Академический Документы
Профессиональный Документы
Культура Документы
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
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
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.
2851
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 (ESI) ion source
using stainless steel TaperTip needles (New Objective, Woburn
MA) with 50-m inner diameter as electrospray needles.
Operating conditions of the ESI, 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(2ft)
+ sin(4ft - )]DV/3, f ) 750 kHz, ) /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.
(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:
(3)
2853
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
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.
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.
2855
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
2857
(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.
2858
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.
AC0618627