.; Dasei, D. M. J. Chromatogr. 1989, 495, 205. (12) Deng, Y.; Maruyama, W.; Dostert, P.; Takahashi, T.; Kawai, M.; Naoi, M. J. Chromatogr. B 1995, 670, 47. (13) Fujimura, K.; Uedo, T.; Kitagawa, M.; Takayanigi, H.; Ando, T. Anal. Chem. 1986, 58, 2668. S0003-2700(96)00185-0 CCC: $12.00
© 1996 American Chemical Society
additive was reported by several researchers.14-16 However, for some hydrophilic amines, such as Sal, it was difficult to obtain retention on conventional bonded reversed-phase system, and ionpair chromatography has been used for the separation.17,18 This article will demonstrate the enantioseparation of Sal and NMSal with an ion-pair chromatographic system using β-cyclodextrin (βCD) in the mobile phase. In the ion-pair system containing cyclodextrin in the mobile phase, the retention equilibrium of analytes becomes more complex. An enantioseparation model for this system is proposed, and the chromatographic behaviors of the enantiomers are discussed in relation to the theory of chiral ion-pair chromatography. EXPERIMENTAL SECTION Materials. Both (R)- and (S)-salsolinol [(R)- and (S)-Sal] and (R)- and (S)-N-methylsalsolinol [(R)- and (S)-NMSal] were synthesized according to the method of Teitel et al.19 Dopamine was purchased from Sigma (St. Louis, MO), and β-cyclodextrin (βCD) was from Nacalai Tesque (Kyoto, Japan). Ion-pair reagents, sodium 1-butanesulfonate (SBS) and sodium 1-heptanesulfonate (SHS), were obtained from Sigma, and sodium 1-octanesulfonate (SOS) was from Aldrich (Milwaukee, WI). All other chemicals were of analytical grade, and organic solvents were of HPLC grade from Nacalai Tesque. Aqueous standard solutions of enantiomeric Sal and NMSal, prepared at concentrations of 10 mM, were stable for at least 6 months when stored at -20 °C. The 1 µM standard solution was prepared fresh daily from the 10 mM solution. Preparation of Human Brain and Banana Samples. Human brain samples were obtained from patients without neurological history. For biological safety, preparation of sample was carried out with care, using disposal instruments. Human brain gray matter was suspended in 10 vol/wet wt of 0.1 M perchloric acid containing 0.1 mM sodium metabisulfite and disodium ethylenediaminetetraacetate and sonicated in a Branson sonicator (Danbury, CT). The sample was centrifuged at 22000g for 10 min at 4 °C, followed by filtration with a Millipore HV filter (pore size, 0.45 µm). The extraction method reported previously10 was applied for dried banana. Dried banana was homogenized in 10 vol/wt of 0.1 M HCl and centrifuged at 22000g for 10 min at 4 °C. The supernatant was diluted to 100 times with distilled water and then filtered through a Millipore HV filter. HPLC Apparatus and Chromatographic Conditions. The HPLC system comprised of a Shimadzu LC-9A pump (Kyoto, Japan), an electrochemical detector Coulochem-II (ESA, Chelmsford, MA), an autosampler AS-8010 (Tosoh, Tokyo, Japan), and a Shimadzu CR6A chromatopac recorder. The separation was performed using a reversed-phase Inertosil ODS-3 column (4.6 mm i.d. × 250 mm, GL Sciences, Tokyo, Japan). The mobile phase for the enantiomeric separation of Sal and NMSal consisted of 25 mM sodium phosphate buffer, pH 3.0, containing 15 mM β-CD, 1 mM sodium 1-heptanesulfonate, and 0.2% 2-methyl-2-propanol, and the flow rate was 0.7 mL/min. In (14) Armstrong D. W.; Nome, F.; Spino, L. A.; Golden, T. D. J. Am. Chem. Soc. 1986, 108, 1418. (15) Walhagen, A.; Edholm, L.-E. Chromatographia 1991, 32, 215. (16) Pullen, R. H.; Brennan, J. J.; Patonay, G. J. Chromatogr. A 1995, 691, 187. (17) Thomas, H.; Stammel, W.; Brossi, A. J. Chromatogr. Sci. 1983, 21, 481. (18) Naoi, M.; Maruyama, W.; Dostert, P. Neurosci. Lett. 1994, 171, 9. (19) Teitel, S.; O’Brein, J.; Brossi, A. J. Med. Chem. 1972, 15, 845.
addition, the mobile phase compositions used for the study on the retention behaviors of enantiomeric Sal and NMSal are described in the figure legends and below, in the text of the Results and Discussion section. The conditions of the Coulochem-II detector were as follows: a conditioning cell, Model 5021, was set at +300 mV, and the first electrode of an analytical cell, Model 5011, was set at +50 mV and the second electrode at -300 mV. The output of the second electrode was monitored. The retention time of the first peak in the chromatogram was used as the holdup time (t0). Data represent the averages of three or more analyses. Enantioseparation Retention Model. The retention behavior in a reversed-phase chromatographic system containing β-CD as a chiral mobile phase additive can be described by the following equation,15 if only one species of solute is present and 1:1 stoichiometric complexes are produced in the mobile phase:
1/k′ ) 1/k0′ + Kf[CD]m/k0′
(1)
where Kf is the apparent formation constant for the inclusion complex, k′ is the capacity ratio in the presence of CD, and k0′ is the capacity ratio in the absence of CD. As seen in eq 1, the reciprocal of the capacity ratio versus the CD concentration is linear, and the slope represents Kf/k0′ and intercept 1/k0′. The ratio of slope over intercept provides directly a value for Kf. This treatment assumes that the distribution of CD and its complex with solute to the stationary phase can be neglected. In the ion-pair chromatography using β-CD and sodium alkanesulfonate in the mobile phase, the equilibrium of analytes will become more complex, and thus a new retention equation should be considered. When large excess of counterion is added to the mobile phase in a reversed-phase ion-pair chromatographic system containing no CD, only the distribution of an ion-pair complex to the stationary phase is assumed, and thus the distribution of a charged analyte Q+ and its counterion X- is represented by the following expression:20
Q+m + X-m ) QXs
(2)
The extraction constant, Kex, is given by
Kex )
[QX]s [Q]m[X]m
(3)
where QX is the solute-counterion complex, and the subscripts “m” and “s” indicate the mobile and stationary phases, respectively. Thus, the capacity ratio (k0′i) of the solute in ion-pair chromatography in the absence of β-CD is given by
k0′i ) ΦKex[X]m
(4)
where Φ is the phase ratio. If β-CD is used as a chiral selector in the ion-pair chromatographic system, the following other equilibria must be considered: (20) Pettersson, C.; Heldin, E. In A Practical Approach to Chiral Separations by Liquid Chromatography; Subramanian, G., Ed.; VCH: Weinheim, 1994; p 279.
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Kf
Q+m + X-m + CDm ) QXCDm
(5)
K1
CDm + Ls ) CDLs
(6)
K2
QXCDm + Ls ) QXCDLs
(7)
where QXCD is the cyclodextrin inclusion complex, and Ls is the stationary phase adsorption site. Kf is the apparent formation constant in the ion-pair system. K1 and K2 are the equilibrium constants of CD and its inclusion complex between the mobile and the stationary phases, respectively. According to Walhagen,15 eqs 6 and 7 can be assumed to be negligible. Thus, the capacity ratio (k′) based on eqs 2 and 5 is expressed as
[QX]s k′ ) Φ [Q]m + [QXCD]m
(8)
Replacing [Q]m, [QX]s, and [QXCD]m with Kex and Kf, one obtains
Kex[X]m k′ ) Φ 1 + Kf[X]m[CD]m
(9)
From eq 4, ΦKex[X]m is equal to the capacity ratio, k0′i, obtained in the ion-pair system in the absence of β-CD. Equation 9 can be rewritten as
Kf[X]m[CD]m 1 1 ) + k′ k0′i k0′i
(10)
Equation 10 shows that the retention value is influenced by the capacity ratio, k0′i, of ion-pair complex in the absence of β-CD, which in turn is mainly determined by the type and concentration of counterion at the given experimental conditions, according to eq 4. To evaluate the influence of β-CD on chiral separation, if the (R)-enantiomer is first eluted, the separation factor, R(S/R), for (S)-/(R)-enantiomers can be written as
[
Kf(R) R(S/R) )
Kf(S)
1-
Kf(R) - Kf(S) Kf(R) + Kf(R)Kf(S)[X]m[CD]m
]
(11)
where Kf(R) and Kf(S) are the formation constants for the (R)- and (S)-enantiomers. From eq 11, the separation factor is dependent on the difference in the formation constants for the two enantiomers and also on the concentration of β-CD and counterion. RESULTS AND DISCUSSION Comparison of Retention Behavior in the Ion-Pair RPLC with That in Conventional RPLC. Figure 2 shows the reciprocal plot of capacity ratio (1/k′) of enantiomeric Sal and NMSal against the β-CD concentrations in the RPLC without counterion (A) and with the counterion (B). Since the concentrations of solutes are relatively low, the concentrations of free β-CD and free counterion may be equal to their respective total concentration in the mobile 2828 Analytical Chemistry, Vol. 68, No. 17, September 1, 1996
Figure 2. Reciprocal of the capacity ratio plotted against the β-CD concentration (mM). The capacity ratio was measured with reversedphase liquid chromatography without counterion (A) or in the presence of 1 mM sodium 1-heptanesulfonate (B), using as the mobile phase 25 mM phosphate buffer, pH 3.0, containing 0.2% 2-methyl-2propanol. O, (R)-Sal; b, (S)-Sal; 4, (R)-NMSal; and 2, (S)-NMSal. Table 1. Results Obtained by Linear Regression and the Formation Constants Calculated from Eqs 1 and 10 solute-CD complex
solute-SHS-CD complex
solute
γa
stoichiometry
Kfb
γa
stoichiometry
Kfb
(R)-Sal (S)-Sal (R)-NMSal (S)-NMSal
0.989 0.985 0.997 0.993
1:1 1:1 1:1 1:1
176.8 160.6 252.4 222.4
0.996 0.996 0.998 0.997
1:1:1 1:1:1 1:1:1 1:1:1
1.823 × 105 1.678 × 105 2.433 × 105 2.146 × 105
a
Linearity coefficient. b Formation constants.
phase. As predicted by eqs 1 and 10, the good linear relationship of 1/k′ versus β-CD concentration for all four enantiomers was confirmed in the two systems. This indicates that the stoichiometry between the species is predominantly 1:1 for solute-β-CD or 1:1:1 for solute-counterion-β-CD according to Armstrong et al.14 Table 1 gives the results obtained by linear regression and the formation constants (Kf) calculated from the slopes and intercepts of straight lines of Figure 2. The data in Figure 2 were obtained by separate analyses of the individual enantiomeric Sal and NMSal. In reality, the enantioseparation of Sal was not obtained with the reversed-phase system without counterion. The complete separation for both Sal and NMSal enantiomers was achieved with the ion-pair system in addition to β-CD in the mobile phase. Figure 3A is an HPLC pattern of the standard mixture of enantiomeric Sal and NMSal and their precursor, dopamine, with a mobile phase consisting of 25 mM phosphate buffer, pH 3.0, containing 12 mM β-CD, 1 mM SHS, and 0.2% 2-methyl-2-propanol. The elution order for (R)- and (S)-enantiomers of Sal and NMSal was opposite to that observed on the cyclodextrin-bonded column.12 This suggests that formation of an inclusion complex of the solute with β-CD is a predominant mechanism for chiral separation. When only the
Figure 4. Effect of β-CD concentration on the separation factor. The separation factors were calculated from the capacity ratios of (R)- and (S)-Sal. Solid line represents experimental data obtained from Figure 2B, and dashed line is the theoretical line calculated from eq 11.
Figure 3. HPLC patterns of samples prepared from dried banana and human brain. (A) Standards, amount per injection for each standard component, 20 pmol; (B) sample prepared from dried banana; (C) standards, amount per injection for each standard component, 0.5 pmol; and (D) sample prepared from human brain gray matter. The mobile phase was 25 mM phosphate buffer, pH 3.0, containing 12 mM β-CD, 1 mM SHS, and 0.2% 2-methyl-2-propanol. Peaks: 1, dopamine; 2, (R)-Sal; 3, (S)-Sal; 4, (R)-NMSal; and 5, (S)NMSal.
distribution between the enantiomers and the achiral stationary phase is considered, there is no difference between (R)- and (S)enantiomers. However, the difference in the equilibrium of the enantiomers when β-CD is added in the mobile phase may distinguish (R)- from (S)-enantiomers and thus is responsible for their chiral separation. Although the experimental conditions in this assay were different from those in the previously reported method that used a cyclodextrin-bonded column,12 the formation constant of β-CD with the (R)-enantiomer was confirmed to be larger than that with the (S)-enantiomer for both methods. Generally, several factors are required for chiral separation. The formation constants (Kf) must be sufficiently large and different between the two enantiomers, and the retention also must be high enough. Using a counterion in the mobile phase, an increase in the formation constants (Table 1) suggests that not only was the retention of hydrophilic chiral amines, such as Sal, increased, but also the stability of the complexes of the solutes with β-CD, expressed as Kf, was improved. β-CD was found to be retained when it was chromatographed on a reversed-phase column with a mobile phase without methanol and β-CD,15 and this contradicts the above assumptions. Adsorbed β-CD did not affect the solute retention, as indicated by the good linearity (Figure 2), and this may be explained as due to the large excess concentration of β-CD in the mobile phase.
Figure 5. Effect of sodium alkanesulfonates on the capacity ratio and separation factor of enantiomers of Sal and NMSal. The capacity ratio (A) and separation factor (B) were obtained using mobile phase composed of 25 mM phosphate buffer, pH 3.0, containing 12 mM β-CD, 0.2% 2-methyl-2-propanol and 1 mM sodium alkanesulfonate. (A) Bars, left to right: (R)-Sal, (S)-Sal, (R)-NMSal, and (S)-NMSal. (B) Bars, left to right: (S)-/(R)-Sal enantiomers, (R)-NMSal/(S)-Sal, and (S)-/(R)-NMSal enantiomers.
In addition, the effect of β-CD concentration on the chiral recognition for enantiomers of Sal and NMSal is shown in Figure 4. As predicated by eq 11, the separation factors obtained were increased with the concentration of β-CD was increased from 0 to 15 mM. The separation of enantiomers of Sal and NMSal was obtained with β-CD higher than 5 mM, with a separation factor larger than 1.05. The experimental data for the separation factor of Sal enantiomers versus β-CD concentration are reasonably fitted to the theoretical curve. Effect of Counterion on Hydrophobic Retention. In a reversed-phase system with a hydrophobic stationary phase, the capacity ratio of solutes such as Sal, which are cations under acidic conditions, may be dependent on the choice of the counterion. As can be seen in Figure 5, the effects of following three anionic alkanesulfonates on the capacity ratio (A) and separation factor (B) were examined: sodium 1-butanesulfonate (SBS), sodium 1-heptanesulfonate (SHS), and sodium 1-octanesulfonate (SOS). SBS gave a rather low retention level for all four enantiomers, Analytical Chemistry, Vol. 68, No. 17, September 1, 1996
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Figure 6. Double-reciprocal plot of the retention against the SHS concentrations (mM). The mobile phase was composed of 25 mM phosphate buffer (pH 3.0) containing 12 mM β-CD and 0.2% 2-methyl2-propanol. O, (R)-Sal; b, (S)-Sal; 4, (R)-NMSal; and 2, (S)-NMSal.
and (S)-Sal and (R)-NMSal could not be separated. An increase in the size of the hydrophobic chain of the counterions enhanced the capacity factor. SHS gave modest retention and the best separation for all four enantiomers of Sal and NMSal and thus was selected as the counterion in these experiments. The retention of solutes in the presence of counterion was found to be dependent on SHS concentration in the mobile phase. As suggested by eq 10, where k0′i ) ΦKex[X]m, a double-reciprocal plot of the retention against the SHS concentration showed a good linear relationship, as shown in Figure 6. The higher concentration (>1 mM) of the counterion increased the retention markedly but hardly improved the chiral separation. Therefore, 1 mM SHS proved to be the best for this assay. Optimization of pH, Ionic Strength, and Organic Modifier Concentration. The concentration of the ionic form of Sal and NMSal is controlled by the pH of the mobile phase. Therefore, the influence of pH on inclusion complex formation was examined with constant ionic strength (27 mM sodium ion) held by the addition of NaCl. As shown in Figure 7A, a small increase in capacity ratio with increasing pH was observed, and the selectivity between two enantiomers of Sal and NMSal was almost the same. However, it was found that the electrochemical reactivity of these enantiomers decreased dramatically with pH from 3 to 6, and no peak was detected at pH 7.0. The exact reason for this remains unclear. From these results, the pH 3.0 was chosen for this assay. The effect of ionic strength on retention was examined in the range of the salt concentration from 5 to 200 mM at pH 3.0. It was shown that an increase in the sodium concentration at pH 3.0 did not significantly change the retention of all Sal and NMSal enantiomers for this ion-pair system (data not shown). The separation factor also was hardly affected by ionic strength. Figure 7B shows the remarkable decrease in retention of Sal and NMSal enantiomers by an organic modifier, 2-methyl-2propanol. Considering the solubility of β-CD, the effects of the concentrations were investigated with 2-amino-2-propanol below 5%. The enantioseparation of Sal and NMSal was not obtained at modifier concentrations above 2%. However, the separation of (S)Sal and (R)-NMSal was improved upon the addition of 0.2% 2-methyl-2-propanol in the mobile phase, while the enantioseparation was not affected with the high concentration of β-CD (12 mM). Analysis of Sal and NMSal Enantiomers in Food and Human Brain Samples. Generally, Sal has been considered to 2830 Analytical Chemistry, Vol. 68, No. 17, September 1, 1996
Figure 7. Effect of the pH (A) or the concentration of organic modifier (B) on the capacity factor. The effect of pH was examined using as the mobile phase 25 mM phosphate buffer containing 12 mM β-CD, 1 mM SHS and 0.2% 2-methyl-2-propanol. The effect of percentage concentration of 2-methyl-2-propanol was examined using as the mobile phase 25 mM phosphate buffer containing 12 mM β-CD and 1 mM SHS. O, (R)-Sal; b, (S)-Sal; 4, (R)-NMSal; and 2, (S)NMSal.
be generated from dopamine by a nonenzymatic Pictet-Spengler reaction to yield racemic (R,S)-Sal.21,22 Sal is also found in some foods, such as dried banana and port wine, suggesting that the intake of Sal might affect Sal levels in tissues.23 However, in the human brain, only (R)-enantiomers of Sal and NMSal were identified, indicating that the (R)-enantiomers are enzymatically synthesized in situ in the brain.21,24 This assay was applied to measure the enantiomeric composition of Sal and its metabolite NMSal in food and human brain. Typical chromatograms are shown in Figure 3B for dried banana sample and in Figure 3D for a sample prepared from human brain gray matter. Sal was found to be racemic in dried banana, whereas only (R)-NMSal was detected in the sample from the human brain. Identification of peak 4, corresponding to (R)-NMSal in the brain sample, was done by comparison of the retention time and the voltammogram in the voltage range from -100 to -350 mV. Relative ratios of responses to -300/-200 mV and to -300/-250 mV are 3.38 and 1.99 for the authentic standard of (R)-NMSal, and 3.34 and 1.98 for the peak detected in the brain sample, respectively. Further study of the enantiomeric composition of Sal and NMSal in foods and in the human brain is now being undertaken. (21) Dostert, P.; Strolin Benedetti, M.; Dordain, G. J. Neural Transm. 1988, 74, 61. (22) Naoi, M.; Maruyama, W.; Dostert, P. Prog. Brain Res. 1995, 106, 227. (23) Strolin Benedetti, M.; Dostert, P.; Carminati, P. J. Neural Transm.: Gen. Sect. 1989, 78, 43. (24) Naoi, M.; Maruyama, W.; Dostert, P.; Hashizume, Y. Biog. Amines 1996, 12, 153.
To analyze biological samples, high selectivity is required in order to discriminate the solute from matrix components. With the previous method,12 the conditions needed to increase the selectivity were limited due to the use of the chemically bonded β-CD phase. In contrast, when β-CD is used as a chiral mobile phase additive, the selectivity required for chiral separation may be achieved by optimizing the mobile phase and the stationary phase. CONCLUSION A new assay for enantiomers of Sal and NMSal was developed, using reversed-phase ion-pair chromatography containing β-CD as a chiral mobile phase additive. A proposed retention model was characterized as a function of mobile phase conditions and proved to be consistent with the experimental data. Using SHS
as a counterion in the mobile phase, not only was the retention of hydrophilic chiral amines such as Sal increased, but also the stability of the complexes of solutes with β-CD was improved. The Sal and NMSal enantiomers in banana and in human brain samples were determined using this assay. ACKNOWLEDGMENT This work was supported by a Grant-in-Aid for Scientific Research on Primary Area, Ministry of Education, Science and Culture, Japan. Received for review February 27, 1996. Accepted June 6, 1996.X AC960185L X
Abstract published in Advance ACS Abstracts, July 15, 1996.
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