Fragmentation of toxicologically relevant drugs in...

FRAGMENTATION OF TOXICOLOGICALLY RELEVANT DRUGSIN POSITIVE-ION LIQUID CHROMATOGRAPHY–TANDEMMASS SPECTROMETRY

W.M.A. Niessen*hyphen MassSpec, de Wetstraat 8, 2332 XT Leiden, The Netherlands

Received 14 August 2010; revised 5 January 2011; accepted 5 January 2011

Published online in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/mas.20332

The identification of drugs and related compounds by LC–MS–MS is an important analytical challenge in several applicationareas, including clinical and forensic toxicology, doping controlanalysis, and environmental analysis. Although target-com-pound based analytical strategies are most frequently applied,at some point the information content of the MS–MS spectrabecomes relevant. In this article, the positive-ionMS–MS spectraof a wide variety of drugs and related substances are discussed.Starting point was an MS–MS mass spectral library of toxicolo-gically relevant compounds, available on the internet. The posi-tive-ionMS–MS spectra of�570 compounds were interpreted bychemical and therapeutic class, thus involving a wide variety ofdrug compound classes, such benzodiazepines, beta-blockers,angiotensin-converting enzyme inhibitors, phenothiazines, dihy-dropyridine calcium channel blockers, diuretics, local anes-thetics, vasodilators, as well as various subclasses of anti-dia-betic, antidepressant, analgesic, and antihistaminic drugs. Inaddition, the scientific literature was searched for availableMS–MS data of these compound classes and the interpretationthereof. The results of this elaborate study are presented in thisarticle. For each individual compound class, the emphasis is onclass-specific fragmentation, as discussing fragmentation of allindividual compounds would take far too much space. The recog-nition of class-specific fragmentationmay be quite informative indetermining the compound class of a specific unknown, whichmay further help in the identification. In addition, knowledge on(class-specific) fragmentation may further help in the optimiza-tion of the selectivity in targeted analytical approaches of com-pounds of one particular class. # 2011 Wiley Periodicals, Inc.Mass Spec Rev

Keywords: tandem mass spectrometry; systematic toxicologi-cal analysis; fragmentation; drugs

I. INTRODUCTION

The identification of drugs and related compounds is an importantanalytical challenge in several application areas, includingclinical and forensic toxicology, doping control analysis, andenvironmental analysis. Gas chromatography–mass spectrom-etry (GC–MS), and more recently also liquid chromatog-raphy–mass spectrometry (LC–MS), are important tools forconfirmation of identity or identification.

In all these fields, methods have been developed directed atthe analysis of particular target compounds or classes of com-pounds. With respect to clinical and forensic toxicology, severalearly review articles map the possibilities and limitations in thisrespect (Maurer, 1998, 2005; Bogusz, 2000; Van Bocxlaer et al.,2000). In the field of doping control analysis, the simultaneousanalysis of 72 target compounds from different classes, that is,glucocorticoids, diuretics, stimulants, anti-oestrogens, b-adre-nergic drugs and anabolic steroids, in human urine is a niceexample (Mazzarino, de la Torre, & Botre, 2008). For equinedoping control, 75 basic drugs were analyzed in equine plasmawith an analysis time of only 8 min (Leung et al., 2007). Theanalysis of 80 pharmaceuticals from different pharmacologicalclasses used as adulterants in ‘‘natural’’ herbal medicines isanother example of targeted multiresidue analysis (Boguszet al., 2006). In environmental analysis, a wide variety of drugsand antibiotics, metabolites, and drug-like substances, includingendocrine disrupting compounds, have been analyzed in influentsand effluents of water sewage treatment plants as well as insurface waters (Ternes, 1998). They are considered emergingenvironmental contaminants (Richardson, 2008). Extensive tar-geted multiresidue screening methods based on LC–MS havebeen developed for the quantification and confirmation of identityof these classes of compounds in the environment (Kim &Carlson, 2005; Petrovic et al., 2005; Gros, Petrovic, & Barcelo,2006; Kuster, Lopez de Alda, & Barcelo, 2009).

Inmost of these application areas, primarily targeted analysisis performed.However, there also is an increasing need to developand apply untargeted analytical approaches, both in toxicology,doping control, environmental, and food safety analysis. Untar-geted screening for unknown compounds by LC–MS is highlychallenging. Commonly applied analyte ionization methods likeelectrospray ionization (ESI) and atmospheric-pressure chemicalionization (APCI) suffer from selectivity issues: ionization isprimarily based on the ability to protonate or deprotonate thetarget compounds, which in practice excludes many compounds.Matrix effects commonly observed in electrospray ionizationdemand for effective sample pre-treatment procedures (Niessen,Manini, & Andreoli, 2006), which in turn may lead to analytelosses due to too high polarity or non-specific interaction withsolid-phase extraction (SPE) materials. The interpretation oftandem mass spectrometry (MS–MS) spectra is another chal-lenge, because of the limited understanding of the fragmentationand rearrangement reactions involved and the limited numberof fragments generally observed. Multi-platform proceduresinvolving both multistage mass spectrometry (MSn) usingion-trap instruments and accurate-mass determination using

*Correspondence to: W.M.A. Niessen, hyphen MassSpec, de Wetstraat

8, 2332 XT Leiden, The Netherlands. E-mail: [email protected]

Mass Spectrometry Reviews# 2011 by Wiley Periodicals, Inc.

high-resolution time-of-flight or orbitrap mass spectrometers areobligatory to successfully identify unknowns.

Systematic toxicological analysis (STA) or generalunknown screening (GUS) more or less bridges the twoapproaches of targeted and untargeted analysis. The target ana-lytes are typically up to a 1,000 toxicologically relevant com-pounds, including drugs from a wide variety of therapeuticclasses, illicit drugs, pesticides and related compounds, andantimicrobial and antibiotic compounds, and metabolites of allthese compound classes. Generally, the approach followed ispreferably untargeted, but the main focus in data processing isin identification and quantification of the compounds present in alist or database of target analytes, either just a list of compoundswith their formulae and expectedm/z values, or a dedicated massspectral library.

In all these application areas, at some point the informationcontent of the MS–MS spectra acquired becomes relevant. Thisnotionwas the starting point of this study. After a brief discussionon developments and the state-of-the-art in STA, the MS–MSspectra of a wide variety of drugs and related substances arediscussed. Starting point was a mass spectral library of toxico-logically relevant compounds available on the internet (Dresen,Kempf, & Weinmann, 2006; Weinmann, 2005), containing MS–MS spectra of �800 compounds acquired at three differentcollision energies. These MS–MS spectra were interpreted bytherapeutic class, after arranging them in relevant chemicalclasses. In addition, the available scientific literature wassearched for MS–MS interpretation of these compound classes.The results of this elaborate study are presented in this article.For each individual compound class, the emphasis is on class-specific fragmentation, as discussing fragmentation for allindividual compounds would take far too much space. Inaddition, the recognition of class-specific fragmentation maybe quite informative in determining the compound class of aspecific unknown, whichmay further help in the identification. Inaddition, knowledge on class-specific fragmentation may furtherhelp in the optimization of the selectivity in targeted analyticalapproaches of compounds of one particular class (Dresen,Kempf, & Weinmann, 2006).

II. SYSTEMATIC TOXICOLOGICAL SCREENING

Systematic toxicological analysis (STA) is concerned with thescreening of biological samples for the presence of therapeuticdrugs, drugs of abuse, toxins, etc. The biological samples analyzedinclude especially urine, whole blood, plasma, serum, and otherbodyfluids, but also liver and hair.As far as volatile compounds areconcerned, GC–MS is the golden standard for identification andquantification (Maurer, 1992; Polettini, 1999; Valli et al., 2001).For compounds not amenable to GC, identificationmethods basedon the use of liquid chromatography–UV photodiode array spec-trometry (LC–PDA) have been developed (Drummer, Kotsos, &McIntyre, 1993; Tracqui, Kintz, & Mangin, 1995; Lo et al., 1997;Sadeg et al., 1997; Valli et al., 2001). With the development ofreliable and robust instrumentation for LC–MS, the potential ofLC–MS was evaluated for toxicological and forensic applicationsand STA. This topic has been extensively reviewed (e.g., Maurer,1998, 2007;Marquet & Lachatre, 1999; VanBocxlaer et al., 2000;Bogusz, 2000; Marquet, 2002).

Systematic toxicological analysis (STA) is important in bothclinical and forensic toxicology. Clinical toxicology is among

others concerned with the diagnosis or the definite exclusion ofan acute or chronic intoxication. In addition, the monitoring ofpersons addicted to illegal drugs has to be performed. Forensictoxicology is among others concerned with proof of an abuse ofillegal drugs or of a murder by poisoning. Similar analytical pro-cedures are relevant in doping control analysis. A variety of strat-egies has been proposed and developed for STA using LC–MS.

Initially, methods were developed targeted at the analysis ofspecific classes of drugs, for example, illicit drugs, benzo-diazepines, antihypertensives, neuroleptics, pesticides usingsingle or triple quadrupole instruments. An overview of suchtargeted methods was given in several review articles (Maurer,1998, 2005; Bogusz, 2000; Van Bocxlaer et al., 2000). Recentexamples of this approach involving a single quadrupole instru-ment are targeted at, for instance, neuroleptic drugs (Kratzsch etal., 2003), or benzodiazepines (Kratzsch et al., 2004). Numerousexamples are available of the use of triple quadrupole instrumentsfor the targeted analysis of specific classes of drugs, for example,beta-blockers (Umezawa et al., 2008), benzodiazepines, andother drugs of abuse (Villain et al., 2005; Badawi et al.,2009), and antidepressants (de Castro et al., 2008).

Two research groups independently developed an LC–MSapproach for STA based on the use of laboratory-built massspectral libraries (Marquet & Lachatre, 1999; Weinmann etal., 1999; Marquet et al., 2000). The approach is based on theapplication of in-source collision-induced dissociation (CID) inthe atmospheric-pressure–vacuum interface of anESI source on asingle quadrupole instrument (AB Sciex, Foster City, CA(www.absciex.com)API-100). The libraries built containedmassspectra of �400 or �1,000 drugs and other relevant compoundsacquired at two or three alternating orificevoltages (low and high,or low,medium, and high).Mass spectra acquired under identicalconditions in the analysis of patient’s samples were searchedagainst the library to provide drug identification. This approachwas subsequently adopted by others (Hough et al., 2000;Schreiber, Efer, & Engewald, 2000; Lips et al., 2001; Rittner,Pragst, & Neumann, 2001), also using other types of singlequadrupole instruments (Agilent Technologies, Santa Clara,CA (www.home.agilent.com) 1100 Series LC–MSD, ThermoFischer Scientific, Waltham, MA (www.thermofischer.com)SSQ7000). Reproducibility problemswith in-source CID spectrawere recognized (Bogusz et al., 1999). In order to assure theexchangeability of mass spectral libraries between instruments,especially instruments from different vendors, but also instru-ments with different source geometries from the same vendor, aset of tune compounds was proposed (Weinmann et al., 2001a,b;Bristow et al., 2002).

Instead of the use of in-source CID on a single quadrupoleinstrument, CID inMS–MSon a triple quadrupole instrument hasbeen used to build mass spectral libraries to be applied in STA(Weinmann et al., 2000a;Weinmann, Gergov, &Goerner, 2000b;Gergov et al., 2000). Alternatively, the building of amass spectrallibrary for STA on ion-trap instruments has been described(Baumann et al., 2000). Although the CID conditions in anion-trap instrument are easier to reproduce and with the use ofnormalized collision energies also easier to optimize, ion-trapinstrument have not found extensive application in STA. On theother hand, STA based on LC–MS–MS on a triple quadrupoleinstrument has been widely applied ever since.

The building of mass spectral MS–MS libraries and theexchangeability of these libraries between instruments from

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the same or from different manufacturers continues to be a topicof interest (Bristow et al., 2004; Josephs & Sanders, 2004;Milman, 2005; Jansen, Lachatre, & Marquet, 2005; Oberacheret al., 2009a,b).

A somewhat different approach to STA is the targetedanalysis of a wide variety of drugs using selected reactionmonitoring (SRM) on a triple quadrupole instrument. The pro-cedure for the screening of 238 drugs in blood using SRM is agood example (Gergov, Ojanpera, & Vuori, 2003). One SRMtransition per compound is monitored, using a compound-dependent collision energy (20, 35, or 50 eV). With 238 drugsand a dwell time of 25 msec, this amounts to a total cycle time of6 sec. Similar procedures have been reported by others (e.g.,Thieme & Sachs, 2003). The simultaneous analysis of 72 targetcompounds (glucocorticoids, diuretics, stimulants, anti-oestro-gens, beta-adrenergic drugs, and anabolic steroids) in humanurine for doping analysis using one or two SRM transitionsper compound may serve as another example of this approach(Mazzarino, de la Torre, &Botre, 2008). A critical review of LC–MS strategies based on SRM for drug analysis and screeningindicated that the use of only one SRMtransition per compound isgenerally insufficient. Even with two SRM transitions per com-pound, a significant number of false-positive answers may beobtained, especially if no proper attention is paid to the selectionof a truly selective, that is: a highly structure-specific, SRMtransition (Sauvage et al., 2008).

Advances in instrumentation, and especially the introduc-tion of a quadrupole-linear ion trap hybrid instrument (AB Sciex,QTRAP), further extended the possibilities of LC–MS in STA. Inthis instrument, the second mass analyzer can be used as either aconventional quadrupolemass analyzer or a linear ion trap,whichby accumulation of ions provides enhanced full-spectrum sen-sitivity compared to a conventional quadrupole (Hager, 2002).This instrument enables information- or data-dependent acqui-sition (DDA), which is an automatic, data-dependent switchingbetween a survey analysis mode and the (enhanced) full-spectrum MS–MS mode. The full-spectrum MS–MS data maybe searched against mass spectral libraries to assist in the identi-fication of drugs and toxins in STA. In the pioneering research ofthe group ofMarquet, enhanced full-spectrum single-quadrupoledatawere used as the survey analysismode (Marquet et al., 2003).The full-spectrum analysis (and subsequent MS–MS analysis)was performed in alternating positive-ion and negative-ionmode;separate libraries were generated for the positive-ion andnegative-ion mode as well (Sauvage et al., 2006a, 2009). Furtherdevelopments involve the use of MS2 and MS3 libraries, asdemonstrated for the analysis of pesticides in blood (Dulaurentet al., 2010).

An alternative procedure, also involving the use of a quadru-pole-linear ion trap hybrid instrument, was proposed by the groupofWeinmann,where targeted SRMwith up to 700 transitionswasused as a survey analysismode, and enhanced product ion spectrawere searched against a mass spectral library (Mueller et al.,2005; Dresen et al., 2009, 2010). Whereas the latter procedureseems to be a versatile and sensitive approach to STA, one mayargue that the inherent targeted nature of the use of SRM in thesurvey mode excludes to answer the more general clinical ques-tion whether an individual had been intoxicated at all rather thanintoxicated with a compound from a predefined list (Sauvage &Marquet, 2010). The use of the positive-ion mode only alsonarrows the detection window of the SRM-triggered DDA

compared to the alternating positive-ion and negative-ion full-spectrum-triggered DDA.

Yet another approach involves the use of high-resolutiontime-of-flight (TOFMS) instruments, enabling accurate-massdetermination of drugs and their metabolites. Identification isthen based on searching the formula calculated from the accuratemass against a database of relevant compounds. LC retentiontime data may also be added as an additional criterion to thedatabase. Structures and thus formulae of possible metabolitesmay be taken from literature and also added to the database.Initially, an Applied Biosystems, Carlsbad, CA (www.applied-biosystems.com) Mariner TOFMS system was applied (Gergovet al., 2001a; Pelander et al., 2003). This approach actually allowsscreening for drugs and related compounds without the avail-ability of primary reference standards, as is often the case withdrug metabolites and with street drugs (Pelander et al., 2003;Laks et al., 2004). In the case of street drugs, quantification wasperformed using LC coupled to a chemiluminescence nitrogendetector (CLND) with caffeine as a single secondary standard.The CLND provides an equimolar response to nitrogen (Laks etal., 2004). In more recent studies, a Bruker Daltonics (Bremen,Germany (www.bdal.com)) MicrOTOF system was used, pro-viding enhanced resolution, acquisition speed, as well as moreadvanced software tools, including SigmaFIT, which allows thecompound identification to be based on both the measured accu-rate mass and the isotopic pattern (Ojanpera et al., 2006; Kol-monen et al., 2007). The procedure was applied in both STA anddoping analysis. A recent adaptation of the method for dopinganalysis comprises a generic sample pre-treatment based onmixed-mode SPE on two types of sorbents and the use of bothpositive-ion and negative-ion mode in LC–TOFMS detection(Kolmonen et al., 2009). The modifications to the methodextended the applicability from the analysis of 104 to 197 com-pounds according to the acceptance criteria of the World Anti-DopingAgency (WADA) in two times 8 min (separate analysis inpositive-ion and negative-ionmode). A similar approach, involv-ing the use of a database of over 50,000 chemical formulae, hasbeen recently described. Biological samples are analyzed usingcapillary electrophoresis coupled to a MicrOTOF instrument(Polettini et al., 2008).

Recently, STA based on a combination of accurate-massdetermination on a TOFMS system and searching of a massspectral library generated by in-source CID was described.Compound identification was based on retention time, accuratemass, and fragmentation patterns in in-source CID (Lee et al.,2009). Only limited results have been reported for STA using anorbitrap high-resolution MS instrument (Virus, Sobolevsky, &Rodchenkov, 2008). A next step in the development of LC–MSapproaches in STA is the use of an MS–MS library containingaccurate-mass data, developed on an Agilent Technologies Q-TOFMS instrument (Pragst et al., 2010; Broecker et al., in press).Such a library has been developed and commercialized (Wuest,2010). For a limited set of compounds (�40), this library wasused to checked our MS–MS interpretation against accurate-mass data.

III. METHODS

The starting point of this study was a mass spectral library oftoxicologically relevant compounds available on the internet(Dresen, Kempf, & Weinmann, 2006; Weinmann, 2005),

FRAGMENTATION OF TOXICOLOGICALLY RELEVANT DRUGS &

Mass Spectrometry Reviews DOI 10.1002/mas 3

containing MS–MS spectra of �800 compounds acquired onan AB Sciex API365 triple-quadrupole instrument at threedifferent collision energies (20, 35, and 50 V). First, the spectraof some compound classes were removed from the collection:antibiotics and antimicrobial compounds (�45 compounds),steroids (�30 compounds), illicit drugs and related compounds(�26 compounds), and pesticides (�85 compounds). Fragmen-tation of antibiotic and antimicrobial compounds and of pesti-cides was discussed in separate review articles (Niessen, 1998,2005, 2010). MS–MS fragmentation of illicit drugs has beenreviewed (Castiglioni et al., 2008), while a further study on theMS–MS fragmentation of this compound class is in preparation(Bijlsma et al., in preparation). Finally, steroids were left outbecause a reliable interpretation of their MS–MS spectra withoutaccurate-mass information is not possible (see Section VIII.ESteroids).

Most of the MS–MS spectra of the �570 compounds leftwere interpreted by therapeutic class, after arranging them inrelevant chemical classes. As the mass spectral library whichwas taken as a starting point (Dresen, Kempf, & Weinmann,2006; Weinmann, 2005) only contained positive-ion MS–MSspectra, the current discussion is limited to the fragmentationof protonated molecules [M þ H]þ. The interpretation ofthe MS–MS spectra was done manually. A structure drawingprogram, like ACD Labs ChemSketch (Toronto, Canada,www.acdlabs.com) or HighChem Structure Editor (Bratislava,Slovakia, www.highchem.com), was used to help in rapidlycalculating the m/z values for the fragments. In some cases,the manual interpretation was checked against predictionsfrom HighChem Mass Frontier software (vs. 5.0); in mostcases, this did not add to or give new insights in the interpret-ation of the spectra. In addition, the available scientific literaturewas searched for MS–MS interpretation of these compoundclasses. As a result, ion-trap data have occasionally beenadded, as indicated in the text, although most data are fromtriple-quadrupole MS–MS spectra. Whenever possible, theinterpretation was checked against accurate-mass data, eitherfrom literature or from a recently introduced commercial massspectral library (Pragst et al., 2010; Wuest, 2010; Broeckeret al., in press).

IV. FRAGMENTATION OF EVEN-ELECTRON IONS

Prior to the discussion on the fragmentation of the various com-pound classes, somegeneral fragmentation rules are discussed. Inthis respect, it is important to understand the difference betweenanalyte ionization by means of electron ionization (EI) at onehand and ESI andAPCI at the other. In EI, a molecular ionMþ� isgenerated, which is an odd-electron ion, whereas in ESI andAPCI for most compounds even-electron ions are generated,such as protonated molecules [M þ H]þ, deprotonatedmolecules [M � H]�, or adduct ions like [M þ NH4]

þ,[M þ Na]þ, or [M þ HCOO]�. For a known structure, differ-entiation between an even-electron ion and an odd-electron ioncan be made from the nitrogen rule. According to the nitrogenrule, an odd-electron ion with an odd number of nitrogen atomsshould have an odd m/z, whereas the even-electron ion with anodd number of nitrogen atoms should have an even m/z.

The fragmentation of the molecular ion Mþ� has been exten-sively studied and described, for instance in monographs byMcLafferty and Turecek (1993) or Smith (2004). The most

important fragmentation pathways of a molecular ion Mþ� isthe homolytic or a-cleavage, which is initiated by the radical. Inan even-electron ion, a radical is not present. Therefore, the frag-mentation reactions of even-electron ions are significantly differentfrom those of odd-electron ions. Unfortunately, fragmentation ofeven-electron ions is not as extensively studied. Monographs orcomprehensive reviews are hardly available, except for fragmen-tation of biomolecules (Ham, 2008). Recently, some basic rules ofsmall-molecule fragmentation in ESI mass spectra were reviewed(Levsen et al., 2007; Holcapek, Jirasko, & Miroslav, 2010).

The first general rule involving the fragmentation of even-electron ions is the parity rule or even-electron rule, stating thatupon fragmentation the electron pairs remain intact (Karni &Mandelbaum, 1980). As a consequence, the loss of a neutralmolecule from an even-electron ion, thus resulting in an even-electron fragment ion, is far more likely than the loss of aradical, thus resulting in an odd-electron fragment ion. The eve-n-electron rule has recently been evaluated for a series of 100pesticides (Thurman et al., 2007). For these 100 pesticides, only7% of the 432 fragment ions observed were found to be odd-electron ions. The most important deviations from the even- elec-tron rule involve losses of relatively stable radicals like Cl�, Br�,NO�, and NO2

�, mainly from aromatic ring systems, and the loss ofCH3

� orCH3O� radicals fromaromaticmethoxy compounds (Thur-

man et al., 2007;Holcapek, Jirasko,&Miroslav, 2010). In addition,the loss of a side chain as a radical may occur from conjugatedaromatic ring systems with a side chain.

Themost important even-electron fragmentation inprotonatedmolecules [M þ H]þ involves carbon-heteroatom (N, O, S) clea-vages either by inductive cleavage (with charge migration to the a-carbon) or involving a proton rearrangement (with charge retentionon the heteroatom; Niessen, 2010). These two reactions may beconsidered as competitive and complementary reactions. Therelative abundance of the two fragments is primarily determinedby Field’s rule, indicating that charge retention at the fragmentwiththe higher proton affinity is more favorable and thus that the loss ofthe neutral molecule with a low proton affinity is more likely. TheField’s rule can be considered as the even-electron counterpart ofStevenson’s rule from EI. In addition, the relative abundance of thetwo possible fragments is their relative stability. The sum of them/zvalues of these fragments adds up to the m/z of [M þ H]þ þ 1.

The general principles outlined here can be readily illus-trated with the fragmentation of protonated bromhexine,[M þ H]þ with m/z 377 (precursor ion with 79Br81Br selected;see Fig. 1). The two major fragments, the ions with m/z 114 and264, are due to cleavage between the substituted benzyl and thetertiary amino group. Them/z values for the two fragments add upto 378, which ism/z of [M þ H]þ þ 1. Cleavage at the other sideof the nitrogenmay explain the fragment withm/z 83 (protonatedcyclohexene), but because the complementary fragment ion withm/z 295 is missing, the fragment withm/z 83 is most likely due tosecondary fragmentation of the fragment with m/z 114 (loss ofmethyl amine). As the proton affinity at the (2-amino-3,5-dibro-mophenyl)methanaminium fragment withm/z 295 is higher thanthat of the cyclohexene, its occurrence in the spectrum wouldcertainly be expected. The minor peaks in the bromhexine spec-trum are readily interpreted as secondary fragmentation of thefragment ion with m/z 264 (an even-electron ion: even m/z for anion with one nitrogen atom), involving the loss of Br� resulting intwo odd-electron fragment ions with m/z 185 and 183 (odd m/zwith one nitrogen atom), corresponding to the loss of either 79Br�

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or 81Br�, respectively. Further loss of another Br� (79Br� and 81Br�

from the ions withm/z 183 and 185, respectively), which is likelyto occur from an odd-electron ion, results in the even-electron ionwith m/z 104 (even m/z with one nitrogen atom).

This type of fragmentation, involving cleavage of a carbon-heteroatom bond, is not only observed in amines like bromhex-ine, but also for several other compound classes, including ethersand glycosides, thioethers, esters, and amides. The observation oftwo complementary fragments, them/z values of which add up tothe m/z of [M þ H]þ þ 1, is underlying the nomenclature rulesfor b- and y-ions in the fragmentation of peptides and oligosac-charides (Ham, 2008).

Another fragmentation reaction involves the cleavage of theb-C–C bond relative to the heteroatom (N, O) with charge-retention at the heteroatom, thus resulting in iminium and oxo-nium ions. Ample examples are given below.Obviously, yet othertypes of fragmentation reactions of even-electron ions occur,which are generally not easy to summarize in general rules.The current treatment suffices for the discussion below.However,an underlying goal of studying the fragmentation of many differ-ent classes of drugs (and other compound classes, like pesticides(Niessen, 2010)) is in fact to increase the understanding of small-molecule fragmentation in MS–MS.

V. DRUGS FOR CARDIOVASCULAR DISEASES ANDHYPERTENSION

Antihypertonic or antihypertensive compounds are used to treathypertension (high blood pressure) and are also prescribed inrelation to various cardiac diseases. There are many classes ofantihypertensive compounds, which lower blood pressure bydifferent means. In this section, the interpretation of MS–MSspectra of the various compound classes of antihypertensivedrugs as well as other drugs related to cardiovascular diseasesis discussed. After some information on the individual compoundclasses and their therapeutic use, the relevant general chemicalstructures of the compound class are introduced. In general, acompound class can be subdivided in a number of subclasses.Class-specific fragmentation is discussed for each of these sub-classes, often illustrated with an example.

A. Beta-Blockers or Beta-Adrenergic Antagonists

Beta-blockers, also called beta-adrenergic blocking agents, beta-adrenergic antagonists, or beta-antagonists, are a class of drugs

particularly used for the management of cardiac arrhythmias,cardioprotection after myocardial infarction (heart attack), andhypertension. As they reduce hypertension and trembling insports, their use in sports is prohibited by the WADA. Multi-residue methods for beta-blocker analysis have been reported inrelation to therapeutic drug monitoring (Li et al., 2007), toxi-cology (Johnson & Lewis, 2006), doping control (Gergov et al.,2000; Thevis & Schanzer, 2005;Mazzarino, de la Torre, &Botre,2008; Pujos et al., 2009), food safety analysis (Zhang et al., 2009),and environmental analysis (Hernando et al., 2007; Lee, Sarafin,& Peart, 2007).

Beta-blockers have a general structure of 1-alkylamino-3-phenoxypropan-2-ol, with alkyl being isopropyl, like in atenololand propranolol, tert-butyl, like in timolol or talinolol, or other,like in labetalol or fenoterol (see Fig. 2). Individual compoundsshow further substitution of or changes in the phenoxy group, forexample, a naphthoxy rather than a phenoxy in propranolol, or a4-morpholino-1,2,5-thiadiazolyl group in timolol.

General features in the MS–MS fragmentation of all beta-blockers comprise loss of water, cleavage on either side of theamino-function with charge retention on either side, and com-binations thereof. If upon cleavage around the amino function,charge retention occurs on either side, this leads to series ofcomplementary ions, characterized by the sum of theirm/z valuesbeing equal to the m/z of [M þ H]þ þ 1 (Niessen, 2010).Additional cleavage at the ether bond may be observed, withcharge retention at the 1-(alkylamino)-propan-2-ol part of themolecule and, if sufficient proton affinity is present, in the otherpart. These features are illustrated for propranolol and timolol inScheme 1. Please note, that additional compound-specific frag-ments may be observed due to losses from the substituents on thearomatic ring. These types of losses are not discussed here.

With isopropyl-substituted compounds like propranolol([M þ H]þ, m/z 260, see Scheme 1), one expects primary frag-ment ions with m/z 43 (isopropyl cation, (CH3)2C

þH), m/z 60(protonated isopropylamine, (CH3)2CH–N

þH3), and m/z 116(þCH2–CHOH–CH2–NH–CH(CH3)2), and their complementaryions with m/z of [M þ H – 42]þ, [Mþ H – 59]þ, and[M þ H – 115]þ, respectively, that is, m/z 218, 201, and 145for propranolol. Secondary fragmentation in the low m/z rangeinvolves the ion withm/z 116, that is, loss of water to an ion withm/z 98, loss of propylene (C3H6) to an ion with m/z 74, and acombination of these to an ion withm/z 56. At the highm/z end ofthe spectrum, the secondary fragmentation leads to losses of60 Da (water and C3H6) and 77 Da (water and isopropylamine).

FIGURE 1. MS–MS spectrum of protonated bromohexine (precursor ion with 79Br81Br with m/z 377 selected).

Data from hyphen MassSpec mass spectral collection.



The spectra of 14 compounds of this class, available in the library,were searched for the presence of these ions (see Table 1a); asomewhat similar table was reported by others (Lee, Sarafin, &Peart, 2007). From the data in Table 1a, onemay conclude that thefragment ions with low m/z values are observed for most of thecompounds, whereas at the highm/z values, losses of 59 or 60 Daare relatively rare. For most compounds, either the ion with m/z116 or the ion due to the loss of 77 Da is most abundant at lowcollision energy. InTable 1a, the observation of an ionwithm/z 72is also indicated for most compounds. This ion is due to a b-C–Ccleavage relative to the amine group, resulting in relatively stableiminium ion (H2C=N

þH–CH(CH3)2). These observations areconsistent with interpretations based on deuterium labeling stud-ies and accurate-mass determination (Upthagrove, Hackett, &Nelson, 1999; Kumar, Malik, & Singh, 2008; Wuest, 2010).

In the same way, tert-butyl-substituted compounds liketimolol ([M þ H]þ, m/z 317, see Scheme 1) may be considered.The characteristic fragment ions observed for 14 compounds inthis class, available in the library, are summarized in Table 1b. Forthese compounds, ions with m/z 130, structurally similar to the

above-described ion with m/z 116, are not observed, indicatingthat the tert-butyl group is more readily lost than the isopropylgroup. However, the secondary fragment ion with m/z 74, whichis due to the loss of 56 Da from the ion withm/z 130, is observedfor most compounds.

B. Dihydropyridine Calcium Antagonists

Dihydropyridine calcium channel blockers (CCBs) are a class ofdrugs that disrupt Ca2þ conduction of calcium channels. Thisclass of compounds is easily identified by the suffix ‘‘-dipine’’.Their main clinical usage is to reduce systemic vascular resist-ance and arterial pressure in individuals with hypertension.Multiresidue LC–MS methods for this class of compounds havebeen reported by the group of Weinmann (Mueller, Gonzalez, &Weinmann, 2004; Baranda et al., 2005).

The general structure of the dihydropyridine CCBs ordipines and the side groups of 10 representative compounds isshown in Table 2 (Mueller, Gonzalez, & Weinmann, 2004).

InMS–MS,most dipines show losses of the ester side chainsfrom the 3- and 5-positions (cf. Table 2) at the dihydropyridinering as an alcohol or an alkylene, with the loss of the largest sidechain yielding the most abundant fragment in most cases (Muel-ler, Gonzalez, & Weinmann, 2004). The resulting fragment ionsare used as product ions in SRM in multiresidue LC–MS quanti-fication methods (Mueller, Gonzalez, & Weinmann, 2004). Theresulting fragment ion after the loss of the alcohol is an acyliumion. This behavior can be illustrated with the fragmentation ofnimodipine (Qiu et al., 2004;Miglioranca et al., 2005; Scheme 2).Nimodipine ([M þ H]þ, m/z 419) shows the losses of 60 and76 Da, corresponding to the loss of isopropylalcohol from the R1

side chain and the loss of HOCH2CH2OCH3 from the R2 side

FIGURE 2. Structures of some relevant betablockers, as discussed in the

text.

SCHEME 1

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chain, respectively. The latter fragment (m/z 343) is more abun-dant and shows subsequent losses of 42 Da (C3H6) from the R1

side chain to a fragment ion with m/z 301 and water to m/z 283.This sequence of events, from [M þ H]þ with m/z 419 via theions with m/z 343, which supposedly is an acylium ion, and m/z301 to the ion with m/z 283, raises some interesting mechanisticquestions. Whereas the loss of C3H6 from the ion with m/z 343can in principle be explained as a charge-remote fragmentation,resulting in an ion with a –COOH group in the three-position andthe acylium ion –C:Oþ still in the five-position, the subsequentloss of water to an ion with m/z 283 demands for a charge andhydrogen rearrangement, possibly resulting in aromatization ofthe dihydropyridine ring to a pyridine ring (Scheme 2). Accurate-mass data show that the fragment ion with m/z 255 is due to theloss of a NO2

� rather than the loss of HCOOH from the ion withm/z 301 (Wuest, 2010). The side chain losses from three- andfive-position, that is, the fragment ions corresponding to the ionswith m/z 359, 343, and 301 for nimodipine, are specified for alldipines in Table 2b. A fragment ion corresponding to m/z 301 of

nimodipine is not observed if the 3- and/or 5-position is a methylester.

Amlodipine ([M þ H]þ,m/z 409) does show fragments dueto loss of the R1 and R2 ester side chains from the three- and five-position at the dihydropyridine ring as well, but the MS–MSspectrum is dominated by the loss of NH3 from the –CH2OCH2CH2NH2 group at the two-position, and the sub-sequent fragmentation initiated by this loss, leading to the frag-ment ions with m/z 238 and 294 (Yasuda, Tanaka, & Iba, 1996).The mechanism involved in the formation of the fragment ionwith m/z 294, featuring the formation of an 1,4-oxirane ring, isoutlined in Scheme 3, together with the proposed structure for thefragment ion with m/z 238. This fragmentation route is notobserved in analogues of amlodipine where the primary aminogroup is substituted by a dimethyl or acetyl group (Yasuda,Tanaka, & Iba, 1996). This would sterically hinder the formationof the 1,4-oxirane ring. In ion-trap MS2, the fragment ions withm/z 238 and 294 are not observed (Suchanova, Sispera, & Wsol,2006).

TABLE 1. . Fragmentation of betablockers (a) compounds with isopropyl substituent, and

(b) compounds with tert-butyl substituent

m/z values [M+H] m/z

43 m/z 56

m/z 72

m/z 74

m/z98

m/z116

–18–w

–42–iPr

–59–iPrAm

–60–w

–iPr

–77 –w

–iPrAm Acebutolol 337 x x x x x x 319 260 Alprenolol 250 x x x 232 208 190 173 Atenolol 267 x x x x 249 225 208 190 Befunolol 292 x x x 274 250 232 215 Betaxolol 308 x x x x x 266 231 Bisoprolol 326 x x x x x x 308 Carazolol 299 x x x x x x 257 222 Esmolol 296 x x x x x x 278 254 219 Mepindolol 263 x x x x x x 221 186 Me�pranolol 310 x x x x x x 268 233 Metoprolol 268 x x x x x x 226 191 Oxprenolol 266 x x x x x x 248 206 189 Pindolol 249 x x x x x x 207 190 172 Propranolol 260 x x x x x x 242 218 201 200 183 Toliprolol 224 x x x x x x 206 182 164 147

[M+H] m/z 56 m/z 57 m/z 74 –18

–w–56

–tBu–73

–tBuAm–74–w

–tBu

–91–w

–tBuAmBunitrolol 249 x x x 193 175Bupranolol 272 x x x 216 198 181Carbuterol 268 x 250 212 194 177Carteolol 293 x x x 237 202Celiprolol 380 x x 362 324 307 306 289Clenbuterol 278 204 187Levobunolol 292 x x x 236 219 201Penbutolol 292 x x x 236 201Pirbuterol 241 x 223 185 167Salbutamol 240 x 222 166Talinolol 364 x x x 308 273Tertatolol 296 x x x 240 222 205Timolol 317 x x x 261 244Tulobuterol 228 x 210 172 154



C. Angiotensin-Converting Enzyme Inhibitors

Angiotensin-converting enzyme inhibitors (ACE inhibitors) arepharmaceuticals that are primarily used in treatment of hyper-tension and congestive heart failure. Captopril was the first ACEinhibitor developed (1975) and was one of the earliest successesof structure-based drug design. Due to its sulfhydryl group,captopril shows adverse drug reactions. Other classes of ACEinhibitors were designed that do not have the sulfhydryl moiety.

Most ACE inhibitors are given as (ethyl ester) prodrugs toimprove oral bioavailability.

Angiotensin-converting enzyme inhibitors (ACE inhibitors)can be subdivided into three groups based on their molecularstructure: (1) sulfhydryl-containing agents, like captopril, (2)dicarboxylate-containing agents, like enalapril, and (3) phospho-nate-containing agents, like fosinopril.

The fragmentation of captopril ([M þ H]þ, m/z 218) isoutlined in Scheme 4. The most abundant fragment ion with

TABLE 2. . Dihydropyridine calcium channel blockers; (a) structures of some of the compounds studied,

(b) characteristic fragmentation observed

Substance R1 R2 R3 R4 R5

Amlodipine –CH3 –CH2CH3 –CH2O(CH2)2NH2 –Cl –HFelodipine –CH3 –CH2CH3 –CH3 –Cl –ClIsradipine –CH(CH3)2 –CH3 –CH3 =N–O–N=Lacidipine –CH2CH3 –CH2CH3 –CH3 –CHCHCOOC(CH3)3 –HNicardipine –CH3 –(CH2)2N(CH3)CH2Ph2 –CH3 –H –NO2

Nifedipine –CH3 –CH3 –CH3 –NO2 –HNilvadipine –CN –CH2(CH3)2 –CH3 –H –NO2

Nimodipine –CH(CH3)2 –CH2CH2OCH3 –CH3 –H –NO2

Nisoldipine –CH3 –CH2CH2(CH3)2 –CH3 –NO2 –HNitrendipine –CH3 –CH2CH3 –CH3 –H –NO2

Substance [M+H]+ Loss of R2 Loss of R1 Loss of both R1 and R2

m/z m/z Neutral Lost m/z Neutral

Lost m/z

Amlodipine 409 377 MeOH Felodipine 384 356 C2H4 352 MeOH 324 338 EtOH Isradipine 372 330 C3H6 340 MeOH 298 312 iPrOH Lacidipine 456 410 EtOH 410 EtOH 382

Nicardipine 480 315 HO(CH2)2N(CH3)CH2Ph2 448 MeOH Nifedipine 347 315 MeOH 315 MeOH Nivaldipine 386 344 C3H6 354 MeOH 326 iPrOH

Nimodipine 419 343 HOCH2CH2OCH3 359 iPrOH 301

Nisoldipine 389 315 HOCH2CH(CH3)2 357 MeOH Nitredipine 361 315 EtOH 329 MeOH

3 5

NH

O

OO

OR1

R3

R4

R2

R5

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m/z 116 results from the loss of CO and C3H6S. Chargeretention on the complementary (acylium) ion is also observed(m/z 103), as well as a fragment ion due to the loss of CO from theion with m/z 103. Alternatively, the loss of HCOOH from[M þ H]þ is also observed. A combination of these losses leadsto the fragment ion with m/z 70 (the dihydropyrroliumion, C4H8N

þ).The dicarboxylate ACE inhibitors are administered as mo-

no-ethyl-ester prodrugs, for example, enalapril, which is metab-olized to the active compound, for example, enalaprilat. As anexample, the fragmentation of enalapril ([M þ H]þ, m/z 377) isoutlined in Scheme 4 (Bhardwaj & Singh, 2008). The fragmention with m/z 303 is due to the loss of formic acid ethyl ester(HCOOCH2CH3). The fragment ion with m/z 234 is due to acleavage in the –NH–CH(–CH3)–C(=O)– structure element,resulting in charge retention at the N-side, that is –N–CþH–CH3 or –NH

þ=CH–CH3. The fragment ion with m/z 160 resultsfrom a combination of these two losses (cf., Scheme 4). The samefragmentation routes are also observed with quinapril([M þ H]þ, m/z 439) and ramipril ([M þ H]þ, m/z 417) (seeScheme 4). Thus, fragment ions with m/z 130, 134, 160, and 234are also observed, whereas the loss of HCOOH (instead ofHCOOCH2CH3) results in fragment ions with m/z 365 and343 for quinapril and ramipril, respectively. With lisinopril([M þ H]þ, m/z 406), this fragmentation behavior is hiddenbehind the major fragments ions with m/z 84 and 246 (seeScheme 4), due to the lysyl side chain.

The fragmentation of fosinopril ([M þ H]þ, m/z 564)involves the loss of the ester group of the phosphonate, resultingin the fragment ionswithm/z 436 and 418 (Scheme 4). Secondaryloss of HCOOH from the ion withm/z 436 leads to a fragment ion

withm/z 390. At higher collision energies, the ion withm/z 152 ismost abundant (Scheme 4).

D. Diuretic Drugs

A diuretic is any drug that elevates the rate of urination and thusprovides ameans of forced diuresis. Diuretics are frequently usedas antihypertensive agents. Diuretic agents are misused in sportsto achieve rapid weight loss or to mask the use of other bannedsubstances. There are several categories of diuretics. In our set of(positive-ion) library spectra, data are available for three classes:(1) high ceiling loop diuretics, such as torsemide and bumetanide,(2) thiazide-type diuretics derived frombenzothiadiazine, such ashydrochlorothiazide, benzthiazide, butizide, and indapamide,and (3) epithelial sodium channel blockers, such as amilorideand triamterene. Multiresidue analysis of diuretic drugs is prim-arily directed at doping analysis (Deventer et al., 2002, 2009;Goebel, Trout, & Kazlauskas, 2004; Thevis & Schanzer, 2005;Mazzarino, de la Torre, & Botre, 2008; Tsai & Lee, 2008). Mostdiuretics are analyzed in negative-ion mode (Goebel, Trout, &Kazlauskas, 2004). The fragmentation of diuretic drugs in nega-tive-ion mode has been studied extensively (Garcia et al., 2002;Thevis & Schanzer, 2007; Giancotti et al., 2008).

In positive-ion MS–MS, the thiazide-type diuretics showcomplex and extensive fragmentation, which is difficult to inter-pret without accurate-mass data. Some general fragmentationcharacteristics has been illustrated with butizide (or buthiazide,[M þ H]þ,m/z 354) in Scheme 5. At the high-end of theMS–MSspectrum, the loss of NH3 is observed. The major fragment ionsare due to cleavages of the benzothiadiazine ring, resulting in thefragment ions withm/z 269 (see Scheme 5),m/z 253, andm/z 205

SCHEME 2

SCHEME 3



(due to the loss of SO2 from the fragment ion with m/z 269). Thecomplementary ion tom/z 269, with charge retention at the otherside of the cleavage, may be observed as well.

As an example of a high-ceiling loop diuretic, the fragmen-tation of torsemide ([M þ H]þ,m/z 349) is outlined in Scheme 5.Characteristic losses in the urea moiety, with cleavages on eitherside of the carbonyl, result in fragment ions with m/z 290 (anacylium ion, R–C:Oþ) and m/z 264 (a protonated amine, R–NH3

þ). Cleavage of the C–S bond between the pyridine ring andthe SO2 group results in the fragment ion with m/z 183.

Amiloride ([M þ H]þ, m/z 230) is an epithelial sodiumchannel blocker diuretic drug. Its fragmentation in MS–MS isoutlined in Scheme 5. It shows characteristic fragmentation oneither side of the carbonyl, with charge retention on either side.The fragment ion with charge retention on the pyrazine ring (m/z143) shows secondary fragmentation involving the loss of a Cl� toan ion with m/z 108 or the loss of HC:N to an ion with m/z 116.The identity of most of these fragments was confirmed by accu-rate-mass determination on a linear-ion-trap–orbitrap hybridinstrument (Giancotti et al., 2008).

SCHEME 4

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E. Angiotensin II Receptor Antagonists

Angiotensin II receptor antagonists, also known as angiotensinreceptor blockers or sartans, are drugs which modulate the renin-angiotensin-aldosterone system. Their main use is in hyperten-sion, kidney damage due to diabetes, and congestive heart failure.Multiresidue analysis of angiotensin II receptor antagonists usingLC–MS in human plasma has been reported (Ferreiros et al.,2007).

Losartan, candesartan, irbesartan, olmesartan, and valsartanhave a common structure element, the 1-[20-(1H-tetrazol-5-yl)bi-phenyl-4-yl]methyl group. For reference, the structures of los-artan, irbesartan, and valsartan are given in Scheme 6. This groupleads to three common fragment ions withm/z 235, 207, and 192.Structures for these fragments are proposed in Scheme 6 (Zhaoet al., 1999). The identity of these fragments was confirmed usingaccurate-mass determination on a TOF MS instrument (Shah,Sahu,&Singh, 2010). At the high end of theMS–MS spectrum oflosartan ([M þ H]þ, m/z 423), the loss of 18 Da (water) and46 Da (water andN2) is observed (Zhao et al., 1999), whereas theloss of both N2 and HN3 from the tetrazole ring is observed forirbesartan ([M þ H]þ,m/z 429) (Shah, Sahu, & Singh, 2010). Inaddition to these class-specific fragments, various compound-specific fragments are observed, involving the various sidechains. With irbesartan ([M þ H]þ, m/z 429), the complemen-tary fragment to the ion with m/z 235 is observed with m/z 195.With valsartan ([M þ H]þ,m/z 436), structure-specific fragmentions withm/z 306 and 291 are observed, which are due to the lossof the (H3C)2CH–CH2–COOH side chain and either N2 or HN3

from the tetrazole ring (Lu et al., 2009a). An alternativeinterpretation of these fragments, involving the loss of the tetra-zole ring and one of the phenyl rings (Koseki et al., 2007) is mostlikely incorrect, given the observations with related compounds.

F. Other Antihypertensive Compounds

Next to the beta-blockers or beta-adrenergic antagonists, thedihydropyridine calcium antagonists, the angiotensin-convertingenzyme inhibitors, the diuretic drugs, the angiotensin II receptorantagonists discussed in the previous sections, there are a numberof other classes of antihypertensive compounds, including a1-adrenoceptor antagonists, a2-adrenoceptor agonists, centrallyacting adrenergic drugs, and vasodilators. Multiresidue analysisof various classes of antiarrhythmic in human plasma has beenreported (Li et al., 2007). The MS–MS fragmentation of some ofthe chemical classes involved is discussed here.

1. a1-Adrenoceptor Antagonists

The a1-adrenoceptor antagonists comprise of compounds likeurapidil, trimazosin, prazosin, terazosin, tamsulosin, alfuzosin,doxazosin, and silodosin. They are not only used to treat hyper-tension, but also benign prostatic hyperplasia. Except tamsulosin,alfazosin, and silodosin, these compounds are characterized byan N,N0-substituted piperazine ring, which plays an importantrole in the fragmentation. The typical MS–MS fragmentation ofthese compounds is illustrated for terazosin ([M þ H]þ,m/z 388)in Scheme 7. Fragmentation of the piperazine ring results in two

SCHEME 5



complementary fragments with m/z 247 and 142. Alternativecleavage of the ring results in the fragment ion with m/z 221.Similar cleavage of the piperazine ring is observed for prazosin,trimazosin, and doxazosin. Multistage MSn fragmentation oftamsulosin ([M þ H]þ, m/z 409) leads to a series of fragments,outlined in Scheme 7 (Nageswara Rao et al., 2008). Some frag-ments in the MS4 spectrum are not understood.

2. a2-Adrenoceptor Agonists and CentralAdrenergic Drugs

This compound class comprises of a number of compoundswith aguanidine group, such as guanabenz, guanoxan, and guanfacine.Characteristic fragmentation involves the loss of NH3, the loss ofHN=C=NH (42 Da), and the loss of guanidine HN=C(NH2)2(59 Da), as well as the loss of the substituent with charge reten-tion at the guanidine, resulting in an ion with m/z 60.

The MS–MS fragmentation of clonidine and moxonidinedoes not result in structure-informative fragmentation. The mostabundant fragment ion is an ion with m/z 44 (H2N–CH2CH2

þ).Phentolamine ([M þ H]þ, m/z 282) yields an intense fragmentionwithm/z 212, due to the loss of the 4,5-dihydro-1H-imidazolering.

Extensive fragmentation is observed for the adrenergicreceptor antagonist urapidil ([M þ H]þ,m/z 388),mainly involv-ing cleavages in the propylamine chain in the molecule (seeScheme 7). Cleavage at the piperazine ring results in two frag-ments, the odd-electron ion with m/z 190 and the even-electronion with m/z 196. Other major even-electron fragments are

observed withm/z 205, 233, 248, and 141 (see Scheme 7). Thesefragments were confirmed by accurate-mass data (Wuest, 2010).In addition, various less abundant fragments are observed.

3. Vasodilators

Vasodilation refers to the widening of blood vessels, which leadsto a decrease in blood pressure. Compounds that result in vaso-dilation are termed vasodilators. Chemically, vasodilators showgreat differences in structure, from a small molecule like 2-hydroxymethylpyridine ([M þ H]þ,m/z 110) to a largemoleculelike inositol nicotinate ([M þ H]þ, m/z 811). Most of the com-pounds in the mass spectral library show readily interpretablefragmentation. The fragmentation of butalamine ([M þ H]þ,m/z317) may serve as an example of this (see Scheme 7). Initially,two major fragments are formed, that is, an ion with m/z 188,which is due to the loss of dibutylamine, and an ion withm/z 199,resulting from the cleavage in the oxadiazole ring. At highercollision energies, additional fragments are observed (seeScheme 7). A fragment ion with m/z 143 is due to secondaryfragmentation of the ion with m/z 199 (loss of C4H8). Anothersecondary fragment is the ion withm/z 87 (see Scheme 7). Thesefragments were confirmed by accurate-mass data (Wuest, 2010).

The vasodilator ajmalicine ([M þ H]þ, m/z 353), an alka-loid fromCatharanthus roseus roots, shows twomajor fragments,which both result from cleavages in the piperidine ring (seeScheme 7; Lu et al., 2009b; Ferreres et al., 2010). Additionally,both [M þ H]þ and the fragment ion withm/z 210 show a loss of32 Da (CH3OH) from the methyl ester group.

SCHEME 6

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The vasodilators dilazep ([M þ H]þ, m/z 605) and hexo-bendine ([M þ H]þ, m/z 593) have symmetric structureswith propyl-trimethoxybenzoates attached to a diamine, thatis, ethylene diamine in hexobendine and a diazepine ring indilazep. Both compounds show fragment ions with m/z 195(trimethoxybenzene acylium ion, (CH3O)3C6H2–C:Oþ) and253, the propyl-trimethoxybenzoate cation ((CH3O)3C6H2–C(=O)O–CH2CH2CH2

þ).

4. Phosphodiesterase-5 Inhibitors

The vasodilator sildenafil ([M þ H]þ, m/z 475), also known asViagra, is a phosphodiesterase-5 inhibitorwhich is primarily usedfor the treatment of erectile dysfunction. Sildenafil and relatedcompounds like vardenafil (Levitra) and tadalafil (Cialis) arefrequently found as adulterants in ‘‘natural’’ herbal medicines(Bogusz et al., 2006; Gratz, Gamble, & Flurer, 2006). A multi-residueLC–MSmethod for the detection of 80 common syntheticadulterants from various pharmacological classes in herbal rem-edies has been described (Bogusz et al., 2006). The fragmentationof sildenafil and six related compounds was studied with the helpof accurate-mass measurement using a Fourier-transform ion-cyclotron resonance mass spectrometer (Gratz, Gamble, &Flurer, 2006).

Abundant fragments of sildenafil result from cleavages oneither side of the SO2 group (see Scheme 8), involving the loss of1-methylpiperazine sulfinic acid to a fragment ion with m/z 311,or the charge retention on the piperazine part of the molecule tofragment ions withm/z 99, 100 and 163. Both an odd-electron ionwith m/z 100 and an even-electron ion with m/z 99 is observed.Accurate-mass data confirm that the fragment ion with m/z 283results from the loss of C2H4 from the fragment ion withm/z 311(and not from a cleavage between the benzene and the pyrimi-dinone ring with charge retention on the benzene ring). Thefragment ion with m/z 377 shows secondary fragmentationinvolving a rearrangement in the SO2 group, resulting in theloss of SO to a fragment ion with m/z 329 and subsequent lossof C2H6 to a fragment ion with m/z 299 (see Scheme 8). Therearrangement in the SO2 group resulting in the loss of SO is alsoobserved and extensively studied for sulphonamide antibiotics(Niessen, 1998; Klagkou et al., 2003; Sun, Dai, & Liu, 2008).Secondary fragmentation of the 1-methylpiperazine ring (m/z 99or 100) leads to a fragment ion with m/z 58 (C3H8N

þ).Vardenafil (Gratz, Gamble, & Flurer, 2006; Ku et al., 2009)

and aildenafil (Wang et al., 2007) show similar fragmentation tosildenafil, because they also contain the =N–SO2– group,whereas tadalafil due to its different structure shows differentfragmentation routes (Gratz, Gamble, & Flurer, 2006).

SCHEME 7



Pentoxifylline en diprophylline and other methylxanthinesact as nonselective phosphodiesterase-5 inhibitors. Pentoxifyl-line ([M þ H]þ, m/z 279) shows the loss of the 5-oxohexyl sidechain to a fragment ion with m/z 181 as well as the complemen-tary fragment ion with m/z 99, with charge retention at theoxohexyl. The ion with m/z 181 shows the characteristic frag-mentation of the xanthine structure, that is, the formation offragment ions with m/z 138 and 110, due to losses of HNCO,and HNCO and CO, respectively (see Section VI.H).

5. Other Calcium Channel Blockers

Next to the dihydropyridine calcium antagonist (Section V.B),there are a number of other compounds with similar function,especially phenylalkylamines like verapamil and benzothiaze-pines like diltiazem.

Verapamil ([M þ H]þ, m/z 455) shows four fragments(Scheme 8). Two fragments, that is, the ions with m/z 165 and260, are due to cleavages of C–N bonds. The ion with m/z 150 isdue to secondary fragmentation of the ionwithm/z 165, involvingthe loss of a CH3

� radical. The other, less abundant, fragment ionwith m/z 303 is due to a b-C–C bond cleavage relative to thecentral N-atom, resulting in an iminium ion (Rousu, Herttuainen,& Tolonen, 2010). The metabolism of verapamil has been exten-sively investigated (Walles et al., 2003).

Diltiazem ([M þ H]þ, m/z 415) shows subsequent losses ofdimethylamine to an ion with m/z 370 and acetic acid to an ionwithm/z310. The base peak is an ionwithm/z178,which is due tothe loss of all side chains from the benzothiazocinone ring(Scheme 8). Diltiazem is extensively metabolized. MultiresidueLC–MSanalysis of diltiazemand 11 of its Phase Imetabolites hasbeen described. The fragment ion with m/z 178 is a commonfragment for all Phase I metabolites (Molden et al., 2003).

VI. PSYCHOTROPIC OR PSYCHOACTIVECOMPOUNDS

A psychoactive drug, psychopharmaceutical or psychotropiccompound is a chemical substance that acts primarily uponthe central nervous system where it alters brain function, result-ing in changes in perception,mood, consciousness, and/or behav-ior. These drugs may be used recreationally, to purposefully alterone’s consciousness, or therapeutically as medication.

A. Phenothiazines

Phenothiazines are frequently used as neuroleptic (antipsychotic)drugs, for example, chlorpromazine, and fluphenazine, suitablefor the treatment of several mental disorders. They mainly actas inhibitors of dopamineD1- andD2-receptors. In addition, somephenothiazines are used as antihistaminic drugs, for example,promethazine (see Section VIII.B). Multiresidue analysisof phenothiazines in human body fluids has been reported(Kumazawa et al., 2000; Kratzsch et al., 2003).

10H-Phenothiazine is a tricyclic heterocyclic compoundwith general formula S(C6H4)2NH. One of the two benzene ringsmay be substituted at the two-position, for instance with a Cl-group, as in chlorpromazine, prochlorperazine, and perphena-zine, or a CF3-group, as in fluphenazine and trifluoperazine.Mostphenothiazine antipsychotic drugs can be classified into twogroups that differ with respect to the ring-N substituent at thephenothiazine: (a) compounds with a 1-propyl-4-methylpipera-zine substituent, like (prochlor)perazine and trifluoperazine,or with a 2-(4-propylpiperazin-1-yl)ethanol substituent likein (flu)perphenazine, and (b) compounds with an acyclic sub-stituent such as N,N-dimethyl-propan-1-amine, like (chlor)pro-mazine. In addition, some other structures are available, likewith

SCHEME 8

& NIESSEN


an 4-propyl-piperidine substituent (in propericiazine) and with aN,N-dimethyl-propan-2-amine substituent (in promethazine).

Characteristic fragmentation patterns of promazine([M þ H]þ, m/z 285) and perazine ([M þ H]þ, m/z 340) areshown in Scheme 9. Cleavages occur at various sites in thepropyl-chain between the dimethylamine or N-methyl-pipera-zine group and the phenothiazine tricyclic ring (McClean,O’Kane, & Smyth, 2000).

Cleavage of the C–N-bond at the phenothiazine-N occursmostly with charge retention at the aliphatic amine part, thusleading to fragment ions with m/z 86 in promazine, with m/z141 in perazine, and with m/z 171 with perphenazine(–CH2CH2OH instead of –CH3 substituent at piperazine ring).Less abundant ions result from charge retention at the pheno-thiazine side, either as a protonated phenothiazine (m/z 200 þ 2-substituent) or as a sulfonium ion (m/z 198 þ 2-substituent).

The cleavage of ab-C–C-bond relative to the phenothiazine-N results in an iminium ion (m/z 212 þ 2-substituent). The loss ofelemental S is frequently observed from this iminium ion (m/z180 þ 2-substituent, see Scheme 9).

The cleavage of the b-C–C-bond relative to the dimethyl-amine-N or piperazine-N results in abundant iminium ions withm/z 58 (H2C=N

þ(CH3)2) or m/z 113 (Scheme 9). Secondaryfragmentation of the iminium ions containing the piperazine ring(m/z 113) may lead to additional fragment ions with m/z 70(C4H8N

þ) and 98 (C5H10N2).The cleavage of the C–N-bond at the dimethylamine-N or

piperazine-N results in charge retention at the phenothiazine part(m/z 240 þ 2-substituent).

The cleavages leading to the characteristic fragments withm/z 141, 113, and 212 were also described by others (Kumazawaet al., 2000; McClean, O’Kane, & Smyth, 2000). A recentlyreported secondary fragmentation of the ion with m/z 240 to afragment ion with m/z 166 (C9H12NS

þ) was not observed in ourset of library spectra (Wen & Zhou, 2009). With phenothiazinescontaining a 2-Cl-substituent, the loss of Cl� may occur (Wen &Zhou, 2009).

In ion-trap MSn of chlorpromazine ([M þ H]þ, m/z 318),subsequent cleavages occur in subsequent stage of MSn,that is, the loss of 45 Da (dimethylamine) in MS2, of 28 Da(C2H4) in MS3, and of 32 Da (an S atom) in MS4 (Joyce et al.,2004).

Similar fragmentation routes are observed for the pheno-thiazines with different N-substitution.

Phenothiazines are extensively metabolized, involving sul-foxidation, aromatic-ring hydroxylation, N-demethylation, andN-oxidation. Unfortunately, MS–MS spectra of phenothiazinemetabolites were not present in the library collection. Therefore,the influence of metabolism, especially the sulfoxidation, on thefragmentation cannot be discussed.

B. Other Classes of Neuroleptic Drugs

The phenothiazines are one of the three compound classes of theso-called first-generation antipsychotics, next the butyrophe-nones and the thioxanthenes. Subsequently, the second-gener-ation or so-called atypical antipsychotic drugs were developed,including risperidone, and clozapine and other dibenzazepineanalogues. Both first- and second-generation antipsychotics tendto block receptors in the dopamine pathways in the brain,although they often encompass a wide range of receptor targets.Multiresidue LC–MS analysis of various classes of neurolepticdrugs, including phenothiazines, has been described using single-quadrupole MS, using in-source CID and library searching(Kratzsch et al., 2003) andSRM in a triple-quadrupole instrument(Kirchherr & Kuhn-Velten, 2006; Saar et al., 2009). In order toimprove confidence of identity during analysis, three SRM tran-sitions per compounds rather than one have been applied (Saaret al., 2009).

Risperidone [M þ H]þ, m/z 411) is an atypical antipsy-chotic drug used in the treatment of schizophrenia and otherstates associated with bipolar disorder, and irritability in childrenwith autism. In MS–MS, risperidone shows only one majorfragment ion with m/z 191 (see Scheme 10), and a minor sec-ondary fragment ion with m/z 163 due to subsequent loss of CO(McClean, O’Kane, & Smyth, 2000). Further fragmentation maybe observed using MSn in an ion-trap instrument (McClean,O’Kane, & Smyth, 2000). The major metabolites, 7- and 9-hydroxyrisperidone, show the same cleavages, thus leading toa major fragment ion with m/z 207.

Pyritinol ([M þ H]þ,m/z 369), generated by binding of twopyridoxine (vitamin B6) molecules via a disulfide bridge, showsunusual fragmentation: two odd-electron fragments are gener-ated with m/z 153 and 217, respectively, differing S2 (seeScheme 10). Both fragments show loss of water to m/z 135and 199, respectively.

Amisulpride, sulpiride, and tiapride are atypical antipsy-chotics, carrying an SO2 group. The fragmentation of sulpiride([M þ H]þ, m/z 342) and tiapride ([M þ H]þ, m/z 342) is out-lined in Scheme 10. The fragmentation of sulpiride involvescommon fragmentation reactions at amide and amine bonds.Similar fragmentation is observed for tiapride, however secon-dary fragmentation of the ions with m/z 256 and 213, involvingthe loss of H3C–SO2

� results in the formation of two radicalcations with m/z 177 and 134.

The thioxanthene antipsychotics flupentixol ([M þ H]þ,m/z 435) and zuclopenthixol show very similar fragmentation(Scheme 10). Initial fragmentation involves the loss of anSCHEME 9



unexpected HOCH2CH2� radical to a fragment ion withm/z 390.

The same loss is observed for zuclopenthixol. Two major frag-ments are observed, containing the thioxanthene structure; theseare ions with m/z 265 and 305. Other fragments contain thepiperazine ring, such as the even-electron ion with m/z 139and the radical cation with m/z 128 (Scheme 10). Ion-trap frag-mentation of flupentixol has been reported as well (McClean,O’Kane, & Smyth, 2000).

1. Butyrophenones

Butyrophenones are used to treat various psychiatric disorderssuch as schizophrenia. They also act as antiemetics. Typicalexamples include bromperidol, droperidol, fluanisone, haloper-idol, melperone, moperone, trifluperidol. The characteristic frag-mentation of the butyrophenones is illustrated for haloperidol([M þ H]þ,m/z 376) in Scheme 11. Characteristic fragment ionsare observed withm/z 95 due to F–C6H4

þ, withm/z 123 due to F–C6H4–C:Oþ, and with m/z 165 due to F–C6H4–C(=O)–CH2CH2CH2

þ. A fragment ion complementary to the ion withm/z 165 may be observed as well, that is, an ion with m/z 212 (orwith m/z 194 after loss of water) for haloperidol, an ion with m/z100 for melperone ([M þ H]þ, m/z 264), or an ion with m/z 246for trifluperidol ([M þ H]þ, m/z 410).

Pipamperone ([M þ H]þ, m/z 376) is an isobar ofhaloperidol. However, next to the common fragment ionswithm/z 165 and 123, pipamperone shows several other discrim-inative fragments ions, such as the ions with m/z 291 and 98(see Scheme 11).

2. Dibenzazepines and Related Structures

The dibenzazepine structure is a common feature of sometricyclic antidepressants, including imipramine, lofepramine,and trimipramine (see Section VI.C), as well as of second-gener-ation antipsychotics like clozapine ([M þ H]þ,m/z 327). Relatedstructures like loxapine ([M þ H]þ, m/z 328) and quetiapine([M þ H]þ, m/z 384) contain an oxazepine or thiazepine ringinstead of the azepine ring. They have an N0-substituted piper-azine ring attached to the seven-member ring. Characteristicfragmentation involves the piperazine ring and is illustratedfor clozapine in Scheme 11. Loss of methylamine (31 Da), ofH3C–N–CH=CH2 (57 Da), and of 1-methylpiperazine (100 Da)is observed. For both clozapine and loxapine, subsequent loss ofCl� is observed, leading to fragment ions with m/z 192 and 195,respectively.

C. Antidepressants

Tricyclic antidepressants (TCAs) and tetracyclic antidepressants(TeCAs) are heterocyclic compounds that were introduced asantidepressants in the late 1950s (TCAs) and in the 1970s(TeCAs). In clinical use, the TCAs have nowadays been largelyreplaced by newer types of antidepressants such as the selectiveserotonin reuptake inhibitors (SSRIs) and serotonin-norepi-nephrine reuptake inhibitors (SNRIs). Multiresidue LC–MSmethods for the analysis of antidepressants in plasma andother body fluids for therapeutic drug monitoring as well as inwastewater and raw sewage from a sewage treatment plant for

SCHEME 10

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environmental analysis have been described (Kollroser &Schober, 2002; Kirchherr & Kuhn-Velten, 2006; Sauvageet al., 2006b; de Castro et al., 2007, 2008; Lajeunesse, Gagnon,& Sauve, 2008; Breaud et al., 2010).

1. Tricyclic Antidepressants

Two structurally different classes of TCAs can be distinguished,one based on a dibenzazepine tricyclic group, like in trimipr-amine ([M þ H]þ,m/z 295) and lofepramine, and one based on adibenzocycloheptane group, like in amitriptyline ([M þ H]þ,m/z 278) and protriptyline. Alternative structures, such as withan oxepine ring, for example, doxepin, or a thiepine ring, forexample, dosulepin, are also available.

To some extent, the fragmentation of TCAs is similar to thatof phenothiazines. Characteristic to all TCAs with a dimethyl-amine group in the side chain (amitriptyline, trimipramine) is theloss of 45 Da (dimethylamine) (Kollroser & Schober, 2002) andthe formation of an iminium ion with m/z 58 (H2C=N

þ(CH3)2)due to a b-C–C cleavage (see Scheme 12 for amitriptyline). Instructures with methylamine (nortriptyline, protriptyline), theloss of 31 Da (methylamine) is observed instead of that of45 Da. The fragment ion with m/z 58 and those resulting fromthe loss of 31 or 45 Da are generally used in SRM for quanti-fication of these drugs in biological matrices (Kollroser &Schober, 2002; de Castro et al., 2007, 2008; Lajeunesse, Gagnon,& Sauve, 2008). One may question the selectivity of such atransition (Allen, 2006). Cleavage of the side chain at the aze-pine-Nmay lead to two complementary fragments due to possiblecharge retention on either side.

In amitriptyline, fragmentation of the dibenzocycloheptaneskeleton is observed at higher collision energies. Structure pro-posals for the more abundant fragments are given in Scheme 12.The formulae of these fragments are confirmed by accurate-massdata. Similar fragmentation is observed in theMS–MS spectra ofnortriptyline and protriptyline.

With opipramol ([M þ H]þ, m/z 364), which has an N-linked 2-(4-propylpiperazin-1-yl)ethanol side chain (similar to

the phenothiazine perphenazine), the most abundant fragmentions are due to a cleavage of the C–N-bond at the azepine-N (withm/z 171, charge retention at the side chain) and of the b-C–C-bond relative to the piperazine-N (with m/z 143, iminium ion).These fragments were also observed for perphenazine and relatedcompounds (see Section VI.A).

2. Tetracyclic Antidepressants

The fragmentation of the TeCAs mianserin and mirtazepine([M þ H]þ, m/z 266) is characterized by typical losses from amethyl-substituted piperazine ring, that is, losses of 31 Da(H3CNH2), of 43 Da (H3C–N=CH2), and of 57 Da (H3C–N=CH–CH3) (see Scheme 12). For mirtazepine, an additionalfragment ion withm/z 195 is observed, which is due to the loss ofCH2=CH–N(CH3)2. The latter fragment is the most abundantfragment ion of mirtazepine in ion-trap MS2. The formulae ofthese fragment ions were checked using high-resolutionMS on aQ-TOF instrument (Smyth et al., 2006).

The TeCA maprotiline ([M þ H]þ, m/z 278) is isobaric tothe TCA amipriptyline. Although these compounds have a num-ber of fragment ions in common, discrimination appears to bepossible based on the primary loss of 45 Da (HN(CH3)2) to afragment ion with m/z 233 for amitriptyline and the primary lossof 28 Da (C2H4) due to a retro-Diels-Alder fragmentation inmaprotiline.

3. Selective Serotonin Reuptake Inhibitors

Next to the TCAs and TeCAs, there are three other classes ofantidepressant drugs. The selective serotonin reuptake inhibitors(SSRIs) aremodern antidepressants with less adverse effects thanTCAs. SSRIs act upon and increase the levels of serotonin in thebrain which is known to play an important role in mood. From achemistry point-of-view, these drugs do not share clear commonstructural features. Therefore, class-specific fragmentation can-not be defined. Nevertheless, some examples are briefly dis-cussed. Fragmentation of the SSRIs citalopram, fluoxetine,

SCHEME 11



paroxetine and sertraline was studied using ion-trap MSn andaccurate-mass determination on a Q-TOF instrument (Smythet al., 2006).

In ion-trapMS2, citalopram ([M þ H],m/z 325, Scheme 13)generates an abundant fragment ion with m/z 262, and lessabundant ions with m/z 307 and 280 (Smyth et al., 2006; Ramanet al., 2009). The fragment ion with m/z 307 is due to the loss ofwater, which most likely results in ring opening of the furanring (Raman et al., 2009), although an alternative structureinvolving a rearrangement of the N,N-dimethyl-propylaminegroup was proposed as well (Smyth et al., 2006). The fragmentions with m/z 280 and 262 are due to the loss of dimethylaminefrom the ions with m/z 325 and 307, respectively. Further frag-mentation of the ion with m/z 262 results in a fragment ion withm/z 234 due to the loss of C2H4 (in MS3) and withm/z 215 due tothe subsequent loss of F� (in MS4) (Smyth et al., 2006). Theinterpretation is confirmed by Q-TOF data (Smyth et al., 2006).More extensive fragmentation of citalopram is observed in atriple-quadrupole instrument (Jiang et al., 2010). Next to thefragments already reported and a number of additional minorfragments, the most abundant fragment is the ion with m/z 109(F–C6H4–CH2

þ), whereas an ion with m/z 116 (N:C–C6H4–CH2

þ) is also readily observed (see Scheme 13). The SRMtransition m/z 325 > 109 is applied for quantitative analysis ofcitalopram (de Castro et al., 2007, 2008; Jiang et al., 2010). Thelow-m/z fragments observed with the triple quadrupole instru-ment are not observed in the ion-trapMSn spectra, possibly due tothe inability to trap fragment ions with m/z-values less than�25% of the m/z of the precursor ion (Hakala, Kostiainen, &Ketola, 2006).

Paroxetine ([M þ H]þ, m/z 330, Scheme 13) is reported toform product ions with m/z 220 and 70 (Massaroti et al., 2005).The fragment ion with m/z 220 was erroneously attributed to theloss of H2C=CH–C6H4–F (122 Da), whereas in fact it shouldinvolve the loss of H3C–C6H4–F (110 Da). This fragmentationpattern is not in agreement with our library data, which showsions with m/z 192, 178, 163, 151, 123, and 70 as the majorfragment ions. Proposed structures for these fragments are givenin Scheme13; formulae are in agreementwith accurate-mass datafrom different sources (Smyth et al., 2006; Wuest, 2010). Afragment ion with m/z 192 is also the most abundant fragmentin ion-trap MS2, with less abundant ions with m/z 313 (loss ofNH3), 151, and 123 (Smyth et al., 2006).

The MS–MS spectrum of fluoxetine ([M þ H]þ, m/z 310),also known as Prozac, in the library collection most likely is notcorrect, because it shows too many fragments that cannot bereadily explained from the structure; it also differs from spectrapublished elsewhere. Characteristic fragmentation of fluoxetineinvolves the loss of F3C–C6H4–OH (162 Da) to a fragment ionwith m/z 148. At higher collision energies, fragments with m/z117 (C9H9

þ) and 91 (C7H7þ) may be observed. In the low m/z

range, a fragment with m/z 44 (H2C=NþH–CH3) is observed

(Sutherland et al., 2001; Smyth et al., 2006). The formulae wereconfirmed by accurate-mass data (Wuest, 2010).

4. Other Antidepressants

Monoamine oxidase inhibitors (MAOI’s) are only prescribed inspecial cases because of possible dietary and drug–drug inter-actions. Moclobemide is an example of this class. The

SCHEME 12

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fragmentation of moclobemide is outlined in Scheme 14. Itinvolves straightforward cleavages of C–N or amide bonds.

Similar to selective serotonin reuptake inhibitors (SSRIs),the serotonin-norepinephrine reuptake inhibitors (SNRIs) aremodern antidepressants with less adverse effects than TCAsand MAOIs. Fragmentation of the SNRI venlafaxine([M þ H]þ, m/z 278, Scheme 14) was studied using ion-trapMSn and accurate-mass determination on a Q-TOF instrument(Smyth et al., 2006) and in a triple-quadrupole instrument (Bhattet al., 2005; Patel et al., 2008). In ion-trap MSn, a water loss was

observed inMS2, a subsequent loss of dimethylamine inMS3, andfinally the loss of C4H8 in MS4, resulting in an ion with m/z 159(Smyth et al., 2006). The latter ion is hardly observed in triple-quadrupoleMS–MS spectra, indicating that multi-stageMSn andion-trap CID can yield structural information different fromcollision-cell CID in a triple quadrupole instrument. Next towater and subsequent dimethylamine losses, four additional frag-ment ions are observed in the triple-quadrupole MS–MS spec-trum (Bhatt et al., 2005; Patel et al., 2008), that is, ionswithm/z 58(H2C=N

þ(CH3)2), m/z 121 (H3C–O–C6H4–CH2þ), m/z 147 and

173, for which structure proposals are provided in Scheme 14.

D. Benzodiazepines

Benzodiazepines are psychoactive drugs that enhance the effectof the neurotransmitter g-aminobutyric acid (GABA), whichresults in sedative, hypnotic, anxiolytic, anticonvulsant, musclerelaxant, and/or amnesic action. As such, they are useful in avariety of indications such as alcohol dependence, seizures,anxiety, panic, agitation, and insomnia. Benzodiazepines arecommonly misused and taken in combination with other drugsof abuse. When combined with other central nervous systemdepressants such as alcohol and opiates, the potential for toxicityincreases. Multiresidue LC–MS analysis of benzodiazepines hasfrequently been reported, using either selected-ion monitoring(SIM) on single-quadrupole instruments (Kratzsch et al., 2004;Ishida et al., 2009), SRM on triple-quadrupole instruments (e.g.,Villain et al., 2005; Marin et al., 2008; Badawi et al., 2009;Nakamura et al., 2009;Glover&Allen, 2010;Marin&McMillin,2010), SRM on ion-trap instrument (Smink et al., 2004), oraccurate-mass determination on time-of-flight instruments(Hayashida et al., 2009; Nielsen et al., 2010). The benzo-diazepines are analyzed in matrices such as whole blood, plasma,serum, urine, hair, meconium, and saliva.

SCHEME 13

SCHEME 14



The core chemical structure of benzodiazepines is the fusionof a benzene ring and a diazepine ring. The drugs are substituted1,4-benzodiazepines with different side groups attached to thecore structure, which affect the binding of the molecule to theGABA receptor and therebymodulate the pharmacological prop-erties. Many of the benzodiazepine drugs contain a 5-phenyl-1H-benzo[e][1,4]diazepin-2(3H)-one substructure, for example, dia-zepam, whereas others have an additional hydroxy-substituent atthe three-position, for example, oxazepam, or a methyl-triazoloring attached to the diazepine ring, for example, alprazolam.

Fragmentation of benzodiazepines has been systematicallystudied using ion-trap andQ-TOF instruments (Smyth,McClean,& Ramachandran, 2000; Smyth et al., 2004), and using a quadru-pole–linear-ion-trap hybrid instrument (Risoli et al., 2007). TheMS–MS fragmentation of three exemplary benzodiazepines (dia-zepam, oxazepam, and flunitrazepam) is discussed here.

In ion-trap MSn, diazepam ([M þ H]þ, m/z 285, seeScheme 15) shows the loss of 28 Da (CO) by contraction ofthe diazepine ring to an ion withm/z 257 in MS2, followed by theloss of either 35 Da (Cl�) to an ion with m/z 222, or of 29 Da(H2C=NH) to an ion with m/z 228 in MS3. In MS4, the ion withm/z 228 shows the loss of 35 Da (Cl�) to an ion with m/z 193(Smyth, McClean, & Ramachandran, 2000). The fragment ionswith m/z 257, 228, 222, and 193 are also observed in triple-quadrupoleMS–MS spectra. In addition, a fragment ion withm/z154 is observed. Structure proposals for these fragment ions aregiven in Scheme 15 (Risoli et al., 2007). Writing a structureproposal for the ion withm/z 154 may serve as an example of thedifficulties in finding general fragmentation rules for benzo-diazepines. The fragment ion with m/z 154 corresponds to theloss of 131 Da and may thus result from the subsequent loss ofCO and benzonitrile C6H5–C:N (proposal (i) in Scheme 15).Thismay be true for diazepam, but a similar loss of 131 Da is also

observed for analogues that lack the N1-methyl substituent, suchas nordiazepam, prazepam (after the loss of the cyclopropyl-methyl group from N1), and 7-aminonitrazepam (loss of 149 Da,CO, and fluorobenzonitrile) (Badawi et al., 2009; Nakamuraet al., 2009). Therefore, the structure proposal (ii) inScheme 15 seems to be more likely (Risoli et al., 2007).

Oxazepam ([M þ H]þ, m/z 287) is the N1-desmethyl, 3-hydroxy analogue of diazepam. In ion-trap MSn, subsequently,the loss of water to an ion with m/z 269 in MS2, and of CO to anionwithm/z 241 inMS3 is observed. InMS4, the ionwithm/z 241shows losses of benzene to an ion withm/z 163, of benzonitrile tom/z 138, of Cl� to m/z 206, and of HC:N to m/z 214 (Smyth,McClean, & Ramachandran, 2000). In a triple-quadrupole MS–MS spectrum, next to these ions, a protonated benzonitrile withm/z 104, and an ionwithm/z 231, consistentwith the loss of C2O2,is observed (Risoli et al., 2007).

The primary and predominant fragmentation of flunitraze-pam ([M þ H]þ, m/z 314, see Scheme 15), as well as of clona-zepam and nitrazepam, involves the loss of 46 Da (NO2

�), in bothion-trap MS2 and triple-quadrupole MS–MS. In ion-trap MS3, asubsequent loss of 29 Da from the radical cation with m/z 268 isobserved, which is consistent with the loss of HCO�, as demon-strated by accurate mass determination (Smyth et al., 2004). Anadditional fragment in a triple-quadrupole instrument is the ionwith m/z 211, consistent with the losses of NO2

�, CO andH2C=NH (Scheme 15), which is also confirmed by accurate-mass data (Wuest, 2010). Based on the formulae derived fromaccurate-mass data (Wuest, 2010), structure proposals couldhave be given for some other minor fragment ions of flunitraze-pam as well, for example, ions with m/z 119 (C8H9N

þ�), 147(C9H9NO

þ�), 165 (C8H9N2O2þ), and 183 (C13H8F

þ; see Scheme15). The fragmentation of the major metabolites of flunitraze-pam, that is, 7-amino-, 3-hydroxy-, and N-desmethyl-

SCHEME 15

& NIESSEN


flunitrazepam, has been studied as well (Smyth, McClean, &Ramachandran, 2000; Smyth et al., 2004).

The fragmentation of methyl-triazolo-benzodiazepines, likealprazolam, is difficult to understand, even with accurate-massdata. Based on the formulae derived from accurate-mass data(Wuest, 2010) for the fragments of alprazolam ([M þ H]þ, m/z309), structures are proposed for most of the fragments(Scheme 16), but in some cases similar likely alternative struc-tures could be drawn. No reasonable structure could be proposedfor the ion with m/z 251 (most likely formula C15H8N2Cl

þ).Whereas the benzodiazepines seem to belong to a well-

defined compound class, hardly any class-specific fragmentationreactions could be found. Predominant fragmentation appears tobe determined by compound-specific side groups rather thanclass-specific fragmentation of the diazepine ring, for instance.This is also illustrated by the great variety of neutral losses used indefining SRM transitions for this class of compounds (Badawiet al., 2009; Nakamura et al., 2009).

E. Analgesic and Anti-Inflammatory Drugs

Analgesics are drugs used to relieve pain and/or because of theiranti-inflammatory properties. They are also indicated as antipy-retic or antiphlogistic drugs; some of them are used as antirheu-matic drugs. Analgesic drugs act in various ways on theperipheral and central nervous system. Important drug classesare acetaminophen (also called paracetamol), non-steroidal anti-inflammatory drugs (NSAIDs), and opiates. The NSAIDs con-sists of a structurally wide variety of compounds, prohibiting thesearch for class-specific fragmentation. Therefore, just sometypical examples are discussed. The opiates are primarily com-pounds structurally related to morphine; as common drugs ofabuse, this class of compounds was left out completely, as thesecompounds are extensively discussed elsewhere (Castiglioniet al., 2008; Bijlsma et al., in preparation). In addition, thereare selective cyclooxygenase-2 enzyme (COX-2) inhibitors, andvarious other agents with analgesic properties, such as tramadol,

amitriptyline, etc. Multiresidue analysis of analgesic drugs isreported for plasma (Suenami et al., 2006a,b), in bovine muscleand milk (Van Hoof et al., 2004; Dowling et al., 2009; Maloneet al., 2009), and in surface and wastewater (Petrovic et al., 2005;Farre, Petrovic, & Barcelo, 2007).

1. Acetaminophen

Acetaminophen (N-(4-hydroxyphenyl)acetamide, paracetamol,[M þ H]þ, m/z 152) is a widely used over-the-counter analgesicand antipyretic drug. In MS–MS, the loss of the acetyl groupas H2C=C=O to a fragment ion withm/z 110 and its complemen-tary ion with m/z 43 (H3C–C:Oþ) are important fragments.Secondary fragmentation of the ion with m/z 110 with the lossofNH3 orwater leads to ionswithm/z 93 and 92, respectively, andfurther to m/z 65 (C5H5

þ) (see Scheme 17). Acetaminophen isalso widely studied in relation to its ability to form reactivequinoneimine metabolites (Evans et al., 2004).

2. Salicylic Acid Derivatives

Aspirin (acetylsalicylic acid or 2-(acetyloxy)benzoic acid,[M þ H]þ, m/z 181) was the first discovered NSAID. Becauseof its undesirable side effects in gastrointestinal ulcers, it isnowadays primarily used for its antiplatelet effect, inhibitingthe production of thromboxane, which helps to prevent heartattacks, strokes, blood clots, etc. In MS–MS, the protonatedmolecule with m/z 181 shows losses of H2O to an ion withm/z 163, of H2C=C=O to an ion withm/z 139, and a combinationof these to a fragment ion withm/z 121 (see Scheme 17;Williamset al., 2006).

The MS–MS spectra of a number of related compoundsare in the set of library spectra. 5-Aminosalicylic acid([M þ H]þ,m/z 154) shows losses of H2O or HCOOH. At highercollision energies, a fragment ion with m/z 80 is formed, mostlikely C5H6N

þ as a result of the loss of HCOOH and CO.Salicylamide ([M þ H]þ, m/z 138) shows subsequent lossesof NH3, CO, and CO to a fragment ion with m/z 65 (C5H5

þ).The isopropyl analogue of this ([M þ H]þ, m/z 180) first showsthe loss of C3H6 to an ion with m/z 138 and further a similarspectrum. Ethenzamide (2-ethyoxybenzamide, [M þ H]þ,m/z 166) shows subsequent losses of NH3 and C2H4 to an ionwithm/z 121, which shows further H2O and CO loss and ends upwith m/z 65 (C5H5

þ) as well. Salsalate ([M þ H]þ, m/z 259)shows the loss of salicylic acid to an ion withm/z 121 and furtherto m/z 65 (C5H5

þ).Benorilate ([M þ H]þ, m/z 314) is a combination of acet-

aminophen and aspirin. In MS–MS, it first shows the lossof H2C=C=O, either from N-acetyl or O-acetyl. In addition,fragment ions with m/z 121 (see above) and m/z 152, consistentwith protonated acetaminophen, are formed, as well as m/z 110,due the loss of H2C=C=O from the latter.

3. Non-Steroidal Anti-Inflammatory Drugs

A number of NSAIDs can be considered as derivatives of N,N-diphenylamine or N-pyridinyl-N-phenylamine, that is, com-pounds like diclofenac, tolfenamic acid, flunixin, niflumic acid,mefenamic acid, meclofenamic acid, glafenine, and floctafenine.Most of these compounds can be analyzed in both positive-ionand negative-ion mode, although in multiresidue LC–MSSCHEME 16



analysis, the negative-ion mode is mostly preferred (Van Hoofet al., 2004; Farre, Petrovic, & Barcelo, 2007). In negative-ionmode, these compounds predominantly show losses of CO2. Likeacetaminophen, diclofenac is extensively studied in relation to itsability to form reactive quinoneimine metabolites (Evans et al.,2004; Dieckhaus et al., 2005).

In positive-ion MS–MS, meclofenamic acid ([M þ H]þ,m/z 296) shows subsequent losses of H2O and Cl�, niflumic acid([M þ H]þ, m/z 283) of H2O and HF, and mefenamic acid([M þ H]þ, m/z 242) of H2O and CH3

�. These three compoundsare all N-phenyl derivatives of anthranilic acid. Cleavagebetween the N and either ring does not occur, because it wouldrequire H-rearrangement to the N, which is not likely to occurfrom a phenyl ring. Glafenine ([M þ H]þ, m/z 373) and flocta-fenine ([M þ H]þ, m/z 407) are N-quinolinyl derivatives ofanthranilic acid. They first show the loss of the 2,3-dihydrox-ypropyl side chain, predominantly as 1,2,3-trihydroxypropane,followed by the loss of CO or Cl� in glafenine and the loss of COand HF with floctafenine.

Ketoprofen ([M þ H]þ,m/z 255) shows an abundant loss ofHCOOH to an ion with m/z 209, followed by loss of benzene(C6H6) to an ion with m/z 131, and a minor loss of benzene(C6H6). The ion with m/z 209 shows the loss of a CH3

�. Inaddition, ions with m/z 105 (most likely C6H5–C:Oþ) and 77(C6H5

þ) are observed. Naproxen ([M þ H]þ, m/z 231) shows aloss of HCOOH followed by a loss of a CH3

�. Ketorolac([M þ H]þ, m/z 256) shows a predominant loss of HCOOH toan ion with m/z 210, a minor loss of benzene (C6H6), and anabundant ion with m/z 105 (C6H5–C:Oþ).

In the set of library spectra, a number of compounds wereavailable of the pyrazolone class, like propyphenazone, mora-zone, aminophenazone. Propyphenazone ([M þ H]þ, m/z 231)yields three major fragments and a variety of less abundant ones.Based on accurate-mass data (Wuest, 2010), the fragment ionwithm/z 201 is consistent with the subsequent loss of two methylradicals, whereas the ion with m/z 189 is due to the loss of the

isopropyl group as propylene C3H6; the ion with m/z 58 can bedescribed as H2C=N

þH–CH2CH3 due to a cleavage in the pyr-azolone ring. Morazone ([M þ H]þ, m/z 378) yields just onemajor fragmentwithm/z 201 due to the loss of the phenyl-methyl-morpholine ring. The major fragments of aminophenazone([M þ H]þ, m/z 232) are two fragment ions with m/z 113(C6H13N2

þ) and 111 (C6H11N2þ), both due to cleavages in the

pyrazolone ring. At higher collision energies, secondary frag-ments of these two ions are observed. This short discussionindicates that no common trends were found in the fragmentationof these pyrazolone compounds.

4. Selective COX-2 Inhibitors

The selective cyclooxygenase-2 enzyme (COX-2) inhibitors pir-oxicam ([M þ H]þ,m/z 332), meloxicam ([M þ H]þ, m/z 352),tenoxicam ([M þ H]þ, m/z 338), and isoxicam ([M þ H]þ, m/z336) are also consideredNSAIDs belonging to the oxicam group.InMS–MS, these compounds show three characteristic fragmentions with charge retention on either the pyridinyl (piroxicam andtenoxicam) or the 5-methyl-1,3-oxazolyl (meloxicam and iso-xicam) side of the molecule. The fragmentation is outlined forpiroxicam in Scheme 17 (Ji et al., 2005).

F. Local Anaesthetics

A local anaesthetic drug causes reversible local anesthesia and aloss of nociception. There are two classes of local anesthetics: themore frequently used aminoamides like lidocaine and the amino-esters like procaine. Multiresidue LC–MS analysis of the amino-amide anaesthetics bupivacaine, mepivacaine, ropivacaine, andprilovacaine in human serum was described (Koehler, Oertel, &Kirch, 2005).

In MS–MS, the aminoamide anaesthetics show the lossof the N-phenylformamide part of the molecule with chargeretention on the resulting iminium ion, that is,

SCHEME 17

& NIESSEN


on H2C=Nþ(C2H5)2 (m/z 86) in lidocaine ([M þ H]þ, m/z 235;

Scheme 18). Secondary fragments from the iminium ion may beobserved, for instance m/z 58 (H2C=N

þHC2H5) in lidocaine.The aminoester anaesthetics showmore fragments, covering

both sides of the molecule. For procaine ([M þ H]þ, m/z 237),for instance, fragments ions are observed with m/z 92 (H2N–C6H5

þ), 120 (H2N–C6H5–C:Oþ), 164 (H2N–C6H5–C(=O)O–CH2CH2

þ), and with m/z 100 (H2Cþ–CH2–N(C2H5)2), and

m/z 72 (H3C–CH=NþH–C2H5) (Scheme 18).

G. Antiepileptic Drugs

The antiepileptic drugs comprise of a wide variety of compoundclasses used in the treatment of epileptic seizures, whereas theyare also increasingly being used in the treatment of bipolardisorder. Some examples of antiepileptic drugs are barbiturates,benzodiazepines like clobazam and clonazepam (see SectionVI.D), carboxamides such as carbamazepine, valproates andvalproylamides, hydantoins like phenytoin, propionates likebeclamide, pyrimidinediones like primidone, succinimides likemesuximide, and triazines. Based on an amide-iminol tautomer-ism, most barbiturates are analyzed in negative-ion mode and aretherefore not discussed in this review.

Popular antiepileptic drugs are carbamazepine and itsderivative oxcarbazepine. With a dibenzoazepine skeleton, thesecompounds resemble tricyclic antidepressants. The fragmenta-tion of carbamazepine ([M þ H]þ,m/z 237) involves losses fromthe carboxamide side chain, that is, loss of NH3 or loss of HNCOas well as some loss of NH3 and CO. In oxcarbazepine([M þ H]þ, m/z 253), the loss of NH3 and CO results in moreabundant fragment than the loss of HNCO. Further loss of waterto an ion with m/z 180 (or 182) is also observed.

The major fragment ion of beclamide ([M þ H]þ, m/z 198)is the ion withm/z 91 (C7H7

þ) due to the loss of the side chain ofthe benzyl group. Sultiame ([M þ H]þ, m/z 291) shows loss ofNH3, loss of SO2, and a combined loss of SO2 and C3H6 to afragment ion with m/z 185. At higher collision energy, a radicalcation with m/z 105 is observed (C7H7N

þ�; see Scheme 19).Primidone ([M þ H]þ, m/z 219) shows subsequent lossesof H3CNCO (to an ion with m/z 162) and HNCO (m/z 119), aswell as the formation of C7H7

þ (m/z 91) (see Scheme 19).Phenytoin ([M þ H]þ, m/z 253) subsequently shows the lossof CO (to an ion with m/z 225), of HNCO (m/z 182), and ofbenzene (m/z 104) (see Scheme 19). Mesuximide ([M þ H]þ,m/z 204) shows the loss of CO, followed by the loss of

methylamine. Furthermore, fragment ions are observed withm/z 126, 119, and 91, which are due to the loss of benzene,and the formation of C9H11

þ and C7H7þ, respectively (see

Scheme 19).

H. Other Psychotropic Drugs

There are numerous other psychotropic drugs. First of all, thereare thevarious classes of drugs of abuse,many ofwhich are illicit,such as amphetamine and related compounds, cocaine andrelated compounds, opiates. These were deliberately left outof this review, because MS–MS fragmentation of illicit drugshas been reviewed (Castiglioni et al., 2008), and a further study isin preparation (Bijlsma et al., in preparation).

Methylphenidate ([M þ H]þ,m/z 234) or Ritalin is a centralnervous system stimulant that is used in the treatment of attentiondeficit disorders, with or without hyperactivity, and narcolepsy.The major fragment is an ion with m/z 84 (the tetrahydropyr-idinium ion, C5H10N

þ), which at higher collision energies showsthe loss of C2H4 via a retro-Diels-Alder fragmentation to an ionwith m/z 54.

Zolpidem and zopiclone are non-benzodiazepine hypnoticsthat are prescribed for the short-term treatment of insomnia, aswell as some brain disorders. Zolpidem ([M þ H]þ, m/z 308)primarily shows the loss of dimethylamine to an ion withm/z 263followed by the loss of CO to an ion with m/z 235. Zopiclone([M þ H]þ, m/z 389) is easily fragmented, generating fragmentionswithm/z 345, 263, 245, 217, 112, and 99.Accurate-mass datashow that the ion with m/z 345 results from a rearrangementinvolving the loss of CO2 (Wuest, 2010). The ions with m/z 263,245, and 217 are all due to losses of the methylpiperazine sidechain (see Scheme 20). The ion withm/z 112 is due to Cl–C6H4

þ

and the ion with m/z 99 is protonated methyl-tetrahydropyrazine(C5H11N2

þ).Baclofen ([M þ H]þ, m/z 214) is a derivative of gamma-

aminobutyric acid (GABA), primarily used to treat spasticity andunder investigation for the treatment of alcoholism. Upon MS–MS, baclofen generates fragment ionswithm/z 197 due to the lossof NH3, 196 (loss of H2O), 179 (combined loss of NH3 and H2O),151 (loss of CO from the ion withm/z 179), andm/z 116 and 115due to the loss of Cl� and HCl, respectively, from the ion withm/z151.

Caffeine ([M þ H]þ, m/z 195) is probably the most widelyused compound with a central nervous system (CNS) stimulantaction. The fragmentation of caffeine results in two major

SCHEME 18



fragment ions: the ion with m/z 138 is due to the loss of H3C–N=C=Oand the ionwithm/z 110 results froman additional loss ofCO. The formation of the ion with m/z 138 can be written as aretro-Diels-Alder fragmentation. At higher collision energies,additional low m/z fragment ions are observed, including anion withm/z 123 due to loss of a CH3

� from the ion withm/z 138.

VII. DRUGS RELATED TO DIGESTION AND THEGASTROINTESTINAL TRACT

A. Anti-Diabetic Drugs

Anti-diabetic drugs or oral hypoglycemic or antihyperglycemicagents treat diabetes mellitus by lowering glucose levels in theblood. There are different classes of anti-diabetic drugs, whichare prescribed depending on the nature of the diabetes, the ageand situation of the person, as well as other factors. MS–MS data

for two classes anti-diabetic drugs are discussed here: (1) sec-retagogues, including sulfonylureas and meglitinides, and (2)sensitizers, including biguanides and thiazolidinediones. Multi-residue analysis of anti-diabetic agents has been described foranalysis in plasma (Maurer et al., 2002; Wang & Miksa, 2007)and in equine urine (Ho et al., 2004).

First-generation sulfonylurea anti-diabetic agents includecompounds like carbutamide, tolazamide, and tolbutamide.Characteristic fragmentation of these compounds involves clea-vages on either side of the SO2 group of the sulphonamidemoiety.This type of fragmentation is similar to that of sulphonamideantibiotics (Niessen, 1998, 2005). For carbutamide ([M þ H]þ,m/z 272, Scheme 21), this fragmentation results in fragmentions with m/z 156 and 92. A fragment ion with m/z 108 isalso observed, which is due to the loss of SO from the fragmentwith m/z 156, after rearrangement in the SO2 group (Niessen,

SCHEME 19

SCHEME 20

& NIESSEN


1998; Klagkou et al., 2003; Sun, Dai, & Liu, 2008). Fragmentswith charge-retention at the other side of the molecule, forexample, ions with m/z 74 and 57 (H3N

þC4H9 andþC4H9) are

observed as well. The related compounds show similarfragmentation.

The second-generation sulfonylurea agents includeglipizide, glyburide (glibenclamide), glimepiride, gliclazide, gli-quidone, glyclopyramide, glibornuride. As an example, thefragmentation of glibenclamide ([M þ H]þ, m/z 494) isoutlined in Scheme 21. The identity of these fragments has beenconfirmed by accurate-mass determination on a Q-TOF instru-ment (Radjenovic et al., 2008). Again, the cleavage on either sideof the SO2 group in the sulfonamide moiety is an importantfragmentation route. Secondary fragmentation of the fragmention with m/z 352 involving the loss of SO to a fragment ionwith m/z 304 is also observed. Other compounds of this classshow similar fragmentation. Like many sulfonamides, thesecompounds can also be analyzed in negative-ion mode. Thenegative-ion fragmentation of glimepiride ([M � H]�, m/z489) and of some of its degradation products has been reported(Bansal et al., 2008).

Extensive fragmentation is observed for the biguanide anti-diabetic drug metformin, with the fragment ions with either m/z71 ((H3C)2N–C

þ=NH) or m/z 60 (H2N–C(=NH)NH3þ) being

most abundant (Scheme 21). Similar fragmentation is observedfor phenformin (Wang et al., 2004).

The meglitinides repaglinide and nateglinide do not showclear structure similarities, except for the presence of an amidebond and a carboxylic acid group. They show extensive frag-mentation. As an example, the fragmentation of nateglinide isoutlined in Scheme 21. Cleavage at the amide bond results in twocomplementary fragment ions with m/z 166 and 153. Both frag-ments show secondary fragmentation, that is, the ionwithm/z 120due to the loss of formic acid, and the ion withm/z 125 due to theloss of CO. The loss of formic acid directly from [M þ H]þ isalso observed (m/z 272).

The last group of anti-diabetics to be discussed here are thethiazolidinediones, also known as glitazones, such as rosiglita-zone, pioglitazone, troglitazone. In MS–MS, these compoundsshow one major fragment, due to the cleavage of an aliphatic-aromatic ether bond (see Scheme 21 for rosiglitazone,[M þ H]þ,m/z 358). Troglitazone is studied because of its abilityfor form reactive metabolites (Kassahun et al., 2001; Dieckhauset al., 2005).

B. Anti-Ulcer Drugs

There are two major classes of anti-ulcer agents, used in thetreatment of various gastric diseases that is H2-histamineantagonist and proton pump inhibitors. Proton pump inhibitorshave in fact largely superseded the H2-receptor antagonists.Multiresidue analysis of anti-ulcer drugs, both H2-histamineantagonist and proton pump inhibitors has been described (Chunget al., 2004).

1. Proton Pump Inhibitors

Characteristic fragmentation of the proton pump inhibitorsinvolve the cleavage of the C–S bond between the benzoimida-zole group and the S=O group, with charge retention at the S=Oside. The resulting fragments, that is, m/z 198, 242, 200, and 252for omeprazole, rabeprazole, pantoprazole, and lansoprazole,respectively, are generally used in SRM for quantitative analysis.The fragmentation of omeprazole ([M þ H]þ, m/z 346) is out-lined in Scheme 22. Next to the characteristic fragment ion withm/z 198, the complementary fragment ion with m/z 149 isobserved as well.

2. H2-Histamine Antagonists

H2-histamine antagonists are used to reduce the secretion ofgastric acid, and prescribed for the treatment of various gastricdiseases. The multiresidue LC–MS of several H2 antagonists hasbeen reported in horse urine (Chung et al., 2004), in wastewater

SCHEME 21



and surface water (Hernando et al., 2007), and in plasma (Sunet al., 2009). In these studies, target compounds are cimetidine,famotidine, lafutidine, nizatidine, and/or ranitidine. In the historyof LC–MS, the analysis of ranitidine plays an important role,because in the 1980s ranitidine served as a benchmark for LC–MS interface performance (Tomer & Parker, 1989).

Except lafutidine, these compounds contain a thioether link,which is prone to fragmentation. It results in the most abundantfragment ionm/z 159 for cimetidine,m/z 189 for famotidine, andm/z 176 for ranitidine (see Scheme 22). Additional fragments inthe MS–MS spectrum of ranitidine ([M þ H]þ,m/z 315) are dueto losses of HN(CH3)2 to a fragment ionwithm/z 270 followed bythe loss of NO2

� to m/z 224 as well as the loss of NO2� from the

fragment ion with m/z 176 to an ion with m/z 130 (Chung et al.,2004; Hernando et al., 2007). Two other fragments include ionswith m/z 124 due to cleavage at the furan ring and chargeretention at that side and m/z 144 due to cleavage at the otherside of the sulfur atom (see Scheme 22). The formulae of thesefragments are checked against accurate-mass data (Wuest, 2010).The fragmentation of cimetidine ([M þ H]þ, m/z 253) is alsooutlined in Scheme 22. Next to the fragments indicated,additional fragment ions are observed, for example, an ion withm/z 211 due to the loss of HN=C=NH from [M þ H]þ, an ionwith m/z 117 due to loss of HN=C=NH from the fragment withm/z 159, and an ionwithm/z 82 due to the loss ofNH3 from the ionwith m/z 99. Famotidine ([M þ H]þ, m/z 338) show the loss ofHNSO2 to a fragment ion with m/z 259 next to ion with m/z 189(Qin et al., 1994). Further loss of H2S results in the fragment ionwith m/z 155 (see Scheme 22). MSn fragmentation of lafutidineand its metabolites after microsomal incubation has been studiedusing a linear-ion-trap–orbitrap hybrid instrument (Wang et al.,2008).

C. Lipid-Lowering Agents

Hyperlipidemia involves raised or abnormal levels of lipids and/or lipoproteins in the blood. This is currently quite common in thegeneral population. It is regarded as a high risk factor for car-diovascular diseases. General treatment involves, next to dietarymodification, the use of statins and/or fibrates.

1. Fibrates

The fibrates are agonists of the peroxisome proliferator-activatedreceptor alpha (PPAR-a) and are used in the treatment of hyper-cholesterolemia (high cholesterol). Fenofibrate ([M þ H]þ, m/z361) and bezafibrate ([M þ H]þ,m/z 362) show fragments due tocleavages at similar sites in the structure (Scheme 23). Commonfragment ions are observed with m/z 111 (Cl–C6H4

þ), m/z 121,andwithm/z 139 (Cl–C6H4–C:Oþ). The ionwithm/z 121 isHO–C6H4–CH2CH2

þ for bezafibrate, and the isobaric HO–C6H4–C:Oþ for fenofibrate. Another common fragmentation involvesthe cleavage of the ether bond in the 2-methyl-2-phenoxypropa-noic acid part of the molecule (Scheme 23).

2. Statins

Statins (or HMG-CoA reductase inhibitors) are a class of drugsused to lower the plasma cholesterol level by inhibiting theenzyme 3-hydroxy-3-methyl-glutaryl-Coenzyme A reductase,which is the rate-limiting enzyme of the mevalonate pathwayof cholesterol synthesis. Inhibition of this enzyme in the liverresults in decreased cholesterol synthesis and increasedsynthesis of LDL receptors, which in turn results in an increasedclearance of low-density lipoprotein (LDL) from the blood-stream. Members of this class comprise synthetic compounds,

SCHEME 22

& NIESSEN


such as atorvastatin, cerivastatin, fluvastatin, pitavastatin, androsovastatin, and fermentation-derived compounds, such as lov-astatin, mevastatin, and pravastatin. The most widely used statin,simvastatin, is a synthetic derivate of a fermentation product.

The MS–MS fragmentation of simvastatin ([M þ H]þ, m/z419) is studied in detail using triple-quadrupole, ion-trap and Q-TOF instruments (Wang, Wu, & Zhao, 2001; Vuletic, Cindric, &Koruznjak, 2005). The initial fragmentation step involves the lossof the 2,2-dimethylbutanoate side chain to fragment ionswithm/z321 and 303 (Scheme 24). Further fragmentation is best con-sidered as subsequent fragmentation of the ion with m/z 303. Aloss of water results in a fragment ion withm/z 285, which couldeither involve a lactone ring opening or a loss of the hydroxy fromthe ring, resulting in a,b-double bond formation, and a loss ofacetic acid in an ion with m/z 243. The loss of 44 Da (H3CCHO)from the ion with m/z 243 results in a fragment ion withm/z 199,which can be written as a three-member ring (Scheme 24). Theloss of 56 Da (C4H8) fromm/z 199 would result in a fragment ionwithm/z 143 (C11H11

þ) (Wang, Wu, & Zhao, 2001), although analternative structure has been suggested, involving a cleavage of

the C–C bond at the hexahydronaphthalene ring with chargeretention at the lactone ring (C7H11O3

þ; see Scheme 24). Inter-estingly, accurate-mass data indicate that both structure areobserved, that is, the ion with C7H11O3

þ at low collision energy,and the ion with C11H11

þ at higher collision energy, which is inagreementwith expectation based on the fragmentation pathwaysof both ions. Furthermore, fragment ions are observed with m/z267 (loss of water from the ion with m/z 285), m/z 225 (loss ofwater from the ion with m/z 243), and m/z 173 (C13H17

þ, seeScheme 24). Similar fragments are observed for related struc-tures, such as lovastatin, mevastatin, pravastatin.

The other statins have different structures. As an example,the fragmentation of atorvastatin ([M þ H]þ, m/z 559) is out-lined in Scheme 24. Initial fragmentation results in losses ofaniline orN-phenylformamide to fragment ions withm/z 466 and440, respectively. Both fragments show loss of water. Furtherfragmentation may be considered as secondary fragmentation ofthe ion withm/z 440, resulting in fragment ions withm/z 380, dueto the loss of acetic acid, and m/z 292, due to the loss of thecomplete side chain and the formation of an iminium ion

SCHEME 23

SCHEME 24



(Shah, Kumar, & Singh, 2008). The fragment ion with m/z 250results from the subsequent loss of C3H6 (42 Da) from the pyrrolering.

D. Anorectic drugs

Anorectic drugs are appetite suppressing compounds that shouldresult inweight loss. Awide variety a compoundsmay be listed asanorectic drugs, including a number of amphetamine derivatives,such as mefenorex, fenproporex, and dexfenfluramine. Thesecompounds show fragmentation similar to amphetamine, thatis, formation of fragment ionswithm/z 119 due to the phenylethylion (C8H11

þ) and m/z 91 due to the tropylium ion (C7H7þ). With

dexfenfluramine, these fragment ions are obviously found 68units higher, due to the presence of the CF3 group at the phenylring. Norephedrine ([M þ H]þ, m/z 152), a phenyl-a-hydroxyanalogue) first shows the loss of water, and subsequently theformation of fragment ions with m/z 117 (with an additionaldouble bond in the propyl part) and m/z 91. A possible ring-closure fragment withm/z 115 is also observed for norephedrine.Metamfepramone ([M þ H]þ, m/z 178, an N-dimethyl, phenyl-a-keto analogue) shows three major fragment ions, that is, an ionwith m/z 133 due to the loss of dimethylamine, m/z 105 (C6H5–C:Oþ), and m/z 72 due to C4H10N

þ.Sibutramine ([M þ H]þ, m/z 280) provides extensive frag-

mentation. The ions with m/z 125, 139, 153, and 179 are mostabundant. Based on accurate mass data (Wuest, 2010), the ionswithm/z 125, 139, and 153 can be interpreted as Cl–C6H4–C

þH2,Cl–C6H4–C

þHCH3, and Cl–C6H4–CþHCH2CH3, respectively,

thus involving cleavage and/or loss of the cyclobutane ring. Theion with m/z 179 is due to losses of dimethylamine and butanefrom [M þ H]þ. Two minor fragments are due to losses ofdimethylamine, the chlorophenyl group and part of the carbon-hydrogen skeleton, that is, the ions withm/z 97 (C7H13

þ) and 109(C8H13

þ).

VIII. OTHER CLASSES OF DRUGS

A. Beta-Adrenergic Agonists

Structurally very similar to the beta-blockers (see Section V.A)are the beta-adrenergic agonists, like bambuterol, clenbuterol,and salbutamol. These compounds can impair the relaxation ofbronchial muscles and are therefore used against asthma andchronic obstructive pulmonary disease. The general structure ofthe beta-adrenergic agonists is 1-alkylamino-3-phenylpropan-2-ol, with alkyl being predominantly tert-butyl. The benzene maybe substituted, for example, 4-amino-3,5-dichlorobenzene inclenbuterol.

Fragmentation of clenbuterol ([M þ H]þ,m/z 277) involvesthe loss of water to an ion with m/z 259, subsequent loss of C4H8

to an ion with m/z 203, and subsequent losses of Cl� and HCl, toions with m/z 168 and 132, respectively (Debrauwer & Bories,1993; Doerge, Bajic, & Lowes, 1993). Thus, fragmentation ispartly very similar to that of beta-blockers (see Section V.A).

Other structurally related drugs acting as bronchodilators arecompounds like ephedrine, etafedrine, isoprenaline, and dioxe-thedrin. For etafedrine ([M þ H]þ,m/z 194), as an example, nextto the loss of water, two complementary fragments ions areobserved with m/z 135 and 60, due to charge retention eitheron the 3-phenylpropan-2-ol side or at the N-methyl-ethanamineside.

B. Histamine Antagonists

Histamine antagonists are agents that inhibit the release or actionof histamine. The term antihistamine is usually reserved for thecompounds that act upon the H1-histamine receptor. They are usedas treatment for allergies. H2 antagonists are used to reduce thesecretion of gastric acid, and used in the treatment of variousgastric diseases (discussed in Section VII.B). H3 and H4 receptorantagonists are still under investigation: H3 antagonists in relationto the treatment of conditions such asADHD,Alzheimer’s disease,and schizophrenia, and H4 antagonists as anti-inflammatory andanalgesic drugs. No examples of H3 and H4 antagonists are avail-able in the set of library spectra.

Froma structural point of view, theH1-histamine antagonistscover a variety of different structures, including phenothiazines,pheniramines, diphenhydramines, 1-(diphenylmethyl)pipera-zines, N-phenyl-N-methylthiophene-amines, and others. Multi-residue analysis of 18 H1 antihistamine drugs from differentsubclasses in blood using LC–MS in SRM mode was reported(Gergov et al., 2001b).

In MS–MS, phenothiazines with antihistamine properties,like dimetotiazine, isothipendyl, mequitazine, oxomemazine,and oxypendyl, show identical fragmentation behavior to theantipsychotic agents discussed in Section VI.A.

Pheniramine ([M þ H]þ, m/z 241, Scheme 25) is an anti-histamine with anticholinergic properties used to treat allergicconditions such as hay fever or urticaria. Derivatives of phenir-amine include chlorpheniramine ([M þ H]þ, m/z 275), andbrompheniramine ([M þ H]þ, m/z 319). The halogenation ofpheniramine increases its potency. Characteristic fragmentationof this class of compounds involve the loss of 45 Da (HN(CH3)2),and the loss of 73 Da (H3C–CH2– N(CH3)2), resulting in theformation of the secondary carbocation X-Phenyl–CþH–Pyri-dine, with X=H in pheniramine, X=Cl in chlorpheniramine, andX=Br in brompheniramine. In the latter two cases, a subsequentloss of Cl� or Br� is observed.

Related structures are the diphenhydramines, like broma-zine, clemastine ([M þ H]þ,m/z 344, Scheme 25), cloperastine,diphenhydramine, doxylamine, and etoloxamine. Except etolox-amine, all these compounds show the loss the ethanolamine sidechain to generate the secondary or tertiary carbocation X-Phenyl–CþH–Phenyl (with clemastine (m/z 215), cloperastine(m/z 201), and diphenhydramine (m/z 167)) or X-Phenyl–CþH–Pyridine (with bromazine (m/z 245) and doxylamine (m/z 182)).With bromazine and doxylamine, a subsequent loss of Br� orCH3

�, respectively, is observed. With clemastine, an additionalcomplementary fragment ion with m/z 130 is observed due tocharge retention at the 2-(1-methylpyrrolidin-2-yl)ethanol. Eto-loxamine behaves somewhat differently, with the loss of diethyl-amine, and fragments with m/z 100 (þCH2–CH2–N(C2H5)2) and72 (H3C–H2C=N

þC2H5 orþCH2–CH2–NH–C2H5).

Another class of related structures are based on 1-(diphe-nylmethyl)piperazine, that is, for instance cetirizine, cinnarizine,hydroxyzine, and meclozine. In MS–MS, the bond between thediphenylmethyl part and the piperazine ring is cleaved, withcharge retention either on both sides, as shown for cinnarizine([M þ H]þ, m/z 369, Scheme 25) or at the diphenylmethyl partonly, resulting in the secondary carbocation X-Phenyl–CþH–Phenyl (m/z 201, if X=Cl). Subsequently, the loss of a Cl� isobserved from the carbocation, resulting in a radical cation withm/z 166.

& NIESSEN


Methapyrilene and methaphenilene ([M þ H]þ, m/z 261,Scheme 25) both have an N-methylthiophene group. They showcharacteristic fragmentation involving the cleavage of the N–C-bond to the methylthiophene group, with charge retention oneither side, resulting in fragment ions with m/z 97 and m/z of[M þ H�98]þ. Loss of 45 Da (dimethylamine, HN(CH3)2) isalso observed.

The quantitative bioanalysis of loratadine and its activemetabolite desloratadine ([M þ H]þ, m/z 311, Scheme 25) isextensively studied (e.g., Yang et al., 2003). In MS–MS, deslor-atadine shows the loss of NH3 followed by the loss of Cl

�, and theloss of 29 Da (H2C=NH) from the 4-methylenepiperidine ring.Similar losses from the ring are observed for azatadine, thedeschloro-N-methyl analogue of desloratadine. Rupatadine([M þ H]þ, m/z 416), that is, desloratadine with a (5-methyl-3-pyridinyl)methyl group rather than a methyl group at thepiperidine nitrogen, shows similar fragmentation, that is, theloss of 3,5-dimethylpyridine, and subsequent losses of NH3,H2C=NH2, and a combination of NH3 and Cl�. For rupatadine,the protonated 3,5-dimethylpyridine with m/z 108 is observed aswell (Wen et al., 2009). The fragmentation of loratadine, deslor-atadine, and their metabolites has recently been studied in moredetail using MS2 and MS3 on quadrupole–linear-ion-trap hybridand linear-ion-trap–orbitrap hybrid instruments (Chen et al.,2009; Picard et al., 2009).

C. Anticholinergic Agents

An anticholinergic agent is a compound that blocks the neuro-transmitter acetylcholine in the central and the peripheral nervoussystem. A classic example is atropine. Anticholinergics inhibitparasympathetic nerve impulses by selectively blocking the

binding of the neurotransmitter acetylcholine to its receptor innerve cells. They can be subdivided into three categories inaccordance with their specific targets in the central and/or per-ipheral nervous system: antimuscarinic agents, ganglionic block-ers, and neuromuscular blockers. Anticholinergic drugs are usedin treating a variety of conditions, such as gastrointestinal dis-orders like gastritis, pylorospasm, diverticulitis, and ulcerativecolitis, genitourinary disorders, respiratory disorders, Parkin-son’s disease.

This class of compounds contains a high structural variety,including for instance phenothiazine-like structures, like in pro-fenamine, diethazine, and metixene (see Section VI.A). Someother examples are discussed below.

Atropine ([M þ H]þ, m/z 290) shows one major fragmention withm/z 124 due to the 8-methyl-8-azabicyclo[3.2.1]oct-3-ylion. Subsequent loss of methylamine results in a fragment ionwithm/z 93 (C7H9

þ), whereas a fragment withm/z 91 (C7H7þ) is

also observed. At the high m/z end, losses of water and formal-dehyde are found (Chen et al., 2006). With scopolamine([M þ H]þ, m/z 304), the major fragment with m/z 138 resultsfrom the cleavage of the same O-bicyclo-ring bond as atropine,but in this case a fragment due to cleavage at the other side of theO is also observed, thus resulting in a protonatedHO-bicyclo-ring(m/z 156). The related benzatropine ([M þ H]þ, m/z 308) showsjust onemajor fragment ionwithm/z 167 (Phenyl–CþH–Phenyl).

A number of anticholinergic compounds are derived from(hydroxy)phenylacetic acid, with an additional group attached tothe phenyl-a-C. The major fragment ions of drofenine([M þ H]þ, m/z 318) are due to the loss of diethylamine (m/z245) and subsequent loss of cyclohexene (m/z 163). Ions withm/z100 ((H3CH2C)2NCH2CH2

þ), m/z 91 (C7H7þ), and m/z 173

(C13H17þ) are also observed (see Scheme 26). Oxybutynin

SCHEME 25



([M þ H]þ, m/z 358) shows a water loss and cleavages on eitherside of the carbonyl group, resulting in fragment ions with m/z189 due toC6H5–C

þ(–OH)–C6H11 andm/z171 due to subsequentwater loss and fragment ions with m/z 142 and 124 due toprotonated 4-diethylaminobut-2-yn-1-ol and water loss from that(see Scheme 26). Propiverine ([M þ H]þ, m/z 368) also showssimilar fragmentation (see Scheme 26) in addition to losses ofpropene and propanol at the high end (Oertel et al., 2007). Tworelated structures, triperiden and trihexylphenidyl, based on acommon1-phenyl-3-(piperidin-1-yl)propan-1-ol backbone showa major fragment ion due to the 1-methylidenepiperidinium ion(C6H12N

þ).

D. Antimycotic and Antifungal Compounds

An antimycotic or antifungal drug is a medication used to treatfungal infections, such as athlete’s foot, ringworm, candidiasis

(thrush), and other (serious) systemic infections, such as crypto-coccal meningitis. There are different classes of antifungal com-pounds: (1) macrocyclic olyenes, such as natamycin andcandicin, (2) imidazoles and triazoles, that is, conazoles, (3)allylamines, such as naftifine and butenafine, and (4) others.Allylamine antifungals, such as naftifine and terbinafine, showfragment ions due to cleavages on either side of theN–CH3group,as is illustrated for naftifine ([M þ H]þ, m/z 288) in Scheme 27.

Next to antimycotic compounds, several fungicides used inagriculture are of toxicological interest, that is, compounds likebenomyl, carbendazim, and thiabendazole.

1. Imidazole and Triazole Antimycotic Compounds

The azole antifungals inhibit the enzyme CYP51 14a-demethy-lase which converts lanosterol into ergosterol. Depletion of ergo-sterol disrupts the structure and many functions of fungal

SCHEME 26

SCHEME 27

& NIESSEN


membranes leading to inhibition of fungal growth. The LC–MSanalysis of various azole antifungal drugs has been described fortherapeutic drug monitoring. A multiresidue LC–MSmethod forconazoles was recently reported (Alffenaar et al., 2010). Multi-residue environmentalmonitoring of conazoleswas also reported(Jeannot et al., 2000).

The imidazole antifungals, such as bifonazole, bitertanol,isoconazole, and miconazole, show a characteristic loss of imi-dazole (68 Da, C3H4N2) and/or a characteristic fragment ionwithm/z 69. The isomers isoconazole and micoconazole also give afragment ion with m/z 159, Cl2C6H3–CH2

þ with either 2,6-dichloro in isoconazole and 2,4-dichloro in micoconazole. Inaddition, compound-specific fragmentation may be observed.

The triazole antifungals, such as fluconazole, itraconazole,difenoconazole, propiconazole, and triadimenol, show a charac-teristic loss of the 1,2,4-triazole (69 Da, C2H3N3) and/or acharacteristic fragment ion with m/z 70. In addition, compoundspecific fragmentation may be observed, as is illustrated forpropiconazole ([M þ H]þ, m/z 342) in Scheme 27. For propi-conazole, next to the ion with m/z 70, a more abundant ion withm/z 69 is observed. Accurate-mass data (Wuest, 2010) indicatethat this ion is due to C5H9

þ, that is, due to cleavage in thepropyldioxolane group. Loss of the triazole ring (to a fragmentwith m/z 273) and subsequent cleavage in the propyldioxolanering results in the fragment ion withm/z 205. A fragment ion withm/z 159 (Cl2C6H3–CH2

þ) is also observed.

2. Fungicides

Awide variety of compounds is used as fungicides in agriculture,for instance in the conservation of fruit. Among the compoundsfrequently used are some of the conazoles already discussed,pyrimidine fungicides, carbendazim, and thiabendazole.

The fragmentation of the related pyrimidine fungicidesbupirimate and ethirimol is studied using ion-trap and Q-TOFinstruments (Soler, James,&Pico, 2007). Themajor fragments ofbupirimate ([M þ H]þ,m/z 317, see Scheme 27) are the ionswithm/z 272 due to the loss of (H3C)2NH, m/z 237 due to an unex-pected rearrangement involving the loss of a neutral with elemen-tal composition CH4SO2 (rather than the loss of SO3), and m/z210 after the loss of (H3C)2NSO2 (Soler, James, & Pico, 2007).The ion with m/z 210 is the protonated molecule of ethirimolwhich in MS–MS shows fragment ions related to subsequentlosses of parts of the side chains, that is, ions withm/z 194 due tothe loss ofCH4,m/z 180 due to the loss of C2H6,m/z 166 due to theloss of C3H8, m/z 150 due to the loss of two times C2H6 (Soler,James, & Pico, 2007). However, alternative accurate-mass data(Wuest, 2010) indicate that the ion withm/z 237 is actually due tothe loss of SO3 with rearrangement of the N(CH3)2 group to thering (see Scheme 27), and that the formulae of the ions with m/z180 and 150 are C10H18N3

þ and C8H12N3þ, respectively, rather

than C9H14N3Oþ and C7H8N3O

þ (see Scheme 27).In MS–MS, carbendazim ([M þ H]þ, m/z 192) shows sub-

sequent losses of CH3OH, CO, and HCN to yield fragment ionswith m/z 160, 132, and 105, respectively. The identity of thefragmentswas confirmed by accurate-mass determination using aQ-TOF instrument (Pico et al., 2007). The proposed structures ofthese fragment ions given in Scheme 27 differ from the onesproposed elsewhere (Pico et al., 2007), because the formation ofthe structure of the ion with m/z 132 would otherwise requiresignificant (and unlikely) rearrangement.

Thiabendazole ([M þ H]þ,m/z 202) shows major fragmentions with m/z 175 and 131, consistent with the subsequent lossesof HC:N and CS, respectively. Minor fragment ions withm/z 65(C5H5

þ) and m/z 92 (C6H6Nþ) are observed as well.

Fungicides with (methyl-substituted) furan rings, such asmethfuroxam, furalaxyl, and fenfuram, yield an intense fragmention, consistent with the furan–C:Oþ acylium ion, that is, afragment ion with m/z 137 for methfuroxam, with m/z 95 forfuralaxyl, and with m/z 109 for fenfuram. In addition, fragmentsdue to the loss of the furan ring and charge retention on the otherpart of the molecule are also observed for methfuroxam andfenfuram, that is, also involving acylium ions. Furalaxyl rathershows losses from the methyl ester side chain (losses ofCH3OH and CO).

E. Steroids

Steroids are characterized by the sterane core which is composedof seventeen carbon atoms bonded together to form four fusedrings: three cyclohexane rings (designated as the A, B, and Crings) and one cyclopentane ring (the D ring). There are hundredsof steroids that are found in plants, animals and fungi and thatvary by the functional groups attached to the rings and by theoxidation state of the rings. In mammals, steroids acts as hor-mones, classified as (1) sex steroids like androgens, estrogens,and progestagens that produce sex differences or support repro-duction, (2) corticosteroids including glucocorticoids regulatingmany aspects of metabolism and immune function and miner-alocorticoids that help maintain blood volume and control renalexcretion of electrolytes, and (3) anabolic steroids that interactwith androgen receptors to increase muscle and bone synthesis.Cholesterol, which modulates the fluidity of cell membranes andis the principal constituent of the plaques implicated in athero-sclerosis, also contains a sterane core. There are many steroiddrugs, derived from either one of these groups.

A toxicological mass spectral library, like the one we used,contains numerous spectra of steroids, both of endogenoussteroids and of steroid drugs. Interpretation of low-resolutionMS-MS spectra of steroids is difficult. Many C–C-bonds in asteroid are of similar strength,which upon fragmentation can giverise to numerous isomeric and isobaric fragments, for which inturn different structures may be proposed, involving differentparts of the molecule. The complexity of the steroid fragmenta-tion is nicely illustrated in a study on testosterone (Williams et al.,1999). Series of hydroxylated and deuterium-labeled analogueswere needed to elucidate the identity of just three fragment ions(m/z 97, 109, and 123). In this study, it was shown that the identityof the ion withm/z 109 (C7H9O

þ) is not involving the A-ring andC-19, as previously proposed, but rather C-1 to C-7 (Williamset al., 1999).

To some extent, one may be helped in the interpretation ofsteroid MS–MS spectra by extensive tables for structure eluci-dation of steroids, generated for electron ionization (Von Unruh& Spiteller, 1970a,b,c; Von Unruh, Spriteller-Friedmann, &Spiteller, 1970). The recent advent of readily accessible instru-ments for high-resolution mass spectrometry somewhat simpli-fies the interpretation, because at least the elemental compositionof the fragments can unambiguously be determined. An excellentillustration of this is a study on fragmentation characteristics ofanabolic steroids in relation to sports doping control, providinguseful tables with characteristics fragment ions (Pozo et al.,



2008). Structure proposals for the fragments of norethisterone,based on formulae derived from accurate-mass data using an ion-trap–TOF instrument, are given in Scheme 28. These data wereacquired in the course of a metabolic study with on-line activityassessment of themetabolites against the estrogen receptors ERaand ERb (de Vlieger et al., 2010). For most fragments, unam-biguous structure proposals can be made. Further studies onsteroid fragmentation in MS–MS are needed, involving stableisotope labeling and MS–MS and MSn on different platforms.

IX. CONCLUSIONS

This article describes the interpretation of the positive-ion MS–MS mass spectra of a wide number of toxicologically relevantcompounds, mainly referring to triple-quadrupole MS–MS data.Starting point of the discussion was a mass spectral library,available on the internet (Weinmann, 2005), containing at thestart of the project �800 entries. In total, MS–MS spectra for�570 compounds were interpreted. The compounds were classi-fied according their therapeutic class, andwithin this according tochemical structures. Whereas several clear chemical compoundclasses could be identified within the various therapeutic classes,in some cases compounds with (widely) different chemicalstructure are used to achieve the same therapeutic result. Thefragmentation of �200 compounds is discussed in detail in this

text. With some compound classes, only a few examples werediscussed, because other members of that class shows similarfragmentation. Our interpretation of the mass spectra waschecked against the literature and, whenever available, accu-rate-mass data. Reference is made to available literature contain-ing fragmentation data as well as to literature on multiresidueanalysis methods for particular classes of drugs.

One may question the usefulness of the information pro-vided in this article, especially in the light of the increasing use ofmass spectral libraries and other software tools, especially thosebased on accurate-mass information, to retrieve compound iden-tity without actual interpretation of the MS and MS–MS massspectra. To the author, this study primarily aimed at increasing theunderstanding of small-molecule fragmentation of even-electronions like protonated molecules in MS–MS. As such, at a laterstage this information, together with other relevant data, shouldbe compiled in fragmentation rules.

It is not possible to indicate how the information collectedand reviewed may change the common practice of generalunknown screening in the near future. However, the authorbelieves that knowledge on the actual fragmentation of small-molecule compounds in LC–MS applications is needed to assistin identification of unknowns, to improve methodologies andenhance selectivity, and to assist in the identification of metab-olites of these toxicologically relevant drugs.

SCHEME 28

& NIESSEN


ACKNOWLEDGMENTS

I would like to thank Dr. Wolfgang Weinmann (originally at theInstitute of Legal Medicine, University of Freiburg, Germany,currently at Institute of Forensic Medicine, University of Bern,Switzerland) for providing public access to his toxicology libraryvia the internet. I would like to thank Dr. Bernhard Wuest ofAgilent Technologies for help in accessing and using the AgilentBroecker, Herre & Pragst PCDL for Forensic Toxicology.Ben Bruyneel is thanked for acquiring the MS–MS spectra ofsimvastatin, enabling confirmation of the identity of the ion withm/z 143.

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