Chemical standards for ion mobility spectrometry: a review

REVIEW

Chemical standards for ion mobility spectrometry: a review

Gushinder Kaur-Atwal & Gavin O’Connor &

Alexander A. Aksenov & Victor Bocos-Bintintan &

C. L. Paul Thomas & Colin S. Creaser

Received: 29 January 2009 /Revised: 7 May 2009 /Accepted: 8 May 2009 /Published online: 28 May 2009# Springer-Verlag 2009

Abstract Ion mobility spectrometry (IMS), using stand-alone instrumentation and hyphenated with mass spectrom-etry (IM-MS), has recently undergone significant expansionin the numbers of users and applications, particularly insectors outside its established user base; predominantlymilitary and security applications. Although several IMSreference standards have been proposed, there are nocurrently universally recognised reference standards forthe calibration and evaluation of mobility spectrometers.This review describes current practices and the literature onchemical standards for validating IMS systems in positiveand negative ion modes. The key qualities and requirementsan ‘ideal’ reference standard must possess are defined,together with the instrumental and environmental factorssuch as temperature, electric field, humidity and drift gascomposition that may need to be considered. Importantchallenges that have yet to be resolved are also identifiedand proposals for future development presented.

Keywords Ionmobility spectrometry . Reference standards .

Calibration . Standardisation . Calibrants

Introduction

Ion mobility spectrometry (IMS) has undergone a series ofdevelopments in recent years that have enabled the techniqueto be applied in new research and application areas. Indeedthe utility of IMS has expanded well beyond its establishedrole for detecting explosives, narcotics and chemical warfareagents (CWAs). Recent applications include the analysis ofpharmaceutical actives and formulations, and structural andconformational studies in biological and biomedical research[1–4]. Many of these developments arise from the hyphen-ation of IMS with mass spectrometry (IM-MS) [5].

In its simplest form, the drift velocity (vd, m s−1) of an ion ina drift tube (DT-IMS) under the influence of a uniform electricfield gradient is proportional to the strength of the electricfield gradient (E, V cm−1) with the proportionality coefficientbeing ion mobility (K, cm2 s−1 V−1), defined by Eq. (1):

K ¼ vdE

¼ d=tdV=d

¼ d2

tdVð1Þ

Where td is the drift time, the time taken for ions totraverse the distance d (drift length), between the ion shuttergrid and the detector, vd is the ion velocity and V is thepotential difference applied to the drift tube. This relation-ship holds under low field conditions (≤ 2 Td, where 1 Td=10−17V cm2). The relationship between ion size, charge andmobility is described by the Mason-Schamp equation, asimplified form of which is given in Eq. 2:

K ¼ 3ez

16N

� �2pmkT

� �1=2 1

ΩDð2Þ

Where e is the electronic charge (1.602×10−19 C) and zis the charge on the ion, N is the number density of the driftgas, µ is the reduced mass of the ion and drift gas, k is the

Int. J. Ion Mobil. Spec. (2009) 12:1–14DOI 10.1007/s12127-009-0021-1

G. Kaur-Atwal :V. Bocos-Bintintan :C. L. Paul Thomas :C. S. Creaser (*)Centre for Analytical Science, Department of Chemistry,Loughborough University,Leicestershire LE11 3TU, UKe-mail: [email protected]

G. O’Connor :A. A. AksenovLGC,Queens Road,Teddington, Middlesex TW11 0LY, UK

Boltzmann constant (1.381×10−23J K−1), T is the tempera-ture of the drift gas and ΩD is the collision cross section (i.e.size and shape) of the ion. The units of the Mason-Schampequation are nominally C s kg−1, equivalent to m2 s−1 V−1.Provided the conditions within the drift cell, such astemperature and buffer gas pressure are kept constant, themobility of an ion is dependent on reduced mass, chargestate and collision cross section.

It is common practice for comparison purposes and tocorrect for instrumental variations, to express ion mobilityas reduced mobility (K0), normalised to standard conditionsof temperature (T in Kelvin) and pressure (P in Torr) of thedrift gas.

K0 ¼ K273

T

� �P

760

� �ð3Þ

One of the key challenges in IMS is to measure themobility of an ion accurately. Precision and resolution arelimited by uncertainties in measuring the exact distancebetween the ion grid and detector (drift tube length), thetemperature, pressure and composition of the drift gas, andambiguity in timings such as ion injection and ion drift times.These uncertainties arise as a result of variations in instrumentdesign, effects of temperature (causing thermal expansion orcontraction of the drift tube) and pressure within the drift tube.Maintaining an accurately defined homogeneous electricalfield is also non-trivial, and inhomogeneity in the electric fieldstrength results in variations in ion drift times [6]. Thus,determining absolute ion mobilities using Eq. 1 may beproblematic. This practical problem may be addressed by theadoption of mobility standards, where a compound of knownmobility K0ref (Eq. 4) is used to calibrate the drift tube and todetermine the reduced mobilities of other analytes.

K0ref ¼ A:d2

tdV

273

T

� �P

760

� �ð4Þ

The correction factor A is usually incorporated with thedrift length, d, and field defining voltage, V to give the cellconstant, C, which is determined experimentally using theobserved drift time of the mobility standard, Eq. 5.

C ¼ K0ref td273

T

� �P

760

� �ð5Þ

The reduced mobility of an unknown may be calculatedusing the cell constant or from the ratio of the drift times ofthe reference standard and the unknown compounds, Eq. 6.

K0u ¼ K0ref �tdreftdu

ð6Þ

Where K0u is the reduced mobility of the unknown, tdrefis the drift time of the reference standard and tdu is the drifttime of an unknown compound.

In addition to low-field IMS, differential mobilityspectrometry (DMS), also known as field asymmetric ionmobility spectrometry (FAIMS), presents further challengesfor the determination of the relative low field and high fieldmobilities involved with this ion mobility variant. These arealso sensitive to changes to the parameters discussed above,particularly with respect to the drift gas composition.

Several IMS reference standards have been proposed fordetermining cell constants, reduced mobilities and collisioncross section for specific applications. For example,nicotinamide [7] for narcotics analysis, and dipropyleneglycol monomethyl ether (DPGME) [8] and methylsalicy-late [9] for field testing and verification of chemical agentmonitors, as simulants for nerve and blister agentsrespectively. Nevertheless, the adoption of internationallyagreed standards and standardisation procedures for agrowing range of IMS applications and techniques is at alow level, particularly within proteomic and pharmaceuticalanalysis. Indeed there are no internationally acceptedreference standards for the calibration and evaluation ofmobility spectrometers (IMS or DMS/FAIMS systems). Aset of IMS reference standards would allow traceableinstrument calibration, and ensure that the validations ofion mobility spectrometry experiments could be undertakenin a practicable manner.

The widespread adoption of standards would also facilitateand support inter-laboratory exchange of mobility dataobtained from different IMS systems generated by differentoperators and laboratories. The routine use of referencestandards in ion mobility spectrometers would aid thevalidation of IMS methods, reduce ‘false positive’ results,increase the certainty of analyte assignments and lead toimprovements in repeatability, reproducibility and robustnessof IMS measurements, enabling cross examination/inter-comparison of results obtained using a diverse range of IMSsystems and between different users and laboratories. Fur-thermore, IMS standards incorporated into quantitativemobility measurements would enable reliable spectral align-ment; most important when combining very large data setssuch as those generated within clinical and environmentalstudies.

Current status of standardisation in IMS

Several standards, listed in Table 1, have been reported forion mobility-based applications and to test mobilityspectrometers. The majority of the standards listed inTable 1 are readily available from most chemical cata-logues, with the exception of TNT and DMMP, which maylimit their application as reference standards. It is perhapshelpful to categorise mobility standards in terms of drift cellpressure and ion mode. Ambient pressure systems in the

2 Int. J. Ion Mobil. Spec. (2009) 12:1–14

Table 1 Examples of IMS reference standards/calibrants currently being used for positive and negative ion mobility mode (*Denotes a reagentgas internal calibrant, K0 values obtained in air (A) or nitrogen (N))

Compound Chemical Structure Mol. Weight

LiteratureReduced Ion

Mobilities(cm2 V-1 s-1)

Ref.

Positive Ion

MobilityMode

Hydrated proton ion (H2O)nH+

(n=1 to 8) 19.02 –145.12

1.95-2.70 (A)

(37-250 °C) [11, 13]

Lutidine (2,4-dimethylpyridine) (2,4-DMP) 107.15

1.95 (A&N), 1.82-2.05

(78-250 °C)(A) and1.43 (A&N) for (DMP)2H

+

[6],[14,17, 21]

Nicotinamide* 122.12 1.85 (A) [7, 34-37, 39]

Dimethylmethylphosphonate

(DMMP)124.08

1.80-2.08 (50-250 °C)((A)

1.40 for (DMMP)2H

+ (A)

[6, 8]

Dipropylene glycolmonomethyl ether

(DPGME)148.20 - [8, 27]

2,6-di-t-butyl pyridine(2,6-DtBP) 191.31 1.42 (37-250 °C)

(A&N)

[6, 26,30, 31],

[25]

Dibenzylamine (DBA) 197.28 1.407 (A) [40]

Trihexylamine (THA) 269.51 1.060 (A) [42]

Hexaphenylbenzene(HPB) 534.69 - [43]

Tetraalkylammoniumhalides

(C2–C8, C10 and C12)

130.25 - 691.31

1.88 (N), 1.56 (N),1.33 (N), 1.15(N),1.02 (N), 0.92(N),0.84(N), 0.73 (N),

and 0.67 (N)

[32], [33]N+

P OO

O

OHO

O

N

N NH2

O

N

NH

N

(CH2)5CH3

H3C(H2C)5 (CH2)5CH3

Fullerenes 720.64 - [4, 78,79]

NegativeIon

MobilityMode

4-Nitrobenzonitrile 148.12 - [46]

Methyl salicylate 152.15 1.62 (A) [9, 36,44, 45]

Trinitrotoluene (TNT) 227.31 1.45 (A) [24, 44]

Hexachloroethane* 236.74 2.21 & 1.83 (A)

(estimated fromFig. 2 in ref.)

[36]

Iodine* 253.81 - [48]

Dioctylphthalate 390.56 - [43]

I I

OH

O

O

Cl

Cl

Cl

Cl

Cl

Cl

NO2

NO2

O2N

NN+

O-

O

H17C8O

O O

OC8H17

Int. J. Ion Mobil. Spec. (2009) 12:1–14 3

positive mode are generally used for the detection of drugsof abuse (narcotics), nerve agents, and in some pharma-ceutical applications. Ambient pressure applications in thenegative ion mobility mode include the detection ofexplosives, acidic gases and blister agents. Lower pressuredrift tubes (50–100 Pa) are mostly associated with thehyphenation of ion mobility spectrometry to mass spec-trometry in proteomic, metabolomic and pharmaceuticalstudies [4, 5, 10].

Ambient pressure, positive mode

The reactant ion peak (RIP) has been used as an internalstandard, to which other IMS peaks are referenced [11, 12].An RIP is commonly observed with ionisation sources suchas 63Ni and corona discharge, and some researchers haveargued that using the RIP is the most convenient andefficient way of calibrating their IMS systems. Such anapproach was exemplified by Tabrizchi [11] who proposedusing [(H2O)2H]

+, as an internal reference standard.However, investigations by Eiceman and co-workers [6]concluded that an (H2O)2H

+ RIP was an unsuitablechemical standard, since the reduced mobility of this ionwas affected significantly by temperature (as shown inFig. 1). The presence of ammonia or other drift gas dopantsalso affects the RIP response and drift time. Further, thelandmark studies of Kebarle and co-workers described theequilibrium distributions of hydration states with changes inwater concentration as well, making the isolation of(H2O)2H

+ difficult [13].

The use of 2,4-dimethyl pyridine (2,4-DMP), commonlyreferred to as 2,4-lutidine or lutidine, was first proposed asa reference standard in the 1980s [14, 15] and it has sincebecome a widely adopted positive mode standard [16].Lutidine ions produce a single, sharp IMS peak with areduced mobility (K0) value of 1.95 cm2 V−1 s−1 (in air [15]and nitrogen [17]). The lutidine dimer peak can also beobserved with K0 of 1.43 cm2 V−1 s−1 [6, 18]. The mobilityof lutidine has been examined using a range of ionisationtechniques, including; 63Ni, electrospray ionisation [19],photoionisation [17] and matrix-assisted laser desorption/ionisation (MALDI) sources [20], all of which producedcomparable IMS responses. In the early investigations ofLubman [17] and Karpas [14], it was believed that themobility of lutidine was independent of drift cell temper-ature. However, recent observations indicate that the K0 oflutidine increases with temperature [6], see Fig. 1. Manygroups have used lutidine to calibrate their mobilityspectrometers and have compiled reference libraries ofreduced mobility values for a range of compoundsnormalised relative to the standard 2,4-DMP (lutidine)[17, 21, 22]. Thomas et al. [23] introduced a novel methodof measuring drift gas temperature through the use of 2,4-lutidine to determine the cell constant, and demonstratedreliable calibration of an IMS system. Kanu and Hill [24]recently reported the reduced mobility of lutidine in driftgases of differing polarisability. Consistent with otherstudies, the reduced mobility of 1.95±0.01 cm2 V−1 s−1

was obtained for the standard 2,4-lutidine in air andnitrogen. However, the K0 value of lutidine in carbondioxide and nitrous oxide was determined to be 1.20±0.02and 1.18±0.01 cm2 V−1 s−1, respectively. This decrease inmobility was observed as a consequence of the effect on theion collision cross section on increasing the polarisabilityand size of the drift gas. These findings demonstrate thatthe mobility of a reference compound cannot be transferredbetween different chemical regimes.

Eiceman et al. [6] examined the potential of 2,4-lutidine,dimethyl methylphosphonate (DMMP) and 2,6-di-tert-butylpyridine (2,6-DtBP) as chemical standards for IMS using63Ni ionisation. Results from this investigation indicatedthat, of the three compound tested, 2,6-DtBP would be ahighly suitable candidate for standardising reduced mobi-lities, given that its mobility is largely independent of theeffects of drift gas temperature, moisture and electric fieldstrength. DtBP has been used successfully as a chemicalstandard for the calibration of ion mobility spectrometersusing both electrospray ionisation [25] and 63Ni sources [6].DtBP has been reported to generate a well defined IMSpeak with a reduced mobility of 1.42 cm2 V−1 s−1, using air[6] and nitrogen [26] as the drift gas. The effect oftemperature on the calculated reduced mobilities of DMMP,lutidine and DtBP are shown in Fig. 1 (taken from Eiceman

Fig. 1 The effect of temperature on the AP-IMS reduced mobilities ofthe reactant ion peak (RIP, [(H2O)2H]

+), 2,4-lutidine ([2,4-DMP.H]+ andproton-bound dimer [(2,4-DMP)2H]

+), 2,6-di-tert-butyl pyridine ([2,6-DtBP.H]+ and [(2,6-DtBP)2H]

+) and dimethylmethyl phosphonate([DMMP.H]+ and [(DMMP)2H]

+) (reproduced from reference [6])

4 Int. J. Ion Mobil. Spec. (2009) 12:1–14

et al. [6]). These data suggest that the proton-bound dimers[(2,4-DMP)2H]

+ and [(DMMP)2H]+ may also have poten-

tial as IMS reference standards at temperatures below 78°Cfor lutidine and 180°C for DMMP, the temperature rangesat which these proton-bound dimers are stable. In contrast,an increase in reduced mobility was observed at hightemperatures for the protonated [2.4-DMP.H]+ from a K0 of1.82 to 2.05 cm2 V−1 s,

−1 within the temperature range of78 to 250°C, and for [DMMP.H]+, K0 increased from 1.80to 2.08 cm2 V−1 s−1 at 50 to 250°C (values estimated fromFig. 1).

In 2003, the US Environmental Protection Agencycontracted the National Homeland Security ResearchCentre (NHSRC), as part of the Environmental TechnologyVerification (ETV) Program [8], to assess the performanceof commercial portable ion mobility spectrometers for thedetection of toxic industrial chemicals, CWAs and simu-lants. This verification process involved using dipropyleneglycol monomethyl ether (DPGME) [27] and DMMP, toperform operational check tests, under a range of conditionsand practices that mimic the in-field and on-site use of theseportable IMS instruments. Such verification procedureswere carried out by the United Nations Chemical WeaponsConvention (CWC), to monitor mobility spectrometers forthe detection of nerve agents, in particular alkyl methyl-phosphonofluoridates and related chemicals. In this case,the proton bound DMMP dimer (a simulant for chemicalwarfare agents) was used as the reference standard, with areported K0 value of 1.40 cm2 V−1 s.

−1 The DMMP dimerwas found to be invariant of temperature from 20 to 220°Cand water concentrations of up to 2,500 ppm, making thiscompound ideal to test these ion mobility spectrometers. Theeffects of electric field and moisture on the reduced mobilitiesof lutidine, DMMP and DtBP were also explored and foundto be negligible [6]. The same three compounds were used asIMS chemical standards to determine the effect of thereagent gas nitric oxide on reduced mobility [28].

The radical cation [M]+· of DtBP was observed whencompatibility of DtBP calibrant compound with an atmo-spheric pressure photoionisation (APPI) source was inves-tigated [29]. The DtBP radical cation, though of lower mass,drifted more slowly in ion mobility drift tube compared tothe protonated species, possibly due to a change in spatialconformation. Formation of both types of ions leads to peakbroadening and splitting and, therefore, may limit thecompound’s compatibility with APPI.

The potential of employing DtBP as a referencecompound was further investigated by Viitanen et al. [30,31]. In this study, the effective drift tube lengths of ionmobility spectrometers were determined using 2,6-DtBP, toenable inter-comparison between IMS devices by adjustmentof mobility scales. This method of mobility alignment notonly allowed comparison of different instrumentation, with

varying drift tube lengths, but also suggested that DtBPwould be highly suitable to be used as a reference compoundin conjunction with in-field mobility spectrometers.

Another group of promising candidate standardisationcompounds for IMS are the tetraalkylammonium halides(TAAH), which have been examined as chemical standardsfor electrospray ionisation with ion mobility spectrometryby two groups [32, 33]. Viidanoja demonstrated that thereduced mobilities of the C2–C8, C10 and C12 (m/z range130.0–690.8) TAAH were independent of temperature, driftfield and chemical conditions within the drift tube, makingthem promising candidates as IMS reference standards. Anadditional feature of TAAH is the low tendency to formcluster ions with ESI solvent molecules and water mole-cules in the drift gas, due to the steric hindrance effects ofthe alkyl groups around the quaternary nitrogen. Clusterions can often complicate IMS spectra, as several peaksmay be observed for each compound, whereas with theTAAH a single, sharp IMS peak is generated for eachmember of the homologous series, covering a broad massrange, see Fig. 2. Other advantages of these compoundsinclude low toxicity, ready availability and low cost.

The use of nicotinamide has been reported widely as apositive ion mode internal calibrant in commerciallyavailable IMS instruments [7, 34–37]. The selection of thenicotinamide was based primarily on its availability andease of ionization. The IMS behaviour of nicotinamide isknown to be non-ideal, as K0 value has slight dependenceon T, P and drift gas composition. However, the largeamount of IMS data accumulated for these compoundsenables compensation for K0 deviations via softwarealgorithms. In this approach, the K0 values obtained forcalibrants are always set at their tabulated values and themeasurement scale for the instrument is adjusted accord-ingly. The obvious drawback of this approach is non-transferability of the calibration data in between instruments

Fig. 2 ESI-IM-MS mass-selected ion mobility responses of aliphaticC2–C8, C10 and C12 tetraalkylammonium salts at m/z 130.0, 186.1,242.1, 298.4, 354.5, 410.4, 466.5, 578.7 and 690.8 with reducedmobilities of 1.88, 1.56, 1.33, 1.15, 1.02, 0.92, 0.84, 0.73, and 0.67respectively (reproduced from reference [32])

Int. J. Ion Mobil. Spec. (2009) 12:1–14 5

from different manufacturers, as the IMS behaviour can beinstrument-dependant. Nicotinamide has been employedsuccessfully not only as a reference standard, but also as adrift gas dopant for enhanced detection of analyte ions viaproton transfer reactions. Bota and Harrington reported theuse of nicotinamide with a reduced mobility of 1.85 cm2

V−1 s−1 [14] for the direct detection and quantification oftrimethylamine in meat food products using IMS [34].Nicotinamide has been used as an internal calibrant for theanalysis of drugs of abuse including cocaine, heroin andLSD [7]. Vinopal et al. [38] also employed nicotinamide asan internal reference standard in a study to fingerprint anddifferentiate bacterial strains.

Wang et al. [39] constructed a solid-phase microextraction/surface enhanced laser desorption/ionisation-ion mobilityspectrometer (SPME/SELDI-IMS), which was programmedin the positive mode. Nicotinamide was preloaded into theIMS drift gas as the internal standard for the analysis ofdrugs and other non-volatile compounds from urinesamples. Studies by Keller, Miki et al. [40–42] demon-strated rapid screening and semi-quantitation of illicitdrugs (including methamphetamine and ecstasy) directlyfrom human hair and clothing for forensic applicationsusing IMS. In this work, nicotinamide was also introducedinto the drift cell as a calibrant and reactant gas for IMS.In addition, dibenzylamine (DBA) (K0 1.407 cm2 V−1 s−1)and trihexylamine (THA) (K0 1.060 cm2 V−1 s−1) wereemployed as internal standards for these specific applica-tions. A proposed approach for environmental screeningfor organic contamination described hexaphenylbenzene(HPB) as a mobility reference for the positive ion mobilitymode [43].

Ambient pressure, negative mode

A list of calibrants used for negative ion mobility mode isalso presented in Table 1. IMS is most commonly employedin negative ion mode for the detection of explosives andCWAs. In contrast to the range of reference standards usedfor positive IMS mode, few standards exist for negative ionmobility mode.

Methyl salicylate, a CWA simulant is the most frequent-ly used internal calibrant for the detection of negative ionsincluding explosives [9, 36, 44, 45]. In a study by Patchettet al. [36], methyl salicylate was used as an internalcalibrant for the negative ion mode detection of the drug-of-abuse, γ-hydroxybutyrate (GHB), with a hexachloroeth-ane dopant. The authors reported the reduced mobility ofmethyl salicylate to be 1.62 cm2 V−1 s−1 and used this todetermine the relative reduced mobilities of GHB (usingEq. 6). Buxton and Harrington [44] determined the reducedmobilities of pentaerythritol tetranitrate (PETN) and cyclo-tetramethylene tetranitrate (HMX) relative to methyl salic-

ylate in negative ion mode. The K0 of the methyl salicylateion was calculated (using Eq. 6) by running a standardsample of trinitrotoluene (TNT), and comparing theexperimental reduced mobility to the literature K0 valuefor TNT (1.54 cm2 V−1 s−1).

Vinopal et al. [38] examined the potential of IMS innegative ion mode to differentiate between bacterial stains bythe direct analysis of whole cells using 4-nitrobenzonitrile asthe internal mobility standard. 4-nitrobenzonitrile has alsobeen used with hexachloroethane as the dopant, for thedetection of explosives [46], gunpowder stabilisers [47], andto correct for the operating pressure and temperatureconditions of an ion mobility spectrometer [35]. In addition,a calibration procedure was also carried out using 4-nitrobenzonitrile as an internal calibrant and TNT as theexternal calibrant in the negative mode. This two stepcalibration process enabled K0 for the internal calibrant tobe set so that the observed reduced mobility of the externalcalibrant TNT was adjusted to the defined/known K0

literature value of 1.45 cm2 V−1 s−1. [38].An alternative reference standard for negative ion

mobility mode is iodine, introduced as an internal standardin the sample mixture. Iodine has been used successfully asan internal calibrant for the identification and detection oftrace amounts of toxic compounds such as chemical warfareagents, narcotics and explosives [48]. Dioctylphthalate anddi(ethyl-hexyl)phthalate) have also been proposed asnegative ion mode standards for use in environmentalscreening applications; alongside hexaphenylbenzene(HPB) in the positive ion mode (see above) [43].

Testing and validating commercial, securityand military systems

One of the most valuable attributes of stand-alone IMSdevices is their portable and robust nature, a strong reasonfor their widespread adoption for in-field and on-siteoperations. The use of reference standards and theirassociated procedures under such conditions must becompatible with the demanding requirements of the securityand military user [1, 18, 24, 49]. The detection of chemicalwarfare agents, explosives and illicit drugs typicallyinvolves demanding and variable operating environments.Simple-to-use and fast procedures with straightforward datainterpretation are priority specifications for applicationssuch as these. It is essential that IMS devices are accurateand precise over a range of operating environments and thatfalse positive results are minimised without imparting therisk of delivering false negatives.

Other key aspects that must be implemented in thissector are traceable protocols for verifying claims ofenhanced detection capability; increased sensitivity, fasterclear-down of contamination, or faster sampling. The

6 Int. J. Ion Mobil. Spec. (2009) 12:1–14

adoption of “best-practice guides” for IMS users, with theimplementation of protocols on optimising methods wouldallow cross referencing of results and assure high qualitydata.

The result of these operational imperatives is thatcommercial, security and military instrumental applica-tions have led the way in implementing standardisation.For example, nicotinamide and 4-nitrobenzonitrile areembedded internal calibrants in some commercial IMSsystems, used in combination with test and verificationkits based on “lipsticks” impregnated with analytes,enabling the sampling, thermal desorption, and ionmobility operations to be tested. Chemical agent detectorsuse hand held “confidence-testers” containing vapoursources of DPGME and methyl salicylate for in fieldvalidation. Instrument performance in microfabricatedDMS/FAIMS devices is characterised with dimethylmethylphosphonate (DMMP), diisopropylmethylphospho-nate (DIMP) and methyl salicylate [9] or by monitoringthe RIP to test instrument operation.

Quantitative evaluation

The National Institute of Standards and Technology(NIST), part of the federal agency within the U.S.Department of Commerce [50], are currently developingmethods and referencing material standards to calibrate ionmobility spectrometers for the reliable detection of trace-levels of explosives and chemical warfare agents. NIST’sapproach uses piezoelectric inkjet technology to generatefieldable standards for evaluating the performance of IMSsystems in terms of quantitative accuracy and to assess falsepositive and false negative detection rates. Piezoelectricinkjet technology is a reproducible method that allows thepreparation and printing of a range of explosive calibrationstandards. These include TNT, cyclotrimethylene trinitr-amine (also known as RDX) and PETN, as well as plasticexplosives C4, semtex and detasheet [51]. These explosiveswere used to construct calibration curves from IMSresponses at varying concentration levels and samplingtimes.

Concentration relationships and the quantitative evalua-tion of response curves at ambient pressure have a widersignificance than the straightforward characterisation of ananalytical response in ambient pressure ion mobilityspectrometry. The ion mobility spectrum is the result ofinteracting kinetic and thermodynamic processes within thereaction region, and ultimately within the drift tube itself.The relationships between concentration, residence timeand temperature control the distributions of the cluster ionsseen in the mobility spectrum; the exact nature of whichdepends upon the nature of the reactant ion chemistry.Equation 7 shows the formation of an ion cluster whereby

one or more water molecules are displaced from the reactantion cluster by a molecule of the neutral standard (M).

N2ð Þn H2Oð ÞmHþ þM Ð N2ð Þ n�ið Þ H2Oð Þ m�jð ÞMHþ

þ i N2 þ j H2O

ð7Þ

Increasing the concentration of the neutral standard and,or, its residence time in the reaction region will increase thetendency for the protonated monomer ion to go on to forma protonated dimer Eq. 8. Indeed the possibility exists tocontinue to react to form trimers and potentially largercluster ions.

N2ð Þ n�ið Þ H2Oð Þ m�jð ÞMHþ þM Ð N2ð Þ n� iþkð Þð Þ H2Oð Þ m� jþið Þð Þ

M2Hþ þ kN2þ i H2O

ð8Þ

The thermodynamic and kinetic elements underlyinghomogeneous and heterogeneous proton-bound ion clustershave been described [52, 53] and what emerges from thesestudies is the sensitivity of the ion mobility spectrum tochanges in the critical factors of concentration, temperatureand residence time in the reaction region, making the use ofconcentration relationships in the formation of ion clustersan attractive approach in specifying ion mobility standards.

The generation of calibration/standard curves isemployed not only within military and security applica-tions, but is often used for quantitative studies of otheranalytes. For example standard solutions of caffeineanalysed using LC/AP-IMS have been used for perfor-mance evaluation [54], the determination of atrazineconcentration in natural water [55] and quantification ofmorphine in mouse plasma. The analytical value of an IMSdevice in terms of true positive and false positiveprobabilities have been evaluated using a receiver operatorcharacteristic (ROC) curve approach for determining thedetection limit of an IMS chemical sensor [56].

Mobility standards for ion mobility-mass spectrometry

IMS coupled with mass spectrometry (IM-MS) is an area ofmajor growth and has opened up many new applications ofIMS, as a result of the complementary separation character-istics of the two techniques. IM-MS enables the identificationof unknowns through the determination of the mass-to-chargeratio as well as the collision cross section (size and shape) ofan ion. This offers a number of benefits such as simplificationof mass spectral data, separation of isomers and conformers,reduction in spectral noise and chemical interferences, charge-state separation and structure elucidation [57–63]. Theevolution of IM-MS and applications of the technique haverecently been reviewed by Hill et al. [5].

The commercial availability of IM-MS is relatively newand, consequently, there are lack of protocols and standards

Int. J. Ion Mobil. Spec. (2009) 12:1–14 7

for the independent validation of these instruments and in-house constructed spectrometers. When using such newtechnology, it is important that data are validated indepen-dently to allow cross examination of results and todemonstrate instrument performance, so that results willstand up to scientific and legal scrutiny. The WatersSynapt™ high definition (travelling-wave) ion mobility-quadrupole-time-of-flight mass spectrometer [64] and theThermoFinnigan FAIMS interface for the ion trap and triplequadrupole mass spectrometers, based on the concentriccylinder design by Guevremont et al. [65–67], are examplesof IM-MS that have recently been commercialised. TheFAIMS interface is used to provide an extra degree ofspecificity, by allowing transmission of ions of selecteddifferential mobility only, prior to MS and tandem massspectrometry (MS/MS). The ion selection is based ondifferences in the mobility of an ion under high and lowfield conditions. A constant DC voltage, called thecompensation voltage or CV, of the correct magnitude andpolarity needs to be applied to compensate for the iondisplacement resulting from the difference of Kh (K duringthe high-field cycle) and Kl (K during the low-field cycle),in order to enable the ions to travel between the DMS/FAIMS electrodes and be transmitted to the detectiondevice. Each ion will have its characteristic CV under acertain set of conditions. At present, performance evalua-tion of the FAIMS-MS instrumentation is carried out usinga range of compounds (for example; 1, 3, 6 polytyrosinemix, polyethylene glycols (PEG) and reserpine for positiveion mode, and taurocholic acid in the negative ion mobilitymode), selected mainly to establish whether the instru-ment’s performance/compensation voltages (CV) are ac-ceptable. The usual testing procedure involves spiking thecalibrant into the device and measuring its CV value; if noresponse or a reduced or shifted response is observed(calibrants are normally expected to exhibit CV valueswithin ± 1 V of their target range), this is indicative of aproblem in instrument performance. The FAIMS-MSinstrument may also be tested using the mass spectrometrycalibration mixture (tuning solution) consisting of ultra-mark, caffeine and the MRFA peptide for day-to-daychecks and to test the performance of the spectrometer.

The nature of the Waters Synapt™ Tri-wave IM-MSinstrument necessitates its calibration, as there is no ab initiotheoretical description which correlates drift time in thetravelling wave field to the ion’s size or other parameters.The instrument is calibrated either by introduction ofcaesium sodium iodide [68] to ensure mass accuracy, or viacorrelation with libraries of ion mobility data and literaturestandards. The latter approach seems to be the preferredmethod for calibration, most commonly using species ofproteomic origin [69–71]. The collision cross sections andion mobilities data of a wide range of proteins and peptides

are well documented, and therefore can be used to assess theinstrument performance by comparison of known valueswith experimental measurements [70, 72, 73]. So far,mobility data obtained using the Synapt IM-MS instrumenthas shown good correlation with data obtained from othermobility separators for a mixture of peptides standards(containing bradykinin, substance P, bombesin, LHRH, andangiotensin I and II), tryptic peptides (from haemoglobin,horse albumin etc.) and other large protein and biomolecularcomplexes including cytochrome c, lysozyme, α-lactalbuminand myoglobin [64]. Calibration plots of collision crosssection measurements and drift times of tryptic peptides andproteins have also been constructed to estimate collisioncross sections of compounds of interest, for example thecollision cross section of PEG was estimated using calibra-tion plots of published cross sections of tryptic peptide ionsobtained from human haemoglobin [73].

The proteins cytochrome c, ubiquitin and lysozymeenzyme have been proposed as mobility and collision crosssection standards [64, 72, 74]. Lysozyme enzyme is apromising candidate as the presence of di-sulfide bondsenhances the stability of the structure, making lysozymeless susceptible to conformational changes. Several con-formations of ubiquitin exist: compact, partially unfoldedand unfolded, all of which have been well studied [75, 76].The conformation of ubiquitin can be controlled undercertain solvent compositions, and it is known that ubiquitinin the +13 charge state is the most stable conformation,generating a single, resolved IMS peak.

Other examples of candidate compounds that have beenidentified as suitable for use as collisional cross sectionstandards are caesium iodide and sodiated PEG. Thesodiated PEG complexes have stable collision cross sections,particularly in the nonamer conformation [73, 77]; whichadopts a ball-like structure with the metal ion encapsulatedin the centre. Such compounds experience minimal inter-actions with the drift gas, as the carbon and oxygen atomsare present in the inner structure of the molecules.

For peptide and protein analysis, C60 and C70 fullereneshave also been used as internal standards in conjunctionwith MALDI-ion mobility-time-of-flight mass spectrometry(MALDI-IM-TOF-MS). C60/C70 were co-deposited withthe peptide mixture onto the MALDI target plate [4, 78, 79]for ion mobility separation prior to mass analysis. Thefullerenes displayed different mass-mobility distributions tothat observed for peptide ions, and the drift times of thecompact fullerenes were lower than peptide ions of thesame mass, as indicated by Fig. 3. Peptide ions couldtherefore be easily differentiated from the calibrantresponses. In addition, due to the rigid and stable structuralcharacteristics of fullerenes, these molecules possess con-stant collision cross sections which tend to be independentof factors such as temperature [77].

8 Int. J. Ion Mobil. Spec. (2009) 12:1–14

Sysoev et al. [80] used a standard test mixture consisting oflutidine, 2,6-DtBP and tetrabutylammonium iodide, to test thepotential of electrospray ionisation coupled to IM-MSincorporating an atmospheric pressure drift tube. The instru-ment was used for the rapid screening of complex samples,primarily for drug discovery and environmental screening.

IMS spectral libraries

Spectral libraries of ion mobility data have often been usedas references to calibrate ion mobility spectrometers. Anumber of groups have established libraries of drift times,collision cross sections, and reduced mobilities for selectedanalytes under specified conditions, all of which areaccessible to IMS users. Hill et al. [15] presented tablesof reduced ion mobilities for compounds reported duringthe period 1970–1985 using ambient pressure IMS.Additional information, such as K0 of product ions, thecarrier and drift gases used in each case and the temperatureof the drift tube were also defined in this review. Karpas[14] compiled the reduced mobilities (relative to thereference standard lutidine) of aliphatic and aromaticamines (molecular weight range 32–521) in air, at 150°C,200°C and 250°C. More recently, Eiceman and Karpas [22]provided an extensive spectral library (supplied on a CD-ROM with reference [22]) of drift times and reducedmobilities for a range of compounds, including alcohols,amines, organo-phosphates and pyridines. In some cases,the reduced mobilities of these compounds were calculatedrelative to 2,4-lutidine. In a study by Matz et al. [81],reduced mobilities were tabulated for cocaine and itsmetabolites, amphetamines, benzodiazepines, and smallpeptides in four different drift gases (helium, argon,

nitrogen and carbon dioxide). The reduced mobilities of arange of compounds including lutidine, crown ethers, aminoacids and peptides were reported by Bramwell et al. usingnano-ESI, and reduced mobilities compared with valuesobtained by APCI (63Ni) [19]. In order to facilitate thecreation of libraries of IMS spectral data, the IUPACJCAMP-DX electronic data exchange format was adapted[82, 83]. This was developed to allow exchange of IMSspectral data, inter-comparison between IMS users, labora-tories and systems, as well as validation of new ion mobilityspectrometers. The data exchange software can be down-loaded from the IUPAC JCAMP-DX website [84] or via theInstitute for Analytical Sciences (ISAS) [85] website.

In the field of biomolecule analysis, a database ofcollision cross section measurements (in helium) of proteinsand peptides, including ubiquitin [86], cytochrome c [71],polyaminoacids, and tryptic peptides from common pro-teins such as haemoglobin and bovine serum albumin areavailable on the Indiana University (Clemmer group)website [69, 70]. A database of cross sections and reducedmobilities for a range of singly-charged proteolytic peptideions is also available [87, 88]. It is common practice toexpress mobility data of proteins and peptides as collisioncross section measurements, as proteomic analysis usingIMS often involves structure elucidation and conformationstudies. Purves et al. compared collision cross sectionmeasurements of bovine ubiquitin obtained in nitrogenusing the FAIMS instrumentation [76], with literaturevalues in helium reported by Clemmer [89] using drifttube-IMS as a means of instrument calibration. Theseresults indicated a good correlation between collision crosssection measurements, as similar conformation and chargestates were observed on both IMS systems. This work alsodemonstrated the potential of using ubiquitin (particularlyin the higher charge state) as a collision cross sectionstandard for protein measurements.

Bowers and Jarrold have developed computer programssuch as the Sigma program [77, 90, 91], which employs theprojection approximation algorithm, and Mobcal [92, 93],based on trajectory and exact hard sphere scatteringcalculations. These programs are available to determinemobilities and theoretical collision cross sections of ions,thus allowing comparison between experimental cross sectionmeasurements and modelling for structure elucidation.

Selection of ion mobility standards

UK national measurement system initiative in ion mobilityreference materials

The Chemical and Biological Metrology programme, partof the UK National Measurement System (NMS), aims to

Fig. 3 Plot of drift time vs. m/z for six peptides and the fullerenemolecules C60 (m/z=720.6) and C70 (m/z=840.7), which were used asinternal calibrants for MALDI-IM-TOF MS analysis (modified fromreference [78])

Int. J. Ion Mobil. Spec. (2009) 12:1–14 9

provide the necessary metrology infrastructure for theestablishment of validated protocols and standards forcurrent and emerging uses of IMS [94]. The first objectiveof this initiative is the investigation of the feasibility ofproducing a set of IMS reference materials and protocolsfor an extended application base to allow harmonisationand traceability of IMS data. Currently, IMS users andmanufacturers are being consulted about their require-ments for standards. Further studies are also investigatingpossible causes of interferences in the use of IMSstandards, with the intent of improving confidence inanalyte identification and the reliability of IMS results.Candidate compounds (of m/z range 102–105) for use intest and calibration mixes to enable evaluation ofinstrumental performance and mobility and cross-sectiondetermination, as well as quantitation accuracy, are beingdeveloped.

Specifications for ion mobility reference materials

Ion mobility measurements are affected by experimentalconditions (for example ionic charge, drift gas composition,temperature, pressure and humidity), and instrumentationperformance (factors include electric field stability andhomogeneity, timing accuracy and digital signal process-ing). Therefore, standards are required to enable nominallyfixed instrumentation factors to be isolated from variableexperimental factors for instrumental performance evalua-tion and the determination of reduced mobility/collisioncross section. Mobility reference materials for assessinginstrumentation should be insensitive to changes in exper-imental parameters, while complementary standards that arehighly sensitive to changes in experimental conditionsenable the operational parameters and state of a system tobe assessed. The selection criteria for candidate ionmobility reference standards encompass five criteria (someof these specifications are in opposition to each other);therefore the standards should possess the followingcharacteristics:

Stability

The mobility of ions should ideally be resilient toconditions within the drift cell such as, temperature,pressure, electric field strength, and should be reproduciblein a range of drift gas compositions, including increasingpolarisability (He, N2, CO2, SF6). Special attention isneeded for hyphenated systems where the effect of injectionenergy of ions into a low-pressure drift cell needs to becharacterised, as the formation of excessively energetic,metastable ions would lead to fragmentation of clusters,resulting in changes in the collision cross section andobserved mobility.

Concentration

The concentrations of reference standards need to bespecified carefully to ensure that monomer dimer, andpotentially other cluster ion relationships, are defined andexploited. The information on the mobility of the monomerand dimer ions are augmented with data on their relativeintensities which provides useful information on tempera-ture and gas flows within the instrument.

Sensitivity to change

Responsive to changes in temperature, pressure, electricalfield strength and drift gas in a defined and reproducibleway. The levels of water in the drift gas may also influencedrift times and differential mobility behaviour. Increase inhumidity levels of the drift gas can result in changes in theion chemistry (via cluster reactions between waters mole-cules and sample ions, and hydrolysis), which can oftenlead to the formation of additional ion species that maycomplicate the spectra. IMS peak broadening effects andshifts in reduced ion mobilities are also frequently observed[95, 96]. This is an especially important factor for portableIMS systems that are used in-field.

Practicality

Inexpensive, readily available in sufficient purity, with lowtoxicity, and stable under normal storage conditions forextended periods of time.

Relevance to the target analyte

Reference standards should reflect the chemical nature ofthe target analyte. The standard’s characteristics should becomparable to the analyte’s volatility, polarity, mass andconcentration.

Ion formation

Candidate reference standards should form well defined ionspecies with known reduced mobility and m/z (using IM-MS)values, and should display consistent and stable IMSresponses with little variation between users and instruments.

A number of ionisation techniques have been employedfor sample introduction to mobility spectrometers depend-ing on the nature of the analyte of interest. Compounds canbe divided into categories according to whether they aresufficiently volatile (generally small molecules, includingexplosives and CWAs) to be ionised using 63Ni, coronadischarge and photoionisation, or those that are amenable toelectrospray and matrix-assisted laser desorption/ionisation(proteins and peptides for example). Therefore, source

10 Int. J. Ion Mobil. Spec. (2009) 12:1–14

compatibility of potential candidates and parameters affect-ing ionisation (such as cone voltage, gas flow rate,temperature etc.) also need to be addressed. In addition,reference standards need to be developed for use in bothpositive and negative ion mode.

Preliminary findings

There is no single compound that meets these criteria for allapplication areas, IMS systems and ionisation techniques.Consequently groups of candidate reference standards need tobe assessed over a range of IMS systems (DMS, FAIMS,linear-IMS and low-pressure IMS), and in the presence ofvarious reagent gases andmodifiers with the intent of selectinga calibration mixture, or a set of reference compounds suitablefor use as universal IMS standards.

Candidate standards may be categorised into two maingroups: instrumental/performance evaluation referencematerials and standards for the determination of reducedmobility/collision cross section. The K0 and Ω of the lattertype of standards should be well defined, invariant ofexperimental parameters and insensitive to gas impuritiesand moisture content within the drift cell, the former shouldbehave in a predictable way to changes in experimental/instrumental conditions.

As indicated earlier, lutidine, DtBP and the tetraalky-lammonuim halides have already been proposed as IMScalibrants and appear to be highly suitable candidates asinstrumental and K0/Ω standards for low molecular weightanalytes. The growing interest in the use of IM-MS forproteomic analysis means that standards of high molecularweight and biological origin are also required. Lysozymeenzyme and ubiquitin in the +13 charge are attractivecandidates, whilst Group I metal PEG complexes meritfurther scrutiny. Similarly, the fullerenes are very promisingcandidates due to their rigid (stable) structural properties. Asdiscussed earlier, the fullerenes have already been employedas an internal calibrant (C60 and C70) for peptide/proteinanalysis.

An important additional consideration for mobilitystandards for proteomic analysis is the interaction betweendrift gases and the conformation of the ionised biopolymer.Such relationships have yet to be completely described andrequire further study. Furthermore, the distribution ofcharge and conformational/folding states of protein stand-ards may be influenced by the conditions in the ionisationsource, and the effects of pH and solvent compositions haveyet to be comprehensively characterised. Higher chargestates can be observed for some proteins in acidicconditions, as the proteins become denatured and morebasic protonation sites become available [71, 97]. Struc-tures can be stabilised by bound metal ions, so that thecollision cross section remains constant and resists changes

in conformation as a result of factors such as temperature,pressure and electric field.

In addition to providing a set of IMS reference standards,the production of best practice guides on the experimentaldesign and validation of protocols for ion mobility systemswould greatly benefit the community. The use of IMS datamining software for peak clean up, measurement of peakshape (apex determination) and comparison of drift time toa reference standard would enhance the quality of ionmobility data. Such data processing is particularly impor-tant when combining very large data sets (for example inenvironmental and clinical analysis). The development ofcurrent databases and spectral libraries of mobility, m/z, andcollision cross section measurements (under a given set ofinstrumental and environmental conditions) for a range ofcompounds relevant to different application areas wouldalso facilitate calibration and evaluation of mobilityspectrometers.

Conclusions

The range of IMS applications is increasing beyondestablished military and security in-field monitoring roles.For example, the development of new methods andapplications of IMS in bioanalytical and pharmaceuticalscience, the technique seems certain to continue to expandoutside its established user base. Although the use ofseveral IMS reference standards have been reported for arange of IMS and IM-MS systems, including 2,4-lutidine,DtBP, nicotinamide, and TAAHs for positive ion mode and4-nitrobenzonitrile and methyl salicylate in the negative ionmobility mode, the current status of standardisation for thewider application base is at a low level and indeed there areno internationally accepted reference standards for thecalibration and evaluation of mobility spectrometers. TheUK NMS Chemical and Biological Metrology Programme isanticipating and addressing the need for the user communityto adopt the use of traceable standards to expedite the researchand development activity in ion mobility spectrometry.

This review outlines the current status and benefits ofstandardisation in IMS and identifies issues/challenges thatwould need to be addressed in the future. The keyrequirements/characteristics, in terms of instrumentalparameters and environmental conditions (such as temper-ature, electric field, humidity and drift gas composition)that an ‘ideal’ reference standard must possess to be used asa universal standard, have been discussed. There is also aneed to adopt standard testing protocols to facilitate datacomparison between different IMS systems and users. It isimportant to continue to compile databases and spectrallibraries of mass, mobility and collision cross sections for arange of compounds (under various instrumental and

Int. J. Ion Mobil. Spec. (2009) 12:1–14 11

environmental conditions), and should be accessible byIMS users and manufacturers. The ultimate goal of theNMS programme is to foster the adoption of widelyrecognised mobility standards across the IMS research anduser community enabling more comparable measurementson a global scale.

Acknowledgements The work described in this paper was sup-ported under contract with the UK Department for Innovation,Universities and Skills as part of the National Measurement SystemChemical and Biological Metrology programme. We thank JimKapron from ThermoFinnigan and Reno DeBono from SmithsDetection.

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