Mass Spectrometric Parameters Research Articles

Tandem mass spectrometric data–FAAH inhibitory activity relationships of some carbamic acid O‐aryl esters

We have recently described a class of systemically active inhibitors of the intracellular activity of fatty acid amide hydrolase (FAAH) and traced extensive structure-activity relationships. These compounds, characterized by an N-alkyl carbamic acid O-aryl ester structure, exert potent anxiolytic-like effects in animal models. In the present study, possible relationships between mass spectrometric parameters (related to the propensity of the C(O)--O bond to be cleaved) and FAAH-inhibitory potency were tested. With this aim, a set of our products was analyzed by electrospray ionization mass spectrometry and the protonated molecules were decomposed by low-energy collisions. The experiments were performed by ion trap mass spectrometry, which led to a step-by-step energy deposition, thus favouring the lowest critical energy decomposition channels. For all compounds, breakdown curves relative to [MH](+) ions and to the fragment implying C(O)--O bond cleavage were obtained. The crossing point between these curves was related to the energetics of decomposition and the values found for the investigated compounds were linearly correlated (r(2) = 0.797) with their FAAH-inhibitory activity. This indicates that the energetics of the C(O)--O bond cleavage may be relevant in explaining FAAH inhibition.

Multi‐residue liquid chromatography/tandem mass spectrometry method for the detection of non‐steroidal anti‐inflammatory drugs in bovine muscle: optimisation of ion trap parameters

A multi-residue liquid chromatography/tandem mass spectrometry method (LC/MS2) was developed for the detection of the non-steroidal anti-inflammatory drugs acetylsalicylic acid (via the marker residue salicylic acid), flunixin, phenylbutazone, tolfenamic acid, meloxicam and ketoprofen, in bovine muscle. After extraction of the bovine muscle with acetonitrile, the cleanup was performed using a Oasis HLB column. The evaporated eluate was reconstituted and analysed by LC/MS2. To obtain optimal detection of salicylic acid and phenylbutazone, the ion trap mass spectrometric parameters activation q and maximum ion injection time, respectively, were optimised. The activation q for salicylic acid was increased to obtain reliable detection of both salicylic acid and its product ion. The maximum ion injection time for the time segment containing phenylbutazone was decreased since there were not enough scans across the chromatographic peak of this compound. The multi-residue method was able to detect the different analytes below or at the maximum residue limit (MRL) or minimum required performance limit (MRPL) or, in the case of phenylbutazone and ketoprofen, at 100 and 20 microg kg(-1), respectively.

Information-Dependent Acquisition-Mediated LC−MS/MS Screening Procedure with Semiquantitative Potential

The development of a LC-MS/MS general unknown screening procedure for toxicologically relevant substances in blood samples by means of information-dependent acquisition on a Q-TOF is reported. IDA is an artificial intelligence-based product ion scan mode providing automatic "on-the-fly" MS to MS/MS switching. By performing information-dependent scanning at two different fragmentation energies, two collision-induced dissociation product ion spectra for each of the detected compounds are generated. As such, information-rich MS/MS spectra are obtained from precursor ions not known beforehand. In addition, limitation of the MS/MS acquisition time to an acceptable minimum resulted in an almost instantaneous switch back to the MS mode. As such, this approach provided MS chromatograms that still could be of use for semiquantitative purposes. Since the switching intensity threshold, unequivocally related to the background noise, proved a critical parameter, the solid-phase extraction procedure, the liquid chromatographic conditions, and the mass spectrometric parameters all were optimized to the advantage of information-dependent acquisition. Finally, the screening procedure we developed was benchmarked, on one hand, qualitatively against the results obtained from traditional GUS approaches in a number of routine toxicological laboratories (20 samples) and, on the other hand, quantitatively with respect to its potential against established LC-MS/MS methods (7 samples). The procedure performed very well from a qualitative point of view; almost all of the drugs detected by the conventional techniques were identified, as well as additional drugs that were not previously reported. The procedure proved well-suited for an initial semiquantitative assessment, as is customary in, for example, forensic toxicology before accurate intoxication levels are determined using targeted analytical analyses.

New high-resolution mass spectrometric approach for the measurement of polychlorinated biphenyls and organochlorine pesticides in human serum

To increase our analytical throughput for measuring polychlorinated biphenyls (PCBs) and organochlorine (OC) pesticides without sacrificing data quality, we have developed and validated a combined PCB/OC pesticide gas chromatography–high-resolution mass spectrometry (GC–HRMS) analysis. In a single GC–HRMS analysis, both selected PCBs and OC pesticides are detected and quantified. Previously, this has been difficult, if not impossible, because of the major difference in masses of the most abundant electron-impact ions. However, we have identified slightly less abundant ions to monitor that allow us to successfully combine these analytes into a single analysis without sacrificing any analytical sensitivity or instrument reliability. Consequently, we have been able to double our analytical throughput by modification of mass spectrometric parameters alone. Our new methodology has been validated against our current GC–HRMS method, which entails using two separate injections, one for PCB analysis and one for OC pesticide analysis. The two methods differ by less than 4% overall, with no systematic bias. We used this method to analyze approximately 350 serum samples over a period of several months. We found that our new method was as reliable in automated, overnight runs as our current method.

Detection of inhaled clenbuterol in horse urine by GC/MS2.

Clenbuterol, a beta-adrenergic agonist, is used in the treatment of recurrent airway obstruction in horses. It is prohibited by horse racing authorities, because of its stimulating and growth-promoting properties. However, information on detection times of clenbuterol after administration by nebulization is lacking. In this study, a fast, sensitive quantitative GC-MS(2) method for the detection of clenbuterol in urine was developed. Alkaline liquid-liquid extraction was followed by derivatization to a cyclic methyl boronate derivative and analysis on a Finnigan MAT GCQ instrument. Method validation showed good linearity in the range 0.1-2.0 ng/mL, excellent repeatability and specificity. The limit of quantitative detection of the method was 0.1 ng/ml. Different instrumental parameters of the ion trap mass spectrometer were changed to increase the number of diagnostic ions for the cyclic methyl boronate derivative of clenbuterol. The influence of these changes and their applicability within the requirements and the criteria for mass spectrometry set by the responsible regulatory bodies are discussed. Clenbuterol was administered via nebulization to five standardbred mares (0.4 micro g/kg body weight). Analysis of the urine samples resulted in the detection of clenbuterol, as early as 2 h post administration and for up to 36 h post treatment. Generally, maximum urinary concentrations of 1.2 ng/mL were reached after -6-9 h.

Analysis of cyanobacterial hepatotoxins in water samples by microbore reversed-phase liquid chromatography–electrospray ionisation mass spectrometry

A method based on liquid chromatography coupled to mass spectrometry with positive electrospray ionisation was developed for the analysis of cyanobacterial hepatotoxins in environmental samples. The chromatographic separation was performed using two microbore columns, 2 mm and 1 mm I.D. columns, which allowed the coupling of liquid chromatography to mass spectrometry with no flow splitting. Analytes were eluted using two different water–acetonitrile, both acidified with formic acid gradients. Mass spectrometric parameters were optimised in order to maximise sensitivity. Detection limits for the 2 mm I.D. column ranged from 0.077 to 2.057 ng in full scan and from 0.021 to 1.153 ng in SIM mode. However, limits of detection as low as 60–340 pg in full scan and 6–72 pg in SIM mode were achieved for the 1 mm I.D. column. Finally, the proposed method was applied to the analysis of microcystins and nodularins in real samples.

LC-ion trap electrospray MS-MS for the determination of cyclopiazonic acid in milk samples.

An LC-ESI-MS (negative ions) method was developed for the determination of cyclopiazonic acid (CPA), a mycotoxin produced by many Aspergillus and Penicillium genuses, in milk samples. First, the acquisition parameters of the ESI mass spectrometer were optimised for deprotonated CPA and MS-MS measurements were performed, giving a fragmentation pattern. After this stage, LC separation was applied to milk extracts (with or without CPA spikes) by means of an aminic column, using the selective reaction monitoring (SRM) acquisition mode for MS detection. The CPA response was linear over three decades of concentration and an LOD of 5 ng mL(-1) was estimated; moreover, the extraction procedure produced almost quantitative recoveries of CPA from milk. Twenty different milk samples were analysed and three of them were found to be contaminated with CPA to various extents.

Application of column-switching liquid chromatography-tandem mass spectrometry for the determination of pharmaceutical compounds in tissue samples

Information on plasma-tissue distribution which is important for drug development may be obtained by “in silico” prediction tools. To support the validation of computer models, drug concentrations in rat plasma and tissues (brain, liver, kidney, testes, spleen, gut, lung, heart, muscle, skin and fat) had to be determined. In our work, we established analytical assays for a variety of substances including nicardipine, nitrendipine, felodipine and benzodiazepines. Sample preparation had to be simple and method development as well as analytical run time short to allow a high sample throughput and to minimize resources. Column-switching HPLC after homogenization and protein precipitation served as an efficient, easy and rapid sample preparation method, followed by selective MS–MS detection. Optimization of the trapping procedure was performed in order to reduce the influence of endogenous interferences and to obtain good recovery. Chromatographic separation was necessary to increase the selectivity. The use of small analytical column dimensions (2.1×10 mm) was investigated to achieve higher sample throughput without compromising the assay quality. Mass spectrometric parameters, such as ionization modes (positive vs. negative) and ion source types (TurboIonSpray vs. APCI) were screened to find suitable conditions for sensitive analysis of the compounds. Matrix suppression effects were taken into consideration. Calibration samples were prepared in plasma only, whereas quality control samples were prepared in both plasma and tissues to save animals and time. Accuracy and precision were in the range of 84.4–119.1% and 1–16.5%, respectively. Limits of quantification were in the range of 0.5–2.5 ng/ml for plasma and 2–10 ng/ml for tissues. Run times as short as 2.2 min could be achieved.

Optimization of capillary electrophoretic-electrospray ionization-mass spectrometric analysis of catecholamines.

The capillary electrophoretic-mass spectrometric analysis (CE-MS) of catecholamines was optimized with coaxial sheath flow interface and electrospray ionization (ESI). The parameters studied included the sheath liquid composition and its flow rate, separation conditions in ammonium acetate buffer together with the ESI and cone voltages as mass spectrometric parameters. In addition, the effect of ESI voltage on injection as well as the siphoning effect were considered. The optimized conditions were a sheath liquid composition of methanol-water (80:20 v/v) with 0.5% acetic acid, with a flow rate of 6 microL/min. The capillary electrophoretic separation parameters were optimized with 50 mM ammonium acetate buffer, pH 4.0, to +25 kV separation voltage together with a pressure of 0.1 psi. The most intensive signals were obtained with an ESI voltage of +4.0 kV and a cone voltage of +20 V. The nonactive ESI voltage during injection as well as avoidance of the siphoning effect increased the sensitivity of the MS detection considerably. The use of ammonium hydroxide as the CE capillary conditioning solution instead of sodium hydroxide did not affect the CE-MS performance, but allowed the conditioning of the capillary between analyses to be performed in the MS without contaminating the ion source.

Determination of diphenylamine stabilizer and its nitrated derivatives in smokeless gunpowder using a tandem MS method.

A novel method for determination of diphenylamine (DPA) and its nitrated derivatives, which are considered as characteristic components in smokeless powder and gunshot residues, is described. A tandem mass spectrometric method is established and mass spectrometer parameters optimized for each compound to obtain higher sensitivity. Under optimum conditions, quantitative analysis was carried out. The linear ranges are 5.0-200.0, 2.0-200.0 and 5.0-250.0 ng ml-1 and the detection limits are 1.0, 0.5 and 2.5 ng ml-1 for diphenylamine (DPA), N-NO-diphenylamine (N-NO-DPA) and 4-NO2-diphenylamine (4-NO2-DPA), respectively. Intra-assay and inter-assay precision and accuracy of analysis of these three samples were investigated. Based on the regression lines obtained above, smokeless samples were analyzed. It was found that there are 0.952% DPA, 0.384% N-NO-DPA and 0.128% 4-NO2-DPA in smokeless powder. Recovery tests showed that using cotton swabs, 80.3 +/- 4.9% DPA, 79.6 +/- 3.1% N-NO-DPA and 83.1 +/- 5.4% 4-NO2-DPA could be recovered from human hands.

Application of spectral libraries for high-performance liquid chromatography–atmospheric pressure ionisation mass spectrometry to the analysis of pesticide and explosive residues in environmental samples

The coupling of high-performance liquid chromatography (HPLC) and atmospheric pressure ionisation mass spectrometry (API-MS) seems to be the method of choice if good separation and selective detection of semi-volatile, thermolabile, and polar substances is required. Libraries of mass spectra will make the identification of unknown substances in complex environmental samples easier and more user-friendly. Unfortunately, existing GC–MS libraries are not applicable to HPLC–API-MS analysis. Thus, new and extensive mass spectral libraries were constructed. Several investigations of chromatographic (composition and salt concentration of the eluent) as well as mass spectrometric (orifice voltage) parameters and a few applications of real environmental samples are used to discuss the possibilities and limits of these libraries.

Characterization of Hop Acids by Liquid Chromatography with Negative Electrospray Ionization Mass Spectrometry

The analysis of hop acids, humulones and lupulones, with negative electrospray ionization mass spectrometry (MS) is described for two different chromatographic systems. One system focuses on the complete resolution of the ad- and normal homologues implying that no optimal mass spectrometric parameters can be used, whereas the other system makes no compromise to the MS and yields a high sensitivity. The latter system is especially suitable for the fast screening of bitter acids. With this system, the detection limit with selected ion monitoring (SIM)-MS could be estimated to be 10 ng/ml for the humulones and 5 ng/ml for the lupulones. The presence of hop acids in beer was investigated and was found to be positive for humulones, whereas lupulones could not be detected.

Isotope Depletion of Large Biomolecules: Implications for Molecular Mass Measurements

Isotope depletion (or enrichment) of large biomolecules is a procedure already used in high resolution Fourier transform ion cyclotron resonance mass spectrometry for improving the reliability and accuracy of biomolecular mass characterization. In this work, effects of isotope depletion on a number of mass spectrometric parameters are systematically studied. Implementation of the isotope depletion techniques in conjunction with lower resolution mass analyzers is discussed as well. We investigate theoretically the position of the centroid of the isotopic mass distributions (centroid mass) and the shift between the monoisotopic and the centroid masses of biopolymers as a function of the isotope abundance (e.g., 12C: 13C ratio). The behaviour of other additive mass parameters, like the ratio between the monoisotopic and the first isotopic peak, is also discussed. We address by computer simulations the effects of different instrumental parameters like mass resolution and ion statistics as a function of isotope abundances and from there the achievable mass accuracy for high-mass biopolymers. We assess some of the practical issues of the isotope depletion technique, viz., to what degree and with what accuracy the depletion procedure should be performed for achieving the desired mass accuracy.

Quantification of L-tryptophan and L-kynurenine by liquid chromatography/electron capture negative ion chemical ionization mass spectrometry.

In a number of infectious and inflammatory diseases, stimulation of the immune system can lead to increased accumulation of tryptophan metabolites via induction of kynurenine pathway enzymes in extrahepatic tissues. We developed a liquid chromatographic/mass spectrometric (LC/MS) method suitable for tracing the disposition of 13C isotopomers of L-tryptophan and L-kynurenine in various cultured cell, tissue slice, and whole animal model systems used to investigate tryptophan flux through the kynurenine pathway. The method employs extractive derivatization of the analytes and their 2H internal standards with pentafluorobenzyl bromide in order to enhance the negative ion chemical ionization (NICI) mass spectrometric response. Normal-phase liquid chromatographic separation of derivatized analytes was optimized using a silica column with organic solvents, followed by particle beam transfer and NICI-MS. Standard curves were linear over the range 1-250 ng per sample. Particle beam and mass spectrometric operating parameters were optimized with direct flow injections of 1-(methylamino) anthraquinone, which is an ideal test compound for the evaluation of LC/NICI-MS. The developed method was used to quantify the conversion of (13C6)L-tryptophan to (13C6)L-kynurenine by human monocytes (THP-1) stimulated with interferon-gamma, lung and brain tissue slices obtained from gerbils immune-stimulated with pokeweed mitogen. The effect of whole body immune stimulation on the plasma levels of endogenous L-kynurenine in mice stimulated with interferon-gamma was also quantified.

Ion Optical Methods - Means to Improve Performance of Mass Spectrometers Designed for Isotopic or Chemical Analysis

Abstract To improve performances of mass spectrometers dedicated to isotopic or chemical analysis, the methods of the ion optics are to be used. To apply the transfer matrix method of ion optics in mass spectrometer design, the matrix elements of various mass analyzers as Wien filters and crossed field sectors, of energy analyzers as electric prisms, were presented. These elements result from ion trajectory calculations representing solutions of the Euler–Lagrange equations. The basic parameters of an unconventional double focusing mass spectrometer were calculated and compared with those of classical design instruments.

Selective sensitivity of highly chlorinated species in negative ion mass spectrometry: Implications for complex mixture analysis

In attempts to quantify complex chlorinated hydrocarbon mixtures by negative ion mass spectrometry (NI/MS), a selective sensitivity was observed which was not observed using the technique of electron capture detection (ECD) with conventional gas-liquid chromatography. The phenomenon was systematically studied using chlorinated phenols, chlorinated biphenyl isomers and a mixture of chlorinated bicyclic species. Results indicated that for two aromatic series there is an increase in molar response to both ECD and NI/MS with increased degree of chlorination. However, overall an increase in molar response was observed for a series of aliphatic bicyclic compounds only when analyzed by NI/MS; i.e. the ECD molar response did not vary with increasing chlorination of the bicyclic system. A procedure for optimizing mass spectrometer parameters has been developed for quantifying complex chlorinated hydrocarbon mixtures. A simple mixture which mimics the halogenation in a complex mixture is used to determine the response of the instrument to various degrees of chlorination. The recommended calibration procedure optimizes instrumental parameters such as moderating gas pressure, filament current and electron energy so that complex chlorinated mixtures such as those found in toxaphene residues can be reliably quantified.

Determination of isotope ratios of chromium nickel, zinc and copper by gas chromatography-mass spectrometry by using volatile metal chelates

A gas chromatographic-mass spectrometric (GC-MS) method involving thermally stable, volatile chelates was investigated for measurements of isotope ratios of chromium, nickel, zinc and copper. The chelating agents acetylacetone, trifluoroacetylacetone, sodium diethyldithiocarbamate and lithium bis (trifluoroethyl)dithiocarbamate [Li(FDEDTC])] were used. Experimental conditions for the preparation of chelates and the mass spectrometric operating parameters for precise and accurate measurement of isotope ratios were optimized using a general-purpose mass spectrometer. Imprecision values of 1–4% were obtained for measurements of different isotope ratios using chelates containing about 10 ng of metal. The capability of this technique for the accurate determination of natural and altered isotope ratios was also evaluated for these elements using Li(FDEDTC) as a chelating agent. This GC-MS method obviates the need for a more specialized mass spectrometer such as a thermal ionization or inductively coupled plasma mass spectrometer for trace metal determination. The technique gives detection limits down to parts per 109 levels and offers considerable potential for isotope dilution measurements.

Chemical ionization mass spectrometric characteristics of morphine alkaloids

AbstractThe ion‐molecular reaction behavior of ten morphine alkaloids with several commonly used reagent gases are studied under chemical ionization mass spectrometry conditions. These studies emphasize the correlation of the structural characteristics of the 10 alkaloids with the following four mass spectrometric parameters: (i) mass shifts of the protonated ion as a result of replacing ammonia with deuterated ammonia as the reagent gas, (ii) relative tendencies of the adduct ion and the protonated ion to lose molecules of water, (iii) relative intensity ratio of the adduct ion and the protonated ion and (iv) tendency of a compound to undergo a reduction reaction.

Isovalerylglucuronide, a new urinary metabolite in isovaleric acidemia. Identification problems due to rearrangement reactions

Isovaleryl-β- d-glucuronide, a new metabolite in the urine of patients with isovaleric acidemia, is described. Its gas chromatographic and mass spectrometric parameters are presented. In alkaline solution this glucuronide exhibited intramolecular rearrangements, resulting in isomers bearing the acyl moiety on C-2, C-3 and C-4. The isomers showed similar mass spectra but different positions on the gas chromatogram. In the index patient isovalerylglucuronide was a main metabolite, but the excretion was a transient phenomenon. Only traces of isovalerylglucuronide could also be detected in the urine of three other patients with isovaleric acidemia. The significance of this metabolite for the detoxication of isovalerate in isovaleric acidemia is discussed.

Quantitative aspects in the determination of polychlorinated dibenzo- p-dioxins by high-resolution gas chromatography mass—spectrometry

Polychlorinated dibenzo- p-dioxins (PCDDS) are a group of compounds consisting of 75 isomers which are difficult to quantitate because of the lack of sufficient reference standards. A method of quantitation is developed which uses relative response factors for each isomeric group. Variations in gas chromatographic—mass spectrometric parameters do not bias the quantitation method for PCDDS. Testing the method using an environmental sample gives consistent data that can be used to study PCDD content in a number of samples.