Karen W. Phinney
National Institute of Standards and Technol 204 A
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Separating drug enantiomers is ushering in a renaissance of sub- and supercritical fluid chromatography
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s part of the continuing effort to improve the safety and efficacy of pharmaceutical products, the attention of both pharmaceutical companies and regulatory agen-
cies has been focused on chiral drugs. Chemical synthesis of drugs with a stereogenic center generally yields a mixture of enantiomers. Because each enantiomer can produce different therapeutic or adverse effects, and may even be metabolized through different pathways (1, 2), chiral compounds can seem to have split personalities. Nevertheless, some drugs are marketed as racemic mixtures or racemates–equimolar mixtures of the two enantiomers. One example is ibuprofen, a nonsteroidal anti-inflammatory drug (NSAID). The therapeutic activity of ibuprofen resides in the S-enantiomer, and metabolic chiral inversion converts the inactive R-enantiomer into the active S-enantiomer (3 ). Therefore, a complete picture of the pharmacokinetic and pharmacodynamic profile of this drug cannot be developed without considering the fate of each enantiomer. M A R C H 1 , 2 0 0 0 / A N A LY T I C A L C H E M I S T R Y
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The potential differences in enantiomer activities and toxicities have been known for many years. Only recently, however, have advances in enantioselective synthesis made possible the cost-effective development of single-enantiomer drugs (4 ). At the same time, recent improvements in analytical methodology have made it possible to perform stereochemical characterization of chiral drugs (5). The U.S. Food and Drug Administration has issued guidelines for the development of stereoisomeric drugs as a result of these advances (6 ). These guidelines recommend the use of stereoselective identity, stability, and assay methods. Relative contributions of the individual enantiomers to the pharmacological and toxicological activity of the drug candidate should also be examined. The analyst plays a critical role in the chiral drug development process because of these guidelines. If a drug candidate is developed as a single enantiomer, analytical support is needed to assess the viability of enantioselective synthetic methods, to verify achiral and chiral purity of single isomer products, and to monitor stability of the drug in formulations (7). Measurement of drug enantiomers in biological fluids also may be required for clinical studies. If the race-
of chiral analytical methods. First, successful chiral resolution occurs within a narrow range of mobile-phase compositions that may not be suitable for resolving enantiomers from other components of the sample (7). Second, the low efficiency of the chromatographic separation results in broad peak shapes that may preclude reliable determination of enantiomeric purity (12). Third, choosing the best stationary phase remains a trial-and-error process, and optimizing the separation can require a significant investment in time for each analysis and for column equilibration after changes in the mobile phase (13). Finally, chiral columns tend to be more expensive and less rugged than their nonchiral counterparts (14 ). Because of such difficulties, other separation methods have been explored. This article focuses on one alternative, supercritical fluid chromatography (SFC), describing its use for chromatographic separations in general and for enantiomeric separations in particular.
Evolution of SFC The use of supercritical fluids as eluents for chromatographic separations was first reported by Klesper et al. in 1962 (15). They demonstrated the separation of porphyrin mixtures by using supercritical chlorofluoromethanes as eluents. Above the critical point, these substances have solvating strengths approaching those of liquids, yet they retain the low viscosity and high diffusivity of gases (16 ). As noted by Giddings et al. (17 ), these properties provide an opportunity to analyze nonvolatile or thermally labile samples that are not suitable for GC and to achieve higher efficiencies than are possible with LC. However, because GC and LC worked for most separation problems, progress in these areas overshadowed advances in SFC. In fact, the first fundamental review of SFC was not published until 1990 (18 ). Now that LC is a mature technique and its limitations are known, the door has opened for alternative techniques, such as SFC and CE. Supercritical fluids as mobile phases. Carbon dioxide remains the most widely used supercritical fluid because of its low cost, low toxicity, and modest critical parameters. Unfortunately, because of early studies that greatly overestimated the solvent strength of carbon dioxide, SFC was touted as a replacement for LC. In particular, the belief that a wide range of solvent strengths could be achieved by varying the pressure led to considerable confusion and disappointment in early applications of SFC. Actually, supercritical carbon dioxide is similar to pentane in polarity and apparent solvent strength (19), and although the density of carbon dioxide does influence its solvent strength, it is more effective to add
The reemergence of commercial
instrumentation for packed-col -
umn SFC has revitalized the- tech mate is developed, identity testing that is capable of verifying the racemic nature of the drug is recommended (8). Enantiomeric separations can be performed by several techniques, including GC, LC, and CE (9). However, the already widespread use of LC for pharmaceutical analysis has favored this method for chiral drug separations (10). Because enantiomers have identical chemical and physical properties in an achiral environment, the separation process requires an additional chiral species. For example, chiral stationary phases (CSPs) for LC incorporate a chiral ligand into the column-packing material. Interaction of the enantiomers with these chiral moieties results in the formation of transient diastereomeric complexes. Enantiomers separate because one enantiomer forms a more stable complex and is retained longer than the other (11). Many different types of CSPs are now commercially available and are used routinely. The availability of CSPs for LC has dramatically improved enantioselective analytical methodology. Despite the success of this approach, however, several limitations have been encountered during development and validation
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a modifier to the carbon methanol, acetonitrile, or (a) dioxide eluent. methylene chloride, can Despite early troubles, produce a binary fluid that supercritical fluids have sevhas increased elution eral advantages over liquid strength (19 ). Because eluents. The reduced viscosmodifiers raise the critical ity of supercritical fluids temperature and pressure decreases the pressure drop necessary to maintain across the chromatographic supercritical conditions, column, which permits the many separations performed 0 5 10 15 20 25 30 Time (min) use of higher flow rates and as packed-column SFC longer columns or multiple actually take place under (b) columns. In addition, solute subcritical conditions—that diffusion coefficients are is, at near-ambient temperatypically at least an order of tures (T < Tc) (20 ). However, studies have demonmagnitude higher in superstrated that no discontinuity critical fluids than in liquids. of the diffusion coefficients The increased diffusivity occurs during the transition shifts the optimum linear from supercritical to subvelocity to higher values 0 2 4 6 8 critical conditions, and difcompared with liquid-based Time (min) fusion coefficients in modieluent systems (20). Finally, fied carbon dioxide eluents replacing liquid eluents with FIGURE 1. Enantioseparations of metoprolol by (a) LC and (b) SFC on a are still higher than those in supercritical fluids reduces pure liquids (22). Phase the costs associated with sol- Chiralcel OD CSP. separation of modified carvent purchase and disposal. (a) The LC separation was performed with 20% 2-propanol in hexane with 0.1% bon dioxide eluents is rare Capillary versus packed- (v/v) diethylamine at 0.5 mL/min; selectivity (a) = 2.67 and resolution (Rs) = 4.8. (b) The SFC separation was performed with 20% methanol, which contained 0.5% under most operating concolumn SFC. Supercritical isopropylamine, in carbon dioxide at 2.0 mL/min, 15 MPa, and 30 °C; a = 2.77 and ditions in packed-column fluid chromatography can Rs = 12.7. (Adapted with permission from Ref. 31.) SFC. Therefore, improvebe performed by using ments in speed and effieither capillary or packed ciency are still commonly realized when working in the columns; the instrumentation requirements are different subcritical region. for the two types of columns. Capillary columns typically Instrumentation. The re-emergence during the past have internal diameters of 50–100 µm, and the mobile decade of commercial instrumentation for packed-column phase is usually pure carbon dioxide. The solvating propSFC also has revitalized the technique. Commercial SFC erties of the eluent are modified by changing its density instruments for packed-column operation use many of the through adjustments in temperature and pressure. On the same components as traditional LC systems and, in fact, one hand, capillary SFC has limited sample capacity and is may serve both purposes. But there are some differences not suitable for preparative applications. However, it can between these instruments. For example, the chromatoprovide efficient separations of complex mixtures, includgraphic system must be modified to deliver liquid carbon ing surfactants, lipids, and polymer additives, and it can be dioxide (or another fluid), which is typically supplied in a used in conjunction with flame-ionization detection (21). cylinder. To ensure that the pressurized fluid remains liqOn the other hand, packed-column SFC uses columns uid, the pump head must be chilled. For binary fluids, a originally designed for LC. These columns are larger— second pump is required to deliver the modifier. By varying internal diameters of 2.0–4.6 mm—and are commercially the flow delivered by each pump, the composition of the available from many vendors in a vast array of polarities eluent can be controlled (19). When necessary, mobileand selectivities. Packed-column SFC will be the focus of phase additives can be introduced by adding them to the this article because this approach has dominated SFC for modifier. An injection system capable of introducing the pharmaceutical analysis. sample into a high-pressure environment is also required. As with capillary SFC, carbon dioxide is the most comHowever, the most noticeable difference between LC and mon fluid for packed-column SFC. But because packed SFC instrumentation is the addition of a backpressure regcolumns are inherently more retentive than capillaries, ulator to control the outlet pressure, which prevents expanpure carbon dioxide lacks the solvent strength to elute sion of the eluent into a gas (20 ). Finally, because the syspolar compounds. Adding a more polar modifier, such as
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DZ
FL TM LM
DD
methods (27 ). Higher flow rates can reduce analysis time without sacrificing efficiency. Figure 1 compares the separation of the enantiomers of metoprolol, a NT b-adrenoreceptor blocking agent, on a Chiralcel OD CSP by LC OX LR LR OX and SFC. Complete resolution of the enantiomers is achieved by both techniques, but the separation requires only 6 min in SFC, 0 10 20 30 40 50 60 70 compared with 22 min by LC, Time (min) and resolution is much higher in SFC. Peak symmetry also FIGURE 2. Separation of achiral and chiral benzodiazepines on coupled amino/Chiralcel OD columns. improved in SFC relative to LC, as shown in the figure. Analytes were diazepam (DZ), flunitrazepam (FL), temazepam (TM), lormetazepam (LM), desmethyl diazepam (DD), nitrazepam (NT), oxazepam (OX), and lorazepam (LR). The separation was performed with 10% ethanol, which contained Despite these unique proper0.5% isopropylamine, in carbon dioxide at 2.0 mL/min, 20 MPa, and 30 °C. (Adapted with permission from Ref. 33.) ties, some analysts have been reluctant to add packed-column SFC to their laboratories because tem remains pressurized until after the detector, the detecof the cost of instrumentation and because many enantion cell—usually a UV-detection cell—must be capable of tiomeric separations obtained by SFC have already been withstanding elevated pressures. achieved by LC. Indeed, packed-column SFC is not always the best choice for chiral separations. It sometimes fails to Chiral separations by SFC provide a separation that has already been achieved on the The application of packed-column SFC to enantiomeric sep- same column in LC (28, 29). Furthermore, samples that arations was first reported by Mourier et al. in 1985 (23). require aqueous conditions are not amenable to SFC, They described the resolution of phosphine oxide enanalthough small amounts of water can be tolerated. To tiomers on a Pirkle-type CSP. Since that report, the separadetermine the applicability of SFC for a particular sample, tion of enantiomers has increasingly been identified as an Berger (20 ) has suggested looking at solubility in area in which SFC offers distinct advantages over LC. Some methanol or a less polar solvent. Nevertheless, SFC has of the CSPs that have been used successfully in SFC include become the preferred technique for chiral method develnative and derivatized cyclodextrins, brush-type (Pirkleopment in many laboratories because of the ease and type) CSPs, and derivatives of cellulose and amylose (24 ). speed of method development and the increased resoluEnantioresolutions of many compounds of pharmaceutical tion. Some examples of the unique characteristics and interest have now been reported, and a partial listing is prewide applicability of SFC for chiral separations are outsented in Table 1. As in the nonchiral case, the majority of lined in the following sections. chiral separations use carbon dioxide-based eluents, and modifiers are typically required to elute the solutes. Advantages of SFC Several trends become apparent from the many reports Enantiomeric purity. As more drugs are developed in sinof chiral SFC. The efficiency of many CSPs improves dragle-enantiomer form, the determination of enantiomeric matically when liquid eluents are replaced with sub- or supercritical fluids. The increased efficiency is observed as sharper peaks and improved peak resolution (25 ). Indeed, Table 1. Selected applications of chiral SFC the difference in resolution between LC and SFC can be to pharmaceutical compounds1. significant enough to turn a marginal LC separation into a Chiral stationary phase Compounds resolved viable chromatographic method in SFC. In addition, interCellulose derivatives b-blockers, benzodiazepines, NSAIDs, actions between the eluent and the analyte and/or the barbiturates Amylose derivatives NSAIDs, protease inhibitors, b-blockers, CSP can be different in SFC than in LC and can result in benzodiazepines unique selectivity. In some instances, a separation that is Brush-type CSPs Antimalarials, NSAIDs, b-blockers, readily achieved by SFC is not possible on the same colbronchodilators Cyclodextrins and derivatives Phosphine oxides, NSAIDs, anticonvulsants umn in LC (26 ). Perhaps of most general interest is that Macrocyclic antibiotics Bronchodilators, b-blockers column equilibration occurs within a few minutes, speedFor a more comprehensive listing, see Ref. 19. ing the optimization of chromatographic parameters and 1
NSAID = Nonsteroidal anti-inflammatory drug
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purity of starting materials, intermediates, and bulk drugs also speeds the process of column screening. In addition, has become a necessity. LC methods often do not provide more compounds are typically resolved with a single sufficient peak resolution to permit reliable measurement mobile-phase composition in SFC than in LC, meaning of stereochemical composithat the analyst has a greater tion, particularly when the chance of success on the first enantiomeric impurity is prestry with SFC (30 ). Unat(a) ent at levels
