Enantiomer Separation of Amino Acids by Complexation with Chiral

Feb 28, 2007 - We present a new method for separation of enantiomers with high-field asymmetric waveform ion mobility spectrometry (FAIMS), coupled to...
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Anal. Chem. 2007, 79, 2850-2858

Enantiomer Separation of Amino Acids by Complexation with Chiral Reference Compounds and High-Field Asymmetric Waveform Ion Mobility Spectrometry: Preliminary Results and Possible Limitations Axel Mie,*,† Magnus Jo 1 rnte´n-Karlsson,‡ Bengt-Olof Axelsson,‡ Andrew Ray,§ and Curt T. Reimann†

Department of Analytical Chemistry, Chemical Center, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden, Analytical Development, AstraZeneca R&D, Lund, Sweden, and Analytical Development, AstraZeneca R&D, Loughborough, United Kingdom

We present a new method for separation of enantiomers with high-field asymmetric waveform ion mobility spectrometry (FAIMS), coupled to mass spectrometric detection. Upon addition of an appropriate chiral reference compound to the analyte solution and subsequent ionization of the solution by electrospray ionization, analyte enantiomers formed diastereomeric complexes, which were potentially separable by FAIMS. The methodology being developed is intended to be general, but here amino acid analytes are specifically considered. In the examples presented herein, six pairs of amino acid enantiomers were successfully separated as metal-bound trimeric complexes of the form [MII(L-Ref)2(D/L-A)-H]+, where MII is a divalent metal ion, L-Ref is an amino acid in its L form acting as chiral reference compound, and A is the amino acid analyte. For example, D- and L-tryptophan were separated in FAIMS as [NiII(L-Asn)2(D-Trp)-H]+ and [NiII(L-Asn)2(L-Trp)-H]+. As FAIMS separation typically takes place over a time scale of only a few hundred milliseconds, the presented separation method opens new possibilities for rapid analysis of one analyte enantiomer in the presence of the other enantiomer. Preliminary quantification results are presented, which suggest that fast and sensitive quantitative chiral analyses can be performed with FAIMS. Method limitations are discussed in terms of diverse phenomena, which are not yet understood. Different structural isomers and stereoisomers of molecules often feature different physical, chemical, and biological properties. One group of stereoisomers, the enantiomers, is of specific interest because two enantiomers have different chemical properties only in asymmetric environments. Life itself offers one of the key examples of asymmetric environments, and many cases are known for which two enantiomers have very different effects on biological systems. One example is drug substances, one enantiomer of * Corresponding author. E-mail: [email protected]. † Lund University. ‡ AstraZeneca R&D, Lund, Sweden. § AstraZeneca R&D, Loughborough, UK.

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which has a therapeutic effect (eutomer) while the other enantiomer has no therapeutic effect, or an adverse effect (distomer). It is a challenge for analytical chemists to develop techniques that can either separate enantiomers or else reliably detect one such enantiomer in the presence of the other, even when there is a large concentration difference. A number of techniques have been developed with the aim of quantifying enantiomers. All of them have in common that they provide some kind of local chiral environment that offers stereoselective interaction with the enantiomers, in order to distinguish between them. Probably most widespread today are liquid chromatographic (LC) methods,1 either employing chiral stationary phases or using derivatization steps in order to transform the pair of enantiomers into a pair of more easily separable diastereomers. Another chromatographic technique used is supercritical fluid chromatography1 (SFC). Another common technique is capillary electrophoresis (CE) with chiral selectors in the mobile phase.1 Also, NMR with chiral shift reagents is commonly employed.2 The kinetic method3 is a tandem mass spectrometric (MS/ MS) technique in which diastereomeric complex ions are formed from the enantiomers and chiral enantiopure reference compounds, these complexes then being subjected to fragmentation. Differences in the structures and dissociation energies of these complexes or their dissociation products result in different fragment branching ratios (i.e., different relative intensities of the fragments), making it possible to measure the amounts of the two enantiomers. A number of different mass spectrometric approaches toward chiral recognition have been reviewed.4 Unlike LC, SFC, and CE, neither NMR nor the kinetic MS/ MS method involves physical separation of the enantiomers; rather, the two enantiomers are measured simultaneously. LC, SFC, CE, and NMR are rather time-consuming; NMR and the kinetic method normally have limited sensitivity; and none of the (1) Gubitz, G.; Schmid, M. G. Biopharm. Drug Dispos. 2001, 22, 291-336. (2) Wenzel, T. J.; Wilcox, J. D. Chirality 2003, 15, 256-270. (3) Tao, W. A.; Zhang, D.; Wang, F.; Thomas, P. D.; Cooks, R. G. Anal. Chem. 1999, 71, 4427-4429. (4) Schug, K. A.; Lindner, W. J. Sep. Sci. 2005, 28, 1932-1955. 10.1021/ac0618627 CCC: $37.00

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mentioned techniques provides a general solution for quantifying enantiomers in the presence of each other. Thus, the need to develop alternative methodologies for the fast and sensitive analysis of enantiomers remains. A way to separate ions in the gas phase is ion mobility spectrometry (IMS),5 which has long been employed for discrimination of different kinds of isomers. In IMS, ions subjected to an electric field E drift through a bath gas with a velocity v ) KE, where K is the ion mobility. K is dependent on several factors, including ion structure.5 IMS has been employed for showing that C59+ and C61+ each appear in dramatically different structural forms, while C60+ forms the famed fullerene (“magic number” 60 and compact spherical structure) virtually exclusively.6 Leucine and isoleucine, structural isomers with identical mass, have been distinguished with IMS.7 It has been shown that tryptic peptides containing one or more proline residues exhibit traces of cistrans forms in IMS experiments.8 Diastereomeric ions are generally separable by IMS as well: Zn-ligand-hexose diastereomers have been separated with IMS aiming ultimately at a structural characterization that could explain fragmentation spectra observed in tandem MS of these ions.9 A pair of olefin-linked bisparacyclophane diastereomers has also been separated by IMS.10 For the most part, separation of enantiomers may be considered as impossible by IMS (a typical gas-phase environment does not offer the asymmetry needed to sense chiral intermolecular contacts). However, by providing chiral molecules to the drift gas, enantiomers can undergo formation of short-lived clusters with the chiral gas molecules in a stereoselective way and can thus be separated by IMS. It has been proposed to provide “selectively interactive gaseous particles” in the gas phase of the IMS, and thereby a rough separation of chiral components of fluoxetine has been demonstrated using a “chiral collision gas”, 2-butanol.11 Very recently, this concept has been further investigated and extended to the separation of amino acids, carbohydrates, and drug compounds.12 Recently, the capabilities of IMS were extended with the development of high-field asymmetric waveform ion mobility spectrometry (FAIMS).13,14 FAIMS exploits the fact that, at sufficiently high electric fields, most ion species display a dependence of their ion mobility on the electric field strengths the mobility is no longer electric-field-independent, K ) K(E). If we express K(E) as K0[1 + h(E)], the function h(E) will typically vary depending on the structure and identity of the ion, while K0 constitutes the ion mobility at the low-field limit. FAIMS separates ions characterized by differing ratio of high-field mobility to lowfield mobilitysi.e., ions are separated according to the quantity 1 + h(E). This is done by alternating a high electric field of one (5) Eiceman, G. A.; Karpas, Z. Ion Mobility Spectrometry, 2nd ed.; Taylor & Francis/CRC Press: Boca Raton, FL, 2005. (6) von Helden, G.; Hsu, M. T.; Gotts, N.; Bowers, M. T. J. Phys. Chem. 1993, 97, 8182-8192. (7) Asbury, G. R.; Hill, H. H., Jr. J. Microcolumn Sep. 2000, 12, 172-178. (8) Counterman, A. E.; Clemmer, D. E. Anal. Chem. 2002, 74, 1946-1951. (9) Leavell, M. D.; Gaucher, S. P.; Leary, J. A.; Taraszka, J. A.; Clemmer, D. E. J. Am. Soc. Mass Spectrom. 2002, 13, 284-293. (10) Baker, E. S.; Hong, J. W.; Gidden, J.; Bartholomew, G. P.; Bazan, G. C.; Bowers, M. T. J. Am. Chem. Soc. 2004, 126, 6255-6257. (11) Karas, M. PCT Int. Appl 2002096805, 2002. (12) Dwivedi, P.; Wu, C.; Matz, L. M.; Clowers, B. H.; Seims, W. F.; Hill, H. H., Jr. Anal. Chem. 2006, 78, 8200-8206. (13) Guevremont, R.; Purves, R. W. Rev. Sci. Instrum. 1999, 70, 1370-1383. (14) Guevremont, R. J. Chromatogr., A 2004, 1058, 3-19.

Figure 1. Cylindrical FAIMS interface.

polarity during a short time with a low electric field of the opposite polarity during a longer time. In such a scheme, ions would oscillate and return to their starting positions after each cycle, if it were not for the dependence of the ion mobility on the electric field strength. This dependence gives the ions a net nonzero displacement during each cycle. Typically, FAIMS separation takes place between two parallel plates or two concentric cylinders with a spacing of a few millimeters. A longitudinal (or axial) gas flow sweeps the ions through the plate system, while the electric field applied between the plates causes transverse ion motion perpendicular to the gas flow. When cylindrical electrodes are employed (as shown in Figure 1), FAIMS features an additional ion focusing effect because the electric field between the cylinders possesses a gradient.13 Unfortunately, the functions h(E) are not known in advance for analytes of interest, so that FAIMS separation needs to be determined empirically. FAIMS separation is driven by the dispersion voltage (DV), a high-voltage asymmetric waveform that is applied between the two electrodes. By applying an additional constant but adjustable compensation voltage (CV), the net displacement of a certain ion species during each DV waveform cycle can be compensated, resulting in those ions being transmitted through the FAIMS while noncompensated ions are lost in collisions with the electrode surfaces. By scanning CV and measuring the transmitted ions at the ion outlet, data can be obtained in the form of a “CV scan” or “CV spectrum”. Alternatively, the CV can be kept constant or can be cycled over a few predetermined values, so as to transmit only ions of interest. FAIMS, a relatively new technique, has not yet been applied in many analytical areas, but the method shows considerable promise in enhancing our ability to resolve various isomers in critical applications. The structural isomers o-, m-, and p-phthalic acid have been baseline-separated using FAIMS.15 Anomers, linkage, and position isomers of disaccharides have also been (15) Barnett, D. A.; Purves, R. W.; Ells, B.; Guevremont, R. J. Mass Spectrom. 2000, 35, 976-980.

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shown to be separable in FAIMS.16 Three pairs of ephedrinerelated small-molecule diastereomers have been successfully separated.17 Recently, the partial separation in FAIMS of the drinking water contaminants D- and L-lactic acid by forming complexes with L-tryptophan18 was demonstrated. In the work presented below, we have investigated the separability by FAIMS of diastereomeric complex ions composed of chiral amino acids and reference compounds, where the ultimate goal is unequivocal determination of chirality. The complexes have the form [MII(LRef)2(D/L-A)-H]+, where MII is a divalent metal ion, L-Ref is a chiral reference compound in its L form, and A is the analyte. These analytes were chosen because they are readily available in enantiopure form. Moreover, the chirality of the analyte for such complexes was previously detected indirectly by Tao and Cooks using the kinetic MS/MS method,19 giving a good comparison point for our experiments. Here the focus is on development and optimization of a qualitative gas-phase FAIMS-based separation methodology for such complexes. The separation is confirmed through both use of enantiomerically pure amino acids as test compounds and by applying the kinetic MS/MS method in conjunction with FAIMS. Preliminary quantitative data are also presented. EXPERIMENTAL SECTION All experiments were performed using a beta unit of a “Selectra” FAIMS system (Ionalytics Corp., Ottawa, Canada; Thermo Finnigan, San Jose, CA) coupled with a custom-built PEEK support to an Agilent 1100 series SL MSD ion trap mass spectrometer. Samples were supplied using either an Agilent 1100 series autosampler, a syringe pump, or the autosampler together with a syringe pump, both coupled together via a T-junction. All samples were supplied to the ion source through direct infusion, without prior liquid chromatographic or other separation. The ion source was a custom-built microelectrospray (µESI) ion source using stainless steel TaperTip needles (New Objective, Woburn MA) with 50-µm inner diameter as electrospray needles. Operating conditions of the µESI, FAIMS, and MS were as follows: The spray voltage applied to the microelectrospray needle was +3400 V; the FAIMS front plate voltage was +900 V; the FAIMS outer electrode was set to ground potential; and the FAIMS inner electrode DV was -4000 V (zero-to-peak voltage of the asymmetric waveform) following the equation U ) [2 sin(2πft) + sin(4πft - φ)]DV/3, f ) 750 kHz, φ ) π/2. The CV scan speed was 2 V/min unless otherwise specified. The CV was varied within an overall range of 0 to -16 V. The MS inlet capillary tip was set to ground potential. As the focus of this work was on the FAIMS separation, generic values were used as MS and MS/MS tune parameters. The MS capillary exit was set to 128.5 V; the skimmer was kept at 40 V; lens 1 was set to -5 V; the octopole 1 dc offset was set to 12 V; the octopole 2 dc offset was kept at 1.7 V; and the octopole rf amplitude was set to 178.1 Vpp. The partition was kept at 6.8 V; lens 2 was kept at -60 V; and the trap drive was 46.7 V. The following MS/MS (16) Gabryelski, W.; Froese, K. L. J. Am. Soc. Mass Spectrom. 2003, 14, 265277. (17) McCooeye, M.; Ding, L.; Gardner, G. J.; Fraser, C. A.; Lam, J.; Sturgeon, R. E.; Mester, Z. Anal. Chem. 2003, 75, 2538-2542. (18) Sultan, J.; Gabryelski, W. Anal. Chem. 2006, 78, 2905-2917. (19) Tao, W. A.; Cooks, R. G. Anal. Chem. 2003, 75, 25A-31A.

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Figure 2. Spectral features determining peak resolution in FAIMS. For discussion see the text.

parameters were used throughout all experiments if not otherwise specified: the isolation width was 4 m/z and the fragmentation amplitude was 0.8 V. The actual FAIMS interface (see Figure 1) was a domed-type FAIMS interface20 (ion inlet lateral, ion outlet axial) with 10.0mm diameter of the inner electrode, 14.0-mm inner diameter of the outer electrode, and thus with a 2.0-mm electrode spacing in the cylindrical section, and adjustable axial electrode position (tip distance). Optically pure D- and L-amino acids, tryptophan (Trp), arginine (Arg), phenylalanine (Phe), methionine (Met), glutamine (Gln), asparagine (Asn), isoleucine (Ile), valine (Val), proline (Pro), and lysine (Lys), were obtained from SigmaAldrich. NiCl2‚6H2O, CuCl2‚5H2O, MgAc2‚4H2O, and ZnAc2‚2H2O were obtained from SigmaAldrich. Methanol (LC-grade) was obtained from VWR Intl. (Poole, England). Water was taken from a Millipore water supply (Millipore, Billerica MA; Solna, Sweden). Stock solutions of the amino acids (10 mM) were prepared in 50:50 methanol/water. Stock solutions of the metal salts (25 mM) were prepared in water. For the optimization experiments, samples were prepared containing 0.033 mM metal ions, 0.133 mM reference amino acid, and 0.067 mM concentrations each of the D and L forms of the analyte. Samples were infused at 0.5 µL/min with a syringe pump into the electrospray ion source. For the screening experiments, samples were prepared containing 0.133 mM reference amino acid and 0.067 mM concentrations of each of the D and L forms of the analyte. Samples were supplied by the autosampler at 10 µL/min. Metal solutions at 2.5 mM were delivered by the syringe pump at 8 µL/h. The sample and metal solution flows were combined in a T-junction, resulting in the same molar ratio of metal, analyte, and reference compound as for the optimization experiments. The combined flow was split 1:10 to result in a flow rate of 1 µL/min at the electrospray ion source. For the confirmation experiments, samples were prepared containing 0.033 mM metal ions, 0.133 mM reference amino acid, (20) Guevremont, R.; Thekkadath, G.; Hilton, C. K. J. Am. Soc. Mass Spectrom. 2005, 16, 948-956.

and 0.067 mM of either the D or L form of the analyte, or both. Samples were delivered at 10 µL/min from the autosampler, and the flow was split 1:10 to result in a flow rate of 1 µL/min at the electrospray ion source. In electrospray ionization, a variety of cluster ions are formed from these solutions. After ions have passed the FAIMS interface, clusters with m/z corresponding to the general formula [MII(LRef)2(D/L-A)-H]+ (where MII is a divalent metal ion, L-Ref is a chiral reference compound in its L form, and A is the analyte) were subjected to tandem mass spectrometry. Although the method presented herein is not dependent on the use of tandem mass spectrometry, the mass spectrometer was operated in MS/MS mode unless otherwise specified, in order to confirm the molecular identity of peaks in the CV spectrum. In certain cases, the branching ratio of fragmentation by loss of either analyte or reference was used in order to confirm enantiomeric identity according to the kinetic method.19 We evaluated peak resolution in FAIMS with two different measures (Figure 2; see also Results and Discussion below). The discrimination factor Rdf is defined by

Rdf ) 100% × (hpeak - hvalley)/hpeak

(1)

where hpeak is the signal intensity of the smaller peak and hvalley is the signal intensity at the position of the valley between the two peaks.21 Thus, Rdf ) 100% means perfect (baseline) separation. The peak overlap factor Rof of a peak 1 is defined as

Rof,1 ) Itail,2/Ipeak,1

(2)

where the Ipeak,1 is the peak signal intensity of peak 1 and Itail,2 is the signal intensity of the tail of peak 2 at the CV position of peak 1. Two peaks, peak 1 and peak 2, have two overlap factors; we define the minimum overlap factor Rmof as being the smaller value of Rof,1 and Rof,2:

Rmof ) min(Rof,1, Rof,2)

(3)

We express Rof and Rmof as percent values. Thus, Rmof ) 0% indicates perfect separation. Throughout this work, all Rdf and Rmof values calculated refer to cases where equal concentrations of both analyte enantiomers were used. For all calculations of resolution of peaks appearing in the CV spectrum, the intensities of all fragments appearing at significant intensities, which could be assigned to the loss of one analyte molecule or one reference molecule from the parent cluster ion of interest, were added. In certain cases where the collision energy was too low to induce fragmentation, the intensity of the parent ion was used instead. RESULTS AND DISCUSSION The aim of this study was to investigate whether enantiomers can be physically separated by FAIMS, when they are ionized as metal-bound trimeric diastereomeric clusters with enantiopure chiral reference compounds. Six pairs of amino acid enantiomers (21) El Fallah, M. Z.; Martin, M. Chromatographia 1987, 24, 115-122.

Figure 3. ESI-MS/MS measurements of (a) [NiII(L-Asn)2(L-Trp)-H]+ and (b) [NiII(L-Asn)2(D-Trp)-H]+ showing different branching ratios.

were chosen as model compounds to address this question. We now present our results in three stages: results of optimization, demonstration of separation in several cases, and finally discussion. I. Optimization. As a first step, the relevant experimental parameters for the separation were identified and optimized. In order to generally optimize the FAIMS experimental parameters, one pair of cluster ions was chosen for comparative purposes: [NiII(L-Asn)2(L-Trp)-H]+ and [NiII(L-Asn)2(D-Trp)-H]+. In addition to being separable by FAIMS, these clusters are known to display different branching ratios (i.e., different relative intensities of the fragments) in MS/MS experiments22 (Figure 3). The experimentally determined branching ratios R were RL ) 0.136 and RD ) 0.905, resulting in a chiral selectivity Rchiral ) RD/RL ) 6.65. This is in good agreement with previously published results,22 where the respective values were RL ) 0.140 and RD ) 1.10, with Rchiral ) 7.86. Thus, the FAIMS separation can be confirmed by MS/ MS studies with the kinetic method. FAIMS factors that were investigated for optimization of performance in terms of signal intensity and resolution along the CV axis included carrier gas composition, longitudinal position of the inner electrode relative to the output orifice (referred to as the tip distance), and makeup gas flow. The choice of drift gas composition is important for obtaining a successful separation of ions in many cases.15,23 Based on previous work,24 the evaluated gas compositions were nitrogen/helium mixtures containing 0-50% helium. Recent work20 has investigated the dependence of the resolving power of domed FAIMS devices on the tip distance, indicating that this is an important factor in optimization. Finally, the makeup gas decreases the gas flow rate through the FAIMS interface; prolonged ion residence time of ions in FAIMS can in some cases lead to improved resolution, but too high makeup gas flow can hinder entrance of ions into the FAIMS interface. Other factors, such as ambient pressure, FAIMS interface and carrier gas temperatures, and interface design features, have been disregarded in the experiments presented herein, because they were not accessible as experimental variables due to hardware limitations. The influences of tip distance and gas composition on ion throughput and separation have been evaluated in a multivariate (22) Zhang, D.; Tao, W. A.; Cooks, R. G. Int. J. Mass Spectrom. 2001, 204, 159169. (23) Ells, B.; Barnett, D. A.; Purves, R. W.; Guevremont, R. Anal. Chem. 2000, 72, 4555-4559. (24) Barnett, D. A.; Ells, B.; Guevremont, R.; Purves, R. W. J. Am. Soc. Mass Spectrom. 2002, 13, 1282-1291.

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Figure 5. Response surface of peak resolution Rdf of [NiII(L-Asn)2(L-Trp)-H]+ and [NiII(L-Asn)2(D-Trp)-H]+ as a function of FAIMS tip distance and carrier gas composition.

Figure 4. Dependence of peak resolution and intensities of [NiII(LAsn)2(L-Trp)-H]+ (left peak) and [NiII(L-Asn)2(D-Trp)-H]+ (right peak) on FAIMS tip distance and carrier gas composition. Visually, acceptable signal intensities and peak separations are achieved in the area around 40% helium/2.4 and 2.6-mm tip distance.

way in the form of a full factorial design. Finally, the makeup gas flow rate was optimized manually in order to find out whether additional improvements in resolution could be obtained without loss of ion transmission. Figure 4 shows results of studying FAIMS resolution as a function of tip distance and gas composition, using a solution of D- and L-Trp, L-Asn, and Ni, varying the tip distance between 2.0 and 3.0 mm in increments of 0.2 mm, and varying the gas composition between 0 and 50% helium in nitrogen in increments of 10%. In those cases where two peaks appeared in the CV spectra, the left peak was associated with the presence of [NiII(L-Asn)2(L-Trp)-H]+, while the right peak was associated with the presence of [NiII(L-Asn)2(D-Trp)-H]+. Of note is the observation that the relative intensities of the peaks associated with D-Trp and L-Trp in Figure 4 are strongly dependent on the carrier gas composition. With the data available from the experiments presented herein, it is not possible to understand this phenomenon. However, unlike in, for example, liquid chromatographic techniques in which normally nothing is lost and everything sooner or later elutes from a column, the effectiveness of ion transmission in FAIMS is dependent on favorable ion focusing conditions, which in turn depend on the high-to-low-field mobility ratio function. FAIMS can thus favor a certain ion species’ transmission over that of another ion species, by providing different favorable focusing conditions for certain ions under certain experimental conditions. There is still no widely accepted measure for the resolution of two peaks in a FAIMS CV scan, largely due to the fact that the 2854 Analytical Chemistry, Vol. 79, No. 7, April 1, 2007

peaks, unlike in chromatography, do not generally have Gaussian shape.20,25 Many peaks observed in the CV scans displayed shapes that visually could be regarded as being roughly Gaussian; however, fitting of the experimental data to Gaussian curves showed that the wings (tails) of these peaks were consistently of higher intensity than expected for a Gaussian model. As the tails are important for the calculation of peak overlap, we decided not to use chromatographic models for peak resolution. Instead, we chose the discrimination factor21 (Rdf) to evaluate peak resolution. The discrimination factor does not make any assumptions on the peak shapes, and it is readily accessible from the experimental data. However, all information on the peak separation that may be associated with the peak shape is disregarded; the discrimination factor must therefore be regarded as a rather crude measure of peak resolution. Figure 5 shows the response surface of the peak resolution as a function of tip distance and gas composition. The response surface is somewhat noisy, due to the fact that only a few data points comprise the CV spectra of Figure 4. Nevertheless, it is clearly visible that, within the experimental domain, good separation is achieved at tip distance values around 2.6 mm and 40% He in N2 carrier gas composition. Ion transmission has been disregarded here, although it was affected as well, as can be seen from the peak heights in Figure 4. The optimization has been continued on a finer level in much the same way, varying the tip distance between 2.5 and 2.7 mm at 0.1-mm increments and varying the carrier gas composition between 20 and 50% helium in nitrogen. The results, presented in Figure 6, allowed selection of tip distance 2.5 mm and carrier gas composition 40% He/60% N2 as the optimum; separation as well as ion transmission have been weighed in making this decision. Figure 7 shows the effect of makeup gas. A small flow of makeup gas (0.2 L/min) resulted in a slightly enhanced resolution, while a stronger makeup gas flow had a strong detrimental effect on the ion transmission. CV spectra of the separation of the two complexes, as well as CV spectra of the individual complexes, using the parameters determined above, are presented in Figure 8. Though lacking (25) Guevremont, R.; Purves, R. J. Am. Soc. Mass Spectrom. 2005, 16, 349362.

Figure 6. Dependence of peak resolution and intensities of [NiII(LAsn)2(L-Trp)-H]+ (left peak) and [NiII(L-Asn)2(D-Trp)-H]+ (right peak) on FAIMS tip distance and carrier gas composition, optimizing at a finer level than in Figure 4. Figure 8. Optimized FAIMS separation of L-Trp and D-Trp as trimeric cluster ions [NiII(L-Asn)2(D/L-Trp)-H]+, with DV ) -4000 V, carrier gas composition 60% N2/40% He, FAIMS tip distance 2.5 mm, and makeup gas 0.2 L/min N2. (a) Mixture of L-and D-Trp; (b) L-Trp individually; (c) D-Trp individually. Table 1. Successful Separationsa Figure 7. Dependence of peak resolution and intensities of [NiII(LAsn)2(L-Trp)-H]+ (left peak) and [NiII(L-Asn)2(D-Trp)-H]+ (right peak) on makeup gas flow.

predictive knowledge about the behavior of these complexes in FAIMS, it appears reasonable to employ the experimental settings determined above in other cases as well. II. Separation of Various Enantiomeric Amino Acids. For all six pairs of enantiomeric amino acids, all combinations of nine reference compounds and four metals were screened for their ability to form pairs of [MII(ref)2(D/L-analyte)-H]+ complexes that are separable in FAIMS, using the above optimized FAIMS parameters. In case the screening suggested separation, by resulting in two distinct peaks in the CV spectrum that have a discrimination factor Rdf of more than 50%, confirmation experiments were performed by infusing each of the enantiomers separately with the respective combination of metal ion and reference substance. Successful separations according to this screening and confirmation procedure are presented in Table 1. We observed that in about half of the cases in which two or more peaks appeared in a CV scan of a solution containing both of the enantiomers, it turned out that all peaks had contributions from both enantiomers. In these cases, not included in Table 1, no chiral separation occurred. It should also be mentioned that, of the 23 successful FAIMS separations, we found in only 4 cases a substantial distinction between D- and L-analyte using the kinetic method. These four cases were [(NiII(L-Asn)2(D/L-Trp)-H]+ Rchiral ) 6.65, compared to Rchiral ) 7.86;22 [CuII(L-Pro)2(D/L-Phe)-H]+ Rchiral ) 6.43, compared to 7.4;26 [(ZnII(L-Gln)2(D/L-Trp)-H]+ Rchiral ) 4.05; and [(CuII(L-Gln)2(D/L-Trp)-H]+ Rchiral ) 6.21, compared to Rchiral ) 5.7 or 6.8, depending on collision energy.26 (26) Tao, W. A.; Zhang, D.; Nikolaev, E. N.; Cooks, R. G. J. Am. Chem. Soc. 2000, 122, 10598-10609.

m/z fragments used reference metal analyte (D/L)- substance (L-) (M2+) complex ion for evaluation Trp

Pro Phe

Val Arg

Lys

Gln Pro Arg Asn Gln Lys Asn Gln Val Pro Pro Lys Pro Gln Ile Trp Trp Met Val Gln Lys Ile Val

Zn Zn Mg Ni Cu Cu Zn Ni Mg Mg Ni Ni Cu Cu Mg Cu Mg Ni Ni Cu Cu Cu Cu

559 497 575 525 558 558 443 446 422 418 452 514 457 519 402 587 605 529 465 528 528 470 443

355 + 413 382 371 321 + 393 354 + 412 354 310 + 327 349 422 418 287 + 337 349 292 + 342 354 402 470 401 + 464 380 348 382 382 339 325

Rmof (%)b 0.04* 0.91 0.24* 0.37 0.73 0.91* 0.68 1.51 2.16* 2.24* 0.85 3.08* 0.44 0.58* 1.29* 0.19 3.23 1.39 4.65* 4.53* 0.92* 5.24 3.88

a Note that FAIMS settings were optimized for the case [NiII(LAsn)2(D/L-Trp)-H]+. The factor Rmof characterizing peak overlap can thus probably be improved (decreased) in all other cases by optimizing the individual separations with respect to tip distance, gas composition, and makeup gas. b These “/” cases involve multiple peaks, in such a way that some peaks appear at the same CV for both enantiomers and others do not. See the examples in Figures 10 and 11.

One of the more interesting applications of the method described herein is the sensitive analysis of one enantiomer in the presence of a large excess of the other enantiomer. After having developed a successful separation method, it would not be necessary to perform CV scans in order to do such analyses; rather, it would be sufficient to monitor the signal intensities at Analytical Chemistry, Vol. 79, No. 7, April 1, 2007

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two distinct CVs only, namely, the two CVs corresponding to the peak maximums of the two chirality-linked diastereomers. It is therefore of interest to evaluate what contribution the tail of one peak makes to the intensity of the main portion of the other peak (Figure 2). If this contribution is negligible, then the separation may be considered as perfect for the purposes of measuring enantiomeric excess. Obviously, the simplest and ideal case is a full separation between two peaks in the CV spectrum (Rof,1 ) Rof,2 ) 0%), each one originating from a complex ion containing one and only one analyte enantiomer. While we found some cases that are quite close to this situation, e.g., the separation of D/L-valine with copper and tryptophan as reference compound, there are other cases that are not as favorable. One of the best cases we found is shown in Figure 8b and c. If a sensitive analysis of an L-Trp contamination in a sample of D-Trp is required, it is beneficial if Itail,D, representing the signal intensity of the tail of the peak related to D-Trp at the CV of the peak related to L-Trp, be as small as possible, in order not to obscure very small signals that could arise from a minority component L-Trp. In fact, a ratio Rof,L ) Itail,D/Ipeak,L, calculated from separate measurements of L-Trp and D-Trp as in Figure 8b and c, could serve as a measure for sensitivity of a specific separation method. In this case, the ratio 0.37% is quite low, making a sensitive analysis possible. In the opposite case, when a sensitive analysis of a D-Trp contamination in a sample of L-Trp is required, the separation is somewhat different: The ratio Rof,D ) Itail,L/Ipeak,D is quite large, because the main peak in Figure 8b has a smaller satellite peak at CV ) -7.8V, which is close to the CV ) -7.6V of the main peak in Figure 8c (see also the discussion below). A small amount of D-Trp in the presence of a large excess of L-Trp would thus probably be more difficult to measure using this method. However, if the reference compound L-Asn is exchanged for its enantiomer D-Asn, the peaks of Figure 8 should swap their positions, so that a sensitive analysis of D-Trp would then be possible. This peak position interchange is expected, because, for example, the complexes [NiII(L-Asn)2(L-Trp)-H]+ and [NiII(D-Asn)2(D-Trp)-H]+ are themselves enantiomers and thus should behave identically in FAIMS. This has been confirmed experimentally (data not shown). As becomes clear from these considerations, it is the smaller of the ratios Rof,L and Rof,D that determines the quality of this separation. We report this value as Rmof in Table 1. Compared to Rdf, Rmof is more suitable for characterizing a separation, because the broad flanks of each peak (peak tailing) and the potential contribution to the other peak are considered. Compared to chromatographic measures of resolution, Rmof has the advantage that calculating it does involve making any assumptions about the peak shape. Rmof should be regarded as a “purity of separation” rather than as a statement about our ability to quantitate. It implies the existence of a background or baseline for quantification; the noise with which this baseline is afflicted is really what will determine the ultimate sensitivity of a quantification method. However, Rmof is not as easily accessible as either Rdf or chromatographic resolution, because it cannot be determined from a single experiment. Also, it is susceptible to contaminations of each pure analyte enantiomer with the other enantiomer and thus requires very clean substances. 2856 Analytical Chemistry, Vol. 79, No. 7, April 1, 2007

Figure 9. Separation of D/L-valine as [CuII(L-Trp)2(D/L-Val)-H]+. (a) D-Val; (b) L-Val.

Figure 10. Separation of D/L-arginine as [CuII(L-Gln)2(D/L-Arg)-H]+. (a) D-Arg; (b) L-Arg.

Figure 9 shows a case where only one peak related to the presence of each enantiomer appears in the CV spectrum. Here, in principle, it is directly possible to sensitively measure either analyte enantiomer in the presence of a large excess of the other. As has already been shown in Figure 8, it is possible that a single type of cluster gives rise to more than one peak in the CV spectrum. Another such example is shown in Figure 10. The complex [CuII(L-Gln)2(D-Arg)-H]+ seems to appear in two different forms that are separable by FAIMS. This phenomenon is referred to as “multiple peaks” throughout this work. Similar behavior was observed in more than 10 cases (*’s in Table 1). In this case, we could measure a small concentration of D-Arg in a large excess of L-Arg; measuring a small concentration of L-Arg in a large excess of D-Arg would require exchanging the reference compound for its enantiomer. An even more complex situation can be seen in Figure 11. Here, each enantiomer gives rise to two distinct peaks in the CV spectrum, one pair appearing at the same CV for the two enantiomers and the other pair being clearly separated. The arrangement of the peaks indicates that, despite the complexity of the CV spectra, each enantiomer could be determined in the presence of a large amount of the other. Figure 12 shows results from a quantification of L-Trp in the presence of a large excess of D-Trp. The measurements for the calibration curve of Figure 12 have been performed by monitoring the CV values -5.9 and -7.6 V, corresponding to peak maximums

Figure 11. Separation of D/L-valine as [MgII(L-Ile)2(D/L-Val)-H]+. (a) D-Val; (b) L-Val.

Figure 12. Calibration curve for L-Trp in presence of a large excess of D-Trp. Separation as [NiII(L-Asn)2(D/L-Trp)-H]+ (see Figure 8). r2 ) 0.9989. Error bars show ( estimated standard deviation of two experiments with one replicate each, performed on different days, with samples freshly prepared from stock solutions each day. ICV,1 and ICV,2 denote signal intensities at CV ) -5.9 and -7.6 V, respectively. These CV values correspond to peak maximums in Figure 8.

of [NiII(L-Asn)2(L-Trp)-H]+ and [NiII(L-Asn)2(D-Trp)-H]+ (Figure 8), rather than by performing CV scans. The response for each sample is the ratio of the signal intensities at -5.9 and -7.6 V. The offset of the calibration curve is due to imperfect separation of the two complexes. It should be noted that these quantitative results are of preliminary nature, as the effect of factors such as sample pH, solvent content, ionic strength, or changed relative concentrations of analyte, metal, and reference compound have not yet been addressed. Also, repeatability has not yet been investigated in a rigorous way. Still, these results suggest that our approach to separate enantiomers has potential for the determination of enantiomeric excess, even in cases where there is a large excess of one enantiomer present. In this specific case, it appears that less than 0.1% L-Trp of total Trp could be detected. III. Discussion. All six pairs of amino acid enantiomers have been separated successfully using the experimental settings determined above (see Table 1). Of a total of 216 different combinations of analyte, reference compound, and metal ion, 23 have been successfully separated, averaging just over 10% successful separation attempts. Our current experimental setup allows for four CV scans 0 to -16 V, each taking 8 min. The remaining time is needed for liquid transport, loading MS method, and instrument synchronization. Although we do not want to generalize our findings on the basis of our limited number of analytes

from only one compound group, this would suggest that on average a pair of “unknown” enantiomers could be separated after 10 attempts using different combinations of metal and reference compound, taking ∼2.5 h. In an instrumental system more integrated than ours, probably this time could be substantially shortened. This compares favorably to widespread screening approaches for suitable HPLC columns and conditions, typically taking ∼24 h. Also worth noting is the fact that the number of possible combinations of chiral reference compounds and metals is very high and is by no means limited to the metals and reference compounds used in the present work. Though our data set is limited, it appears that analytes that comprise an aromatic system (tryptophan, phenylalanine) were more easily separated than aliphatic analytes. However, the same aromatic compounds were hardly represented among the reference compounds of successful separations. Here, L-Gln and L-Pro were represented 5 and 4 times out of 23 separations, respectively. Copper appeared to be the most successful metal ion with 9 separations, compared to nickel (6), magnesium (5), and zinc (3). It can also be seen that, in the few cases zinc was successful, it yielded very good separations. The only combinations of metal and reference compounds that appeared several times as successful were Cu and L-Gln (three times) and Cu and L-Lys (twice). We would like to stress that these observations are as yet of only empirical nature and do not provide rules of general validity. We found that only about one-third of cases where we obtained good separation by FAIMS were also amenable to treatment by the kinetic method, owing to lack of suitable fragment branching in about two-thirds of the cases. This result suggests that these two methods generally probe different complex ion properties (which was expected) and that these two methods are complementary. It should be noted that both methods share the need for well-defined standard solutions of the relevant enantiomers, which in some cases could be seen as disadvantageous. For this and other cluster types,26,27 results of the kinetic method fragmentation experiments are interpreted in terms of each diastereomer having one structure of partly but not completely known character. From the results presented herein, it has become clear that many clusters of the form [MII(L-Ref)2(A)H]+, where A is present in only one enantiomeric form, are resolved into at least two “species” transmitted at different CVs (Figures 8, 10, and 11). It is tempting to attribute these features to the presence of different conformers or constitutional isomers of the complex ions. This could possibly include complex ions with (a) different binding sites to the metal ion, (b) different binding sites between analyte and reference molecule or between reference molecules, (c) different sites of deprotonation, or (d) identical constitution but sterically hindered intramolecular rotation (conformers). While FAIMS separation of different conformations of protein ions has been reported before,28-30 this would be, to our knowledge, the first time that FAIMS separation of different (27) Augusti, D. V.; Carazza, F.; Augusti, R.; Tao, W. A.; Cooks, R. G. Anal. Chem. 2002, 74, 3458-3462. (28) Borysik, A. J. H.; Read, P.; Little, D. R.; Bateman, R. H.; Radford, S. E.; Ashcroft, A. E. Rapid Commun. Mass Spectrom. 2004, 18, 2229-2234. (29) Purves, R. W.; Barnett, D. A.; Ells, B.; Guevremont, R. J. Am. Soc. Mass Spectrom. 2000, 11, 738-745. (30) Robinson, E. W.; Williams, E. R. J. Am. Soc. Mass Spectrom. 2005, 16, 14271437.

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conformers or constitutional isomers of such small ions could be suggested. On the other hand, FAIMS and the kinetic method are carried out at completely different parts of the experimental chain. FAIMS is carried out at atmospheric pressure just after electrospray ionization, whereas the kinetic method is carried out after ions have passed through the collisional desolvation region of the MS and into the mass analyzer. This implies the possibility that multiple peaks in the CV spectrum may represent different molecular species, such as complexes with solvent adducts or complex dimers. In this context, it may be worth pointing out that our electrospray-FAIMS interface has no provision for heated dry gas. After transmission through FAIMS, these species would be subjected to declustering or other gas-phase reactions and then detected as identical ions by the mass spectrometer. One experimental observation that would point toward this interpretation is the fact that, out of 19 cases where multiple peaks were observed and where the kinetic method was useable, none showed a distinction of these multiple peaks by the kinetic method. But from the experimental data available, the interesting question of what different FAIMS-separated “species” actually represent remains unsolved. Use of enantiomerically pure standard substances still allows the method to be used for qualitative and quantitative purposes, however. It appears that several issues have to be addressed before this method can be routinely used for chiral separation and quantification of enantiomers. However, the preliminary quantitation data we show in Figure 12 seem to be encouraging. The calibration was performed by mixing the pure enantiomers of tryptophan in known concentrations. This approach requires that pure enantiomers, or at least one pure enantiomer and the racemate, have to be available; this could in some cases be regarded as disadvantageous and is a limitation of our method. However, in principle, the calibration curve can be calculated from the relative heights (or response factor) of the two peaks of a racemate. This approach would require that each of the two peaks of a mixture of enantiomers be associated with the presence of one and

only one enantiomer. We thus believe that it is of relevance for the ultimate value of our method, if the detailed identity of the multiple peaks in the CV spectrum and their origin can be revealed, and preferably if the formation of multiple peaks could be avoided.

(31) Eiceman, G. A.; Krylov, E.; Krylova, N.; Douglas, K. M.; Porter, L. L.; Nazarov, E. G.; Miller, R. A. Int. J. Ion Mobility Spectrom. 2002, 5, 1-6. (32) Krylov, E.; Nazarov, E. G.; Miller, R. A.; Tadjikov, B.; Eiceman, G. A. J. Phys. Chem. A 2002, 106, 5437-5444.

Received for review October 4, 2006. Accepted January 5, 2007.

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CONCLUSIONS We have successfully demonstrated the ability to separate six pairs of amino acid enantiomer complexes by FAIMS-MS. It seems to be difficult to draw any conclusions as to which combinations of metal and reference compound should be tested first in case an unknown analyte should be separated, from the available experimental data. It can be concluded, though, that the screening approach described herein is a promising one even for other analytes. While computational tools exist for calculating the gas-phase structure of diastereomeric complexes of the kind we used in our experiments, it is still unclear how these structures correlate to high electric field mobility. Although progress has been made in this subject,31,32 the prediction of FAIMS behavior from the ionic structure or other characteristics is still not generally possible. Upcoming improvements in FAIMS instrumentation, such as temperature control and increased dispersion voltage, would make it even easier to find good separations or else improving existent separations. We believe it is also of interest to apply the method described herein for analytes other than amino acids. Work on separation and quantitation of drug compound enantiomers is in progress. ACKNOWLEDGMENT The authors acknowledge the loan of a FAIMS beta unit from Ionalytics Corporation, now part of Thermo Electron Corporation. Acknowledged are also Crafoordska Stiftelse, Carl Tryggers Stiftelse. and Stiftelsen J. Gust. Richerts Minne, and AstraZeneca, Lund/Sweden, for providing funding for this work, the Faculty of Sciences at Lund University for providing funding for a Ph.D. position. We also thank Ulrika Nilsson at the Department of Analytical Chemistry at Stockholm University for providing the mass spectrometric instrumentation and laboratory facilities for this work.

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