Chiral
L hirality, or optical activity, is a significant characteristic of many synthetic and most biological organic compounds. Various rightand left-handed molecular entities are fre quently associated with the precise control of biological processes, and numerous examples from the areas of protein structure and function, enzyme action, and even polynucleotide biochemistry attest to the fundamental and far-reaching importance of chirality in nature. Molecular “handedness” is primarily confined to the occurrence of carbon-atom asymmetries in small molecules. These are, however; often the building blocks of important biopolymers: Amino acids in proteins occur characteristically as the L (levo) forms, whereas the D (dextro) forms are typical for sugars incorporated in both nucleic acids and various glycoconjugates. As we gradually improve our ability to recognize specific optical activities, we also are able to identify an increasing number of “exceptions” in biological materials. . Optical activity arises from an asymmetric element, which may be a center, an axis, or a plane, present in a molecule. Racemates (equal mixtures of D- and Lforms) are common in many synthetic products (e.g., numerous pharmaceuticals, herbicides, and pesticides) and some
Milos Novotny Helena Soini Morgan Stefansson Indiana University 646 A
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CZE and EKCC are increasingly complementing and competing with other chiral separation methods natural products (certain alkaloids ana terpenes). In a symmetric environment, various physical and chemical properties of enantiomers in a racemic mixture are nearly identical. Among the few physical differences, purified enantiomers in solutions rotate plane-polarized light in opposite directions. Currently, however, researchers regard optical rotation measurements as too gross for obtaining the chiral impurity assessments needed in modern science and technology. As we expend effort to design better methods for chirality measurements, perhaps the best lessons can be learned from nature, where the “right” chirality in very complex mixtures is routinely recognized. Most often, only one enantiomer is responsible for a compound’s activity, as is the case with many pharmaceuticals and xenobiotics such as pesticides and herbicides. In exceptional situations, both isomers may be active and, in some cases (e.g., in perception of pheromones by certain insects), the cor-
Analytical Chemistry, Vol. 66, No. 11, June 1, 1994
rect ratio of enantiomers is necessary for maximum response. The task of resolving optical antipodes has occupied scientists since 1848,when Louis Pasteur succeeded in physically separating diastereomeric crystals of different forms out of a racemate. During the early years of research in this area the usual approach to resolving different chiral forms was the introduction of an additional chiral center into molecules of interest, that is, preparation of diastereoisomers with distinctly different physical properties. More recently, the possibility of directly separating enantiomers has fascinated separation scientists. In 1966, Gil-Av and co-workers (1) demonstrated for the first time such a direct separation of enantiomeric compounds (volatile derivatives of the amino acids) by capillary GC using an optically active stationary phase. Subsequent studies using GC were aimed at improving optically active stationary phases in terms of thermal stability and selectivity. During the 198Os, the search for optimum systems in enantiomeric separation increasingly included HPLC and suprcritical fluid chromatography (SFC),Studies designed to obtain a better understanding of the molecular causes of chiral recognition have become more common. The exploration of various separation methodologies for direct enantiomeric resolution has made it increasingly clear that the selectivity of solute/stationaryphase interactions plays a crucial role and that optimization at the level of favorable 0003- 2700/94/0366-646A/$04.50/0
0 1994 American Chemical Society
sorption-desorption kinetics (efficiency in terms of theoretical plates) is also desirable. Because differences in the free energy, -A ( A G O ) , of interaction between D- and L-solutes and a solvent environment typically are small, a proper choice of chiral selectors with sufficient selectivities (avalues) is the key to success. And because separation selectivity is related to the difference in interaction energies through a simple thermodynamic relationship, -A(AG”) = RTlna, temperature represents yet another variable in the separation process. The selection of temperature is particularly crucial in capillary GC and capillary SFC, methods that feature high theoretical plate numbers. The usual strategy of optimizing chiral resolution in HPLC seeks unusually large a values, because the numbers of theoretical plates with this methodology (in its conventional mode) are nominally an order of magnitude less than in GC and SFC. The modern electromigration methods, capillary zone electrophoresis (CZE) and electrokinetic capillary chromatography (EKCC) , are high-plate-number (kinetically favorable) processes much like GC and SFC. Most important, however, CZE and EKCC share with HPLC an enormous freedom of choice among various chiral selectors, without direct limitations on thermal stabilities of either solutes o r chiral recognition agents. A considerable number of stereoselective principles can be tapped for enantiomeric separation with electromigration methods: hydrogen bonding, metal coordination, ionic attraction, charge transfer complexation, biopolymer affinity, and host-guest inclusion phenomena. In spite of the relatively short time since modern capillary electromigration techniques were first advanced (2-5), CZE and EKCC are increasingly complementing and competing with other chiral separation methods. Capillary GC and SFC using extremely well-developed chiral phases (optically active polymers and modified cyclodextrins) may, however, remain best suited for relatively volatile solutes. Likewise, electromigration methods cannot compete with HPLC when scale-up separations are required. In this Report, we will review the recent advances in chiral recognition by analytical electromigration processes and 648 A
indicate new possibilities for research in this rapidly developing field. We will also illustrate these advances with appropriate examples using amino acids, carbohydrates, pharmaceuticals, and agricultural chemicals. Modes of electromigration separations
The area of separation science that recently has been dubbed “high-performance capillary electrophoresis” (HPCE) is used for an ever-increasing number of practical applications, despite the fact that some remarkable separations are serendipitously achieved because of somewhat ill-defined mixed mechanisms. Chiral separations are a case in point. What is often globally described as “capillary electrophoresis” in the literature or at scientific meetings may in fact be various crossbreeds between electrophoretic and other separation processes, which (if practitioners ever care) must be clarified by further research. Most capillary electromigration principles advanced during the past decade can be used in chiral resolution. The presence or absence of capillary electroosmosis (3) may determine whether one deals primarily with an electrophoretic process, electrokinetic-chromatographic phenomena, electrochromatography, or various combinations of these. In CZE, the solutes migrate primarily according to their chargeto-mass ratios. When a chiral selector is used as the buffer additive, the chiral analytes may form (in a dynamic equilibrium process) diastereomeric pairs that are sufficiently distinct electrophoretically. In agreement with the theory of CZE (3), high separation efficiencies (plate numbers) generally are encountered. Figure 1 shows such a situation, in which a 16-component mixture of monosaccharide enantiomers is resolved with a roughly 400,000 plate/m column efficiency (6).The effect of electroosmosis in CZE is usually minimized by chemical treatment of the capillary wall (7, 8) or, alternatively, by dynamic adsorption of a suitable buffer additive. The electroosmotic effect also is routinely minimized in isotachophoresis (ITP) , otherwise known as displacement electrophoresis (9),currently a less popular electromigration technique. Capillary
Analytical Chemisfry, Vol. 66, No. 1 I, June I, 1994
me (min)
Figure 1. High-efficiency separation of a complex enantiomeric mixture using CZE. Electrolyte: 12.5 mM p-CD, 2% tetrahydrofuran, and 0.5 M borate (pH 8.2). Peak assignment: 1, D-ribose; 2, D-xylose; 3, L-arabinose; 4, D-fucose; 5, D-glucose; 6, L-xylose and reagent; 7, L-ribose; 8, D-galactose; 9, L-mannose; 10, D-lyxose; 11, L-xylose; 12, D-mannose; 13, L-glucose; 14, D-arabinose; 15, L-fucose; and 16, L-galactose. (Adapted from Reference 6.)
ITP was used in one of the early chiral separations involving cyclodextrin additives (10);although it is not a high-efficiency technique, its routine use as a rapid means of resolving simple mixtures and for solute preconcentration prior to CZE will undoubtedly receive attention in the future. Gel-filled capillaries also can be used in conjunction with chiral selectivity (11, 12). Many HPCE chiral separations appear to fall under the description of EKCC, in which the electroosmotic effect is the means of solute transport toward the point of detection. In the classical arrangement of EKCC, pioneered by Terabe and associates (5),the charged micelles provide the pseudo stationary phase that readily interacts with the analyte molecules. The use of optically active micelles (13-15), mixed-micellar aggregates (16-18), and other means of incorporating chiral selectivity into the micellar systems appears to be a logical extension of this generally powerful approach. Another variation of the EKCC approach is the use of microemulsions (19,20).
THEY SAY SEPARATING IS NEVER EASY WHAT DO THEY KNOW.
. ..
Direct separation of racemic mixtures is readily accomplished with Daicel" Chiral columns from Chiral Technologies, Inc." Chiral HPLC is a separation process which provides a rapid, reliable and easy method for preparing and analyzing enantiomers in quantities ranging from milligrams to grams, even kilograms.
To accommodate the scale of your separation, Daicel columns are available in a variety of sizes ranging from analytical (0.46 cm i.d.) to semi-preparative(1and 2 cm i.d.1 to Preparative (57 10 and 20 cm i-d-). When multi-gram quantities of enantiomers are required for continuing R&D investigations, the 2 cm semi-preparativecolumn is the column of choice.
Our chromatographers at Chiral Technologies can assist in analytical method development, or custom separation on a contract basis. Call us for more information. For a free copy of Chiral Technologies' Application Guide f o r Chiral Column Selection, call our Marketing and Technical Center in Exton, PA at 1-800-6-CHIRAL, or circle the Reader Service Number below.
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Racemic Mixture
Daicel Chiral Column
(R)-Enantiomer
(S)-Enantiomer
h I
EKCC represents a “bridge” between electrophoresis and chromatography. Its unique and desirable feature is high separation efficiency (compared with HPLC) in a relatively short analysis time. The kinetic advantages of EKCC result from highly favorable mass transfer properties of the dispersed micellar system and a relatively flat flow profile (5).Therefore, efficiencies of an order of magnitude greater than those encountered in HPLC are quite common in EKCC. Capillary electrochromatography, first recognized by Tsuda and co-workers (21), is the final major technique that falls into the scope of our discussion. Similar to previously explored open-tubular HPLC (22), a chiral selector attached to the wall of the separation capillary constitutes the stationary phase. The chiral solutes must therefore be transported to the column wall to interact with the stationary phase by means of radial diffusion, aided by the flat velocity profile. In addition, the column inner diameter plays a crucial role in capillary electrochromatography, which is inherently less efficient than EKCC. Given the frequency with which researchers and analysts use unmodified (adsorptive) fused-silica tubes as separation capillaries, the electrochromatographic process must occur at least partially (unintentionally)in some chiral separations intended as either EKCC or CZE. Chiral mechanisms The selectivities of appropriate chiral selectors, in combination with the favorable kinetics of capillary separations, are at the heart of recent successful solutions to difficult chiral separation problems. AIthough the search for new types of interactions remains a high priority, numerous viable options exist. Additional demand for certain “selectivities” may occur in applicationsto biological materials in which discrimination against endogenous (background) metabolites and proteins is essential. Various buffer additives and organic m o d ~ e r are s often used advantageously in such separations. Ligand exchange. The first separation of enantiomers by HPCE was reported by Zare and co-workers in 1985 (23). Adopting the strategy used previously in HPLC by Davankov and Rogozhin (24),Zare’s group used optically
active copper (1Q complexes of L-histidine as additives to the buffer system to separate dansylated amino acids (Figure 2). Copper (11)-aspartame (25) and copper(I1)didecylalanine (26) also were described as useful chiral selectors for derivatized amino acid mixtures. As with many other chiral selection processes, separations resulting from ligand exchange may be aided by the incorporation of micellar entities into the separation medium (26). Micellar systems and microemulsions. The addition of various detergent molecules (cationic, anionic, or neutral) to buffer media in HPCE can have different beneficial effects. Even below the critical concentrations at which micelles form, the detergents may bind to the capillary wall, to the sample molecules, or to other buffer additives, often improving the peak shapes or modifying migration of ballast proteins in natural samples. When aggregates form at the critical micellar concentration (CMC), such organized molecular assemblies present the primary sites of solute interaction in the EKCC mode of electromigration. In chiral recognition, the micelles themselves can become the major site of interaction, or they can harbor various chiral selectors (including ligand exchange selectors) added to the separation medium. Many biological surfactants are chiral themselves; thus, the formed micellar pseudo stationary phase will naturally assume chiral recognition properties. In an even more complicated case, mixed micellar systems (16-18) can intentionallybe formed to optimize chiral separations. Researchers often find it more profitable to optimize their separations empirically; not all chiral recognition mechanisms are completely understood. Various uses of micellar electrokinetic chromatography in chiral separations were recently reviewed by Otsuka and Terabe (27). The use of bile acid salts as micellar entities for separation of chiral pharmaceuticals represents a good example of the use of natural chiral surfactants in this capacity. Deoxycholic acid and taurodeoxycholic acid were found to be effective in resolving racemic mixtures of derivatized amino acids (13) and the pharmaceuticals diltiazem and trimetoquinol(14). Organic modifiers were shown to improve further the effectiveness of the bile salt
650 A Analytical Chemistry, Vol. 66, No. 11, June 1, 1994
Figure 2. Electropherogramsof D, L-dansyl amino acids with (a) Cu(11)-L-histidine electrolyte and (b) 1:l Cu(ll) P and L-histidine electrolyte. Tyr, tyrosine; Phe, phenylalanine; Asp, aspartic acid; Glu, glutamic acid (each at -lop4 M); pH 7.0. (Adapted with permission from Reference 23.)
systems (15).Even in this relatively simple case, the recognition mechanism remains somewhat unclear. It has been suggested that the verified helical arrangement (28) of bile acids interacts discriminately with enantiomers containing a planar and rigid structure (14). Mixed micellar media typically involve common micelle-forming detergents, such as anionic sodium dodecyl sulfate (SDS) or cationic quaternary compounds, used in conjunction with chiral surfactants or cyclodextrin derivatives.As shown in Figure 3, the general complexity of such systems results in both highly specific and more general (achiral) interactions that add to the overall success of enantiomeric resolution. Several literature cases exemplify the resolving power of mixed micellar systems. SDS and N-dodecanoyl-L-valinate were used to form a mixed system for the separation of N-3,5-dinitrobenzoylated amino acid isopropyl esters. The chiral
Figure 3. Solute interaction in a mixed micellar system using SDS and CD. (Adapted with permission from Reference 29.)
recognition mechanism relies on the hydrophobic entanglement within the micelle’s inner core. Differential hydrogen bonding is thought to be responsible for stereochemical discrimination (16). Other examples of comicellar systems include combinations of charged SDS with neutral and chiral digitonins (17) or saponins (18). Most of these systems seem to be restricted to a relatively narrow application range. Cyclodextrin-based micellar systems, on the other hand, are becoming increasingly popular in applications of more hydrophobic compounds (29-31). Microemulsions. Microemulsions, which consist of tiny droplets (approximate size 100 A) of an oily mixture in a buffer medium, can be combined effectively with lipophilic chiral selectors, as demonstrated recently by Aiken and Huie’ (32) for ephedrine separation. Because they form much larger aggregations than do micellar systems, microemulsion systems are capable of solubilizing hydrophobic compounds with a greater capacity than micellar systems. Crown ethers. Inclusion complexation with a macrocyclic ether (chiral18crown-tetracarboxylic acid) was used recently to resolve several chiral amines (33,34) and aminoalcohols (35).The interaction of primary amines (guest molecules) and the crown ether (host) cavity is based on a three-pointbonding between the hydrogen atoms of the amine group and the oxygen dipoles within the polyether ring. Chiral recognition is achieved through lateral electrostatic interactions of the host and guest molecules (33).
Cyclodextrins and other oligosaccharides. Cyclodextrins (CDs) are currently the most powerful chiral selectors because of their applicability to a wide range of organic compounds. Cyclodextrins are chiral, cyclic oligosaccharides, consisting of D-(+)-glucopyranosideunits arranged in a truncated cone shape. Dissolution of CDs in water is mediated by their exterior hydrophilicity, whereas the inner hydrophobic cavity becomes the primary site of interaction with organic analyte molecules (chiral or nonchiral) . Inclusion of various guest molecules is determined primarily by the cavity’s size, which in turn is determined by the number of glucopyranoside units: six sugar units for a-cyclodextrin, seven for p-cyclodextrin, and eight for y-cyclodextrin. The inclusion phenomena also can be affected by derivatizing the secondary hydroxyl groups to form derivatives (e.g, di-0-methyl,tri-0-methyl,or hydroxypropyl) and thus contribute to the snugness of the geometrical fit. Derivatization of a cyclodextrin with ionic groups also can affect its buffer solubility and the electrophoretic mobility of the aggregates. Although various HPCE buffer additives, such as detergents or organic solvents, are often beneficial to the separation, they may actually obscure a precise understanding of the guest-host relationships. The use of cyclodextrin additives is not limited to chiral separations; molecular size and geometric discrimination phenomena also are useful. Chiral recognition processes associated with the use of cyclodextrins have been studied in HPLC (36,37), but drawing analogies to HPCE
is not always appropriate. In a successful chiral recognition with cyclodextrins, hydrophobic interactions between the cavity interior and an organic solute are assumed, whereas hydrogen bonding at the cavity edge presumably determines a compound’s access at the cavity entrance. Intimate stereochemical interactions can express themselves at this level, assuming that a given analyte molecule fits the cavity. An example is the chiral recognition of D- and L-monosaccharides derivatized with a naphthalene sulfonate moiety through the Schiff base mechanism shown in Figure 4. NMR and fluorescence measurements (6) carried out on this system support the notion of the naphthyl groups positioned deep in the CD cavity. The chiral recognition is governed by the carbohydrate moiety extending away from the torus and interacting with the C-3 hydroxyl group on the CD molecule via its own C-2 hydroxyl group. During a kinetically favorable HPCE separation process, even slightly different stability constants of the dynamic inclusion complexation for each enantiomer result in appreciably different mobilities in an electric field. High temperatures have
HpUH
Figure 4. Proposed complex between 5-aminonaphthalene2. sulfonate-D=galactose and p-CD. For simplicity, hydroxyl groups of only one glucose unit are shown. (Adapted from Reference 6.)
Analytical Chemistry, Vol. 66, No. 11, June 1, 1994 651 A
‘ p been reported to worsen the outcome in such separations (34, 38, 39). Unusual effects relating to the exterior part of CDs in chiral recognition remain under consideration for some compounds (40,41). Solute complexation with CDs most typically occurs in a 1:lratio, but 1:2 complexes can also be observed in some cases, as suggested by Li and Purdy (41), on the basis of ‘H NMR, UV-vis, and circular dichroism spectroscopic studies. An interesting twist to the cyclodextrin “success stories” is the use of linear oligomers that may in some ways mimic the widely studied CD inclusion phenomena. An example is shown with a mixture of maltodextrins [ (1-4)-a-D-sugar oligomers] that can be used to resolve various derivatized monosaccharide enantiomers similarly to the CDs, which are also a-(1-4)oligosaccharides (6). Numerous other oligosaccharides, with (1-6)-a-Dor (1-3)PD structural arrangements, were ineffective. As seen in Figure 5, even relatively small maltose oligomers were effective in chiral recognition. Starting with maltotetraose, separation selectivity increases with the increasing size of the oligomer chain. Under favorable buffer conditions, the linear dextrins seem to favor a flexible helix (42) into which a variety of mole-
cules can readily complex. The flexibility of these formations, in contrast to the more rigid CDs, can be beneficial in some applications, as has been demonstrated recently for acidic pharmaceuticals (43). Protein interactions. During the development of enantiomeric separations in HPLC a variety of protein chiral selectors, including bovine serum albumin, a,-acid glycoprotein, conalbumin, and cellulase, have been used. Various proteins are known to interact with small molecules, and most chiral recognition effects are serendipitous attributes that may have little to do with the biological function of such proteins. When used in HPLC, the protein chiral selectors are typically immobilized to silica-based carriers, though they may also be used as mobile-phase components (44). In a similar capacity, HPCE-based separations also can be developed. The degree of chiral separation attributable to a protein additive depends mainly on migration of the solutes in their free and complexed forms. Because the ionization and conformation of the protein’s active groups are strongly dependent on pH, proper selection of the buffer conditions is extremely critical in chiral recognition. A common problem with pro-
Figure 6. Separation of selected P-blockers based on interaction with cellulase. (a) ( R ) , (S)-propranolol; (b) ( R ) ,(S)-pindolol; (c) ( R ) ,(S)- metoprolol; (d) (RRlSS)- labetolol; (e) (RSISR)-labetolol. Buffer: 0.4 M sodium phosphate (ph 5.1) supplemented with 2-propanol. (Adapted with permission from Reference 45.)
tein-based enantioseparations is that the peaks are typically broad (and sometimes asymmetrical), presumably because of slow kinetics and low capacity of the protein-ligand interactions. Proteins also tend to adsorb on the capillary wall. When using UV absorbance as the means for detecting enantiomers, a high background signal resulting from a protein additive may further complicate analysis. One of the more successful literature examples of chiral resolution attributable to a protein is that of common P-blockers (Figure 6). Cellulase, a hydrolytic enzyme, has been used as a buffer additive (45). An alternative approach developed by Birnbaum and Nilsson (12)uses protein gels formed after cross-linking bovine serum albumin with glutaraldehyde. Applications
Figure 5. Influence of malto-oligomer chain length on enantioselectivity. Conditions: 0.4 M borate at pH 8.3 and 405 V/cm. (Adapted from Reference 6.)
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Analytical Chemistry, Vol. 66, No. 11, June 1, 1994
The chiral separation techniques described above can be used to analyze numerous types of racemic mixtures, such as those composed of pharmaceuticals, agricultural chemicals, and biologically active compounds. Pharmaceuticals. The pharmaceuticals occurring as racemic mixtures com-
prise a set of diverse organic molecules. Typical compounds to which HPCE has been applied (see box below) include the anesthetic thiopental (29), the bronchodilator terbutaline (46), the antibiotic chloramphenicol (47), the anti-inflammatory ibuprofen (43,48, 49,54), the P-blocker pindolol (45, 50, 51), and the anticoagulant warfarin (43,51,52,54). Basic compounds are more common than acidic ones. Thus far the most successful chiral selectors in pharmaceutical analysis a p pear to be cyclodextrins. Because of limitations of the cavity size, p- and y-CDs and their alkylated derivatives seem more appropriate than the smaller a form. For most separations of this type, a proper choice of pH (to ensure a charge on the drug molecules) and control of electroosmosis at the capillary wall are essential. The addition of organic solvent also can be useful in fine-tuning the component resolution, presumably through various competitive interactions (6,46). Suppression of electroosmosis is now commonly accomplished through capillary wall modification (coated capillaries) or through addition of cellulose derivatives to the buffer medium (47,53). Because even a small percentage of the polysaccharide is capable of reducing electroosmosis dramatically, the ordinary neutral CD molecules move very slowly under these conditions. The positively charged analytes move toward the negative electrode (detector end) and are allowed to form inclusion complexes dynamically. Cationic detergents, in combination with methylated CD derivatives, have been applied extensively to the separation of various basic chiral pharmaceuticals in our laboratory. Hexadecyltrimethylammonium bromide (HTAB) and cetylpyridinium chloride (CPC) were selected because low CMC values permit their effective use at concentrations for which low current values are encountered and thermal effects appear minimal. By using a methylated p-CD and methylhydroxyethylcellulose (MHEC) as further buffer additives, optimum resolution of the enantiomeric pairs of verapamil and fluoxetine was found at an HTAB concentration near its CMC value. At very low detergent concentrations, HTAB improved the system's stability in terms of migration time and
peak-height reproducibility (50). Maltodextrins (linear oligosaccharides) have been used successfully in chiral separations of various acidic and basic drugs in our laboratory. In some instances, the use of certain buffer constituents appears crucial. Zwitterionic buffers and organic modifiers were found to be beneficial. The use of polymer additives
improved chiral separations with maltodextrins (54). One of the most common determinations needed in the pharmaceutical industry is the assessment of enantiomeric purity of products and synthetic intermediates. Impurity quantification below 0.1%will undoubtedly be enforced by regulatory agencies in most countries, which
3 H3)zCHC H2
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Figure 7. Separation of (I?) and (S) = bupivacaine. Concentrations: (R)-bupivacaine, 333 pg/mL; (S)-bupivacaine, 0.34 pg/mL. Capillary: 75 pm x 57 cm, uncoated. Buffer: 10 mM di-0-methyl-P-CD, 18 mM Tris, pH 2.9, 0.1% MHEC, 0.03 mM HTAB. Detection at 200 nm, temperature 16 "C. (Adapted with permission from Reference 55.)
Analytical Chemistry, Vol. 66, No. 11, June I, 1994 653
4
necessitates a more pronounced component resolution than what typically has been demonstrated in the literature. Baseline separation between two isomer peaks of approximately the same size is hardly adequate when one of the components is present in great excess. Conditions must be found that allow adequate quantification, as illustrated the example of bupivacaine (Figure 7) (55). Although numerous demonstrations of HPCE separations with pure enantiomers can be found in the literature, very few applications to biological materials have been reported to date (50,52,56).The challenges to this type of analysis come from several directions. First, physiological fluids such as blood, urine, or cerebro-
spinal fluid contain a number of endogenous metabolites that can interfere with the zones of separated enantiomeric drugs. Second, biological matrices contain large quantities of certain components that may disrupt the analytical system during attempts at direct analysis. Electrolytes in the urine and proteins in serum or plasma are the most typical examples of such interfering compounds. The third type of complication results from low levels of pharmaceuticals and their metabolites in pharmacokinetic studies. This represents a challenge to HPCE detectors, making the UV detector of marginal utility; therefore, more sensitive detectors based on fluorescence principles, electrochemistry, and MS become highly desirable. Sample preconcentration before HPCE separation provides yet another remedy to the sensitivity problems in drug metabolism studies.
MaBy herbicides aBd pesticides are produced as racemic mktures.
Figure 8. Racemic bupivacaine samples extracted from supplemented serum. (a) 3.8pg/mL, (b) 0.57 pg/mL, (c) serum blank. (Adapted with permission from Reference 50.)
654 A
Figure 8 shows an example of drug determination in human serum. Bupivacaine was added to a 1.0-mL serum sample at typical therapeutic levels (0.24 pg/mL of racemate), extracted with hexane-diethyl ether, and run in a buffer consisting of 10 mM di-Omethyl-P-CD and 0.03 mM HTAB. This simple procedure provides a practically interferencefree analysis (50).In our laboratory, entangled polymer networks using two different polymers simultaneously were found to be appropriate as buffer matrices in a direct analysis of pharmaceuticals in urine. In combination with CDs, the polymer network matrix was suitable for chiral separations of some basic pharmaceuticals (57). Agricultural chemicals. Many herbicides and pesticides are produced as racemic mixtures. However, often only one
Analytical Chemistry, Vol. 66, No. 11, June 1, 1994
enantiomer is biologically active, and the other can be viewed as an impurity (58). The extent to which various agricultural chemicals are being applied in the environment will soon make it urgent to monitor these potential ecological hazards enantioselectively. In the first reported chiral separation of herbicides by HPCE, Nielen (59) used p-CD as a buffer additive to determine enantiopurity of a phenoxyacid herbicide. Thus far, capillary electromigration techniques have not been applied to chiral resolution of pesticides. Carbohydrates. Various glycoconjugates have received attention recently because of their involvement in key biochemical and physiological processes. Although most naturally occurring sugars have the D-configuration, L-forms have been found in algal mucilages and bacterial polysaccharides (60).The possibility of varying the carbohydrate configuration in biopolymers and their synthetic analogues is believed to have significant pharmaceutical potential (61, 62). We have shown (6) that optical isomers of fluorescently tagged monosaccharides can be separated as complexes with borate and dextrins. Factors affecting enantioselectivity were investigated, including a fluorescent tag dextrin form (cyclodextrin forms, derivatives of p-CD, linear dextrins), complexing anions and their concentrations, and the presence of uncharged organic modifiers. Enantioselectivity was observed only with underivatized CDs and hydroxypropyl-P-CD, indicating that hydroxyl groups in a CD are indispensable for chiral recognition. The future
Capillary electromigration methods are eminently suited to the task of efficient and rapid separation of optical isomers. Significant progress already has been made toward the necessary understanding of the mechanisms of chiral recognition in such separation systems. Because of the rapidly increasing interest in biochemical and pharmaceutical aspects of chirality, additional chiral selectors undoubtedly will be developed in the near future. The development of highly sensitive and precise detection techniques will become essential to expand the scope of investigations to metabolic studies.
This study was supported by Grant 24349-13 from the National Institute of General Medical ‘‘ Sciences, US. Dep,artment Or and‘ -man Serv‘ices, and a grant-in-aid from Astra/ ’ ’ ‘I. Hassle (hoinaai, aweaen). r S. is a recipient of a graduate fellowship fromI the Finnish Academy of Sciences. Fellowship5;for postdoctoral studiesal)road have ‘been provided to M. S. . from the aweaisn-merican * Foundation, the foundation “Stiftelsen Blance)-flourBoncampagni-Ludovisi, fodd Bildt,” . . -and . 1the Swedish Academy or Yharmaceutical bciences. ’
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Milos Novotny (Department of Chemistry, Indiana University, Bloomington, IN 47405-4001) received his Ph.D. in biochemistry from the University of Brno (Czechoslovakia). After postdoctoral positions in Czechoslovakia, Sweden, and the United States, he joined the faculty of Indiana University, where he is the James H. Rudy Professor of Chemistry. His research interests include chromatography and CE of biological molecules and bioanalytical aspects of proteins and glycoconjugates.
Helena Soini received her M.S. degree in structural organic chemistry from Oulu University (Finland) in 1982. She worked as a research chemist and laboratory manager for 10 years at Orion Pharmaceutics and is currently a doctoral candidate in analytical chemistry at Indiana University. Her research interests include chiral separations and pharmaceutical analysis using LC and CE. Morgan Stefansson received his Ph.D. in analytical /pharmaceutical chemistry from Uppsala University (Sweden) in 1992. He recently completed a postdoctoral appointment at Indiana University and returned to Sweden to join the Institute of Analytical Chemistry at his a h a mater. His research interests include LC and CE of complex carbohydrate mixtures.
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