An integrated method for metabolite detection and identification using a linear ion trap/Orbitrap mass spectrometer and multiple data processing techniques: application to indinavir metabolite detection

A new strategy using a hybrid linear ion trap/Orbitrap mass spectrometer and multiple post-acquisition data mining techniques was evaluated and applied to the detection and characterization of in vitro metabolites of indinavir. Accurate-mass, full-scan MS and MS/MS data sets were acquired with a generic data-dependent method and processed with extracted-ion chromatography (EIC), mass-defect filter (MDF), product-ion filter (PIF), and neutral-loss filter (NLF) techniques. The high-resolution EIC process was shown to be highly effective in the detection of common metabolites with predicted molecular weights. The MDF process, which searched for metabolites based on the similarity of mass defects of metabolites to those of indinavir and its core substructures, was able to find uncommon metabolites not detected by the EIC processing. The high-resolution PIF and NLF processes selectively detected metabolites that underwent fragmentation pathways similar to those of indinavir or its known metabolites. As a result, a total of 15 metabolites including two new indinavir metabolites were detected and characterized in a rat liver S9 incubation sample. Overall, these data mining techniques, which employed distinct metabolite search mechanisms, were complementary and effective in detecting both common and uncommon metabolites. In summary, the results demonstrated that this analytical strategy enables the high-throughput acquisition of accurate-mass LC/MS data sets, comprehensive search of a variety of metabolites through the post-acquisition processes, and effective structural characterization based on elemental compositions of metabolite molecules and their product ions.     

Hybrid triple quadrupole-linear ion trap mass spectrometry in fragmentation mechanism studies: application to structure elucidation of buspirone and one of its metabolites

The use of a hybrid triple quadrupole-linear ion trap (QqQ(LIT)) mass spectrometer system for a comprehensive study of fragmentation mechanisms is described. The anxiolytic drug, buspirone, was chosen as a model compound for this study. With the advent of a QqQ(LIT) instrument, both the traditional quadrupole and the new linear ion trap scans (LIT) could be performed in a single LC run. In the past, a sample had to be run on two different instruments, namely, a triple quadrupole instrument (QqQ) and a 3D ion trap (3D IT) to obtain similar information. With the new QqQ(LIT) technology, collision-induced dissociation (CID) occur in a quadrupole collision cell, q2, and fragment ions are trapped and analyzed in Q3 operated in LIT mode. In this work, high-sensitivity product ion spectra of buspirone were obtained from the one-stage 'Enhanced Product Ion' scan (EPI) with rich product ions and no low mass cut-off. Furthermore, detailed fragmentation pathways were elucidated by further dissociation of each of the fragment ions in the EPI spectrum using MS(3) mode in the same run. The MS(3) scan was performed by incorporating CID in q2, and trapping, cooling, isolation, and resonance-excitation in Q3 when operating in LIT mode. This approach allowed unambiguous assignment of all fragment ions quickly with fewer experiments and easier interpretation than the previous approach. The overall sensitivity for obtaining complete fragment ion data was significantly improved for QqQ(LIT) as compared with that of QqQ and 3D IT mass spectrometers. This is beneficial for structure determination of unknown trace components. The method allowed structure determination of metabolites of buspirone in rat microsomes at 1 microM concentration, which was a 10-fold lower concentration than was needed for QqQ or 3D IT instruments. The QqQ(LIT) instrument provided a simple, rapid, sensitive and powerful approach for structure elucidation of trace components.     

Use of a quadrupole linear ion trap mass spectrometer in metabolite identification and bioanalysis

A new type of quadrupole linear ion trap mass spectrometer, Q TRAP trade mark LC/MS/MS system (Q TRAP trade mark ), was evaluated for its performance in two studies: firstly, the in vitro metabolism of gemfibrozil in human liver microsomes, and, secondly, the quantification of propranolol in rat plasma. With the built-in information-dependent-acquisition (IDA) software, the instrument utilizes full scan MS in the ion trap mode and/or constant neutral loss scans as survey scans to trigger product ion scan (MS(2)) and MS(3) experiments to obtain structural information of drug metabolites 'on-the-fly'. Using this approach, five metabolites of gemfibrozil were detected in a single injection. This instrument combines some of the unique features of a triple quadrupole mass spectrometer, such as constant neutral loss scan, precursor ion scan and multiple reaction monitoring (MRM), together with the capability of a three-dimensional ion trap. Therefore, it becomes a powerful instrument for metabolite identification. The fast duty cycle in the ion trap mode allows the use of full product ion scan for quantification. For the quantification of propranolol, both MRM mode and full product ion scan in the ion trap mode were employed. Similar sensitivity, reproducibility and linearity values were established using these two approaches. The use of the product ion scan mode for quantification provided a convenient tool in selecting transitions for improving selectivity during the method development stage.     

The application of 2-D dual nanoscale liquid chromatography and triple quadrupole-linear ion trap system for the identification of proteins

2-D nanoscale LC combined with a triple quadrupole-linear ion trap mass spectrometer was applied to the analysis of a complex peptide mixture. A 2-D dual nanoscale LC-MS/MS system was compared to a conventional one. Peptides were separated with a strong cation exchange (SCX) microcolumn in the first dimension and two C18 nanocolumns were used as second dimension. MS experiments were performed using information-dependent data acquisition, where two precursor ions were selected from an enhanced MS (EMS) or an enhanced multicharged ion (EMC) as survey scan. The major benefit of EMC instead of EMS was a two-fold reduction of the data file and a 15% increase of characterized proteins. The advantage of the 2-D dual nanoscale LC-MS/MS system versus the conventional 2-D nanoscale LC-MS/MS system was reflected in the significant increase of peptides which were successfully identified within the same time frame. The first factor contributing to this increase was that the mass spectrometer was collecting twice the number of relevant MS/MS data. The second factor is the use of twice the number of SCX salt fractions in the first dimension, allowing a better sample fractionation, thereby reducing the number of peptides transferred to the second chromatographic dimension per salt fraction.     

Atomic collision induced projectile excitation—Its application to identification of heavy ion fusion products

Collision induced X-rays are shown to be a useful tool to determine atomic numbers and yields of heavy ion fusion products. In the reaction⁵⁶Fe(¹³²Xe; xn, yp) the elements Hg, Au, and Pt are produced as primary fusion products. A resolving power Z/ΔZ of 72±7 has been obtained. Improvements and restrictions of the method are discussed.     

An empirical approach to estimation of critical energies by using a quadrupole ion trap

A simple energy-resolved mass spectrometric technique is described for the estimation of critical energies for dissociation of ions via threshold collisional activation measurements in a quadrupole ion trap. The method is calibrated by using compounds with well-defined dissociation energies, and separate calibration curves must be constructed for radical ions that are bound by covalent bonds versus hydrogen-bonded complexes. For these sets of experiments, the threshold point is defined as the activation voltage required for the fragment ion intensity to be 10% of the total ion intensity. A plot of threshold activation voltage of the calibrant versus literature critical energies shows a near-linear function, and accuracies are estimated as better than ± 6 kcal/mol. The q _z value during activation seems to have little effect on the threshold voltages as long as very low q _z values that cause ion ejection are avoided. Activation periods that are substantially longer than 10-ms result in nonlinear behavior in the calibration curves for ions that have critical energies above 30 kcal/mol. This energy-resolved method was also useful for the estimation of critical energies of complexes bound by electrostatic forces, such as hydrogen-bonding interactions. A quantitative evaluation of proton-bound polyether-amine complexes showed that the number of available hydrogen-binding sites, the gas-phase basicities of the polyether and amine components, and the ability of the complex to attain the most favorable near-linear hydrogen bonds correlate with the threshold values.     

Multi-stage collisionally-activated decomposition in an ion trap for identification of sequences, structures and bn --> bn-1 fragmentation pathways of protonated cyclic peptides

Cyclic penta-, hexa- and heptapeptides have been designed, synthesized and their fragmentations induced by multistage tandem mass spectrometry have been studied. Under low-energy collisionally activated decomposition (CAD), the protonated cyclic peptides mainly dissociate via ring opening pathways and the corresponding bn --> bn-1 pathways to form several sets of b ions as oxazolone rings (and b1 ions as aziridinone rings). Through repeated observation of these b ions in multistep CAD experiments, accurate sequencing and head-to-tail ring structure of cyclic peptides can be determined. The mistaken assignments of these b ions can be avoided by this sequencing method. Semiempirical molecular orbital calculations have been utilized to provide insight into the proposed dissociation mechanism. In addition, for cyclic peptides that include an Asn residue, the nitrogen of the Asn side chain is observed to be preferentially protonated, which can induce a unique ring-opening pathway with a loss of ammonia that competes with the conventional ring opening pathway.     

Quantitative determination of paroxetine and its 4-hydroxy-3-methoxy metabolite in plasma by high-performance liquid chromatography/electrospray ion trap mass spectrometry: application to pharmacokinetic studies

A high-performance liquid chromatography (HPLC) method with tandem mass spectrometric detection is described for the determination of paroxetine, an antidepressant drug, and its metabolite (3S,4R)-4-(4-fluorophenyl)-3-(4-hydroxy-3-methoxyphenoxymethyl)piperidine (HM paroxetine) in human plasma. Plasma samples were hydrolysed with hydrochloric acid and then analytes were extracted with ethyl acetate at alkaline pH. Extracts were analysed by HPLC coupled to an atmospheric pressure ionisation-electrospray (ESI) interface and an ion trap mass spectrometer. Chromatography was performed on a reversed-phase column using acetonitrile/0.02% formic acid (66:34, v/v) as a mobile phase. The mass spectrometer was operated in the multiple reaction monitoring mode. The method was validated over concentration ranges of 0.75-100 microg/L and 5-100 microg/L for paroxetine and HM paroxetine, respectively. Mean recoveries of 77% for paroxetine and 76% for HM paroxetine were found, with precision always better than 15%. The limits of detection and quantification were 0.20 and 0.70 microg/L for paroxetine, and 0.70 and 2.20 microg/L for its metabolite. The method was applied to the analysis of plasma samples obtained from nine healthy male volunteers administered with a single oral dose of 20 mg paroxetine. After the 20-mg dose, the mean peak plasma concentration was 8.60 microg/L for paroxetine and 92.40 microg/L for HM paroxetine showing a tenfold ratio between the metabolite and the parent drug along the entire time-concentration curve.     

Strategy for structural elucidation of drugs and drug metabolites using (MS)n fragmentation in an electrospray ion trap

Triple-stage quadrupole (TSQ) electrospray ionization (ESI) tandem mass spectrometry (MS/MS) and ion trap ESI-MS/MS can be used to cleave protonated molecules to produce carbocations and neutral molecules in the positive ion mode. Dissociation products which correspond to protonated forms of neutral fragment molecules can also be trapped and detected. These protonated molecules in turn can cleave via carbocation cleavage, ipso cleavage, onium cleavage or McLafferty or related rearrangements. One can elucidate the structures of metabolites from the differences in m/z ratios of the fragments arising from the original drug compound and its metabolite. This strategy for structural elucidation is further facilitated by estimates of the reactivity of drugs with oxygen diradicals involved in cytochrome P-450 cycles.     

Electrospray ionization ion trap multiple-stage mass spectrometric fragmentation pathways of leucine and isoleucine: an ab initio computational study

We recently demonstrated the possibility to distinguish between leucine and isoleucine in several tryptic peptides by means of consecutive tandem mass steps (Armirotti et al. J. Am. Soc. Mass Spectrom. 2007; 18: 57), exploiting a gas-phase rearrangement of the immonium ion of Ile. In the present paper we explore the tandem mass spectrometric behaviour of the two amino acids. We propose a plausible structure for the diagnostic m/z 69 ion of Ile, that was reported for the first time in 1996 (Hulst and Kientz J. Mass. Spectrom. 1996; 31: 1188), and we explain why its formation is favoured with respect to Leu. Our conclusions are supported by ab initio quantum chemistry calcultations and isotope-labelled standards experiments.     

The use of a triple quadrupole linear ion trap mass spectrometer with electrospray ionisation for fragmentation studies of selected antifungal drugs

Fragmentation studies of three antifungal drugs, clotrimazole, fluconazole and clioquinol ,were performed. A triple quadrupole linear ion trap mass spectrometer was used for this purpose. This type of equipment enables MS(3) spectra to be obtained which lead to better understanding of fragmentation pathways. Nevertheless, it is rarely used for fragmentation studies. The results obtained here for the antifungal drugs gave further insight into fragmentation pathways of clotrimazole and fluconazole. Moreover, fragmentation of clioquinol was investigated which had not been presented before.     

Picolinyl ester fragmentation mechanism studies with application to the identification of acylcarnitine acyl groups following transesterification

Acyl picolinyl esters provide excellent data to identify the structures of acyl groups. However, the mechanisms for the formation of fragment ions from picolinyl esters are unsettled. Proposed structures for fragment ions have focused on long-chain groups and may not accommodate results from medium- and short-chain acyl groups. Using deuterium-labeled organic acids, we have investigated the mechanisms for the formation of fragment ions. Based on these studies, we propose a new mechanism that is consistent with the experimental data. We then tested the mechanisms by analyzing selected acylcarnitines. Transesterification of acylcarnitines was performed by reaction with 3-pyridylcarbinol and potassium tert-butoxide in dichloromethane to produce acyl picolinyl esters. The picolinyl esters were separated and detected by gas chromatography/electron ionization-mass spectrometry. Each mass spectrum contained a series of peaks with m/z differences of 12, 13, or 14 u depending on the acyl group's chemical structure. The position of an unsaturated bond or branched methyl in the acyl group of acylcarnitine can be readily determined.     

The rapid identification of drug metabolites using capillary liquid chromatography coupled to an ion trap mass spectrometer

Capillary liquid chromatography (LC) using a 320 microns column and a flow rate of 10 microL/min has been coupled to an ion trap mass spectrometer using electrospray ionisation (ESI) to enable the rapid and effective identification of metabolites in urine, following oral administration of a novel human neutrophil elastase inhibitor, GW311616. Metabolites were identified from their mass (MS) spectra and tandem (MS/MS) mass spectra using minimal sample (1 microL of urine) and no sample pretreatment. Sensitivity assessment has shown that both molecular weight and structural information is obtainable on as little as 5 pg of compound, making the capillary LC/ion trap system as described an ideal analytical tool for the detection and characterisation of low level metabolites in biofluids (particularly when sample volume is limited). This level of detection was unattainable using a triple quadrupole mass spectrometer operating in full-scan mode, although 200 fg on column was detected using selected reaction monitoring target analysis.     

A structure-based design approach for the identification of novel inhibitors: application to an alanine racemase

We report a new structure-based strategy for the identification of novel inhibitors. This approach has been applied to Bacillus stearothermophilus alanine racemase (AlaR), an enzyme implicated in the biosynthesis of the bacterial cell wall. The enzyme catalyzes the racemization of l- and d-alanine using pyridoxal 5-phosphate (PLP) as a cofactor. The restriction of AlaR to bacteria and some fungi and the absolute requirement for d-alanine in peptidoglycan biosynthesis make alanine racemase a suitable target for drug design. Unfortunately, known inhibitors of alanine racemase are not specific and inhibit the activity of other PLP-dependent enzymes, leading to neurological and other side effects.This article describes the development of a receptor-based pharmacophore model for AlaR, taking into account receptor flexibility (i.e. a `dynamic' pharmacophore model). In order to accomplish this, molecular dynamics (MD) simulations were performed on the full AlaR dimer from Bacillus stearothermophilus (PDB entry, 1sft) with a d-alanine molecule in one active site and the non-covalent inhibitor, propionate, in the second active site of this homodimer. The basic strategy followed in this study was to utilize conformations of the protein obtained during MD simulations to generate a dynamic pharmacophore model using the property mapping capability of the LigBuilder program. Compounds from the Available Chemicals Directory that fit the pharmacophore model were identified and have been submitted for experimental testing.The approach described here can be used as a valuable tool for the design of novel inhibitors of other biomolecular targets.     

High-sensitivity analysis of specific peptides in complex samples by selected MS/MS ion monitoring and linear ion trap mass spectrometry: application to biological studies

Mass spectrometry (MS) is a technique of paramount importance in Proteomics, and developments in this field have been possible owing to novel MS instrumentation, experimental strategies, and bioinformatics tools. Today it is possible to identify and determine relative expression levels of thousands of proteins in a biological system by MS analysis of peptides produced by proteolytic digestion. In some situations, however, the precise characterization of a particular peptide species in a very complex peptide mixture is needed. While single-fragment ion-based scanning modes such as selected ion reaction monitoring (SIRM) or consecutive reaction monitoring (CRM) may be highly sensitive, they do not produce MS/MS information and their actual specificity must be determined in advance, a prerequisite that is not usually met in a basic research context. In such cases, the MS detector may be programmed to perform continuous MS/MS spectra on the peptide ion of interest in order to obtain structural information. This selected MS/MS ion monitoring (SMIM) mode has a number of advantages that are fully exploited by MS detectors that, like the linear ion trap, are characterized by high scanning speeds. In this work, we show some applications of this technique in the context of biological studies. These results were obtained by selecting an appropriate combination of scans according to the purpose of each one of these research scenarios. They include highly specific identification of proteins present in low amounts, characterization and relative quantification of post-translational modifications such as phosphorylation and S-nitrosylation and species-specific peptide identification.     

An accelerating-reporting group for studies of radical heterolysis reactions and its application in an acid-catalyzed fragmentation reaction of an alpha,beta-dimethoxy radical

The 2,2-diphenylcyclopropyl group was employed to accelerate reactions of alpha-methoxy radicals containing beta-leaving groups, to trap the products of either migration or heterolysis of the leaving group, and to provide a useful chromophore for laser flash photolysis kinetic studies. The reporting group biases reactions in favor of heterolytic fragmentation and most likely intercepts radical cations in ion pairs. The 1-methoxy-1-methyl-2-(diethylphosphatoxy)-2-(2,2-diphenylcyclopropyl)ethyl radical (3a) reacted faster than the kinetic resolution of the instrument (k > 2 x 10(8) s(-1)) in all solvents studied, and the 2-acetoxy analogue (3b) reacted much faster than related radicals that do not contain the cyclopropyl group (e.g., k = 1.1 x 10(6) s(-1) in CH3CN at ambient temperature). The rate constants and Arrhenius parameters for reactions of 3b indicated that the rate-limiting step in the reaction was heterolytic cleavage. The 1,2-dimethoxy-1-methyl-2-(2,2-diphenylcyclopropyl)ethyl radical (26) reacted in a general acid-catalyzed heterolysis reaction, and rate constants for protonation of the beta-methoxy group by a series of carboxylic acids were determined. The results suggest that acid-catalyzed reactions of beta-alkoxy radicals might be employed in synthetic conversions.     

A novel approach for identification and characterization of glycoproteins using a hybrid linear ion trap/FT-ICR mass spectrometer

Combining source collision-induced dissociation (CID) and tandem mass spectral acquisition in a pseudo-MS(3) experiment using a linear ion trap results in a highly selective and sensitive approach to identifying glycopeptide elution from a protein digest. The increased sensitivity is partially attributed to the nonselective nature of source CID, which allows simultaneous activation of all charge states and coeluting glycoforms generating greater ion abundance for the mass-to-charge (m/z) 204 and/or 366 oxonium ions. Unlike source CID alone, a pseudo-MS(3) approach adds selectivity while improving sensitivity by eliminating chemical noise during the tandem mass spectral acquisition of the oxonium ions in the linear ion trap. Performing the experiments in the hybrid linear ion trap/Fourier transform-ion cyclotron resonance (FT-ICR) enables subsequent high-resolution/high-mass accuracy full-scan mass spectra (MS) and parallel acquisition of MS/MS in the linear ion trap to be completed in 2 s directly following the pseudo-MS(3) scan to collate identification and characterization of glycopeptides in one experimental scan cycle. Analysis of bovine fetuin digest using the combined pseudo-MS(3), high-resolution MS, and data-dependent MS/MS events resulted in identification of four N-linked and two O-linked glycopeptides without enzymatic cleavage of the sugar moiety or release of the sialic acids before analysis. In addition, over 95% of the total protein sequence was identified in one analytical run.