Anal. Chem. 2000, 72, 1736-1739
Chiral Separations of Polar Compounds by Hydrophilic Interaction Chromatography with Evaporative Light Scattering Detection Donald S. Risley and Mark A. Strege*
Lilly Research Laboratories, A Division of Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285
The chiral separations of drug substances and underivatized amino acids were demonstrated in this study through the use of hydrophilic interaction chromatography (HILIC). The polar character of the model compounds presented challenges for their analysis by traditional modes of chromatography, but through the employment of multimodal chromatography utilizing the HILIC mechanism and cyclodextrin- or teicoplanin-derivatized stationary phases, effective resolution was achieved. The analytes lacked sufficient ultraviolet chromophores, requiring their determination by evaporative light scattering detection. HILIC was demonstrated to represent a novel technique for the facilitation of chiral chromatography by providing an environment of solubility and retention that could not be achieved through the use of the traditional methods of reversed-phase, normal-phase, or polar organic mode. Within the current environment of increased regulatory requirements that exists for drug products with stereogenic centers, the enantiomeric separation and evaluation of drug substances is an area of intense interest within the pharmaceutical industry. It has become necessary to develop analytical methods for the chiral separation of pharmaceuticals to control optical purity and to gain an understanding of the clinical, pharmacological, and pharmacodynamic modes of action. Several recent reviews have highlighted developments in chiral chromatographic separations, including both direct and indirect separation modes, stationary phases, derivatizing agents, and applications to enantioseparations of drugs and other biological substances,1,2 with specific emphasis upon drug stability, stereoconversion, recovery, and concentration determinations.3 The general structure, solubility, and retention characteristics of many pharmaceutical compounds has facilitated the evolution of reversed-phase liquid chromatography (RPLC) as the method of choice for chiral separation and analysis. Through the employment of stationary phases derivatized with amino acids, proteins, cyclodextrins, and more recently macrocyclic antibiotics, many successful RPLC enantiomeric separations have been achieved. However, a major limitation of the use of RPLC for work of this * To whom correspondence should be addressed: (phone) (317) 276-9116; (fax) (317) 276-5281; (e-mail)
[email protected]. (1) Bojarski, J. Chem. Anal. 1997, 42 (2), 139-185. (2) Davankov, V. A. Pure Appl. Chem. 1997, 69, 1469-1474. (3) Ducharme, J.; Fernandez, C.; Gimenez, F.; Farinotti, R. J. Chromatogr., B: Biomed. Appl. 1996, 686, 65-75.
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nature has been the lack of adequate retention of polar molecules, hindering direct chiral separations of these types of compounds. In these cases, the derivatization of the analytes has been required to promote RPLC retention, or alternate chromatography methods such as normal-phase4 or “polar organic mode”5 have been utilized. Sample derivatization has been labor-intensive, however, and the use of normal-phase and polar organic mode chromatography has been limited by the minimal solubility of many polar compounds in the nonaqueous, highly organic mobile phases associated with these techniques. Hydrophilic interaction chromatography (HILIC) is a method first described by Alpert for the separation of proteins, peptides, amino acids, oligonucleotides, and carbohydrates.6 The HILIC technique employs hydrophilic packings in the presence of mixed aqueous/organic mobile phases for the establishment of a stagnant enriched water layer on the surface of the stationary phase, into which analytes may partition on the basis of their polarity. This partitioning mechanism had been previously studied extensively and employed effectively for the separation of carbohydrates.7-9 In contrast to RPLC, HILIC elution is facilitated by the aqueous component of the mobile phase and not the organic. The separation mechanism of HILIC is therefore opposite to that of RPLC and is also different from normal-phase and polar organic mode chromatography by the fact that HILIC mobile phases are relatively high in water content (10-50% aqueous), an environment that can provide significant advantages in regard to the solubility of many biologically active substances. These attributes, together with its compatibility with electrospray ionization mass spectrometry and evaporative light scattering detection (ELSD), have facilitated the successful application of HILIC for the analysis of polar molecules for drug discovery.10 Recently, the use of ion-exchange resins under HILIC conditions (i.e., in a highorganic environment) has also been investigated and demonstrated to provide mixed mode chromatography of small molecules in a manner highly orthogonal to RPLC for liquid chromatography-mass spectrometry.11 (4) Stalcup, A. M.; Chang, S. C.; Armstrong, D. W. J. Chromatogr. 1991, 540, 113-128. (5) Chang, S. C.; Reid, G. L.; Chen, S.; Chang, C. D.; Armstrong, D. W. Trends Anal. Chem. 1993, 12 (4), 144-153. (6) Alpert, A. J. J. Chromatogr. 1990, 499, 177-196. (7) Verhaar, L. A. T.; Kuster, B. F. M. J. Chromatogr. 1982, 234, 57-64. (8) Orth, P.; Englehart, H. Chromatographia 1982, 15, 91-96. (9) Nikolov, Z. L.; Reilly, P. J. J. Chromatogr. 1985, 325, 287-293. (10) Strege, M. A. Anal. Chem. 1998, 70, 2439-2445. 10.1021/ac9911490 CCC: $19.00
© 2000 American Chemical Society Published on Web 03/03/2000
It has been demonstrated that the HILIC mechanism can be generated by a variety of stationary phases possessing significant polar character, including several that potentially may provide chiral resolution through secondary interactions. For example, the HILIC effect has been previously observed in chromatography employing cyclodextrin-based packings when the columns were eluted at high (>60%) concentrations of acetonitrile.10,12 In these analyses, sample retention was presumed to occur as a result of the generation of the HILIC mechanism by the hydroxyl groups on the outer surface of the cyclodextrin functionalities, and no attempts at chiral separations were made. RPLC data obtained using teicoplanin-based packings in the presence of varying levels of acetonitrile or methanol demonstrated significant increases in analyte retention at organic concentrations of >60%, but investigations or explanations of the cause of this “normal-phase-like” phenomenon were not reported.13,14 This study reports the use of multimodal chromatography wherein the HILIC mechanism has been employed for the facilitation of chiral separations of polar pharmaceutical compounds by stationary phases possessing established chiral selectivity. Through the employment of cyclodextrin or macrocyclic antibiotic stationary phases and a set of enantiomeric test compounds that were weakly retained by RPLC and not soluble in an environment suitable for traditional normal phase, chiral separations under HILIC conditions were demonstrated and evaluated. Since HILIC was employed at relatively high mobilephase organic concentrations facilitating rapid solvent evaporation, the additional benefit of high ELSD sensitivity was obtained. EXPERIMENTAL SECTION The acetonitrile, ammonium acetate, and acetic acid were obtained from Fisher Scientific (Fair Lawn, NJ). Water was deionized and filtered through a Millipore Milli-Q water purification system (Bedford, MA). National Formulary-grade nitrogen (>97.0%) was used for the evaporative light scattering detector. All of the test analytes were synthesized at Eli Lilly and Co. (Indianapolis, IN) with the exception of the amino acids, which were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO). The HPLC system consisted of an HP 1050 series autoinjector and pump from Hewlett-Packard Co. (Wilmington, DE) and an Alltech 500 evaporative light scattering detector from Alltech Associates, Inc. (Deerfield, IL). The TSK-Gel Amide-80 column (250 × 4.6 mm i.d.) was obtained from TosoHaas (Montgomery, PA). The Chirobiotic T and Cyclobond I columns (250 × 4.6 mm i.d.) were purchased from Advanced Separation Technologies, Inc. (Whippany, NJ). Buffer pH was determined with a Brinkman model 691 pH meter from Brinkman Instruments, Inc. (Westbury, NY) The evaporative light scattering detector drift tube temperature and nitrogen gas flow rate were set at 95 °C and 2.60 standard L/min, respectively, with a mobile-phase flow rate of 0.7 mL/min and 20-µL sample injections. Buffer was prepared by adjusting the 6.5 mM ammonium acetate to pH 5.5 using acetic acid. (11) Strege, M. A.; Stevenson, S.; Lawrence, S. M., to be submitted for publication to Anal. Chem. (12) Churms, S. C. J. Chromatogr., A 1996, 720, 75-91. (13) Armstrong, D. W.; Liu., Y.; Ekborg-Ott, K. H. Chirality 1995, 7, 474-497. (14) Berthod, A.; Liu, Y.; Bagwill, C.; Armstrong, D. W. J. Chromatogr., A 1996, 731, 123-137.
Figure 1. Structures of the test compounds A-C.
Samples were initially dissolved in 3 mL of water with sonication and then diluted to a 10-mL volume with acetonitrile to result in a concentration of ∼0.2 mg/mL. RESULTS AND DISCUSSION The three pharmaceutical compounds (see Figure 1) and the five underivatized amino acids utilized for this study were chosen as test analytes because their polarity facilitated minimal retention when analyzed using either the Cyclobond I or Chirobiotic T packings in RPLC mode (data not shown). The RPLC chromatograms of methionine, leucine, norvaline, and proline reported previously indicated retention of 700 s for elution (structure B was not detected within the retention window of 60 min). As expected, enantiomeric resolution was not observed within any of the elution profiles obtained using the TSKGel Amide 80 packing. The Cyclobond I packing consisted of a β-cyclodextrin structure bonded to silica gel.16 In the reversed-phase mode, evidence has suggested that cyclodextrin-based packings provide chiral separations through the formation of an inclusion complex in which the hydrophobic portion of the solute is included into the apolar cyclodextrin cavity.17 In the normal-phase and polar organic modes, chiral separations are facilitated through stereoselective hydrogen bonding with the secondary hydroxyl groups on the cyclodextrin molecule through a size-dependent interaction in which the solute acts like a “lid” over the cyclodextrin cavity.5 (R)- and (S)-naphthylethyl carbamate-derivatized cyclodextrins have also been used in the normal-phase mode where both π acid/ base interactions and hydrogen bonding have been exploited.18 The results of the analyses of the test compounds obtained through the use of the Cyclobond I packing in HILIC mode are displayed in Table 1. In comparison to the chromatography obtained with the TSK-Gel Amide 80 packing, the retention of the test compounds by the Cyclobond I packing was generally greater, especially for structure A, which was observed to separate from its undesired enantiomer in this environment. Under these multimodal chromatography conditions, both HILIC and chiral mechanisms contributed to the retention of A. Structure A has (16) Advanced Separations Technologies Chromatography Product Guide, 1994; p 7. (17) Hinze, W. L.; Riehl, T. E.; Armstrong, D. W.; DeMond, W.; Alak, A.; Ward, T. Anal. Chem. 1985, 57, 237-242. (18) Armstrong, D. W.; Hilton, M.; Coffin, L. LC-GC 1991, 9 (9), 646-652.
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Analytical Chemistry, Vol. 72, No. 8, April 15, 2000
Table 1. Isocratic HILIC Retention of Test Compounds by the Cyclobond I Packing retention timea (s) test compd C A aspartic acid B proline leucine methionine norvaline b
50%
ACNb
60% ACN
705 804 985 (1098) 1112 (1261) 676 756 950 1259 348 433 315 362 316 369 322 381
70% ACN
80% ACN
1143 2482 1585 (1836) >3600 (>3600) 1032 2100 2332 >3600 597 1045 449 668 466 714 493 778
a Numbers in parentheses refer to the second eluting enantiomer. ACN, acetonitrile.
also been previously separated into its optical isomers by capillary electrophoresis through the use of cyclodextrin additives in the run buffer.19 The Chirobiotic T packing consisted of a covalently bonded macrocyclic antibiotic, teicoplanin, that, like cyclodextrin-based stationary phases, can provide chiral selectivity in the reversedphase, normal-phase, and polar organic-phase modes.20 Teicoplanin is a complex molecule possessing many moieties that provide multiple types of enantiomeric mechanisms13 but also should be capable of generating significant HILIC retention for multimodal chromatography. Chromatograms demonstrating the enantiomeric resolution of norvaline via elution by 50, 60, 70, and 80% acetonitrile are displayed in Figure 3. Strong retention relative to that attainable via RPLC was achieved, and the chiral selectivity of the (19) Kearns, M. L.; Risley, D. S. Eighth International Symposium of High Performance Capillary Electrophoresis, P249, Orlando, FL, January 23, 1996. (20) Advanced Separations Technologies Technical Bulletin LC95/CHIRO-T, 1995.
Figure 3. Evaporative light scattering chromatograms demonstrating the chiral separation of norvaline by HILIC using a Chirobiotic T packing and isocratic elution by (A) 50, (B) 60, (C) 70, and (D) 80% acetonitrile in 6.5 mM ammonium acetate, pH 5.5. Table 2. Isocratic HILIC Retention of Test Compounds by the Chirobiotic T Packing retention timea (s) test compd C A aspartic acid B proline leucine methionine norvaline
ACNb
60% ACN
70% ACN
80% ACN
186 249 (311) 194 230 (266) 548 (724) 433 (483) 428 (471) 402 (465)
220 297 (395) 258 348 (442) 652 (905) 453 (516) 477 (537) 480 (580)
356 (388) 495 (734) 369 864 (1280) 998 (1498) 621 (733) 680 (789) 691 (879)
1305 (1488) 1791 (3164) 1289 >3600 (>3600) 2222 (3645) 1126 (1411) 1311 (1600) 1344 (1871)
50%
a Numbers in parentheses bACN. acetonitrile.
refer to the second eluting enantiomer.
macrocyclic antibiotic was evident for all of the test compounds with the exception of aspartic acid, which remained unresolved (see Table 2). The multimodal chromatography method utilized chiral stationary phases eluted under HILIC conditions to demonstrate the strong retention and effective resolution of polar enantiomeric compounds which could not be analyzed as effectively via previously established chromatographic techniques. Virtually any chiral stationary phase that exhibits significant hydrophilic character should be useful for this method. As was demonstrated graphically by the data in Tables 1 and 2 and the chromatography of norvaline in Figure 3, effective chiral resolution using the HILIC mode can be obtained simply through the elution of an appropriate packing with mobile phase consisting of ∼70% acetonitrile and then adjusting the percent organic for the achievement of an optimal separation. Since the literature has indicated that the HILIC effect is significant over only a relatively narrow range of (21) Zukowski, J.; Pawlowska, M.; Nagatkina, M.; Armstrong, D. W. J. Chromatogr. 1993, 629, 169-179.
percent organic (such as 60-90% acetonitrile) for a variety of both analytes and stationary phases, the use of 70% acetonitrile (or a slightly higher concentration of methanol) as a starting condition should provide consistently effective chromatography. In the absence of chiral resolution, the three packings evaluated in this study provided HILIC retention for aspartic acid in the order of TSK-Gel Amide 80 > Cyclobond I > Chirobiotic T. These data suggested that the HILIC effect generated by the TSKGel Amide 80 phase was the greatest and that by teicoplanin the weakest. However, in the case of the other test compounds that were resolved by the Chirobiotic T stationary phase, the enhanced retention and superior resolution provided by this packing relative to the Cyclobond I reflected the differences in effectiveness of the columns for chiral interactions with acidic analytes. As is the case in RPLC mode, the Chirobiotic T packing may offer optimal selectivity via HILIC through its presentation of multiple interaction possibilities such as π-π interactions, chiral hydrogenbonding sites, and inclusion complexation.13 Since the significant presence of water in the mobile phases facilitated the formation of an enriched aqueous layer on the surface of the stationary phase in HILIC mode, in these separations, the cyclodextrin and teicoplanin stationary-phase functionalities exist within a highly polar environment. Therefore, as in RPLC, the primary mechanisms responsible for the chiral resolution within the multimodal chromatography system are most likely those of ionic interaction, selective inclusion, and hydrogen bonding, which become dominant associative forces in the presence of even low concentrations of water.21 Specifically, for structures A, B, and C, which are nonaromatic and therefore weakly included, ionic and hydrogenbonding interactions should be the primary sources of the demonstrated chiral selectivity. Evidence supporting these proposed mechanisms was generated through unsuccessful attempts to separate the enantiomers of A in either the Cyclobond I or the Chirobiotic T packing through the use of the polar organic mode employing a mobile phase consisting of 95% acetonitrile, 5% methanol, 0.2% triethylamine, and 0.2% acetic acid (data not shown). Under these conditions in the absence of water, polar interactions may be significantly weaker. In addition to its provision of enantiomeric resolution, a significant advantage of HILIC for chiral separations is compatibility with ELSD, an attribute crucial for the detection of weakly UV-absorbing analytes such as those employed in this study. In situations where successful chiral separations may be achieved with both RPLC and HILIC, superior ELSD sensitivity will be provided by HILIC through the use of mobile phases of relatively higher organics facilitating optimal evaporation within the instrument.
Received for review October 5, 1999. Accepted January 27, 2000. AC9911490
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