Anal Bioanal Chem (2011) 400:101–117 DOI 10.1007/s00216-010-4450-9 ORIGINAL PAPER Development and practical application of a library of CID accurate mass spectra of more than 2,500 toxic compounds for systematic toxicological analysis by LC–QTOF-MS with data-dependent acquisition Sebastian Broecker & Sieglinde Herre & Bernhard Wüst & Jerry Zweigenbaum & Fritz Pragst Received: 30 September 2010 /Revised: 15 November 2010 /Accepted: 16 November 2010 /Published online: 3 December 2010 # Springer-Verlag 2010 Abstract A library of collision-induced dissociation (CID) data and molecular formulas of more than 7,500 toxicolog- accurate mass spectra has been developed for efficient use ically relevant substances to form the “database and library of liquid chromatography in combination with hybrid of toxic compounds”. For practical evaluation, blood and quadrupole time-of-flight mass spectrometry (LC–QTOF- urine samples were spiked with a mixture of 33 drugs at MS) as a tool in systematic toxicological analysis. The seven concentrations between 0.5 and 500 ng mL−1, pre- mass spectra (Δm<3 ppm) of more than 2,500 illegal and pared by dichloromethane extraction or protein precipita- therapeutic drugs, pesticides, alkaloids, other toxic chem- tion, and analyzed by LC–QTOF-MS in data-dependent icals and metabolites were measured, by use of an Agilent acquisition mode. Unambiguous identification by library 6530 instrument, by flow-injection of 1 ng of the pure search was possible for typical basic drugs down to 0.5– substances in aqueous ammonium formate–formic acid– 2ngmL−1 and for benzodiazepines down to 2–20 ng mL−1. methanol, with positive and negative electrospray- The efficiency of the method was also demonstrated by re- ionization (ESI), selection of the protonated or deproto- analysis of venous blood samples from 50 death cases and nated molecules [M+H]+ or [M−H]− by the quadrupole, and comparison with previous results. In conclusion, LC– collision induced dissociation (CID) with nitrogen as QTOF-MS in data-dependent acquisition mode combined collision gas at CID energies of 10, 20, and 40 eV. The with an accurate mass database and CID spectra library fragment mass spectra were controlled for structural seemed to be one of the most efficient tools for systematic plausibility, corrected by recalculation to the theoretical toxicological analysis. fragment masses and added to a database of accurate mass Keywords Accurate mass spectra library. Collision-induced dissociation . Liquid chromatography. Time of flight mass Published in the special issue Forensic Toxicology with Guest Editors spectrometry. Peak identification . Systematic toxicological Frank T. Peters, Hans H. Maurer, and Frank Musshoff. analysis S. Broecker : S. Herre : F. Pragst (*) Institute of Legal Medicine, University Hospital Charité, Turmstraße 21, Building N, 10559, Berlin, Germany Introduction e-mail: [email protected] Systematic toxicological analysis is the general search for B. Wüst toxic compounds in a biological sample, for instance in Agilent Technologies, Hewlett-Packard-Straße 8, human blood, urine, organ tissues, or hair, without any 76337, Waldbronn, Germany information about presence and kind of poisons. It is one of the most difficult tasks of analytical chemistry because of J. Zweigenbaum the huge number of possible poisons and poison metabo- Agilent Technologies, Inc., 2850 Centerville Road, BL3-2 3L11, lites which may occur in low and very low concentrations Wilmington, DE 19808-1610, USA in the complicated matrix. It includes toxic gases, volatile 102 S. Broecker et al. substances, metal ions and, as the largest group, organic sition” is restricted to a list of preselected precursors compounds with low volatility such as illegal and thera- included in the method. peutic drugs, pesticides, chemical reagents, and alkaloids. Mass spectra libraries for LC–MS with fragmentation by Up-to-date methods for systematic toxicological analysis of in-source collision and LC–MS–MS with fragmentation in organic compounds consist of suitable sample preparation the collision cell between both MS units have been which is able to extract as many poisons as possible from described in several papers and contain between 301 and the matrix, and a combination of chromatography and 1,253 substances [13–16]. molecular spectrometry in order to separate the extracted The availability of time-of-flight mass spectrometers mixture and to characterize the components. Widely used with much improved mass resolution and mass accuracy as method combinations are capillary gas chromatography– detectors in liquid chromatography (LC–TOF-MS) provid- mass spectrometry (GC–MS) [1] and high-performance ed new possibilities in the use of LC–MS for toxicological liquid chromatography with photodiode array detection screening in blood, urine, hair, meconium, and vitreous (HPLC–DAD) [2]. There is not yet any possibility of humor [19–36]. The working principle of these instruments determining the exact structure from these spectra. Therefore, enables comprehensive recording of all data. Therefore, substance identification is always based on comparison of the there is no a priori limitation or prediction of the substances unknown spectrum with those in a library of spectra of included in the search procedure. The increased mass toxicologically relevant compounds. resolution also provides high selectivity for overlapping In the last decade, several approaches have been made to peaks and high matrix burden. The most important use liquid chromatography in combination with mass advantage is that the molecular formula of an analyte is spectrometry (LC–MS or LC–MS–MS) with electrospray directly available from the accurate molecular mass and the ionization (ESI) or atmospheric pressure chemical ioniza- isotope peak pattern. For substance identification by use of tion (APCI) for systematic toxicological analysis [3–36]. the molecular formula, theoretical databases of toxicolog- The application of single-stage quadrupole or ion-trap mass ically relevant compounds with up to 50,500 substances spectrometers for this purpose is limited because of including metabolites [29] or in-house databases with 100 disturbances by high matrix burden and co-eluting peaks. to 869 substances have been created [19–22, 24–28, 30, By use of triple-quadrupole mass spectrometers or, more 33–35]. favorably, hybrid triple-quadrupole linear ion-trap mass Different search procedures are used in TOF methodology. spectrometers these problems have been solved but the The group of Ojanperä and Pelander used reversed target search can only be performed as multi-targeted screening search, which means the TOF file is searched for target masses [7–18]. This means that substances can be detected only if included in the library [18–27]. Polettini et al. used forward they are a priory included in the method. The number of basepeak search, which means the base mass peak of the substances in such a procedure is limited by the minimum unknown chromatographic peak in the analysis file was dwell time for each multi reaction monitoring (MRM) searched after proton subtraction in their large database of transition included in one measurement cycle. Nevertheless, 50,500 compounds of toxicological interest and many phase a powerful screening procedure with 700 substances in one I and phase II metabolites [31, 32]. chromatographic run has been developed by Dresen et al. However, the molecular formula of an eluted unknown with a hybrid triple-quadrupole linear ion-trap mass substance in a chromatogram is only a first step of the spectrometer by limiting the detection time of every analyte identification, because of the huge number of possible to a chromatographic time window of 2 min, information- isomers, as can easily be shown by use of chemical dependent acquisition (IDA) using the sensitive enhanced software. For instance, according to Molgen (molecular product ion scan of the instrument, and uniting the fragment structure generation [37]) for the nominal molecular mass ions from three collision energies in the trap to obtain one M=149, 27 different molecular formulas are theoretically mixed mass spectrum (collision energy spread) [17]. possible if only the elements C, H, N, and O are included. Generally, LC screenings with MS–MS identification TOF-MS with mass accuracy <3 ppm can clearly distin- consist of a survey scan to detect the analytes and a guish between these 27 possibilities. One is C9H11NO with dependent scan for measurement of the corresponding MS– the accurate molecular mass 149.084060. Based on the MS spectra which are submitted to library search for rules of chemical bonds between these atoms, the software identification. Survey scan and dependent scan can be theoretically calculates 25,895,621 structural isomers accomplished within the same analytical run by automatic (stereoisomers not included). From these, 724 substances selection of precursor ions and measurement of the MS–MS are recorded in the Beilstein database and only 45 are spectra immediately after their detection in the survey scan. included in the NIST register. The software “Chemspider” In “data-dependent acquisition” this is determined only by [38] shows structural formulas of 829 compounds with the the actual MS data whereas “information-dependent acqui- molecular formula C9H11NO which are all reasonable, Development and practical application of a library of CID accurat 103 among them, for instance, cathinone, N,N-dimethylbenza- masses and isotope pattern are