A Comparison of Data Analysis Methodsfor Determining Gas Phase Stabilitiesby CID: Alkali Metal Complexesof Polyether Ionophore Antibiotics

Matthew W. Forbes and Dietrich A. VolmerInstitute for Marine Biosciences, National Research Council, Halifax, Nova Scotia, Canada

Gregory J. Francis and Diethard K. BöhmeDepartment of Chemistry, York University, Center for Research in Mass Spectrometry, Toronto,Ontario, Canada

The gas phase stabilities of Group I metal complexes of the polyether ionophore antibioticslasalocid and monensin were investigated by collision induced dissociation mass spectrome-try. Electrospray ionization was used with a triple quadrupole mass spectrometer for thedetermination of threshold dissociation energies upon application of increasing collisionenergies. Various data analysis techniques for the determination of dissociation energies arediscussed to assess the most suitable method for determining the stabilities of the ionophore-metal complexes studied here. In all cases only the relative stabilities of different complexesmay be obtained by the method presented in this study, which does not assess absolute gasphase dissociation energies. Correction factors have been applied, however, to account for theenergy conversion during collisions of different metal complexes and the varying degrees offreedom of different sized ligands, allowing for the comparison of the stabilities of differentionophores with like-metals. The measured threshold dissociation energies were comparedwith respect to the ionic radius of the metal cation, revealing a maximum stability for the K�

complexes of both lasalocid and monensin. A striking decrease in the stabilities of the Rb� andCs� complexes was observed and is believed to be related to a decreasing degree ofcoordination that the ionophores can accomplish with the larger metals. (J Am Soc MassSpectrom 2005, 16, 779–791) © 2005 American Society for Mass Spectrometry

The polyether ionophores comprise a class of com-pounds first isolated from bacteria in the 1950s [1,2] that has grown to consist of approximately 75

base molecules [3]. Of these base structures, thousandsof active derivatives have been either isolated or syn-thesized. Yet all of the compounds owe their potentantimicrobial activity to the intrinsic affinity for cations,specifically the alkali metals that are biologically signif-icant. The biological activity stems from their commonability to encapsulate metal cations by coordination viainternal oxygen functional groups while maintaining alipophilic outer surface of methyl and ethyl substi-tutents. For survival, bacteria maintain elevated concen-trations of Mg2� and K� inside the cell while selectivelyextruding Ca2� and Na� [4]. Metal cations found insubstantial abundance in aqueous biological environ-ments are transported through lipid membranes onlythrough the aid of active carrier proteins, without

Published online March 17, 2005Address reprint requests to Dr. D. A. Volmer, Institute for Marine Bio-

sciences, National Research Council, 1411 Oxford St., Halifax, Nova ScotiaB3H 3Z1, Canada. E-mail: Dietrich.Volmer@nrc-cnrc.gc.ca

© 2005 American Society for Mass Spectrometry. Published by Elsevie1044-0305/05/$30.00doi:10.1016/j.jasms.2005.01.025

which no trans-membrane movement would be possi-ble. The antimicrobial properties of the ionophores area result of their ability to move the otherwise insolublecations through cell membranes, upsetting the naturalcation gradient and ultimately causing cell death. Bothgram-negative and gram-positive bacterial strains aresensitive to polyether ionophores; however, gram-pos-itive species are typically more affected [5].

By the 1980s, the crystal structures and solutionconformations of many ionophores had been well es-tablished by X-ray crystallography [6] and NMR tech-niques [7–12], respectively. It was shown that both insolution and in crystalline form, the ionophores favor acyclic conformation, initiated by complexation with acation. In addition, the antibiotics also contain a carbox-ylic functional group at one end of the molecule and canvery often form intramolecular bonds to further facili-tate and strengthen the cyclic structure. Computationalstudies have been widely applied to further elucidatethe conformation and stabilities of ionophores both insolution [13, 14] and the gas phase [15]. The structuresof two of the most commonly used ionophores, lasalo-

cid A (Las) and monensin A (Mon), are depicted in

r Inc. Received December 5, 2004Revised January 29, 2005

Accepted January 30, 2005

780 FORBES ET AL. J Am Soc Mass Spectrom 2005, 16, 779–791

Scheme 1. The internal oxygen atoms have been shownto be arranged in a distorted octahedral coordinationabout the central Na�.

The binding stabilities and selectivities for differentmetals have been investigated extensively by potentio-metric techniques [16 –25]. Numerous ion transportstudies have also been reported, discussing the relativemetal-transport abilities of the various antibiotics tobiological membranes [26, 27]. Polyether ionophoreshave found their most common use in the agriculturalsector as anticoccidiostats in poultry as well as growthpromoters in ruminant animals [28]. Their widespreadproduction and sale as feed additives has promptedinterest in monitoring the increase in microbial resis-tance in both humans and livestock [5]. There is alsoconcern regarding the allowable residual levels foundin dairy, poultry and beef products.

As a result, considerable efforts have been undertakenin recent years to develop accurate and sensitive methodsfor the characterization and quantitation of ionophores.Because of their polarity, high performance liquid chro-matography (HPLC) methods are used in quantitativeassays. Detection has been achieved by mass spectrometrycoupled to various ion sources including electrosprayionization (ESI) [29 –34], chemical ionization (CI) [35] fastatom bombardment (FAB) [36] and matrix assisted laserdesorption ionization (MALDI) [37]. In addition to thedevelopment of analytical methods, numerous studieshave described fragmentation mechanisms of ionophoresusing collision induced dissociation (CID) spectra. Thesodium complexes of lasalocid [38] and monensin [39 – 43]have received considerable attention, as have salinomycin[43, 44] and tetronasin [45]. Furthermore, significant inter-est has been raised concerning the CID properties ofdifferent metal complexes with ionophores, includingGroup I, Group II, and several transition metals [46].

Recently, our group has prepared an account of theperiodic nature of cation affinities for the ionophoreslasalocid and monensin, in addition to investigating themetal-induced dissociation pathways of Group I com-plexes undergoing CID in a triple quadrupole (QqQ)mass spectrometer [47]. Complexes of monensin andlasalocid with Li, Na, K, Rb, and Cs were prepared inmethanolic solution, and the relative gas-phase affini-ties of the different cations were measured by compar-ing the onset energies of the free metal. A clear linearrelationship was shown to exist between cationic radiusand the metal affinity for the antibiotics. In this study,as opposed to determining the metal affinity for lasalo-cid and monensin, the relative gas phase stabilities ofGroup I complexes are investigated by examining thethreshold dissociation energies of the complexes under-going CID.

The determination of gas phase stabilities of ionicspecies has been accomplished by a variety of methodsincluding infrared multi-photon dissociation (IRMPD),blackbody infrared radiative dissociation (BIRD), andCID. The common principle on which each method is

based is that the vibrational or internal energy of the ion

can be systematically increased by either the absorptionof radiative energy or the conversion of translationalenergy to internal energy via collisions. With eachmethod the analyst is charged with developing a crite-rion by which the threshold dissociation energy isdefined in order to assign relative stabilities to thevarious species under investigation. In this study, gasphase stabilities of the various ionophore-metal com-plexes were determined by performing collision energyramping experiments in a QqQ instrument and analyz-ing the data using a method suitable for the ionophore-metal complexes investigated here.

Experimental

Chemicals

Sodium salts of lasalocid and monensin were acquiredfrom Sigma-Aldrich (Mississauga, ON, Canada) as werethe acetate salts of Li, Na, K, Rb, and Cs and HPLC-grade methanol.

Sample Preparation

One mg·mL�1 stock solutions of the ionophores wereprepared in methanol and stored at 4 °C. Serial dilu-tions of the stock solutions were made to preparesamples at suitable concentrations for infusion into themass spectrometer. Simultaneously, appropriate quan-tities of 20 mM stock solutions (in H2O) of each of themetal salts were added to the ionophoric solutions togive final concentrations of 0.1 �g·mL�1 for each of theantibiotics with corresponding 10-fold molar excessesof each of the metals. The final compositions of thesolutions were approximately 1% H2O in methanol,serving to normalize any variations in hydration of themethanol from atmospheric or preparatory contamina-tion. In many cases, a much lower concentration of themetal salt was required to obtain a strong signal thanwas used. However, the larger metals have lowerbinding constants than sodium (present in the reagentsat substantial levels) so a significant excess must be

Scheme 1. (a) Las·Na� and (b) Mon·Na� showing the cyclicnature of the molecular conformation when complexed with acentral metal cation. The hydrogen boding believed to strengthenthe head-to-tail cycle is also indicated.

added.

781J Am Soc Mass Spectrom 2005, 16, 779–791 GAS PHASE STABILITIES OF POLYETHER IONOPHORES

Instrumentation

Collision energy ramping experiments (discussed be-low) were performed using an API 4000 triple quadru-pole mass spectrometer (MDS Sciex, Concord, ON,Canada) equipped with a turbo ionspray source. Dataacquisition methods were optimized for the sodiumsalts of the two ionophores utilizing the followingpertinent instrument settings.

Lasalocid. Curtain gas (20), drying gas (0), nebulizer(50), needle voltage (5000 V), temperature (80 °C), de-clustering potential (80 V).

Monensin. Curtain gas (20), drying gas (0), nebulizer(60), needle voltage (5500 V), temperature (75 °C), de-clustering potential (100 V).

Solutions were introduced by direct infusion into theion source at a flow rate of 5 �L·min�1. MSn experi-ments (n � 1 to 5) were conducted using an Agilent(Palo Alto, CA) 1100 Series LC/MSD SL quadrupole iontrap equipped with a low-flow ESI source. The samesolutions from above were infused at a rate of5 �L·min�1.

The precursor ions were preselected in Q1 and sub-jected to progressively more energetic collisions in q2

while several fragment ions were monitored in Q3. Thecollision energy was controlled by varying the potentialdifference of the inter-quadrupole lens voltages be-tween q0 and q2 in the range 5–130 V. As will bediscussed below, ionophore-metal complexes areknown to exhibit numerous fragmentation pathwaysincluding small neutral losses of H2O at lower collisionenergy, cleavages of the “backbone” of the macro-cycleat medium collision energies, and finally expulsion ofthe free metal cation beyond an intrinsic thresholdenergy for each specific complex. Replicates of thecollision energy ramping experiments were performedin quintuplicate by recording transitions of the precur-sor ion to the characteristic fragment ions of eachspecies in multiple reaction monitoring (MRM) mode.Typically, three to five fragment species were moni-tored (depending upon the degree of fragment varietyarising from different metals), including the free metalcation and any precursor ions surviving transmissionthrough the collision cell.

CID Spectra and Energy Conversion

To date, descriptions of fragmentation mechanismscombined with accurate mass assignments to productions in CID spectra have primarily focused upon thesodiated ionophoric species, given that the sodiumcomplexes are the predominant species observed in ESIspectra from both analytical standards and biologicalextracts. A summary of the commonly observed frag-

ment ions arising from complexes with different metals

has appeared in the literature and a qualitative expla-nation of the relative intensities observed in ESI spectrawas offered by Shen and Brodbelt [46]. Additionally, intheir study, the genealogy of the fragmentation path-ways of sodiated lasalocid and monensin were eluci-dated using double-resonance stored-waveform inverseFourier transform (SWIFT) experiments. Recently,Lopes et al. reported detailed assignments of fragmentions observed for Ag�, Ca2�, and Ba2� complexes ofmonensin that closely resembled the sodium analog[48]. The authors also observed several new fragmentsfollowing CID of [Mon � H� � Cu2�]� that did notfollow the same pathways as have been described forthe other complexes: one being hydride abstraction byCu2� and the other involving reduction of Cu2� to aCu� species.

The first complete account of CID analysis of iono-phores was presented by Kim et. al. [36]. The authorsdescribe a system of classifying two types of fragmentions, A-type and F-type. In general, A-type ions ofionophore-metal complexes comprise the metal cationbound to some fragment with the terminal carboxylgroup, whereas F-type ions contain the metal cation anda terminal hydroxyl group. In addition to the COCbond cleavages giving rise to the A- and F-type frag-ments, the ionophores are also well known to undergomultiple losses of H2O. The number and intensities ofthese fragments are thought to be charge-remote in-duced processes [49] that may give some indication ofthe resilience of the “backbone” structure of the mole-cule resulting from more stable gas phase geometrieswhen the ionophore is coordinated to a suitable metal.In developing the relative abundance curves, the F-ion,the A-ion, and the free metal were monitored.

The fragmentation pathways of the sodium complexof lasalocid (m/z 613) are given in Scheme 2. In additionto the A- (m/z 359) and F-fragments (m/z 377), threewater losses were observed in the CID analysis at m/z595, 577, and 559 (Figure 1). A closer look at thestructure of lasalocid, however, does not reveal easilywhich portions of the molecule give rise to the observedwater losses. To isolate the water losses, MSn wasperformed using a quadrupole ion trap. The individualwater-loss fragments were isolated and submitted toresonance excitation, yielding intact backbone com-plexes of lasalocid undergoing four consecutive waterlosses, the last of which was not observed in the CIDspectrum from the triple quadrupole. Figure 1 alsoshows product ion spectra of Las·K�, Las·Rb�, andLas·Cs� at the same collision energy showing the effectof the metal on the degree of fragmentation of thedifferent complexes.

Analogous fragmentation pathways were observedfor monensin. The sodium complex (m/z 693) exhibits atleast three significant backbone cleavages to yield F-type ions at m/z 617, 501, and 479. As with Las·Na�,three water losses were observed for Mon·Na�, the firsttwo of which are highly abundant in the product ion

spectrum, the third being less so.

782 FORBES ET AL. J Am Soc Mass Spectrom 2005, 16, 779–791

Having defined species of interest to be investigatedby the CID experiments, a further consideration mustbe made regarding how collisional energy is convertedinto vibrational energy, if the relative stabilities ofionophore-metal complexes are to be compared. Twomain adjustments are widely applied to the laboratory

Scheme 2. Fragmentation pathways of lasaloions.

Figure 1. Product ion CID spectra of [Las·Li]�,

� 40 V showing the relative abundance of the fragm

frame collision energies (Elab). Following a collisionevent, only a fraction of the laboratory frame potentialenergy is actually converted to internal energy. Inaddition, the masses of the ion and the neutral alsoaffect the conversion of collision energy. Eq 1 describesthe maximum fraction (f) of energy available to be

howing formation of A- and F-type fragment

·Na]�, [Las·K]�, [Las·Rb]� and [Las·Cs]� at E

cid s

[Las

Lab

entation routes of the complexes.

783J Am Soc Mass Spectrom 2005, 16, 779–791 GAS PHASE STABILITIES OF POLYETHER IONOPHORES

converted as a function of mn and mion, the masses of theneutral collision gas and the precursor ion, respectively[50]:

f �mn

mn � mion

(1)

The relationship indicates that more massive ions willhave less internal energy transferred than lighter ions.Thus, in order to compare stabilities of complexes withdifferent metals, a correction factor (�) must be appliedand is defined as the ratio of energy conversion frac-tions for the species of interest and a reference com-pound.

� �fcomplex

freference

(2)

In this case, the lightest of the complexes investigated isLas·Li� (mion � 597 Da) and will be defined as thereference complex. Thus, �LasLi � 1.00 and the labora-tory frame collision voltages of the other complexes willbe adjusted according to eqs 1 and 2. Applying thecorrection factor (�) allows the comparison of collisionenergies from instruments using different collisiongases and represents the maximum fraction of thelaboratory energy that can be converted to internalenergy. The correction factor applied here is not in-tended to represent an estimate of the actual quantity oftranslational energy converted to internal energy sinceeq 1 is overly simplistic and for which more rigorousmodels have been discussed at length in the literature[51]. Most importantly, for the assignment of relativedissociation energies, the mass correction is employedto take into account the differences in the energyconversion between ionophores of different masses orthose bound to different metals. For example, if weconsider the heaviest and the lightest Group I metalsexamined in this study, the more massive Cs complexwill have a lower effective transfer of energy from acollision than the analogous Li complex. In the case ofLas·Li� and Las·Cs� in collision with N2, the maximumpossible energy conversions are approximately 2.3 and1.9% of the laboratory collision energy, respectively.Although small, the differences are not insignificantand should not be neglected.

A second adjustment that has been made was de-scribed by Ryzhov et al. [52, 53], in which the size of thecomplex undergoing CID is taken into account to reflectthe quantity of collisional energy converted to internalenergy. Following a collision event, an ergodic processtakes place where the energy deposited in the ion isdistributed rapidly and evenly among all of the bonds.The average vibrational energy of a single bond result-ing from a given collision will decrease when morebonds are available. The number of degrees of freedom(DOF) of a system may be calculated from eq 3, where

N is the number of atoms in the ionophore.

DOF � 3N � 6 (3)

� �DOFref

DOFcomplex

(4)

A correction factor (�) can then be calculated fordifferent complexes with respect to the number ofdegrees of freedom for a reference complex. In thisstudy, lasalocid is the smaller of the two ionophore so itwas selected as the reference compound.

�Las �DOFLas

DOFLas

� 1

�Mon �DOFLas

DOFMon

�(3.97) � 6

(3.110) � 6� 0.88

(5a,b)

When the correction factor is calculated for com-plexes of monensin, the laboratory collision energy willbe reduced to the effective collision energy by a factor�Mon � 0.88, appropriate for the DOF of monensin. Itfollows that threshold dissociation energies (ED) calcu-lated for like-metal complexes of lasalocid and monen-sin may be compared when the correction factor fordegrees of freedom is applied.

Data Analysis

Herein is presented a summary of different methods bywhich threshold dissociation energies have been deter-mined, particularly for noncovalent complexes, and adiscussion of the suitability of these methods for theionophore-metal complexes of interest to us. The ulti-mate goal of the experiments was to develop a system-atic means whereby the relative gas phase stabilities ofthe various metal complexes can be assigned. The basicprocess that is being observed is the decomposition of aprecursor ion complex following CID. By tracking therelative abundance of the precursor with respect toincreasing collision energy, differences in the stabilitiesof the various metal complexes may be evaluated.Breakdown plots of the abundances of the ion speciesrecorded in the multiple reaction monitoring (MRM)mode of the QqQ experiments were constructed withrespect to collision energy. By means of various dataanalysis techniques, the stabilities of the ionophore-metal complexes were determined. Specifically, it is thethreshold dissociation voltage (ED) that was measuredas a function of the laboratory collision voltage. There-fore, we must select a set of criteria that, when appliedconsistently to the data sets, will yield a chemicallyrelevant value for the threshold dissociation energy ofthe complex. The following sections briefly summarizepreviously developed conventions for the determina-tion of dissociation energies of other host–guest sys-tems and describe the application of each method to thedissociation curves of the ionophore-metal complexes.

In each case the results have been examined to evaluate

784 FORBES ET AL. J Am Soc Mass Spectrom 2005, 16, 779–791

the suitability of the definition of the threshold dissoci-ation energy.

E1/2 Method

The first method for determination of threshold disso-ciation energy was recently described by Brodbelt andcoworkers for analysis of host–guest systems of crownethers and several amine compounds [54]. The abun-dances of the crown-amine complexes were monitoredand the dissociation energy was defined as the excita-tion amplitude corresponding to a 50% survival rate ofthe precursor complex. This method of analysis isillustrated in Figure 2a, using data recorded for the CIDdissociation of the Las·Rb� complex in the triple quad-rupole MS.

A variation of the E1/2 method was described byRyzhov et al. [52], in which the dissociation energies ofHeme complexes with small peptides were evaluated. Invirtually identical experiments as those conducted byBrodbelt et al., the appearance of the [Heme]� ion wasmonitored simultaneously with the decomposition of theHeme-peptide complex. The difference between the twostudies lies in the analysis of the E1/2 energy; whereasBrodbelt et al. defined the dissociation energy at 50%abundance of the precursor complex (Figure 2a), Ryzhovet al. characterized the E1/2 value as the collision energy atwhich the abundances of the precursor ion and the frag-ment ion were equal. A graphical analysis of the sameLas·Rb� complex appears in Figure 2b and yields aslightly, though not insignificantly, higher value for ED.Figure 2c and d serve to further illustrate the divergence ofthe values that are obtained applying the E1/2 methodswhen Las·K� and Las·Na� are examined.

For clarification, Ryzhov et al. point out in theirstudy that solitary [Heme]� fragment ions are obtainedfor virtually all of the different peptide complexes,providing justification for the E1/2 method. However,Figure 2 does illustrate that the ionophore-metal com-plexes may not be suitable candidates for analysis in thesame way. As a result of significant alternate fragmen-tation pathways exhibited by ionophore complexes,such as the loss of 236 Da observed for lasalocid(Scheme 1), the disappearance of the Las·Metal� speciesdoes not necessarily proceed directly via expulsion ofthe metal ion. The onset of the metal cation is indicativeof the affinity of the metal for an intermediate speciesand it is not explicitly related to the gas phase stabilityof the ionophore-metal complex.

Tangent Method

In a previous paper, we have described measurement ofthe onset energies of metal cations from ionophore-metal complexes [47]. From similar experiments tothose presented in Figure 2, the portion of the disap-pearance curve where the abundance of the species isbetween 0.25 and 0.75 was analyzed. A least squares

regression analysis was applied, yielding a regression

line tangent to the linear portion of the curve where arapid decrease in the abundance of the complex occurs.By extrapolation of the tangent line to the ordinate, avalue for ED is obtained as indicated in Figure 3a.

It becomes apparent, when Figure 3b is scrutinized,that blindly applying the tangent method to multiplecomplexes may yield results that are misleading. First,with the exception of the Las·Rb� complex, all of thedisappearance curves have fairly similar shapes and thetangent method yields consistent results for ranking ED.The stability for the Group I complexes of lasalocidwould be:

Cs � Li � Rb � Na � KThis result is slightly different from the results that

are obtained from the E1/2 method (50% abundance) andclose examination of Figure 3b reveals:

Cs � Li � Na � Rb � KRegion III highlighted in Figure 3b illustrates the

discrepancy that occurs as a result of differences in theshapes of the curves. Although the abundance of the Rbcomplex remains higher prior to the region of the rapiddecay, it is actually the magnitude of the slope of thedecay curve that causes the tangent method to return avalue that is lower; i.e., the regression line intersects theordinate at lower collision energy than would be ex-pected for one of the other complexes with a slope oflower magnitude. Regions I and II also point outintersections of other decay curves that may not neces-sarily affect the validity of the determination of disso-ciation energies but do serve to illustrate that the

Figure 2. (a) Graphical representation of the determination of thedissociation energy by the E1/2 method corresponding to the 50%abundance of the precursor Las·Rb� complex. (b) Implementationof the E1/2 method utilized by Ryzhov et al. for the same Las·Rb�

complex whereby the dissociation energy is defined by the inter-section of the disappearance and onset curves. (c) A species plot ofthe Las·K� complex illustrating the further divergence of the twoE1/2 methods used to evaluate the dissociation energy. (d) Anexample of the extreme case where the intersection of the disap-pearance and onset curves occurs at negligible relative abundanceof both species.

various complexes are undergoing different fragmenta-

785J Am Soc Mass Spectrom 2005, 16, 779–791 GAS PHASE STABILITIES OF POLYETHER IONOPHORES

tion processes, or more precisely, are undergoing anal-ogous fragmentation pathways in different proportions.

In real terms, this can be rationalized by an under-standing of the fragmentation pathways available toionophore complexes. Figure 1 shows product ion spec-tra of the Na and Rb complexes of lasalocid at a labcollision energy (CE) of 40 V. Although there are neutrallosses of water and a low intensity signal for anF-fragment from the Las·Rb�, the dominant pathway isthe expulsion of the bare Rb� ion which becomes thesole mechanism at CE � 50 V and the abundance of theprecursor Las·Rb� is rapidly depleted. In contrast, theformation of Na� is not favored at CE � 40 V and doesnot become a dominant fragmentation mode until muchhigher collision energies �100 V. Thus, there are morealternate routes available to the Las·Na� complex andthe abundance of the precursor decays more slowlythan its Rb counterpart. If strict adherence to thetangent method were applied, one would conclude thatthe Rb complex is significantly less stable than eitherthe Na or K complexes, when in fact its decay curveshows that its relative abundance remains quite strongafter the other complexes have begun to show reduc-tions in abundance.

Critical Slope Method

A third means of analysis of the decay curves involvesthe use of a method that was originally developed forthe determination of appearance potentials or ioniza-tion energies. Traditionally, the determination of ion-ization energies was carried out by irradiating gasphase species with a finely tuned, energy-resolvedbeam of electrons and monitoring, by mass spectrome-try, the abundance of the molecular ion with respect tothe energy of the impinging electrons [50]. A difficultysimilar to that which we have faced in the analysis ofthe dissociation curves in these experiments was en-countered by Honig when he analyzed the appearancepotentials of hydrocarbons [55]. A thorough discussionof different methods used by his predecessors waspresented in addition to a description of a scenario in

Figure 3. (a) Determination of ED for Las·Rbregression line was used to calculate the intercepof the Group I complexes of lasalocid.

which the tangent method provided erroneous results

[56]. In response, Honig suggested a method wherebythe appearance curves were replotted on a semi-loga-rithmic scale to better represent the exponential onset ofthe ionized species. Figure 4 shows an example of atypical onset curve plotted in both linear (Figure 4a)and semi-logarithmic (Figure 4b) scales. The criticalpoint (EC) is defined as the energy below which theonset of the ionic species is logarithmic (linear in asemi-log scale), that is, the energy beyond which theappearance of the ionic species no longer increasesexponentially. This corresponds to the point on theappearance curve where the rate of change of the slopereaches a maximum and continues to decrease thereaf-ter.

A direct comparison may be made between Honig’smethod applied to onset curves and the exponentialdecay curves of the precursor ionophore-metal com-plexes in this study. It is suggested that the point on thedisappearance curve where the decay rate is no longerincreasing (in other words, the point at which the rate ofchange of the slope reaches a minimum) would bestmeasure, without bias, the threshold dissociation en-ergy. Mathematically, this point is defined as the min-

ing the tangent method; the equation of thee ordinate. (b) Plots of the disappearance curves

Figure 4. (a) Linear and (b) semi-log plots of an appearancecurve. The critical point (E ) is indicated in plot (b), corresponding

� ust of th

C

to the voltage below which the curve is approximately linear.

786 FORBES ET AL. J Am Soc Mass Spectrom 2005, 16, 779–791

imum of the gradient function d2A/dE2, where A is theabundance of the ionic species and E is the collisionenergy.

To identify the threshold dissociation energies of theionophore-metal complexes according to the definitiondescribed above (the point where the gradient is at aminimum), the data were processed by means of adigital filter using the software package Matlab(Mathworks, Mattick, MA). The filtering algorithm waswritten based upon a simplified least squares method togenerate the second differential of the discrete datapoints from the dissociation curves. These methods,originally introduced to analytical chemists in 1964,have been widely applied to various data processingapplications and have since become known as SG filtersowing to the now-famous paper by Savitzgy and Golayin Analytical Chemistry [57]. A detailed discussion ofthese methods will not be presented herein, however,the reader is directed to a tutorial on digital datafiltering methods [58]. The use of matrix algebraicmethods by Matlab allows the calculation of differentialfunctions for discrete data points with ease. In general,the algorithm examines a “window” of data points (thelength of which can be specified) and returns thesolution to the least squares, nth order polynomiallinear regression. The solution is an (n � 1) x p matrixwhere n is the order of the fitting equation and p is thenumber of points. For example, a 2nd order SG filterfitting five moving points to the eq f(x) � a � bx � cx2,has a least squares solution matrix (A) containing threerows of coefficients; the first row of the matrix containsthe linear regression coefficients, the second row con-tains the first differential coefficients and the third rowcontains the second differential (gradient) coefficients.

A �

��0.0857 0.3429 0.4857 0.3429 �0.0857

�0.2000 �0.1000 0 0.1000 0.2000

0.1429 �0.0714 �0.1429 �0.0714 0.1429�

(6)

The new filtered points are then calculated from thedot product of the third row of A (a3n) and the windowof five points (di) from the dissociation data. Thecalculation is carried out for every consecutive set offive points in the sliding window to generate a vector offiltered data, in this case the gradient. In general eachfiltered point (pr) is given by eq 7a and the filteredgradient vector (G) is made up of the N filtered pointsas in eq 7b.

pr � [ a31 a32 a33 a34 a35 ] ·�di

di�1

di�2

� � (7a)

di��p�1)

G ��pr1

pr2

�prN

� (7b)

It is pointed out that the solution matrix A is dependentonly upon the order of the fitting equation and thenumber of points in the sliding window when the dataare equally spaced. The filtered matrix G, can then becalculated for numerous decomposition curves usingthe same coefficients for A. In this way, the thresholddissociation energies were determined for each of thelasalocid and monensin metals complexes by calcula-tion of the gradient curve and determination of thegradient minima for each complex. The methodology isbest summarized graphically in Figure 5 showing dis-appearance curves for the monensin-metal complexesand their associated gradient curves.

The comparison serves to point out two characteris-tics of the measurements; first, that the laboratory framedissociation energies are not absolute and can be usedonly to assess the relative stabilities of the differentmetal complexes. Table 1 summarizes the differentlaboratory dissociations voltages that one would obtainfor the Las·K� complex applying the four methodsdescribed above.

Figure 5. (a) Disappearance curves for monensin-metal com-plexes and (b) their associated gradient functions as determinedusing the SG digital filter. The minimum of the gradient functioncorresponds to the critical point on each disappearance curve atwhich the rate of dissociation of the complex is no longerexponential. This energy is defined as the threshold dissociation

Table 1. A summary of the laboratory dissociation voltage forthe Las · K� complex, as determined by both E1/2 methods, thetanjent method and the critical slope method

Critical slope Tanjent E1/2 (50%) E1/2 (equal)

45 V 57 V 48 V 52 V

energy for the complex.

6

787J Am Soc Mass Spectrom 2005, 16, 779–791 GAS PHASE STABILITIES OF POLYETHER IONOPHORES

The impetus for selecting a particular method is basedprimarily upon the suitability of its application to thefragmentation pathways exhibited by the complex. Thecritical slope method is sensitive to the collision energy atwhich the disappearance of the parent complex is pro-ceeding at the highest rate and thus takes into account thecontributions of all of the significant fragmentation routes.Whereas the E1/2 methods are primarily suited to systemsthat exhibit only one fragment ion, the tangent methodhas been shown to be of utility in disseminating appear-ance potentials when the shapes of the onset curves are allsimilar. However, the selection of any one method isbased purely upon the differences in stabilities betweencomplexes and not the magnitudes of the measurements.The critical slope method is the most appropriate fordetermination of dissociation energies of the ionophore-metal complexes, not because of the absolute values of thecollision energies but because this method best reflects thenature of the decomposition of the various metal species.

Of additional interest is the application of the criticalslope method to data from different CID conditions suchas those encountered in quadrupole ion traps or Fouriertransform ion cyclotron resonance mass spectrometers. Aswe have reported, different relative abundances of frag-ment ions have been observed for the ionophore-metalcomplexes in the QIT than were observed on the QqQ,resulting from differences in the frequency and energeticsof collision events. David and Brodbelt reported [54] thedetermination of relative gas phase stabilities using CIDby resonance excitation in a QIT in which the collisionenergy was reported as a function of the amplitude of theexcitation potential. Under similar collision conditions, thedifferences in gas phase stabilities will still be apparent fordifferent complexes and the threshold dissociation ener-gies can be evaluated using the critical slope method.However, no experiments have been completed at thispoint to determine whether a different trend in stabilityfor different complexes will be observed under differentcollision conditions. We do hypothesize that the thresholddissociation energy is an intrinsic property of the complexthat should be unaffected by collision conditions.

Results and Discussion

Disappearance curves were constructed for the Group I

Table 2. A summary of dissociation voltages (ED) as determinedvoltages are adjusted with scaling factors (�) and (�) to account fupon conversion of translational energy to internal energy. The rLas · Li�, the smallest of the complexes investigated

Lasalocid

ELab (V) � � ED

Li� 34 1.00 1 3Na� 38 0.97 1 3K� 45 0.95 1 4Rb� 40 0.89 1 3Cs� 31 0.83 1 2

complexes of lasalocid and monensin according to the

experimental procedures described previously. Eachcurve was processed using a seven point, second orderSG digital filter to generate the gradient functions. Thecollision voltages at the minimum of the second orderdifferential curves were then identified as the criticalpoints of the disappearance curves of each of theionophore-metal complexes. Table 2 summarizes all ofthe threshold stability energies determined via thecritical point analysis of the decay curves for lasalocidand monensin. The correction factors for mass (�) anddegrees of freedom (�) were applied to the laboratorycollision voltages to obtain the scaled dissociation en-ergies (ED).

The laboratory collision voltages also include uncer-tainties that are simply taken to be the increment bywhich the collision energy was stepped—in this case by1 V. The rationale for assigning �1 V was based uponthe fact that the dissociation energy was defined from apoint on the gradient curve and is a filtered represen-tation of the abundance data recorded by the massspectrometer. Thus, although the standard errors ofabundance of five replicate experiments were deter-mined to be only a fraction of a percent, a more rigorouscondition was applied to estimate the uncertainty bysaying that we could not be certain in the assignment ofthe threshold dissociation energy than being one of thepoints that were measured. This represents a relativeuncertainty of 1–3% (depending upon the magnitude ofthe laboratory voltage) and is considered reasonabledespite being an over-estimate. The insignificant rela-tive errors in the measured abundance and the repro-ducibility of the disappearance curves upon replicationlend a good degree of confidence to the determinationsof the relative stabilities of the ionophore metalcomplexes.

The first observation that can be made is based on atraditional means of comparison to another class ofmacro-cyclic ionophores, namely crown ethers that alsoshow high tendencies to complex metal cations. It haslong been established that there exists a strong relation-ship between the stability of crown ether-metal com-plexes and the ionic radius of the cation [59], and insolution the stability series for 18-Crown-6 bound to thealkali metals is:

Li � Na � K �� Rb � Cs

the critical slope method. The laboratory frame collisionfferent masses and degrees of freedom of the various complexesnce complex to which the correction factors are compared is

Monensin

ELab (V) � � ED (V)

53 0.88 0.88 4158 0.86 0.88 4463 0.85 0.88 4750 0.79 0.88 3534 0.75 0.88 22

byor diefere

(V)

4735

In the previous paper, where we investigated the

788 FORBES ET AL. J Am Soc Mass Spectrom 2005, 16, 779–791

Group I cation affinities for lasalocid and monensin,the appearance energies of the free metals werecompared with respect to ionic radius revealing anegative correlation with increasing ionic radius as aresult of polarizability effects. A similar comparisonhas been made in Figure 6 to investigate the relation-ship between cation size and the stability of thecomplex.

It is clear from Figure 6 that there is a differenttrend in the stabilities of the metal complexes thanthe linear trend that was observed for the metalaffinities measured previously. Maximum stabilitieswere determined for the K� complexes of both Lasand Mon and there appears to be a positive linearrelationship of increasing stability with cation sizethrough Li�, Na�, and K�. A sharp decline in stabil-ity occurs for both ionophores when the ionic radiusreaches a threshold between K� and Rb�, at whichpoint there appears to be an inverse dependence.These observations point to a phenomenon that canbe explained by the degree of coordination that theionophore is able to accomplish with cations ofvarying size. When the first shell coordination geom-etry most closely matches the conformation of the Oatoms participating in coordination with the antibi-otic, the most stable species is obtained. The sameeffect of cationic size, therefore, can be attributed tothe antibiotic complexes as is known to exist forcrown ethers.

It would also appear that the sharp decrease inthreshold stability of the Rb� and Cs� points to alesser degree of coordination interaction between theionophore and the larger metals. It is not difficult toimagine that beyond a particular size, the ionophoreis no longer able to “encapsulate” the metal but must

Figure 6. Plots of the stabilities of the Group Isquare) monensin versus cationic radius [60]. T

mass differences (m) and vibrational degrees of freed

take on a less favorable geometry, dominated primar-ily by electrostatic interaction. In addition, it may alsobe the case that the hydrogen bonding in the “head-to-tail” conformation of the ionophore is interruptedbeyond a critical diameter of the metal. However, it isfound that this hypothesis is not supported uponexamination of calculated structures. Juillard et al.[11] discuss detailed in vacuo structures of metalcomplexes of lasalocid obtained by semi-empiricalcalculations, indicating that the cyclic structure re-mains intact by virtue of the fact that the inter-nuclear OOH hydrogen bonding distance betweenthe head and the tail remained approximately thesame for complexes of Li�, Na�, K�, Rb�, and Cs�.

The MS2 spectra of the progressively larger metalcomplexes that we observed, however, do not sup-port this conclusion. It would appear that somechange in molecular conformation takes place if weaccept the differences in the threshold stability ener-gies and must deviate from the ideal cyclic confor-mation for larger metals. Further support for thistheory may be taken from the decrease in the relativeabundance of the F-type ions observed in the production spectra as the metal size increases. Returning toFigure 1, the Las·Na� and Las·K� complexes give riseto substantial signals for both A- and F-type ions,whereas Las·Rb� shows a much lower abundance ofthe F-ion with the A-ion being absent altogether.Examination of the Las·Cs� product ion spectrumshows the abundance of the F-fragment further de-pressed to �2% of the total abundance. If the gasphase complexes of the ionophores all have similarconformations and if all tend to coordinate the dif-ferent metals to the same extent, as is suggested bythe calculations, it would seem reasonable to expect

l complexes of (filled circle) lasalocid and (filledold dissociation voltages (E ) are corrected for

metahresh

D

om (�).

789J Am Soc Mass Spectrom 2005, 16, 779–791 GAS PHASE STABILITIES OF POLYETHER IONOPHORES

that similar fragmentation pathways at similarthreshold energies would be observed. In contrast,we suggest that the degree of coordination is inter-rupted by the large size of Rb� and Cs�, leading to adecrease in the gas phase stability.

One further piece of evidence supporting thishypothesis can be found in the convergence of thethreshold dissociation energies for the Rb� and Cs�

complexes of lasalocid and monensin. Although thelaboratory dissociation energies appear to be quitedifferent, after applying correction factors for mass(�) and degrees of freedom (�), the normalized ED

values are virtually identical for Las·Rb� andMon·Rb� and extremely close for Las·Cs� andMon·Cs�. It would appear that the comparativelyhigher stability of the monensin complexes (resultingfrom better coordination geometries than are avail-able to the lasalocid complexes) is diminished whenthe degree of coordination is reduced by the cationsize. In effect, the remaining binding capability islargely electrostatic in nature and is unaided by theadvantages of increased coordination.

Conclusions

The final stability trends for the Group I metals aregiven in Table 3. The marked decrease in the stabili-ties of the Rb and Cs complexes is believed to beattributable to the marginal degree of coordination ofthe ionophore to the metal cation. Further studies arecurrently underway to extend the stability measure-ments for Group II cations and some transition metalsto explore the interaction between charge density,cationic radius, and coordination geometry. The crit-ical slope method developed here to extract thresholddissociation energies from disappearance curvesshould be applicable to a wide range of metal-ligandsystems regardless of the dissociation pathwaysavailable to the system being studied. In particular,we plan to continue our investigations of some of theother commonly available ionophoric antibiotics.

AcknowledgmentsThe authors acknowledge financial support from the NationalSciences and Engineering Research Council of Canada (NSERC)and MDS Sciex. They also thank Dr. Peter Wentzell (DalhousieUniversity, Halifax) for help with developing algorithms to per-form digital filtering using Matlab.

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Table 3. Stability trends for the Group I metal complexes oflasalocid and monensin

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