On the inter‐instrument and the inter‐laboratory transferability of a tandem mass spectral reference library. 3. Focus on ion trap and upfront CID

Mass spectral libraries represent versatile tools for the identification of small bioorganic molecules. Libraries based on electron impact spectra are rated robust and transferable. Tandem mass spectral libraries are often considered to work properly only on the instrument that has been used to build the library. An exception from that rule is the ‘Wiley Registry of Tandem Mass Spectral Data, MSforID’. In various studies with data sets from different kinds of tandem mass spectrometric instruments, the outstanding sensitivity and robustness of this tandem mass spectral library search approach was demonstrated. The instrumental platforms tested, however, mainly included various tandem‐in‐space instruments. Herein, the results of a multicenter study with a focus on upfront and tandem‐in‐time fragmentation are presented. Five laboratories participated and provided fragment ion mass spectra from the following types of mass spectrometers: time‐of‐flight (TOF), quadrupole–hexapole–TOF, linear ion trap (LIT), 3‐D ion trap and LIT–Orbitrap. A total number of 1231 fragment ion mass spectra were collected from 20 test compounds (amiloride, buphenin, cinchocaine, cyclizine, desipramine, dihydroergotamine, dyxirazine, dosulepin, ergotamine, ethambutol, etofylline, mefruside, metoclopramide, phenazone, phentermine, phenytoin, sulfamethoxazole, sulfamoxole, sulthiame and tetracycline) on seven electrospray ionization instruments using 18 different instrumental configurations for fragmentation. For 1222 spectra (99.3%), the correct compound was retrieved as the best matching compound. Classified matches (matches with ‘relative average match probability’ >40.0) were obtained for 1207 spectra (98.1%). This high percentage of correct identifications clearly supports the hypothesis that the tandem mass spectral library approach tested is a robust and universal identification tool. Copyright © 2012 John Wiley & Sons, Ltd.     

Towards a universal product ion mass spectral library - reproducibility of product ion spectra across eleven different mass spectrometers

Product ion spectra produced by collision-induced dissociation (CID) in tandem mass spectrometry experiments can differ markedly between instruments. There have been a number of attempts to standardise the production of product ion spectra; however, a consensus on the most appropriate approach to the reproducible production of spectra has yet to be reached. We have previously reported the comparison of product ion spectra on a number of different types of instruments - a triple quadrupole, two ion traps and a Fourier transform ion cyclotron resonance mass spectrometer (Bristow AWT, Webb KS, Lubben AT, Halket JM. Rapid Commun. Mass Spectrom. 2004; 18: 1). The study showed that a high degree of reproducibility was achievable. The goal of this study was to improve the comparability and reproducibility of CID product ion mass spectra produced in different laboratories and using different instruments. This was carried out experimentally by defining a spectral calibration point on each mass spectrometer for product ion formation. The long-term goal is the development of a universal (instrument independent) product ion mass spectral library for the identification of unknowns.The spectra of 48 compounds have been recorded on eleven mass spectrometers: six ion traps, two triple quadrupoles, a hybrid triple quadrupole, and two quadrupole time-of-flight instruments. Initially, 4371 spectral comparisons were carried out using the data from eleven instruments and the degree of reproducibility was evaluated. A blind trial has also been carried out to assess the reproducibility of spectra obtained during LC/MS/MS.The results suggest a degree of reproducibility across all instrument types using the tuning point technique. The reproducibility of the product ion spectra is increased when comparing the tandem in time type instruments and the tandem in space instruments as two separate groups. This may allow the production of a more limited, yet useful, screening library for LC/MS/MS identification using instruments of the same type from different manufacturers.     

Instrument dependence of electrospray ionization and tandem mass spectrometric fragmentation of the gingerols

The gingerols, including [6]-, [8]-, and [10]-gingerols, a series of chemical homologs differentiated by the length of their unbranched alkyl chains, have been identified as major active components in fresh ginger rhizome. The purpose of this study was to investigate the utility of ion trap liquid chromatography/tandem mass spectrometry (LC/MS/MS) as an online tool to identify and quantify these compounds in raw or processed ginger rhizome samples. Negative mode electrospray ionization (ESI) was used in MS, MS/MS and MS(n) experiments in quadrupole ion trap instruments from two different manufacturers and in high-resolution and accurate mass MS and MS/MS experiments in a Fourier transform ion cyclotron resonance mass spectrometer to elucidate the ionization and fragmentation mechanisms of these compounds in these instruments. Positive mode ESI, which generated many more fragment ions in full scan MS even under gentle ionization conditions, was also used in LC/MS and MS/MS experiments and in direct infusion MS and MS/MS experiments. Consistent and predictable ionization and fragmentation behaviors were observed for all gingerols when analyzed in the same instrument. Instruments from different manufacturers, however, had different ionization mechanisms. The major difference between instruments was their ability to form covalent dimer adducts of the gingerols. Subsequent fragmentation patterns of the precursor ions were essentially identical. These results clearly demonstrate that LC/MS instruments produce data that cannot necessarily be replicated in other laboratories, especially if those laboratories do not have the same instrument model from the same manufacturer. This presents major problems for metabolite target analysis, metabolic profiling and metabolomics investigations, which would benefit from LC/MS mass spectrum libraries as they do from GC/MS mass spectrum libraries, because such libraries may not be valid across platforms.     

Comparison of product ion spectra obtained by liquid chromatography/triple-quadrupole mass spectrometry for library search

Reproducibility of product ion spectra acquired using a liquid chromatography/triple-quadrupole mass spectrometry (LC/MS/MS) instrument over a 4-year period, and with three other LC/MS/MS instruments, one from the same manufacturer and two from a different manufacturer, was examined. The MS/MS spectra of 30 drug substances were generated in positive electrospray ionization mode at low, medium, and high collision energies (20, 35, and 50 eV). Purity and Fit score percentages against a 400-compound LC/MS/MS spectral library were calculated using an algorithm in which fragment intensity ratios and weighting factors were included. The long-term reproducibility study was conducted using a brand A instrument; after 4 years the reproducibility of the product ion spectra was still 94%, expressed as average Purity score. The inter-laboratory study involved two parts. Firstly, two LC/MS/MS spectral libraries, created independently in separate laboratories using brand A instruments, were compared with each other. The average Fit and Purity scores of spectra from one library against the other were better than 93 and 91%, respectively, when the same collision energies were used. Secondly, for the comparison of product ion spectra between brand A and brand B instruments, fragmentation conditions were first standardized for amitriptyline as the standard analyte. The average Fit scores of brand B spectra against the brand A spectral library varied between 79 and 85% at all three collision energies. These results indicate that, after standardizing the instrumental conditions, LC/MS/MS spectral libraries of drug substances are suitable for inter-laboratory use.     

Multiphoton ionization and photodissociation at the second-order space focus in a time-of-flight mass spectrometer: The amino acid tryptophan

Neutral IR-desorbed molecules of the amino acid tryptophan entrained in a supersonic beam were post-ionized by multiphoton ionization (MUPI) in the gas phase. Variations in the intensity of the ionization laser produced different degrees of fragmentation observed in the time-of-flight mass spectra of tryptophan according to the ladder-switching model for MUPI fragmentation. From the number of absorbed photons the upper values for the appearance energies of specific fragment ions were obtained. A complete fragmentation pathway of tryptophan was established by using photodissociation of molecular ions and fragment ions formed by MUPI. Photodissociation was successfully performed by secondary excitation with a UV laser at the second-order space focus of a reflection time-of-flight instrument. A fragmentation tree for a laser-desorbed molecule obtained in a laser tandem time-of-flight instrument is shown for the first time.     

Evaluation of protocols for reproducible electrospray in-source collisionally induced dissociation on various liquid chromatography/mass spectrometry instruments and the development of spectral libraries

Mass spectral libraries provide a tool for identifying unknown compounds using both molecular weight and fragmentation information. Mass spectrometers with electrospray ionisation (ESI) and atmospheric chemical ionisation (ApCI) sources have the capability to produce data of this type using in-source collisionally induced dissociation (CID), and in-source CID libraries can be created. Due to the variation in electrospray source design from different instrument manufacturers, the production of reproducible in-source CID spectra that can be used in libraries for all instrument types is not a trivial task. To date, the evaluation of the production of in-source CID libraries has tended to focus on similar instruments from one manufacturer. The studies have also tended to focus on specific compound classes, with a limited molecular weight range.This report describes the findings from the investigation of protocols for the creation of mass spectral libraries using ESI in-source CID on six instruments from four different manufacturers. The overall goal was to create a spectral library for the identification of unknowns. The library could then be applied across all manufacturers' electrospray instruments.Two different experimental protocols were attempted. The first used a tuning compound to establish standard ESI source conditions, with fixed fragmentation potentials. The second involved the attenuation of the [M + H](+) ion to a known degree. A diverse range of compounds (pharmaceutical, photographic, pesticides) was tested to establish the reproducibility of the spectra on the six instruments. Both protocols produced spectra on the various instruments that in many cases were very similar. In other examples, the spectra differed not only in their relative ion abundances, but also in terms of the spectral content. Important observations regarding the effect of ion source design are also reported.The degree of spectral reproducibility was calculated off-line by comparing the five most abundant ions (20% for each ion that matches) from each spectrum on each instrument. This approach was adopted, as we do not possess a software package that met our requirements for spectral comparison. Match factors (% fit) were calculated by comparing each spectrum against the spectra recorded for the same compound and then for all other compounds, on each instrument. The % fit values derived by the off-line approach gave a clear view of the spectral reproducibility from instrument to instrument and also discriminated the spectra of the various compounds from each other. The applicability of this approach was tested using a blind trial in which several compounds were presented as unknowns, their in-source CID spectra recorded and the five-ion approach used for identification.     

Collision-induced dissociation pathways of anabolic steroids by electrospray ionization tandem mass spectrometry

Anabolic steroids are structurally similar compounds, and their product-ion spectra obtained by tandem mass spectrometry under electrospray ionization conditions are quite difficult to interpret because of poly-ring structures and lack of a charge-retaining center in their chemical structures. In the present study, the fragmentation of nine anabolic steroids of interest to the racing industry was investigated by using triple quadrupole mass spectrometer, Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer, and a linear ion trap instrument. With the aid of an expert system software (Mass Frontier version 3.0), accurate mass measurements, and multiple stage tandem mass spectrometric (MS(n)) experiments, fragmentation pathways were elucidated for boldenone, methandrostenolone, tetrahydrogestrinone (THG), trenbolone, normethandrolone and mibolerone. Small differences in the chemical structures of the steroids, such as an additional double-bond or a methyl group, result in significantly different fragmentation pathways. The fragmentation pathways proposed in this paper allow interpretation of major product ions of other anabolic steroids reported by other researchers in a recent publication. The proposed fragmentation pathways are helpful for characterization of new steroids. The approach used in this study for elucidation of the fragmentation pathways is helpful in interpretation of complicated product-ion spectra of other compounds, drugs and their metabolites.     

A high-performance matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometer with collisional cooling

A high-performance orthogonal time-of-flight (TOF) mass spectrometer was developed specifically for use in combination with a matrix-assisted laser desorption/ionization (MALDI) source. The MALDI source features an ionization region containing a buffer gas with variable pressure. The source is interfaced to the TOF section via a collisional focusing ion guide. The pressure in the source influences the rate of cooling and allows control of ion fragmentation. The instrument provides uniform resolution up to 18,000 FWHM (full width at half maximum). Mass accuracy routinely achieved with a single-point internal recalibration is below 2 ppm for protein digest samples. The instrument is also capable of recording spectra of samples containing compounds with a broad range of masses while using one set of experimental conditions and without compromising resolution or mass accuracy.     

Bacterial analysis by MALDI-TOF mass spectrometry: An inter-laboratory comparison

Bacterial analysis by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry has been demonstrated in numerous laboratories, and a few attempts have been made to compare results from different laboratories on the same organism. It has been difficult to understand the causes behind the observed differences between laboratories when different instruments, matrices, solvents, etc. are used. In order to establish this technique as a useful tool for bacterial identification, additional efforts in standardizing the methods by which MALDI mass spectra are obtained and comparisons of spectra from different instruments with different operators are needed. Presented here is an extension of our previous single-laboratory reproducibility study with three different laboratories in a controlled experiment with aliquots of the same bacterial culture, matrix stock solution, and calibrant standards. Using automated spectral collection of whole-cell bacteria and automated data processing and analysis algorithms, fingerprints from three different laboratories were constructed and compared. Nine of the ions appeared reproducibly within all three laboratories, with additional unique ions observed within each of the laboratories. An initial evaluation of the ability to use a fingerprint generated within one laboratory for bacterial identification of a sample from another laboratory is presented, and strategies for improving identification rates between laboratories is discussed.     

Comparison of negative ion electrospray mass spectra measured by seven tandem mass analyzers towards library formation

A library of negative ion electrospray ionization mass spectra and tandem mass spectra (MS/MS) of sulfonated dyes has been developed for fast identification purposes. The uniform protocol has been elaborated and applied to the measurements of more than 50 anionic dyes. Three collision energies are selected in our protocol which ensures that at least one of them provides a suitable ratio of product ions to the precursor ion. The robustness is investigated with altered values of tuning parameters (e.g. the pressure of the nebulizing gas, the temperature and the flow rate of drying gas, and the mobile phase composition). The results of the inter-laboratory comparison of product ion mass spectra recorded on seven different tandem mass spectrometers (three ion traps, two triple quadrupoles and two hybrid quadrupole time of flight instruments) are presented for four representative anionic dyes--azo dye Acid Red 118, anthraquinone dye Acid Violet 43, triphenylmethane dye Acid Blue 1 and Al(III) metal-complex azo dye. The fragmentation patterns are almost identical for all tandem mass analyzers, only the ratios of product ions differ somewhat which confirms the possibility of spectra transfer among different mass analyzers with the goal of library formation.     

Electrospray ionization mass spectrometry and tandem mass spectrometry of clodronate and related bisphosphonate and phosphonate compounds

The bisphosphonate family with a P-C-P structure is a broad class of drugs, widely investigated as potential inhibitors in bone diseases and calcium metabolic disorders. In this study, the mass spectrometric (MS) behavior and fragmentation of clodronate and related bisphosphonate and phosphonate compounds was studied by using negative ion electrospray ionization (ESI) with triple quadrupole and ion trap instruments. The effect of pH on the degree of deprotonation of the polyprotic bisphosphonic and phosphonic acids in negative ion ESI-MS was investigated, and the degree of deprotonation in the ESI mass spectra and the dissociation in the liquid phase were compared. The results provide evidence that the measured ESI mass spectra do not correlate with the chemistry in the liquid phase owing to the decrease in the pH of the solvent droplets during the ion evaporation process and the charge state neutralization in the gas phase. Ion trap MS(n) provided useful information on the fragmentation study of clodronate and related bisphosphonate and phosphonate compounds, in which interesting fragmentation pathways including the direct elimination of carbon monoxide from deprotonated bisphosphonates and formation of a P-P bond were observed. Reactions between the product ions with a -PO(2) group and residual water in the ion trap or in the high-pressure region of the triple quadrupole instrument formed other unexpected fragmentation paths for all the bisphosphonates studied.     

Effects of liquid chromatography mobile phase buffer contents on the ionization and fragmentation of analytes in liquid chromatographic/ionspray tandem mass spectrometric determination

The effects of liquid chromatography mobile phase buffer contents on the ionization and fragmentation of drug molecules in liquid chromatographic/ionspray tandem mass spectrometric (LC/MS/MS) determination were evaluated for simvastatin (SV) and its hydroxy acid (SVA). The objective was to improve further the sensitivity for SV by overcoming the unfavorable condition caused by the formation of multiple major adduct ions and multiple major fragment ions when using ammonium as LC mobile phase buffer. Mobile phases (70:30 acetonitrile-buffer, 2 mM, pH 4.5) with buffers made from ammonium, hydrazine or alkyl (methyl, ethyl, dimethyl or trimethyl)-substituted ammonium acetate were evaluated. Q1 scan and product ion scan spectra were obtained for SV in each of the mobile phases under optimized conditions. The results showed that, with the alkylammonium buffers, the alkylammonium-adducted SV was observed as the only major molecular ion, while the formation of other adduct ions ([M + H](+), [M + Na](+) and [M + K](+)) was successfully suppressed. On the other hand, product ion spectra with a single major fragment ion were not observed for any of the alkylammonium-adducted SVs. The affinity of the alkylammoniums to SV and the basicity of the alkylamines are believed to be factors influencing the formation and abundance of molecular and fragment ions, respectively. Methylammonium acetate provided the most favorable condition among all the buffers evaluated and improved the sensitivity several-fold for SV in LC/MS/MS quantitation compared with that obtained using ammonium acetate buffer. Better precision for SV in both Q1 and SRM scans was observed when using methylammonium buffer compared with those using ammonium buffer. The mobile phase buffer contents did not seem to affect the ionization, fragmentation and chromatography of SVA. The results of this evaluation can be applied to similar situations with other organic molecules in ionspray LC/MS/MS determination.     

On the zirconium oxide neutral cluster distribution in the gas phase: detection through 118 nm single photon, and 193 and 355 nm multiphoton, ionization

Zirconium oxide clusters are generated in the gas phase by laser ablation of the metal into a flow of ca. 5% O2/95% He at 100 psig and supersonic expansion into a vacuum chamber. Mass spectra of neutral gas phase zirconium oxide clusters are obtained through photoionization at three different laser wavelengths: 118, 193, and 355 nm. Ionization of the clusters with 118 nm laser radiation is through a single photon ionization mechanism, while ionization by 193 and 355 nm laser radiation is through a multiphoton (three or more photon) mechanism. Fragment ion features are observed in the mass spectra of ZrmOn+ for only the 193 nm and 355 nm ionization schemes. The true neutral ZrmOn cluster distribution is obtained only through 118 nm single photon ionization, as verified by mass spectral peak linewidths and calculations of the cluster binding energies, ionization energies, and fragmentation rates. The neutral cluster distribution consists mainly of the series ZrmO2m and ZrmO(2m+1) for m = 1,..., approximately 30.     

Production of Reliable MALDI Spectra with Quality Threshold Clustering of Replicates

We present the first application of the quality threshold (QT) clustering algorithm to mass spectrometry (MS) data. The unique abilities of QT clustering to yield precision nodes that are commensurate with the mass measurement precision of the instrument are exploited to generate a consensus spectrum out of multiple replicate spectra. The spectral dot product and confidence intervals are used as a tool for evaluating the similarity and reproducibility between the consensus and replicates. The method is equally applicable to high and low resolution measurements. This paper demonstrates applications to linear spectra from a matrix assisted laser desorption ionization (MALDI) time of flight (TOF) instrument as well as peptide fragmentation data obtained from a TOF/TOF after unimolecular decomposition. The advantages of clustering to mitigate the inherent precision the shortcomings of MALDI data are discussed.     

Determination of glycopeptide structures by multistage mass spectrometry with low-energy collision-induced dissociation: comparison of electrospray ionization quadrupole ion trap and matrix-assisted laser desorption/ionization quadrupole ion trap reflectron time-of-flight approaches

Multistage mass spectrometry, as implemented using low-energy collision-induced dissociation (CID) analysis in three-dimensional (3D) quadrupole ion traps (QITs), has become a powerful tool for the investigation of protein glycosylation. In addition to the well-known combination of QITs with electrospray ionization (ESI), also a matrix-assisted laser desorption/ionization--quadrupole ion trap--reflectron time-of-flight (MALDI-QIT-rTOF) mass spectrometer has recently become available. This study systematically investigates the differences between these types of instrument, as applied to characterization of glycopeptides from human antithrombin. The glycopeptides were obtained by tryptic digestion followed by lectin-affinity purification. Some significant differences between the ESI-QIT and MALDI-QIT-rTOF approaches appeared, most of them are causally related to the desorption/ionization process. The combination of a vacuum MALDI source with an ion-trap analyzer accentuates some characteristic differences between MALDI and ESI due the longer time frame needed for the trapping process. In contrast to ESI, MALDI generated ions that exhibited considerable metastable fragmentation during trapping. The long time span of the QIT process (ms range) compared with that for conventional rTOF experiments (micros range) significantly magnified the extent of this metastable fragmentation. With the investigated glycopeptides, a complete depletion of the terminal sialic acids of the glycopeptides as well as a variety of other fragment ions was already found in the MS1 spectra from the MALDI-QIT-rTOF instrument. The positive ion low-energy CID spectra (MS2) of the selected glycopeptides obtained using the two different QIT equipped instruments were found to be quite similar. In both approaches, fragmentation of the glycan and peptide structures occurred sequentially, allowing unambiguous sequence determination. In the case of ESI-QIT-MS, fragmentation of the glycan structure occurred at the MS2 stage and fragmentation of the peptide structure was obtained only at the MS3 stage, which indicates the necessity of multistage CID experiments for complete structure elucidation. The MALDI-QIT-rTOF instrument yielded both kinds of fragments at the MS2 stage but without mutual interference.     

Effect of mobile phase pH, aqueous-organic ratio, and buffer concentration on electrospray ionization tandem mass spectrometric fragmentation patterns: implications in liquid chromatography/tandem mass spectrometric bioanalysis

Liquid chromatography/tandem mass spectrometry (LC/MS/MS) based on selected reaction monitoring (SRM) is the standard methodology in quantitative analysis of administered xenobiotics in biological samples. Utilizing two SRM channels during positive electrospray ionization (ESI) LC/MS/MS method development for a drug compound containing two basic functional groups, we found that the response ratio (SRM1/SRM2) obtained using an acidic mobile phase was dramatically different from that obtained using a basic mobile phase. This observation is different from the well-established phenomenon of mobile phase affecting the [M+H](+) response, which is directly related to the amount of the [M+H](+) ions produced during the ionization. Results from follow-up work reported herein revealed that the MS/MS fragmentation patterns of four drug or drug-like compounds are affected not only by the pH, but also by the aqueous-organic ratio of the mobile phase and the buffer concentration at a given apparent pH. The observed phenomenon can be explained by invoking that a mixture of [M+H](+) ions of the same m/z value for the analyte is produced that is composed of two or more species which differ only in the site of the proton attachment, which in turn affects their MS/MS fragmentation pattern. The ratio of the different protonated species changes depending on the pH, aqueous-organic ratio, or ionic strength of the mobile phase used. The awareness of the mobile phase dependency of the MS/MS fragmentation pattern of precursor ions of identical m/z value will influence LC/MS/MS-based bioanalytical method development strategies. Specifically, we are recommending that multiple SRM transitions be monitored during mobile phase screening, with the MS/MS parameters used for each SRM optimized for the composition of the mobile phase (pH, organic percentage, and ionic strength) in which the analyte elutes.     

Dissociation of different conformations of ubiquitin ions

The fragmentation pathways of different conformations of three charge states of ubiquitin ions are examined using ion mobility/collisional activation/time-of-flight techniques. Mass spectra for fragments for different conformers of a single charge state appear to be identical (within the experimental reproducibility). These results are consistent with a mechanism in which different conformers of each charge state rearrange to similar dissociation transition states prior to fragment formation.     

Approach to the analysis of diuretics and masking agents by high-performance liquid chromatography-mass spectrometry in doping control

The application of thermospray and plasmaspray high-performance liquid chromatography-mass spectrometry to the analysis of diuretics and probenecid has been investigated. The latter method gave better ionization efficiency than the former, and its response was optimized by altering the solvent composition: best results were obtained with water-methanol-acetonitrile-trifluoroacetic acid. Using different proportions of these solvents, three isocratic systems were developed to separate the compounds under study. The principal characteristic of plasmaspray positive-ion mass spectra was a protonated molecular ion and very little fragmentation was evident. In the negative ionization mode, the plasmaspray method gave mass spectra showing more fragmentation, which resulted in additional structural information. The ability of trifluoroacetic acid to form negative cluster ions precluded its use as a mobile phase component. The minimum detectable amounts determined by the analysis in the positive-ion mode was compound-dependent, but generally ca. 10-150 ng. In many cases the compounds could be detected in urine extracts.     

Mass spectral fragmentation reactions of a therapeutic 4-azasteroid and related compounds

Mass spectra were acquired for a therapeutic 4-azasteroid (dutasteride), and some related compounds, using various ionization conditions (EI, CI, APCI and ESI) in both positive and negative ion modes. The ionization and fragmentation behavior of the compound dutasteride, its precursors and several analogs is reported. Positive atmospheric pressure chemical ionization (APCI+) and positive electrospray ionization (ESI+) produced distinctive collision-induced dissociation (CID) spectra for the respective [MH]+ ions of dutasteride. The spectral differences are attributed to ion populations having either different structures or different internal energy distributions (as a consequence of the method of ionization). Irrespective of their origin, the protonated molecules undergo interesting fragmentation reactions when collisionally activated. The identity of the major fragmentation products was confirmed by accurate mass measurement. The negative APCI mass spectrum of dutasteride displays extensive dehydrohalogenation, apparently due to the thermal component of the APCI process. Some of the resulting radical anions display remarkable stability toward collisional decomposition. Details of the fragmentation behavior for the negative ion species and their relationship to the positive ion results are discussed.     

Multi-photon ionization in the mass spectrometry of polyatomic molecules: Cross sections

Multi-photon ionization (MPI) with tunable visible/UV laser light is shown to be a sensitive tool for analysis of traces in gas mixtures when combined with a mass spectrometer. Mass spectra of six different organic molecules, obtained with low intensity laser light, are presented and demonstrate the facility of ionization without fragmentation (soft ionization) under proper experimental conditions. Quantitative values for the cross sections for both two photon steps are obtained from the measured intensity dependence and the absolute ion numbers. Such quantitative data help in the evaluation and definition of this new ionization technique in mass spectrometry. Efficiencies of ionization for some molecules are as high as 25% leading to 10⁶ ions in a single pulse from the dye laser (1 kW). Detectability as low as 2 parts in 10⁹ is thus predicted.