485 a 2009 John Wiley & Sons, Ltd. c Birthe Schubert, a and In contrast to biopolymer MS/MS, the c s, University Departments at the Vienna Biocenter, [17,18] [email protected] The kinetics of a single ion-molecule-reaction [19] Institute of Legal Medicine, Innsbruck Medical University, Innsbruck, Austria Center for Analytical Chemistry, DepartmentResources and IFA-Tulln, Applied Life University Sciences, of Vienna, Austria Natural Max F. Perutz Laboratorie Department of Biochemistry, University Vienna, Vienna, Austria Core Facility for MassUniversity of Graz, Spectrometry, Graz, Austria Center for Medical Research, Medical Correspondence to: Herbert Oberacher, InstituteMedical of University, Legal Muellerstrasse Medicine, 44, 6020 Innsbruck Innsbruck, Austria. E-mail: herbert.ober Edina Csaszar ion-energy values for fragmentation. In the course of the ∗ c a d b sonance mass spectrometer. In each participating laboratory, pectral library search approach is to a large extent platform mass spectral libraries. is an intrinsic,investigated system. generic, It is and important to thusset note, of however, experimental that transferable conditions a may given property only be applicable of to a limited the outcome of CID ofwell a known that small the moleculeion observed is is intensity controlled difficult of by to a kineticgas predict. (e.g. potential and type fragment It and is center-of-mass density(e.g. of energy) the specifics collision and ofof instrumental fragment the parameters ion appliedthe collection, ionization detector). and technique, mass discrimination efficiency effects of ferent laboratories, where 418 tandem mass spectra were b atching algorithm, which is presented in detail in a companion outinely applied workflows. No standardization procedure was adrupole-quadrupole-linear ion trap, quadrupole-quadrupole- MS spectra. The acquired tandem mass spectra were matched Kathrin Libiseller, mined. The library consisted of 3759 MS/MS spectra collected f a tandem mass spectral reference library originally built on a [6,7] proteins, [4,5] (MS/MS) summa- Biopolymers con- MS/MS-techniques [14–16] [9–11] [1–3] Marion Pavlic, ∗ a Rainer Schuhmacher, d 2009 John Wiley & Sons, Ltd. b c , 485–493 Copyright 44 , ¨ and oligonucleotides. ofeler precursor ion) and its dissociation to generate To a large extent fragment ion mass spectra are [8] = 2009 [12,13] tandem mass spectrometry; library; transferability product ion tandem mass spectrometry

Characterization of the structure of small molecules represents another important fieldextensively used for of target-specific analysis and applicationfor general to unknown a screening for lower procedures employing extent searchable MS/MS, which is predictable and can be used fornovo derivation database of search a as biopolymer’s well sequence. as for de are widely used for the fragmentationatmospheric of pressure ions ionization gathered (API) from methods. ‘soft’ Incases, collision-induced the dissociation majority (CID) of experiments are used to obtain structural information from ation precursor of ion. MS/MS-techniques The with computational combina- data interpretation routines represents a valuableidentification tool of for biopolymers the characterization such and as peptides, J. Mass. Spectrom. oligosaccharides, Introduction The term from 402 reference compounds applying several different collis quadrupole, and linear ion trap-Fourier transform ion cyclotron re rizes mass spectrometric methods concerned with the selectiona of particular ion ( The inter-instrument and inter-laboratory transferability o multicenter study Michael Sulyok, a quadrupole-quadrupole-time-of-flight instrument was exa multicenter study, 22acquired test using compounds fourmass were spectrometers: different quadrupole-quadrupole-time-of-flight, sent qu instruments to from three two dif optimized manufacturers. instrumental parameters were The gathered solely study from r covered the following types of tandem reference library: 1. Results of an Austrian Herbert Oberacher, transferability of a tandem mass spectral (www.interscience.com) DOI 10.1002/jms.1545 On the inter-instrument and inter-laboratory applied to increase theagainst inter-instrument the comparability established reference of library using MS/ apaper. sophisticated m Correct answers,For meaning the that remaining the 1.9%correct correct of assignments clearly compound spectra, suggests the was that correct retrieved the compound developed as was mass top s matched hit, at second were rank. obtained The in observed 98.1% high of percentage cases. of Research Article Received: 14 April 2008 Accepted: 16 November 2008 Published online in Wiley Interscience: 22 January 2009 independent. Copyright Keywords: sist of a limitedare number preferably of broken buildingsame blocks during fragment and CID the ions are bonds areirrespective well that obtained of known. from the Almost aexperiment. instrumental the certain platform precursor used ion for the MS/MS- characteristic secondary fragment ions. Harald C. K . et al , 485–493 44 , 2009 H. Oberacher g/ml. Samples were µ ratory, optimized instrumental second quadrupole). The third ass spectrometer. The QqLIT was = J. Mass. Spectrom. To verify that the set of test compounds was [32] [31] 0 amu from one of the test compounds’ masses were taken . 1 suited to qualifythe the library performance was of surveyed thewith for at least mass entries one test that spectral compound during were library, reference library compounds search likely with (Table a to 1). molecular mass All deviation interfere of± less than into account (Table 1).surveyed Furthermore, for the compounds databaseto entries that one were show of some theto structural groups test of compounds similarity pharmaceuticals (Tablemembers that 1). in were All the represented but library.example, by are five Sulfamethoxazole two several of belonged nine and structurally closely sulfamoxole, relatedthat sulfanilamides for are part of(nearly the 30% spectral of library.probable all A cause for total database misleading of entries) searchparticipating 118 results. were laboratories, compounds Before the shipping identified to chemicalwas as identity checked highly in of the the referencepreviously. samples laboratory by GC/MS as described suitable for identification. The remaining mass peaks werefrom deleted the reference spectra. Test compounds The sample setlaboratory transferability for of the library checking searchof22compounds(Table approach the consisted 1),ofwhich19compoundswererandomly inter-instrumentselected and from inter- the collectionlaboratory. of Only the drug legal status standards of a ofsort compound of was selection the considered criterion. as The reference ergot alkaloids (dihydroergotamine, ergotamine, and methysergide) weredue added to to a themycotoxins. pronounced sample set scientific interest of one laboratory in Instrumentation and data acquisition Three different laboratories participated in(Table the 2). multicenter Tandem mass study spectra were acquiredinstruments using from four different two manufacturers. Thelowing types study of covered tandem mass the spectrometers: fol- QqTOF,quadrupole-linear quadrupole- ionquadrupole trap (QqQ), (QqLIT), andcyclotron resonance (LIT-FTICR) m linear quadrupole-quadrupole- ionoperated in two trap-Fourier different scanning modes: in transform ‘product ionand scan’ in (pi) ion ‘enhanced product ion scan’ (epi) mode.modes, In both precursor operational ions were selectedfragmented in in the the collision first cell quadrupole ( quadrupole and was either operated as quadrupole (pi)trap or (LIT, as epi) linear ion and was usedFTICR to instrument, product scan ions the were fragment generated in ions. the On LITeither and the analyzed were LIT- at low resolution inthe the LIT Fourier or transform at ion high cyclotron resolution resonancetrometer. in In (FTICR) each mass participating labo spec- parameters were gathered from routinely appliedexperimental workflows. parameters are The summarized in Table 2.were Test weighed samples and dissolved in 0.1%containing aqueous acetic 50% acid (v/v) solution acetonitrilethe before performance analysis. Depending of on thecompound-specific different ionization instruments efficiencies, as thethe well concentrations sample as of solutions varied on from the directly 0.02 to infused 10 into thestrumental mass platform, spectrometer. tandem On masscompound each spectra were single specific acquired in- for at three aues. different certain Additionally, collision-energy on val- the QqLIT in epi mode a single spectrum [31] 2009 John Wiley & Sons, Ltd. 0amu . c 4 ± The results Inthecourseof [29] Copyright [31] Nevertheless, still the inter- The use of tune compounds has Thus, the common doctrine is that [21] [19,21–24] of the precursor ion were deleted from z / [23,25–28] m In that particular study, 3126 MS/MS spectra, Therehavebeenattemptstoproducelibrarieswith [30] [20] In the present report, the examination of the inter-instrument either taken from four large collections orused from the literature, to were construct asearches with library. a subcollection By of performingspectra, the it library a was defined number as shown ‘unknown’ of thatfirst correct rank library in answers 60% were of obtained the search as results. the and inter-laboratory transferabilityreference of library a originally tandemtime-of-flight (QqTOF) built instrument mass is on presented. spectral a quadrupole-quadrupole- instrument differences in thein relative intensities MS/MS of spectra ionsstatistically present are evaluated. significant when strictly measured and spectra recorded on awhich number of have mass been spectrometric reviewed. platforms, ‘‘MS/MS libraries are mostly in-house libraries successfully running only on a single apparatus or apparatus type’’. been proposed for the ‘normalization’ of experimentalwhich conditions, could helpMS/MS to spectra reduce and thelibrary to inter-instrument increase search variability the procedures. of success rate of automated www.interscience.wiley.com/journal/jms For each reference compound, product-ion spectraacquired were typically at ten differentand collision-energy 50 (CE) eV. Because values of possible between saturationpositive effects 5 and to matching avoid false of thenating precursor from alternative ion compounds, all with signals product within a ions origi- Reference library The reference library was recently developed in the referenceratoryonaQqTOFinstrument(QstarXL,AppliedBiosystems,Foster labo- City,CA)andcontained3759 MS/MSspectraof402compounds. Experimental Section window around the the reference spectrareference obtained. spectra were To filtered. increase Onlyintensity those specificity above signals 0.01%, with further, a and relative whichspectra collected were at observed different at collision least energies in were two regarded as range of compoundsTherefore, due to the variations collectionand in of high collisional collision-energy spectra stabilities. settings acquired hascreation. been at The proposed low, for impact library medium, of of instrument-specific a parameters smallthe on molecule creation CID is of difficult achallenging to platform-independent task. control mass A and spectralfrom number library predict diverse of a making manufacturers differentbe have classified types either MS/MS-capabilities of asinstruments. and ‘tandem-in-space’- instruments or can as ‘tandem-in-time’ of a recentlyMS/MS published libraries study, might however, beof suggested sufficiently unknowns that efficient and single forspectrometers. suitable identification for use with different tandem mass a multicenter study, 418 tandemwere mass collected spectra in of three 22 differentinstruments compounds laboratories using from four two different manufacturersestablished and reference matched library againstThe with the extent the of samespectrometric platform library search independence in of algorithm. combinationstrategy with the was the established evaluated developed mass and search is discussed. no As other study far on as thereference we transferability library of available know, a that there tandem approximates massin the spectral comprehensiveness presented and work completeness.

486 487 ; sulfanilamides – : hexoprenaline; mescaline; : carbamazepine; : : epinephrine; octopamine; : ergotamine; methysergide :dihydroergotamine; :dihydroergotamine; : sulfadiazine; sulfaguanidine; : sulfadiazine; sulfaguanidine; related compounds chlortetracycline; doxycycline; The principles of the applied : bumetanide; chlortalidone; : bumetanide; chlortalidone; Similar or structurally : aminopyrine; : moclobemide; sulpiride : mephenytoin : cetirizine; cinnarizine; hydroxyzine; : caffeine; pentifylline; theobromine; [31] -methyl-3,4-methylenedioxyamphetamine oxytetracycline indapamide; mefruside; probenecid sulfameter; sulfamethoxazole; sulfamethoxypyridazine; sulfametrole; sulfaphenazole; sulfathiazole; sulfisoxazole ergotamine ketophenylbutazone; oxyphenbutazone; phenylbutazone 2,5-dimethoxy-4-bromoamphetamine; 2,5-dimethoxy-4-methylamphetamine; 3,4-methylenedioxyamphetamine; amphetamine; metamphetamine; N sulfameter; sulfamethoxypyridazine; sulfametrole; sulfamoxole; sulfaphenazole; sulfathiazole; sulfisoxazole sulthiame indapamide; probenecid; methysergide oxilofrine; phenylephrine; terbutaline theophylline clomipramine; imipramine; lofepramine; opipramol; trimipramine meclizine methoxamine; octopamine; oxyfedrine; suloctidil; verapamil www.interscience.wiley.com/journal/jms Amphetamines Dibenzazepines Tetracyclines: Xanthines Pyrazolones Ergot alkaloids Ethanolamines Ergot alkaloids Sulfonamides Sulfonamides Sulfanilamides Benzamides Hydantoins Sulfanilamides Ergot alkaloids Piperazines Phenylethylamines ell as a summary of putative interfering reference compounds 0u the authors upon request. Allestablished spectra reference were matched library. against the library search procedure are described in the companion paper. Results and Discussion Characterization of the applied instrumental platforms A total ofcompounds 418 were collected tandem in three mass different laboratories spectra on four corresponding to 22 test . 1 ± 2009 John Wiley & Sons, Ltd. within c ine; desipramine; sulfisoxazole; lizine; sulfamoxole; sulfisoxazole Reference compounds ; benzoylecgonine; atropine; esipramine; sulfamoxole; ; mebendazole; dibenzepin – ; acenocoumarol; yohimbine; catechin; trimethoprim metoprolol codeine; buphenin; clobazam; temazepam sulfamethoxazole tizanidine; triamterene; tolpropamine vincamine sulfisoxazole terbuthylazine hydrocodone; codeine; clobazam; temazepam; carazolol + H] + 1605 Doxycycline Theoretical mass , 485–493 Copyright (amu) of [M 44 , 2009 List of compounds with corresponding precursor ion masses as w ) and the corresponding relative intensities). The files were z / Tetracycline 445. Sulthiame 291.0467 Norcocaine MetoclopramidePhenazone 300.1400Phentermine Carazolol; chlordiazepoxide; hydrocodone; 189.1022 150.1277 Cathinone; metamphetamine Sulfamoxole – 268.0750 Atenolol; cycliz PhenytoinSulfamethoxazole 254.0593 253.0971 Oxcarbazepine; phenytoin; cimetidine; Oxcarbazepine; cimetidine; tizanidine; Methysergide 354.2176 Butizide EthambutolEtilefrineEtofyllineMefruside 205.1910 182.1175 Bufotenin; dexpanthenol 225.0982 Oxilofrine 383.0496 Ethamivan; nifenalol; mesoranil – – DihydroergotamineDosulepinDyxirazine 584.2867Ergotamine 296.1467 428.2366 582.2710 Diclofenac – – – – Desipramine 267.1855 Atenolol; cyc CinchocaineCyclizine 344.2332 267.1855 Tetrahydrocannabinolic acid Atenolol; d – Compound Amiloride 230.0551 Clonidine; propazine; sebuthylazine; Table 1. Buphenin 300.1958 Chlordiazepoxide; metoclopramide; m On the transferability of a tandem mass spectral reference library sent to the reference laboratory and are available for review from J. Mass. Spectrom. Data handling Acquired mass spectra were centroidedEach and txt-file exported contained as information txt-files. aboutand the precursor a ion list mass of( the observed fragment ions (mass-to-charge ratios under ‘collision-energy spread’ conditions wasnumber measured. of A 418 fragment total ion mass spectra were collected. . et al , 485–493 44 ,

2009

otaeAaytQ . P nls 1.4. Analyst SP8 1.0 QS Analyst Software H. Oberacher 2.0 Xcalibur 2.0 Xcalibur 1.1 QS Analyst 1

custo ie10mn10mn10mn10mn10min 1.0 min 1.0 min 1.0 min 1.0 min 1.0 time Acquisition

3microscans

oto vrg of average control amu/s 325 s 1.0 time: amu/s 1000 s 1.0 time:

uoai gain automatic speed: scan accumulation speed: scan accumulation

cn5–0 amu 50–700 Scan 070amu 50–700 amu 50–700 amu 50–700

070amu 50–700 J. Mass. Spectrom.

rcro o slto ntrslto ntresol Unit resolution Unit isolation ion Precursor to o eouinUi eouinWdh . amu 1.0 Width: resolution Unit resolution Low ution

2 2 2 4abtayuis r(. Tr)He mTorr) (1.5 Ar units) arbitrary (4 N ‘high’) (status N units) arbitrary (5 olso a N gas Collision

Ultramark Ultramark

afie RAand MRFA caffeine, and MRFA caffeine, ALILTLVS peptide polypropylenglycols reserpine and

airto xenlwt caffeine with External Calibration xenlusing External using External the using External of mixture a with External

/l .–0P oe . p oe .20111 10 10 0.1–1 0.02 mode: epi 0.2 mode: Pi 0.1–10 g/ml) ( concentration Sample µ

/ / / < / / min l 10 min l 10 min nl 100 min l 10 min l 3 rate Flow µ µ µ µ

C C C 200 200 200

◦ ◦ ◦

temperature: temperature: temperature:

150 o rnfrtube transfer ion tube transfer ion C source

◦

oretemperature: source eprtr:50 temperature: C C eprtr:50 temperature: ub a:4 units 40 gas: turbo

◦ ◦

ub a:5 psi 50 gas: turbo a vaporization gas a vaporization gas units

euie a:3 psi 30 gas: nebulizer hahadsweep and sheath hahadsweep and sheath euie a:1–3 gas: nebulizer

2009 John Wiley & Sons, Ltd. otg:40kV 4.0 voltage: otg:45kV 4.5 voltage: otg:45kV 4.5 voltage: V 850 voltage: otg:40kV 4.0 voltage:

c oiiemode Positive oiiemode Positive oiiemode Positive mode Positive o oreprmtr oiiemode Positive parameters source Ion

o oreTrooSryTurboIonSpra TurboIonSpray source Ion aopa -S oreHEIsource H-ESI source H-ESI Nanospray y ieoption time

Copyright scan(epi)withdynamicfill

nacdpoution product enhanced

lentv cnmd rdc o cn(i or (pi) scan ion Product modi scan Alternative I rLIT-FTICR or LIT

rd aeQTRX Ta 00QTRPla S unu lr T-TUltra LTQ-FT Ultra Quantum TSQ i Pulsar QSTAR 4000 QTrap XL QSTAR name Trade

Scientific Scientific

opn ple issesApidBiosy Applied Biosystems Applied Company tm ple issesTem Fisher Thermo Biosystems Applied stems hroFisher Thermo

ntuetQTFQLTQTFQQLIT-FTICR QqQ QqTOF QqLIT QqTOF Instrument

eeec aoaoyLaborat laboratory Reference r aoaoy2Lbrtr 3 Laboratory 2 Laboratory 1 ory

oprsno xeietlstig sdo ifrn in different on used settings experimental of Comparison 2. Table tuetlpafrsfraqiigtne asspectra mass tandem acquiring for platforms strumental www.interscience.wiley.com/journal/jms

488 489 (c) QqLIT - pi (d) QqLIT - epi (e) QqTOF - epi (f) LIT m/z www.interscience.wiley.com/journal/jms simply by eliminating all cyclizine-specific [31] 100 150 250 300 ... observed in all MS/MS-experiments ... observed only via "tandem in space" - experiments ... precursor ion removed 30% (g) LIT-FTICR 30 eV 35 eV 25 eV 16% 30 eV20 eV (a) reference (b) QqQ 50 200 Comparison of buphenine-specific tandem mass spectra col- 0 0 0 0 0 0 0

50 50 50 50 50 50 50

100 100 100 100 100 100 100 relative signal intensity [%] intensity signal relative Figure 1. lected on different instrumental platforms. were also detected inon all the desipramine-specific QqLIT spectra inrevealed collected epi that mode. besides The desipramine-specificof visual ions cyclizine-specific inspection ions a were of present large (Fig. the number cyclizine 3(c)). have spectra Desipramine identical and empirical formulas andmonoisotopic therefore identical masses (Table 1)fragment but ions different (Fig. compound-specific 3(a) andare present (d)). in Hence, one if sampleto both solution carry-over effects, the due fragment ions to compounds corresponding contamination towill both or species due appear in thespectra, fragment cyclizine ion was mass obtained spectrum.the as sample For top spectra all hit. affected After ‘purification’ of ions, desipramine was retrieved as(Fig. the 3(b)). best matching compound Evaluation of theAustrian library multicenter study search results obtained fromLibrary the search wasusing performed a against sophisticated a matchingalgorithm 402-compound algorithm. library The are principles described of in the the companion paper. The library 2009 John Wiley & Sons, Ltd. c 176 ppm; LIT-FTICR. ± < 44 ppm). The . 0 ± 102 ppm; QqTOF: QqTOF 440 ppm ± − < 00 ppm . fragment ions that were very QqLIT z imized instrumental parameters / < m LIT < , 485–493 Copyright 44 , 7 ppm; LIT-FTICR: 0 139 ppm; QqLIT: 70 ppm standard deviation (QqQ: . 2009 ± ± 32 ± 30 ppm 8 ppm − . Compound-specific mass spectra acquired on the different in- 10 On the transferability of a tandem mass spectral reference library Visual check of the collected fragment ion mass spectra Before matching theeach collected spectrum sample wasreference compared spectra spectra visually to to with uncover theputative the noticeable mefruside library, corresponding discrepancies. spectra In collectedare Fig. on depicted. 2 Visual the inspection revealed QqLIT that theat in spectra 20 epi collected eV mode (Fig. 2(c)) and(Fig.2(e))donothaveanyfragmentionincommonwithmefruside- under ‘collision-energy spread’ conditions specific reference spectra (Fig.did 2(a)). not Thus, retrieve bothspectra mefruside sample was spectra as unclear. match. Mosteither The probably, during origin the the of spectratxt-files extraction got to these process the mixed two or reference up laboratory. during Furthermore, the abnormalities transfer as The magnitude of the average masscate measurement the error presence can indi- ofimproper some calibration. kind For of all instrumental systematicthe platforms error but calculated arising the QqQ, from standardmass deviations deviations. were Obviously, largerproperly all than calibrated. instruments the Besides mean but thement the precursor ion QqQ ion masses were masses, were alsoof the the affected frag- QqQ. by All the erroneous improperdata masses processing. calibration were corrected prior to further strumental platforms were comparedinter-instrument to differences estimate in the fragmentationportant extent behavior. to of note It in is this im- contextwas that applied no standardization to procedure increaseeach the participating laboratory, comparability opt of MS/MSwere solely spectra. gathered from In routinely appliedtain workflows. Thus, fragment cer- ions were detectedwhereas on others were all preferentially experimental produced platforms, byinstrument. Typically, a more extended certain fragmentation was type observed of on ‘tandem-in-space’ instruments than on LIT/LIT-FTICR (anple exam- is depicted in Fig. 1). Moreover, ‘tandem-in-space’favored experiments the production of low- weak or absent in the LIT (Fig. 1).spectra Comparison of clearly ‘tandem-in-space’ showed thatfragmentation pathways differences were minor regarding (Fig. the 1(a)–(e)). observed intensities, Relative signal however, varied significantly. Particularly,collision-energy the settings applied were foundrelative to signal have intensities. In a this major respect,were impact ‘tandem-in-time’ found spectra on to be less affected than ‘tandem-in-space’ spectra. J. Mass. Spectrom. LIT: standard deviation indicatescertain the instrument type inherent and enables masstrometers a as accuracy ranking follows: of QqQ of the mass a spec- different instruments (QqQ, QqLIT,QqLIT, QqTOF, and and LIT-FTICR). QqTOF QqQ, ments were and classified LIT-FTICR as aswas ‘tandem-in-time’ ‘tandem-in-space’ operated instrument. instru- The in QqLIT mode. two On the different LIT-FTICR instrument, scanningalyzed product ions at modes: were low either ‘pi’ an- resolutionFTICR. and in Depending the ‘epi’ on LIT thecal or type characteristics of at of mass high eachrestricted. analyzer resolution instrument To and in used, estimate the the the mass physi- relative performance accuracy differences between of was measured the and instruments,ion theoretical precursor masses the were determinedstatistically for evaluated. all The results spectra were per reportedsurement error as instrument average and mea- − . et al , 485–493 is observed 44 cut-off) , 3 > 40 eV 30 eV 30 eV 30 eV 2009 H. Oberacher ipramine-specific spectrum for m/z J. Mass. Spectrom. le spectra (relative intensity ... desipramine-specific fragment ions ... cyclicine-specific fragment ions ... precursor ion removed 100 150 200 250 300 reference - desipramine reference - cyclizine QqLIT - epi - "desipramine" - cleared QqLIT - epi - "desipramine" (a) (b) (c) (d) of compound-specific fragment ions z / Visual inspection of a putative des 50 m 0 0 0 0

50 20 10 50 50

100 100 100 relative signal intensity [%] intensity signal relative for both compounds (nominal fragment ion mass: 133; Table 3(a)). spectra (two mefruside-were and four excluded desipramine-specificfragment spectra) from ion mass matching. spectra,incorrect Among an assignments unexpected the high wassample percentage remaining obtained spectra. of for For 412 instruments, cathinone phentermine-specific nine was obtained spectra asand top acquired match. cathinone Phentermine on havenominal different precursor low-resolution ion masses empirical (Table 3(a)). The formulas, two compoundsstructurally are but related. In identical MS/MS experiments loss of NH Figure 3. the presence of cyclizine-specific fragment ions. of fragment ions observed in samp z / m 2009 John Wiley & Sons, Ltd. c . Of the initially 20 eV 10 eV 20 eV 30 eV 10, 20, 30 eV Copyright of precursor ion uside-specific spectra collected of precursor ion z correct answer / z m/z / m m (a) Reference (b) QqLIT - epi (c) QqLIT - epi (d) QqLIT - epi (e) QqLIT - epi ... mefruside-specific fragment ions ... precursor ion removed 100 150 200 250 300 350 400 Summary of fragment ions observed (a) for phentermine and cathinone in selected reference spectra as well as (b) for phentermine on Visual inspection of putative mefr 50 0 0 0 0 0

50 50 50 50 50

100 100 100 100 100 relative signal intensity [%] intensity signal relative QqQ – 6QqQ eV – 12QqQ eV – 18QqLIT eV – epiQqLIT – – 10 epi eVQqLIT – – 20 epi eVQqLIT – – 30 epi eVLIT – – 10, 20, 15% 30LIT eV – 17%LIT – 20% 150.12 150.12 150.16 150.12 150.16 150.16 150.16 133.12 133.12 133.12 133.12 – – – – – 150.11 – 150.11 – 150.11 – – – – – – 133.11 – 133.08 105.09 – 105.04 – – 133.08 – 91.09 105.04 – 91.04 – 91.09 105.04 91.04 – – – – 105.04 – 91.04 90.00 – – 91.04 91.09 90.08 – – – – 90.08 – – – – – – – – – – – – – – – – – – – – – – – – – – – (b) Instrument – CE Reference compound PhentermineCathinone 150.13 150.09 133.10 133.06 – 132.08 117.06 – 105.07 91.05 105.07 90.05 91.05 77.04 – 65.04 – 65.04 Table 3. different instrumental platforms (a) www.interscience.wiley.com/journal/jms at different collision-energy settings on the QqLIT in epi mode. search results areenergy summarized settings are in specified Fig. byDifferent 4. the background The numbers colors given are applied in usedlibrary each collision- search to box. results. designate Here, the any obtained librarycompound search as resulting top in hit the test wascollected called set a of 418 spectra, the above-mentioned questionable Figure 2.

490

491 correct assignments correct

97% 99% 99% 95% 100% 100% tetracycline

30 eV At certain 10, 20, sulthiame [19]

30 eV 10, 20, sulfamoxole

30 eV

10, 20, sulfamethoxazol g/ml) were responsible µ 30 eV 10, 20,

02 phenytoin .

15%18% 15%21% 20% 15% 25% 20% 10% 25% 14% 10% 18% 13% 16%

15 eV 20 eV 20 eV 16 eV 20 eV 10 eV 10 eV 10 eV 10 eV 10 eV 30 eV 10, 20, phentermine

15% 17% 20% 30 eV

12 eV 10 eV 10, 20, phenazone 40 eV 20, 30,

www.interscience.wiley.com/journal/jms methysergide ct MS/MS spectra. The applied scan modes and

50 eV 20, 35, metoclopramide

30 eV 35 eV 30 eV 20 eV 20 eV 20 eV 20 eV 20 eV 20 eV 30 eV 10, 20, mefruside

20 eV 30 eV 10, 20, Identification of ‘artifacts’ within a cathinone-specific reference etofylline

… wrong substance

10 eV 10 eV 20 eV 20 eV 20 eV 30 eV 10, 20, etilefrine for the somewhat reduced comparability of tandemHundred mass percent correct spectra. search results were achieved with spectra acquired on the LIT-FTICR instrument. Impact of the comprehensiveness of theon mass the spectral search library efficiency It is well known thation the observed is intensity of controlled aof certain by fragment the kinetic collision parameters gas (e.g. and type center-of-mass and density energy). experimental conditions, usuallyfragmentation only pathways is a observed. subset Thus, for of the all creation of possible a Figure 5. spectrum. obtained in 98.1% of search results.classified For all as eight incorrect, spectra the that were correctrank.Thelowestpercentageofcorrectresults(95.0%)wasobtained compound matched at second for the QqLIT operated in epithe mode. Nevertheless, QqLIT we operated believe in that epi modefragment is ion as suitable spectra for as thethe all collection very of low other sample examined concentrations (0 platforms. Probably,

10 eV 10 eV 30 eV

10, 20, ethambutol

30 eV

10, 20, ergotamine 2009 John Wiley & Sons, Ltd.

c

60 eV 30, 45, dosulepin

40 eV 20, 30, dixyracine our different instruments were used to colle

… incorrect match

40 eV 20, 30, dihydroergotamine

10 eV

20 eV 20 eV35 eV eV 20 30 eV 30 eV 30 eV 10 eV 45 eV 20 eV 20 eV 20 eV 50 eV 50 eV 40 eV 40 eV 60 eV 30 eV 30 eV 30 eV 30 eV 65 eV eV 50 40 eV 30 eV 30 eV 30 eV 30 eV 30 eV 30 eV 20, 35, desipramine 10 eV 20 eV 30 eV 30 eV slot of 0.1 amu was used for 10, 20,

z cyclizine / m

30 eV

10, 20, values close to 105.07 and 133.06 cinchocaine z /

m 40 eV

20, 30, buphenin , 485–493 Copyright … correct match after the elimination of artifacts from reference spectra … correct match

45 eV 44 25, 35,

, amiloride 20% 12% 14% 12% 15% 12% 13% 15% 13% 14% 12% 15% 12% 13% 14% 25% 25%30% 16% 20% 18%25% 22%30% 15% 25%35% 18% 30% 20% 20% 35% 25% 25% 15% 20% 30% 18% 25% 17% 20% 30% 21% 25% 20% 15% 30% 25% 20% 15% 20% 25% 18% 25% 18% 25% 30% 22% 30% 16% 14% 35% 20% 19% 23% 25% 24% 30% 30% 14% 25% 35% 18% 30% 17% 25% 35% 21% 30% 18% 14% 35% 22% 19% 30% 25% 24% 35% 30% 25% 35% 30% 35% 35% 40% 15% 45% 17% 25% 20% 30% 25% 35% 30% 25% 35% 30% 15% 35% 20% 10% 25% 15% 20% 10 eV 10 eV 10 eV 10 eV20 eV 10 eV 20 eV 5 eV 20 eV 12 eV 10 eV 20 eV 15 eV 30 eV 20 eV 30 eV 15 eV 25 eV 20 eV 30 eV 8 eV eV 16 15 eV 5 eV 20 eV 10 eV 18 eV 12 eV 20 eV 10 eV 25 eV 20 eV 30 eV 20 eV 6 eV 5 eV 10 eV 10 eV 10 eV 10 eV 30 eV 30 eV 30 eV20 eV 20 eV 20 eV30 eV 30 eV 20 eV 30 eV 40 eV40 eV 20 eV 30 eV 40 eV 40 eV 10 eV 30 eV 35 eV 40 eV 20 eV10 eV 20 eV 40 eV 60 eV 20 eV 25 eV 30 eV20 eV 24 eV 30 eV 20 eV 20 eV 30 eV 35 eV 25 eV 40 eV 20 eV 10 eV 30 eV 30 eV 30 eV 40 eV eV 10 30 eV 20 eV 24 eV 40 eV 10 eV eV 20 30 eV 40 eV 10 eV 20 eV 30 eV eV 30 10 eV 20 eV 40 eV 30 eV 10 eV 20 eV 18 eV 30 eV 20 eV 20 eV 25 eV 30 eV 20 eV 30 eVeV 25 30 eV 10 eV 30 eV 30 eV 40 eV 10 eV 20 eV 22 eV 40 eV 10 eV 20 eV 30 eV 30 eV 10 eV 20 eV 30 eV 10 eV 20 eV 30 eV 10 eV 20 eV 30 eV 20 eV 30 eV 30 eV 25 eV 20 eV35 eV 20 eV 25 eV45 eV 10 eV 30 eV 30 eV 15 eV 15 eV 35 eV 20 eV 20 eV 20 eV 20 eV 30 eV 25 eV 20 eV 30 eV 40 eV 20 eV 30 eV 40 eV eV 10 30 eV 40 eV 10 eV 20 eV 40 eV 20 eV 20 eV 30 eV 20 eV 30 eV 30 eV 20 eV 30 eV 40 eV 20 eV 30 eV 40 eV 25 eV 30 eV 40 eV 15 eV 30 eV 40 eV 20 eV 20 eV 35 eV 20 eV 25 eV 25 eV 20 eV 25 eV 30 eV 20 eV 25 eV 30 eV 30 eV 25 eV 35 eV 35 eV 35 eV 40 eV 30 eV 45 eV 40 eV 30 eV 30 eV 10, 20, 2009 -difference was less than 0.05 amu and subsequent

z scan mode scan / pi epi LIT m FTICR

Results of the Austrian multicenter study. F instrument QqQ - QqLIT QqTOF LIT-FTICR On the transferability of a tandem mass spectral reference library elimination of the fragment ionOwing exhibiting lower to signal filtering, intensity. spectrum six phentermine changed spectra theirassigned and (Fig. status one 4). etilefrine The fromfrom the number incorrectly initially of 3.6% to down incorrect to correctly matches only 1.9%. decreased Correct answers were collision-energy settings are given. J. Mass. Spectrom. Moreover, the twocommon compounds (Table have 3(a); 105.07, severalhowever, 91.05, fragment exhibited and ions several 65.04 in fragment amu).this ions Cathinone, compound that and werecathinone can unique from for be phentermine usedand (Table to 3(a); 77.04 unequivocally 132.08, 117.06, distinguish amu).one 90.05, The to four mismatchedmass different sample spectra fragment spectra acquired ionsthree on contained possible (Table fragment 3(b)). the ions QqLIT Within thatfor represented in three cathinone unique identifiers (Table epi-mode 3(b);to one 90.08 the of amu) appearance was the Although of observed none cathinone giving of rise as thein best cathinone-unique one matching signals of were compound. thewere observed remaining spectra preferentially listed matched inassignable to cause Table 3(b), for cathinone. the all prevalence ofvisualinspectionofthecathinone-specificreferencespectra(Fig. In of them cathinone these was 5) cases, missing.revealed A that an artifacts with Figure 4. amu were present and were found to representsource the most of probable overestimation. Thefragment mass ion and differences its between neighboring artifactions a were matched so true to small the that corresponding both which signal increased in the total a number sample of spectrum, as ‘matching the fragments’ corresponding ‘match as probability’ well values artificially. For low- resolution mass spectra where a signal assignment, cathinone was retrieved as top match instead of phentermine.Werecognizedthattheartifactsarosefromimproper centroiding and bypassedTo the eliminate already the installed artificiallyspectra, filtering produced filtering was extended. fragments The steps. newly from developed filteringincluded step reference scanning each reference spectrum forions whose pairs of fragment , , , , . 67 , et al 2001 2005 2000 Journal Journal 1995 Trends in , 485–493 44 , 510. , 200. , 15 13 , , , 135. 24 2009 AnalyticalChemistry , H. Oberacher 2004 2002 , 211. 2005 74 , , 3533. 70 , 6250. , Analytical Chemistry rometry: issues and strategies. ctrometry to the automated , 235. arged parent ions via ion/ion 77 2002 Rapid Communications in Mass liquid chromatography-tandem , Mass Spectrometry Reviews e ion trap and triple-quadrupole 25 Jr. Simplification of product ion g-chain oligonucleotides. , Journal of Mass Spectrometry G. Huber. Comparative sequencing 1998 rmack, D. Schieltz 3rd. Method to , 493. , 2594. J. Mass. Spectrom. 2005 uencing by mass spectrometry. 2006 24 73 , , , 2337. , 32. ¨ Journal of the American Society for Mass ¨ uller, R. D. Appel. Automated protein Rapid Communications in Mass Spectrometry Osterreichisches Sicherheitsforschungs- 2005 2001 17 15 , , Analytical Chemistry Mass Spectrometry Reviews Journal of the American Society for Mass Spectrometry 2003 2004 Rapid Communications in Mass Spectrometry Analytical Chemistry , 743. , 3202. Analytical Chemistry , 599. . 18 67 3 n , , , , 1053. , 566. , 1069. spectral libraries using quadrupol mass spectrometers. 2004 G. Lajoie. PEAKS: powerful software for peptide deby novo sequencing tandem massSpectrometry spectrometry. sequencing of nucleicmass acids spectrometry. by Spectrometry Analytical Chemistry 1426. of triplemass quadrupole, spectrometers quadrupolespectrometry in ion of electrospraysequencing. trap oligonucleotides. ionization and 2. tandem15 sector Suitability mass field for dede novo novoAnalytical peptide Chemistry sequencing by tandem mass spectrometry. correlate tandem mass spectra of modifiedsequences peptides in to the amino acid protein database. strategies for carbohydrate sequencing. 1. Mining structuralby details MS 24 correlating tandem masspeptides to sequences spectra in nucleotide databases. of1995 modified and unmodified of nucleicspectrometry. acids byApplicability liquid of tandemcomparative chromatography-tandem sequencing mass of spe mass lon of the American Society for Mass Spectrometry of ions by collisional activation. spectrometry. 35 produced in electrospray sources. identification by tandem mass spect Mass Spectrometry Reviews ab initio oligonucleotide seq of the American Society for Mass Spectrometry spectrometry. 1992 spectra derived fromchemistry. multiply ch [8] D. Ashline, S. Singh, A. Hanneman, V. Reinhold. Congruent 4 ..ae,JKEg ..comc r.Mnn genomes: 3rd. Mining A.L.McCormack J.K.Eng, J.R.Yates, [4] [5] P. Hernandez, M. M [2] A. K. Shukla, J. H. Futrell. Tandem mass spectrometry: dissociation [7] J. Laskin, J. H. Futrell. Activation of large ions in FT-ICR mass [3] V. Gabelica, E. De Pauw. Internal energy and fragmentation of ions [6] J. L. Stephenson, S. A. McLuckey [9] J. Rozenski, J. A. McCloskey. SOS: a simple interactive program for [1] S. A. McLuckey. Principles of collisional activation in analytical mass [18] J. L. Josephs, M. Sanders. Creation and comparison of MS/MS [15] B. Ma, K. Zhang, C. Hendrie, C. Liang,[16] M. Li, H. Oberacher, A. Doherty-Kirby, B. M. Mayr, C. G. Huber. Automated[17] B. de L. Milman. Identification novo of chemical compounds. [13] A. Premstaller, C. G. Huber. Factors determining the performance [14] J. A. Taylor, R. S. Johnson. Implementation and uses of automated [12] J. R. Yates, J. K. Eng, A. L. McCo [10] H. Oberacher, B. Wellenzohn, C. [11] H. Oberacher, W. Parson, P. J. Oefner, B. M. Mayr, C. G. Huber. ¨ orderprogramm KIRAS – eine Initiative des Bundesministeriums ¨ ur Verkehr, Innovation, Technologie (BMVIT), Projekt 813786) is acknowledged. Acknowledgements The authors wishsupport. to Furthermore, financial thank support by AppliedPromotion the Austrian Agency Biosystems/MDS Research (FFG, SciexF for f References 2009 John Wiley & Sons, Ltd. c [27,33,34] To determine [31] Copyright www.interscience.wiley.com/journal/jms Conclusions The tandem mass spectralinstrument reference and library its tested3759 for inter-laboratory spectra its transferability inter- collected consistedQqTOF from of instrument. 402 Intandem reference the mass compounds course spectra of of ondifferent 22 laboratories a a compounds using were multicenter four acquiredmanufacturers study, different and in instruments matched 418 three from against two thecated peak library matching using algorithm. a Thus, sophisti- the a comprehensive inter-instrument study and on thetandem inter-laboratory mass transferability spectral of reference a point library out that has the results been were authentic, problems conducted. werethat discussed To may occur inalso the in course laboratories of during a thenumber use multicenter of of study such and spectra, a maybe gadget. however,(0.5%) Only were a were of small sorted unknown origin out.fragment and ion Two four masses of out others two (1.0%) of species.for contained All 418 statistical other evaluation spectra were of useful theapproach. performance The of overall the high library(98.1%) search percentage can be of cited as correct evidence for search thethe platform presented results independence tandem of mass spectral libraryfollowing search approach. factors The are obligatoryresult: to (1) For obtain the such libraryplatform an is creation, encouraging necessary a that tandem enablesmeasurement the mass of accurate fragment spectrometric and ion reproducible masses. (2) Compound-specificerence ref- spectra need to beenergies. collected (3) Reference at spectra several must differentthe be library collision filtered to eliminate before unspecific signals. storage (4) in The instrumentused that to is collect sample spectra must be properlysearch calibrated. (5) algorithm The must exhibitwithin a the high intensity tolerance toward distributionpathways changes among (for details different on fragmentation thispaper). topic Future please work refer will to show the ifber the companion of ongoing database increase of entries the will num- haveand a transferability major of the impact mass on spectral the library. efficiency We have recently proposedacquired at the ten different collection collision-energy of values. reference spectra comprehensive MS/MS-spectral library, theacquired collection at of spectra several differentrecommended.Theuseofthreedifferentcollision-energylevelsfor collision-energy settings hasacquiringcompound-specificreferencespectraiscommon. been the influence ofspectra stored the in a number library oncollection search of efficiency, the was compound-specific sample spectra matched reference entire to library, one the subset of following thecollected entire three library at consisting three databases: of spectra different the collisionand energies another (20, 35, subset andcollected 50 of eV), at the a entireThe single performance library of collision consisting the library energy of searchcomprehensiveness decreased of spectra of with a decreasing the meanthe mass entire level spectral library, (35 for eV). libraryspecific the searched. spectra, library and For consisting for of98.1, the three 97.3, library compound- and consisting ofresults. 91.9% The one of decline spectrum, matches, of themoderate, respectively, which search clearly suggests yielded efficiency, that the however, developed correct library was search procedure rather has reached a high degree ofsuboptimal development. Thus, collections even of referenceapplied for spectra unequivocal compound can identification. be successfully

492 493 , , ¨ a. 38 , 2005 , 1039. Clinical 18 2005 , , 2649. 2004 20 , , 1399. 2006 41 , , 409. , 2265. 2006 Clinical Biochemistry 72 844 f toxicologically relevant drugs. , , orensic toxicology and in doping r the generation of searchable 9 mycotoxins in wheat and maize. acher. Combined use of ESI-QqTOF- 2000 a, R. Schuhmacher. Development and Analytical and Bioanalytical Chemistry 1999 , 1735. www.interscience.wiley.com/journal/jms 52 , Rapid Communications in Mass Spectrometry 2006 , 69. Analytical Chemistry Journal of Mass Spectrometry Rapid Communications in Mass Spectrometry 386 , , 2833. P. Marquet. Screening ofchromatography-lineariontrap drugs tandemmass and spectrometry. toxic compoundsChemistry with liquid 19 MS and ESI-QqTOF-MS/MSqualitative with analysis of mass-spectral drugs. library2006 search for Testing ofcompounds. a library containing 3126 MS2 spectra of 1743 of electrospray transportlibraries. CID fo validation of a liquid chromatography/tandem massmethod spectrometric for the determination of 3 Rapid Communications in Mass Spectrometry 362. indispensable tools in clinical and f control. toxicological analysis: comparison of MS/MS spectradifferent obtained with instruments and settings. Comparison ofchromatography/triple-quadrupole mass spectrometry product forsearch. library ion spectra obtained by liquid pressure ionization mass spectra o Journal of Chromatography A [34] F. L. Sauvage, F. Saint-Marcoux, B. Duretz, D. Deporte, G. Lachatre, [31] M. Pavlic, K. Libiseller, H. Ober [32] M. Sulyok, F. Berthiller, R. Krsk [30] B. L. Milman. Towards a full reference library of MS(n) spectra. [33] J. M. Hough, C. A. Haney, R. D. Voyksner, R. D. Bereman. Evaluation [29] H. H. Maurer. Hyphenated mass spectrometric techniques- [28] R. Jansen, G. Lachatre, P. Marquet. LC-MS/MS systematic [27] M. Gergov, W. Weinmann, J. Meriluoto, J. Uusitalo, I. Ojanper , , 9 , 16 , 2009 John Wiley & Sons, Ltd. Rapid c 1995 Journal of 2002 , 2162. 62 , 1990 ,8. 2 , Rapid Communications in 1988 ionization/in-source collision- ectrometry: triple quadrupoles Rapid Communications in Mass rospray in-source collisionally Vogt, M. Svoboda, A. Schreiber. y of product ion spectra across Kelley, D. C. Bradford. Tandem-in- , 1779. , 1013. Analytical Chemistry 22 36 , , , 1447. , 485–493 Copyright 18 2008 , 2001 44 , Rapid Communications in Mass Spectrometry 2004 2009 Rapid Communications in Mass Spectrometry and quadrupole ion traps. space and tandem-in-time mass sp J. Herniman, J. Langley.spectral Towards library – a reproducibilit eleven universal different product massMass ion Spectrometry spectrometers. mass medications byspectrometry. liquid chromatography/electrospray mass M. L. Miller.Poorreproducibility ofin-source collisionalatmospheric 1270. protocols for reproducible elect 2374. induced dissociationspectrometry on instruments variouslibraries. and liquid chromatography/mass the development of spectral database for XQQ instruments: The dynamical prerequisites. Communications in Mass Spectrometry Mass Spectrometry product-ionchromatography/mass tandemdevelopment spectrometry mass of spectral libraries. Spectrometry instruments spectra for on the various liquid induced dissociation and mass spectra library searching. Tuning compounds for electrospray [20] J. V. Johnson, R. A. Yost, P. E. [21] C. Hopley, T. Bristow, A. Lubben, A. Simpson, E. Bull, K. Klagkou, [22] J. L. Josephs. Characterization of over-the-counter cough/cold J.Romeril, K.S.Webb, K.D.Kruger, R.D.Maier, M.J.Bogusz, [26] [23] A. W. Bristow, W. F. Nichols, K. S. Webb, B. Conway. Evaluation of [24] W. Weinmann, M. Stoertzel, S. [19] R. I. Martinez. Instrument-independent tandem mass spectrometry [25] A. W. Bristow, K. S. Webb, A. T. Lubben, J. Halket. Reproducible On the transferability of a tandem mass spectral reference library J. Mass. Spectrom.