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Findings of illegal hormone preparations such as syringes, bottles, cocktails, and so on, are an important information source for the nature of the current abuse of anabolic steroids and related compounds as growth-promoting agents in cattle. A new screening method for steroids in cocktails is presented based on liquid chromatography (LC) with diode-array UV-absorbance detection and electrospray ionization time-of-flight mass spectrometry (ESI-TOFMS). Accurate mass measurements were performed at a mass resolution of 4000 using continuous introduction of a lock mass through a second (electro)sprayer. Similar experiments were carried out using dual-sprayer quadrupole time-of-flight mass spectrometry (ESI-QTOFMS/MS) at a mass resolution of 10 000 with data-dependent MS/MS acquisition; i.e. beyond an intensity threshold for the [M + H](+) ions, MS/MS spectra were automatically acquired at three different collision energies. Elemental compositions were calculated for precursor and product ions and it is shown that the combined information from LC retention behavior, UV spectra, elemental compositions, and accurate mass MS/MS spectra yield a fast impression of the steroids present in the complex mixture. Using a new software tool for structure elucidation of MS/MS spectra, an additional non-steroidal additive was identified as well.

The molecular structure of a series of homo- and copolyesters was studied using sustained off-resonance irradiation collisionally activation dissociation on a Fourier transform ion cyclotron resonance mass spectrometer. Electrospray ionization was used as an ionization technique. The most important fragmentation pathways of the homopolyesters poly(dipropoxylated bisphenol-A/adipic acid) and poly(dipropoxylated bisphenol-A/isophthalic acid) were studied. Six different dissociation mechanisms were observed which are very similar to the mechanisms found to occur during pyrolysis of these compounds. Four of these mechanisms are a result of cleavages of the ester bond and the others are due to cleavages of the ether bond or bisphenol-A unit. Some of the fragments expected are not present in the spectrum, indicating that each fragment has a specific sodium affinity. Sequence-specific fragments of two of the three copolyester sequences that theoretically can exist were experimentally observed. Fragments that originate from the third sequence are not unique and can also be formed from other sequences. Therefore, it was not possible to determine the presence of the third sequence. Copyright 2000 John Wiley & Sons, Ltd.

Hyphenation of liquid chromatography (LC) techniques with electrospray ionization (ESI) orthogonal acceleration time-of-flight (oa-TOF) mass spectrometry (MS) provides both MS-based structural information and LC-based quantitative data in polymer analysis. In one experimental setup, three different LC modes are interfaced with MS: size-exclusion chromatography (SEC/MS), gradient polymer elution chromatography (GPEC/MS), and liquid chromatography at the critical point of adsorption (LCCC/MS). In SEC/MS, both absolute mass calibration of the SEC column based on the polymer itself and determination of monomers and end groups from the mass spectra are achieved. GPEC/MS shows detailed chemical heterogeneity of the polymer and the chemical composition distribution within oligomer groups. In LCCC/MS, the retention behavior is primarily governed by chemical heterogeneities, such as different end group functionalities, and quantitative end group calculations can be easily made. The potential of these methods and the benefit of time-of-flight analyzers in polymer analysis are discussed using SEC/MS of a polydisperse poly(methyl methacrylate) sample, GPEC/MS of dipropoxylated bisphenol A/adipic acid polyester resin, LCCC/MS of alkylated poly(ethylene glycol), and LCCC/MS of terephthalic acid/neopentyl glycol polyester resin.

Mass spectrometry of intact synthetic polymers has been limited to field desorption (FD) MS for many years. More recently, soft ionization techniques such as electrospray (ESI) and matrix-assisted laser desorption/ionization (MALDI) and the revival of time-of-flight analyzers created new opportunities for the characterization of polymers. In this review emphasis is put on MALDI time-of-flight mass spectrometry of polymers. The selection of an appropriate MALDI matrix, cationization salt and sample preparation techniques are critical success factors for obtaining a reliable mass spectrum and to infer structural information such as monomer mass(es) and end-groups. However even under optimized conditions mass discrimination in the analysis of polydisperse polymers and specific oligomer discrimination might occur. Hence hyphenated techniques such as size exclusion chromatography (SEC)/MALDI have been developed. Many different polymer applications appeared in recent literature, but most studies deal with homopolymers. Many challenges remain, particularly in the fields of halogenated polymers, polyolefines, copolymers, blends, and in the sequencing of block-copolymers. © 1999 John Wiley & Sons, Inc., Mass Spec Rev 18: 309–344, 1999

Polydisperse synthetic polymers cannot be characterized reliably by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI MS) alone. Coupling of size-exclusion chromatography (SEC) with MALDI MS provides both absolute mass calibration of the SEC column based on the polymer itself and determination of monomers and end groups from the mass spectra. Now a micro-scale SEC (μSEC) system has been developed in which the MALDI matrix solution is coaxially added to the column effluent and directly spotted onto the MALDI targets in a robotic interface. Each spot corresponds with a 10-s elution window from the μSEC column, thus yielding a narrow polymer distribution which is amenable to characterization by MALDI MS. This μSEC/MALDI MS system is less laborious and time-consuming and features very low solvent consumption and reduced solvent emission during the evaporation step as compared to the conventional-sized SEC/fraction collector systems applied in the past. The performance is demo...

The crosslinking of alkyd resins has been studied using ethyl linoleate and methyl ricinoate as model compounds. With quantitative13C NMR it was established that ether- and peroxy-crosslinks were formed in roughly equal amounts. Double bonds reacted to give epoxides, endoperoxides, and β-scission into aldehydes. Using SIMS it was established that dimers through pentamers were formed having included several oxygen atoms. After reduction of peroxide (crosslinks) with stannous chloride, the higher oxygen homologues decreased. In the NMR spectrum of the reduced material, peroxide and epoxide signals completely disappeared. High resolution electrospray ionization mass spectra (ESI-MS) yielded evidence regarding the crosslink mechanism. The non-conjugated linoleic acid was found to crosslink by combination of radicals: dimers were having masses 2(M-1). The conjugated linoleic acid crosslinked by addition of radicals to the double bond and disproportionation, yielding masses 2M as well. (M = mass of ethyl linoleate or methyl ricinoate, respectively, including several oxygen atoms.) EIS-MS of oligomers after isotopic exchange enabled estimation of OH and OOH groups.

Synthetic polymers having a polydispersity greater than 1.1 cannot be reliably characterized by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry alone. This complication has been overcome by off-line coupling of size-exclusion chromatography (SEC) with MALDI. Data are presented for a wide variety of synthetic polymers having polydispersities from 1.7 up to 3.0. Polystyrene (15 kDa and 48 kDa), polybutylacrylate (62 kDa), polycarbonate (28 kDa), aromatic polyester resin (8 kDa) and a methyl methacrylate–methacrylic acid copolymer (34 kDa) were injected on a conventional SEC system with refractive index (RI) or UV absorbance detection and 40 fractions collected over the polymer distribution. Ten fractions were analyzed by a MALDI time-of-flight mass spectrometer operated in the continuous extraction linear mode. The m/z thus obtained were used as calibration points for the SEC-RI system and allowed accurate calculation of the molecular weight averages Mw, Mn, Mz and the polydispersity for all the polymers tested. The data for these entirely different polydisperse polymers were in good agreement with reference data from, for example, SEC with intrinsic viscosity detection. In addition, the choices of matrix and cation for individual types of polymers are commented on. The end groups of the polymers were determined from isotopically-resolved oligomer spectra recorded by MALDI time-of-flight in the delayed-extraction reflectron mode. End groups proposed are compared with reference data from, amongst others, NMR and MALDI Fourier transform ion cyclotron resonance-MS measurements. © 1997 John Wiley & Sons, Ltd.

The hardening of alkyd paints for do-it-yourself purposes is an autoxidation reaction with air oxygen. Since the crosslink reactions are hard to follow in an actual coating system, use is being made of model compounds. These are (m)ethyl esters of unsaturated fatty acids (linoleic and ricinoic) which contain the reactive substructures responsible for crosslinking of an alkyd. In this work, the oligomeric mixture resulting from crosslinking was studied with various mass spectrometric (MS) techniques : direct chemical ionization (DCI), fast atom bombardment (FAB), field desorption (FD), electrospray ionization (ESI) and secondary ion (SI) MS. Consistent results were obtained with all techniques : oligomers yielded signals consisting of groups of peaks 16 mass units apart, pointing to a series of oxygenated homologues. Differences were encountered, however, in the number of oligomers that could be seen with the various techniques. Using ESI-MS and SIMS, oligomer formation up to the tetramer (linoleic) or hexamer (ricinoic) was observed. DCI, FAB and FD generally yielded signals from dimers and trimers only. From the detailed ESI and DCI spectra, a difference in the crosslinking mechanism between conjugated and non-conjugated fatty acids becomes apparent (radical addition to the double bond or recombination of radicals, respectively). Also, using a mixture of the linoleic and ricinoic esters, mutual reaction between these two types of fatty acids can be demonstrated.

Three complementary techniques were applied to the characterization of a complex (methoxymethyl)melamine mixture: gradient reversed-phase liquid chromatography/mass spectrometry (LC/MS), size-exclusion chromatography/mass spectrometry (SEC/MS) and capillary zone electrophoresis/mass spectrometry (CZE/MS). The LC/MS system was used with an atmospheric-pressure chemical ionization interface and the spectra obtained showed characteristic fragmentation patterns and detailed structural information of the monomeric components: up to 18 individual monomeric and 7 dimeric components could be identified. The SEC/MS system was used with a (pneumatically-assisted) electrospray interface, thus providing soft ionization and molecular information about the trimers, the dimers, and the monomers, indicating that the total number of components exceeds one hundred. In addition, the applicability of CZE/MS and H/D-exchange CZE/MS was demonstrated using a tricoaxial sheath-flow electrospray interface. The CZE separation turned out to be governed by the number of unreacted melamine positions only. Detailed structural information could be deduced simply by combining the CZE migration behaviour, the mass spectrometry data and the total number of exchangeable hydrogen atoms as calculated from the H/D exchange experiments

Capillary zone electrophoresis was coupled on-line via a tricoaxial sheath flow interface with a benchtop quadrupole mass spectrometer (CZE-MS) equipped with a (pneumatically assisted) electrospray ionization source. Experimental parameters were optimized for operation in the negative-ion scan mode. The system was applied to the fingerprinting of an alkylsulphate detergent, to the identification of an impurity in a chloramine-T disinfectant, to the characterization of a complex polyethoxylated alkylphosphate emulgator, to the analysis of phenoxy acid herbicides and to the impurity profiling of MCPP. The optimized system was found to fully maintain the CZE separation performance, showing up to 1 million theoretical plates for the low-molecular-mass analytes studied. The sensitivity in the full-scan mode was typically in the picogram range and sufficient for the separation and identification of minor impurities in these industrial chemicals, down to the 0.1% level. In addition, the feasibility of on-column transient isotachophoresis (ITP-CZE-MS) under high electroosmotic flow conditions has been demonstrated for sample preconcentration and subsequent identification of trace impurities.

The last 2 years multi-compound methods are gaining ground as screening methods. In this study a high-resolution liquid chromatography combined with time-of-flight mass spectrometry (HRLC-ToF-MS) is tested for the screening of about 100 veterinary drugs in three matrices, meat, fish and egg. While the results are satisfactory for 70-90% of the veterinary drugs, a more efficient sample preparation or extract purification is required for quantitative analysis of all analytes in more difficult matrices like egg. The average mass measurement error of the ToF-MS for the veterinary drugs spiked at concentrations ranging from 4 to 400 microg/kg, is 3.0 ppm (median 2.5 ppm) with little difference between the three matrices, but slightly decreases with increasing concentration. The SigmaFit value, a new feature for isotope pattern matching, also decreases with increasing concentration and, in addition, shows an increase with increasing matrix complexity. While the average SigmaFit value is 0.04, the median is 0.01 indicating some high individual deviations. As with the mass measurement error, the highest deviations are found in those regions of the chromatogram where most compounds elute from the column, be it analytes or matrix compounds. The median repeatability of the method ranges from 8% to 15%, decreasing with increasing concentration, while the median reproducibility ranges from 15% to 20% with little difference between matrices and concentrations. The median accuracy is in between 70% and 100% with a few compounds showing higher values due to matrix interference. The squared regression coefficient is >0.99 for 92% of the compounds showing a good overall linearity for most compounds. The detection capability, CCbeta, is within 2 times the associated validation level for >90% of the compounds studied. By changing a few conditions in the analyses protocol and analysing a number of blank samples, it was determined that the method is robust as well as specific. Finally, an alternative validation strategy is proposed and tested for screening methods. While the results calculated for repeatability, within-lab reproducibility and CCbeta show a good comparison for the matrices meat and fish, and a reasonable comparison for the matrix egg, only 27 analyses are required to obtain these results versus 63 analysis in the traditional, 2002/657/EC, approach. This alternative is suggested as a cost-effective validation procedure for screening methods.

The potential for coupling technologies to deliver new, improved forms of bioanalysis is still in its infancy. We review a number of examples in which coupling has been successful, with special emphasis on combining surface-plasmon-resonance biosensors with mass spectrometry. We give an overview of current progress towards combining biosensor-based bioanalysis with chemical analysis for confirmation of paralytic shellfish poisons that are marine toxins. This comprehensive approach could be an alternative to the official methods currently used (e.g., animal testing and high-performance liquid chromatography with fluorescence detection) and could serve as a model for many more such applications.

Ambient mass spectrometry has been used for the analysis of strobilurin residues in wheat. The use of this novel, challenging technique, employing a direct analysis in a real time (DART) ion-source coupled with a time-of-flight mass spectrometer (TOF MS) and a desorption electrospray ionization (DESI) source coupled with a linear ion trap tandem MS (LIT MS(n)), permitted a direct screen of the occurrence of target fungicides in treated grains in less than 1 min. For quantification purpose by DART-TOF MS, an ethyl acetate extract had to be prepared. With the use of a prochloraz as an internal standard, the performance characteristics obtained by repeated analyses of extract, spiked at 50 microg kg(-1) with six strobilurins (azoxystrobin, picoxystrobin, dimoxystrobin, kresoxim-methyl, pyraclostrobin, and trifloxystrobin), were in the following range: recoveries 78-92%, repeatability (RSD) 8-15%, linearity (R(2)) 0.9900-0.9978. The analysis of wheat with incurred strobilurin residues demonstrated good trueness of data generated by the DART-TOF MS method; the results were in a good agreement with those obtained by the conventional approach, i.e., by the QuEChERS sample handling procedure followed by identification/quantification employing high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Tandem mass spectrometry using DESI-LIT MS(n) provided a sufficient number of product ions for confirmation of the identity of azoxystrobin and pyraclostrobin in incurred wheat samples.

The use of beta-agonists as growth promoters in cattle breeding is forbidden in many countries for reasons of fair trade and consumer protection. In recent years the use of liquid chromatography (LC) tandem mass spectrometry (MS/MS) has been shown to be the method of choice for the control of beta-agonists. In this study an LC-MS/MS multiresidue analysis method is presented for trace analysis of 22 beta-agonists. A truly generic concept has been designed based on mixed-mode solid-phase extraction and positive electrospray ionisation LC-MS/MS operated in the multiple reaction monitoring mode. This method allows application to a wide variety of sample matrices such as urine, feed and hair, following minor modifications to the analysis procedure only. The method features fit-for-purpose sensitivity in urine as shown by CCalpha and CCbeta values of less than 0.2 and less than 0.5 microg/l respectively, for all beta-agonists studied (terbutaline and reproterol, less than 0.3 and less than 1.0 respectively). Similar but semiquantitative application to feed and hair showed CCbeta values of less than 10.0 and less than 5.0 microg/kg, respectively. A further simplification and improvement is demonstrated using Ultra Performance LC (UPLC) and fast-switching MS/MS. The successful validation of this method following the latest EU requirements and its application to real samples demonstrate that a new versatile tool has been achieved for veterinary control of beta-agonists.

A liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) method was developed using the latest high-resolution LC column technology, the ultra performance liquid chromatography (UPLC (TM)), and electrospray ionization (ESI) in the positive ion mode. Gradient UPLC separation conditions were optimized for a group of 22 analytes comprising 17 glucocorticosteroids, specific designer steroids such as tetrahydrogestrinone (THG) and specific beta(2)-agonists such as formoterol. The UPLC/TOFMS separation obtained required 5.5 min only for all the substances tested. Even the critical pair of dexamethasone and betamethasone isomers was almost completely resolved. Thanks to the over 10,000 full-width at half maximum (FWHM) mass resolution and high mass accuracy features of TOFMS 50 mDa window accurate mass chromatograms could be reconstructed for the individual analytes. Sensitive screening in human and calf urine samples fortified at the glucocorticosteroids minimum required performance limit (MRPL) of 30 mu g L-1 (human urine, sports doping) and 2 mu g L-1 (calf urine, veterinary control) could be obtained. The potential of UPLC/TOFMS for confirmatory analysis was shown by determining the accurate mass of all compounds and fragment ions upon in-source collision-induced dissociation (CID) at different energies. The exact mass measurement errors for all glucocorticosteroids were found to be within 6 ppm. Considering veterinary control, limits of detection (LOD) and limits of quantification (LOQ) were determined for most of the analytes in calf urine and found to range from 0.1 to 3.3 and from 0.4 to 4.4 mu g L-1, respectively. The method can be easily extended with other banned substances of interest, as demonstrated by the addition of 21 beta(2)-agonists to the original analyte mixture in urine, without causing any interferences. (c) 2006 Elsevier B.V. All rights reserved.

A new combined doping control screening method for the analysis of anabolic steroids in human urine using liquid chromatography/electrospray ionization orthogonal acceleration time-of-flight mass spectrometry (LCoaTOFMS) and gas chromatography/electron ionization orthogonal acceleration time-of-flight mass spectrometry (GCoaTOFMS) has been developed in order to acquire accurate full scan MS data to be used to detect designer steroids. The developed method allowed the detection of representative prohibited substances, in addition to steroids, at concentrations of 10 ng/mL for anabolic agents and metabolites, 30 ng/mL for corticosteroids, 500 ng/mL for stimulants and fl-blockers, 250ng/mL for diuretics, and 200ng/mL for narcotics. Sample preparation was based on liquid-liquid extraction of hydrolyzed human urine, and the final extract was analyzed as trimethylsilylated derivatives in GCoaTOFMS and underivatized in LCoaTOFMS in positive ion mode. The sensitivity, mass accuracy, advantages and limitations of the developed method are presented. Copyright (C) 2007 John Wiley & Sons, Ltd.

New anabolic steroids show up occasionally in sports doping and in veterinary control. The discovery of these designer steroids is facilitated by findings of illicit preparations, thus allowing bioactivity testing, structure elucidation using NMR and mass spectrometry, and final incorporation in urine testing. However, as long as these preparations remain undiscovered, new designer steroids are not screened for in routine sports doping or veterinary control urine tests since the established GC/MS and LC/MS/MS methods are set up for the monitoring of a few selected ions or MS/MS transitions of known substances only. In this study, the feasibility of androgen bioactivity testing and mass spectrometric identification is being investigated for trace analysis of designer steroids in urine. Following enzymatic deconjugation and a generic solid-phase extraction, the samples are analyzed by gradient LC with effluent splitting toward two identical 96-well fraction collectors. One well plate is used for androgen bioactivity detection using a novel robust yeast reporter gene bioassay yielding a biogram featuring a 20-s time resolution. The bioactive wells direct the identification efforts to the corresponding well numbers in the duplicate plate. These are subjected to high-resolution LC using a short column packed with 1.7-microm C18 material and coupled with electrospray quadrupole time-of-flight mass spectrometry (LC/QTOFMS) with accurate mass measurement. Element compositions are calculated and used to interrogate electronic substance databases. The feasibility of this approach for doping control is demonstrated via the screening of human urine samples spiked with the designer anabolic steroid tetrahydrogestrinone. Application of the proposed methodology, complementary to the established targeted urine screening for known anabolics, will increase the chance of finding unknown emerging designer steroids, rather then being solely dependent on findings of the illicit preparations themselves.

The influence of the ionization efficiency on the measured copolymer sequence distribution is presented. Large differences in ionization efficiency were observed for mixtures of homopolyesters containing dipropoxylated bisphenol A/adipic acid and dipropoxylated bisphenol A/isophthalic acid and the corresponding copolyester dipropoxylated bisphenol A/isophthalic acid/adipic acid. The adipic acid structure has a higher affinity for the sodium cation, which results in more intense peaks for adipic acid containing oligomers. Relative sodium affinities of the oligomers were found to increase with an increasing number of acid end groups in favor of adipic acid containing oligomers. The ESI response of the oligomers depends on the polymer concentration in the sprayed mixture. This makes it impossible to correct for the ionization efficiency necessary for copolymer analysis. If differences in ionization efficiency are not corrected, the ion intensities in the copolymer mass spectra will show large deviations from the real composition and no conclusion can be drawn about the chemical (in)homogeneity of the MWD nor the random or block structure of the copolymer. This will also be valid for other cationization techniques like MALDI and FAB.

The existence of polythiocar- bamatozinc complexes, species pre- sumed to be involved in catalyzing sulfur vulcanization, has been studied by com- putational and mass-spectrometric tech- niques. Density functional calculations reveal that the sulfuration energy of bis(trithiocarbamato)zinc(ii) is compara- ble to that of bis(phenyltrithiolato)- zinc(ii), a stable sulfurated complex. Interestingly, the analogous nonsym- metrically sulfurated complex (dithio- carbamato)(tetrathiocarbamato)zinc(ii) is energetically slightly more favorable. The calculations indicate that polythio- carbamatozinc complexes may indeed be involved in the sulfur vulcanization cascade, a conclusion corroborated by ensuing laser-desorption ionization (LDI) mass-spectrometric measure- ments on mixtures of dithiocarbamato- zinc(ii) complexes and elemental sulfur. These demonstrate the presence of poly- thiocarbamatozinc ions that are sulfu- rated with up to eight additional sulfur atoms.

A method is described for the identification of monomers and end groups in low molecular weight synthetic polymers using electrospray ionization and an orthogonal accelerated time-of-flight mass spectrometer. Thanks to the high mass accuracy the elemental composition of the monomer and end group of an unknown alkylated poly(ethylene oxide) was determined with an error of 1 mDa. The present method compares well with literature data for polymer characterization by Fourier transform ion cyclotron resonance mass spectrometry and might be a simple alternative provided that the inherent lower resolution is not critical.

This study describes an evaluation of different clean-up methods for the determination of polychlorinated dibenzo-p-dioxins and-dibenzofurans (PCDD/F extracts of fly-ash. The PCDD/F fractions obtained were additionally investigated for the presence or absence of polychlorinated benzenes (PCBz), polychlorinated biphenyls (PCB) and polychlorinated phenols (PCP) to detect any interference, and to test for determinability in the same fractions. Three different methods were compared, viz. solid phase extraction (SPE) using disposable benzenesulphonic acid and silicagel cartridges, multilayer silicagel plus alumina column chromatography (MLA); and an active carbon method plus multilayer silicagel plus alumina column chromatographic (ACM) method. The fractions obtained were analyzed by capillary gas chromatography and mass selective detection using the isotope dilution method. Both the variances and the levels of PCDD/F showed good agreement between the different clean-up methods investigated. The SPE-method, being fast and simple, should be preferred for the determination of PCDD/F in fly-ash; it also allows the determination of PCBz and PCB in the fractions. The presence of PCBz and PCB in the clean-up fractions does not interfere with the determination of PCDD/F provided that the mass spectra of PCDD/F are checked for [M-COCI] fragments.

Matrix-assisted laser desorption/ionization (MALDI) and Fourier-transform ion cyclotron mass spectrometry (FT-ICR-MS) are combined for the characterization of polyoxyalkyleneamines. 1H and 13C NMR data are used to confirm and quantify structural assignments. Characterization of the molecular weight distribution, chemical composition distribution, and end group distribution of amine-terminated (co-)polymers of ethylene oxide and propylene oxide is rather complex because the convolution of these three entities yields an ensemble of structurally related molecules which is hard to characterize by conventional analytical techniques. In this study MALDI FT-ICR-MS is used to resolve intact, Na+ cationized, oligomer ions in the mass range from m/z 500 to 3500. This revealed the presence of various compositional distributions in the polyoxyalkyleneamines. The individual compound masses in the molecular weight distributions were measured with a mass accuracy of

Several molecular imaging techniques are available to study and understand biological objects, like positron-emission tomography, fluorescence, and magnetic resonance imaging. These techniques often require chemical probes and image in a targeted approach. As many biological questions can only be answered in a systems approach, molecular imaging methods that can simultaneously measure many molecules are desired. Mass spectrometry imaging (MSI) is capable of measuring many molecules simultaneously without the use of chemical probes. MSI experiments often require sample stage vacuum conditions and extensive sample pretreatment such as matrix application. Vacuum conditions can disrupt or damage biological samples, and sample pretreatment prevents real-time analyses and can cause analyte losses, analyte delocalization and denaturation of proteins. Ambient ionization was introduced to measure samples under ambient conditions without any sample pretreatment, and ambient MSI followed rapidly after that. A next generation of ambient MSI techniques is desired to improve its sensitivity, molecular mass range, and spatial resolution. This work aims to improve the capabilities and broaden the scope of laser ablation electrospray ionization (LAESI) MSI. In order to broaden the scope and increase the understanding of ambient LAESI-MS(I), polymer materials and synthetic fibers were investigated. The direct analysis of synthetic fibers under ambient conditions is highly desired to identify the polymer, the finishes applied and irregularities that may compromise its performance and value. In Chapter 2 LAESI ion mobility MS was used for the analysis of synthetic polymers and fibers. The key to this analysis was the absorption of laser light by aliphatic and aromatic nitrogen functionalities in the polymers. Analysis of polyamide (PA) 6, 46, 66, and 12 pellets and PA 6, 66, polyaramid and M5 fibers yielded characteristic fragment ions, enabling their unambiguous identification. Synthetic fibers are, in addition, commonly covered with a surface layer for improved adhesion and processing. The same setup, but operated in a transient infrared matrix-assisted laser desorption electrospray ionization mode, allowed the detailed characterization of the fiber finish layer and the underlying polymer. Differences in finish layer distribution may cause variations in local properties of synthetic fibers. In Chapter 2, also the feasibility of mass spectrometry imaging (MSI) of the distribution of a finish layer on the synthetic fiber and the successful detection of local surface defects was shown. Reactions in confined compartments like charged microdroplets are of increasing interest, notably because of their substantially increased reaction rates. When combined with ambient MS, reactions in charged microdroplets can be used to improve the detection of analytes or to study the molecular details of the reactions in real time. In Chapter 3, we introduce a reactive LAESI time-resolved MS method to perform and study reactions in charged microdroplets. This approach was demonstrated with so-called click chemistry reactions between substituted tetrazines and a strained alkyne or alkene. Click reactions are high-yielding reactions with a high atom efficiency. Although click reactions are typically at least moderately fast, in a reactive LAESI approach a substantial increase of reaction time is Summary 149 required for these reactions to occur. This increase was achieved using microdroplet chemistry and followed by MS using the insertion of a reaction tube between the LAESI source and the MS inlet, leading to near complete conversions due to significantly extended microdroplet lifetime. This novel approach allowed for the collection of kinetic data for a model click reaction and showed in addition excellent instrument stability, improved sensitivity, and applicability to other click reactions. In Chapter 3, reactive LAESI was also demonstrated in a mass spectrometry imaging setting to show its feasibility in future imaging experiments. In drug discovery it is important to identify phase I metabolic modifications as early as possible to screen for inactivation of drugs and/or activation of prodrugs. As the major class of reactions in phase I metabolism are oxidation reactions, oxidation of drugs with TiO2 photocatalysis can be used as a simple non-biological method to initially eliminate (pro)drug candidates with an undesired phase I oxidation metabolism. Analysis of reaction products is commonly achieved with mass spectrometry coupled to chromatography. However, sample throughput can be substantially increased by eliminating pretreatment steps and exploiting the potential of ambient MS. Furthermore, online monitoring of reactions in a time-resolved way would identify sequential modification steps. In Chapter 4 we introduce a novel (time-resolved) TiO2-photocatalysis LAESI-MS method for the analysis of drug candidates. This method was proven to be compatible with both TiO2-coated glass slides as well as solutions containing suspended TiO2 nanoparticles, and the results were in excellent agreement with studies on biological oxidation of several drugs. Additionally, a time-resolved LAESI-MS setup was developed and results for verapamil showed excellent analytical stability for online photocatalyzed oxidation reactions within the set-up up to at least one hour. Identification and confirmation of (bio)chemical entities in ambient MS mostly involves accurate mass determination, often in combination with MS/MS work flows. However, an accurate mass only provides the elemental composition of the (bio)molecule, still resulting in numerous possible structures. MS/MS procedures are often insufficient in differentiating between the hundreds possible candidate substances in database searches. Obtaining additional information and thereby improving structural assignment as well as reducing the vast number of possible candidates is thus of high importance in any ambient MS(I) study. In Chapter 5 we present an ambient hydrogen/deuterium exchange (HDX) LAESI-MS method for structure elucidation and confirmation of (bio)molecules. The concept was demonstrated with small molecules, peptides, and proteins. Moreover, the same approach could be applied to MSI as shown by the ambient MSI of arginine and oligosaccharides on an orange slice. Eventually, this approach will allow spatially resolved MSI of different protein conformers and may have a major impact in the life sciences. The main achievements that are described in this thesis offer insights on sample compatibility, hardware improvements to enable online time-resolved reactions and structure elucidation approaches. The outcome of the research chapters shows that LAESI-MS(I) is a highly versatile technique applicable to many research areas. Although the technique is highly 150 dependent on endogenous water in samples for analysis of intact molecules, LAESI can also be exploited for the analysis and identification of (water free) polymer materials. Unfortunately, LAESI sensitivity relative to electrospray ionization is weak, and therefore the technique can currently not live up to the status of the next generation of ambient MSI. Analytes that are present in high abundance are feasible for imaging by LAESI-MS. For low abundance analytes, however, several hardware improvements are required to substantially increase the sensitivity of the results. When the hardware improvements are developed and implemented, the road is open for many end users in, e.g., microbiology, pathology, and botany, to make significant breakthroughs in their fields.