Evaluation of World Anti-Doping Agency criteria for anabolic agent analysis by using comprehensive two-dimensional gas chromatography–mass spectrometry

This work presents the validation study of the comprehensive two-dimensional gas chromatography (GC×GC)–time-of-flight mass spectrometry method performance in the analysis of the key World Anti-Doping Agency (WADA) anabolic agents in doping control. The relative abundance ratio, retention time, identification and other method performance criteria have been tested in the GC×GC format to confirm that they comply with those set by WADA. Furthermore, tens of other components were identified with an average similarity of >920 (on the 0–999 scale), including 10 other endogenous sterols, and full mass spectra of 5,000+ compounds were retained. The testosterone/epitestosterone ratio was obtained from the same run. A new dimension in doping analysis has been implemented by addressing separation improvement. Instead of increasing the method sensitivity, which is accompanied by making the detector increasingly “blind” to the matrix (as represented by selected ion monitoring mode, high-resolution mass spectrometry (MS) and tandem MS), the method capabilities have been improved by adding a new “separation” dimension while retaining full mass spectral scan information. Apart from the requirement for the mass spectral domain that a minimum of three diagnostic ions with relative abundance of 5% or higher in the MS spectra, all other WADA criteria are satisfied by GC×GC operation. The minimum of three diagnostic ions arises from the need to add some degree of specificity to the acquired mass spectrometry data; however, under the proposed full MS scan method, the high MS similarity to the reference compounds offers more than the required three diagnostic ions for an unambiguous identification. This should be viewed as an extension of the present criteria to a full-scan MS method.     

Application potential of gas chromatography high speed time-of-flight mass spectrometry system in analysis of pesticides. Part 1

Analysis of pesticides and polychlorinated biphenyls in food matrices was provided by the method of complete two-dimensional gas chromatography/time-of-flight mass spectrometry. It was shown, that using of technology GC/GC is significantly improves separation of target compounds and decrease the limits of quantification in comparison with the common “one-dimensional” chromatography. Also, the study describes briefly the use of rapid separation for the analysis of trace contamination in food samples. High-speed temperature program, coupled with the possibility of deconvolution of spectra, using the TOF-type detector with the appropriate software, significantly reduces the total time of analysis compared with traditional methods.     

Combined use of ESI–QqTOF-MS and ESI–QqTOF-MS/MS with mass-spectral library search for qualitative analysis of drugs

The potential of the combined use of ESI–QqTOF-MS and ESI–QqTOF-MS/MS with mass-spectral library search for the identification of therapeutic and illicit drugs has been evaluated. Reserpine was used for standardizing experimental conditions and for characterization of the performance of the applied mass spectrometric system. Experiments revealed that because of the mass accuracy, the stability of calibration, and the reproducibility of fragmentation, the QqTOF mass spectrometer is an appropriate platform for establishment of a tandem-mass-spectral library. Three-hundred and nineteen substances were used as reference samples to build the spectral library. For each reference compound, product-ion spectra were acquired at ten different collision-energy values between 5 eV and 50 eV. For identification of unknown compounds, a library search algorithm was developed. The closeness of matching between a measured product-ion spectrum and a spectrum stored in the library was characterized by a value called “match probability”, which took into account the number of matched fragment ions, the number of fragment ions observed in the two spectra, and the sum of the intensity differences calculated for matching fragments. A large value for the match probability indicated a close match between the measured and the reference spectrum. A unique feature of the library search algorithm—an implemented spectral purification option—enables characterization of multi-contributor fragment-ion spectra. With the aid of this software feature, substances comprising only 1.0% of the total amount of binary mixtures were unequivocally assigned, in addition to the isobaric main contributors. The spectral library was successfully applied to the characterization of 39 forensic casework samples. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible to authorized users.     

Tunable single-photon ionization TOF mass spectrometry using laser-produced plasma as the table-top VUV light source

Here we report on a laser plasma-based tunable VUV photoionization time-of-flight (TOF) mass spectrometer conceived mainly to study complex gaseous mixtures. Ionizing photons at tunable vacuum UV (VUV) wavelengths are generated by a gas-target laser-produced plasma, spectrally dispersed in the range 100–160 nm and efficiently focused onto a sample molecular beam. As a test case, we studied the exhaust gas of a four-stroke moped, a typical example of a complex gaseous mixture. Due to the VUV “soft” ionization, the mass spectra are less congested and more easily interpretable. Substituted benzene derivatives are found to give the most intense signals. Several aliphatic hydrocarbons are also detected. The use of tunable VUV radiation allowed the investigation of the contribution of isomers in the mass spectrum from the onset and shape of the photoionization efficiency spectra. Semiquantitative analysis was performed using known literature data detailing the photoionization cross sections. Our findings suggest that using combined data on the mass/photoionization efficiency spectra may be very helpful for a comprehensive analysis of complex gaseous mixtures.     

High-resolution mass spectrometry for integrated qualitative and quantitative analysis of pharmaceuticals in biological matrices

Quantitative and qualitative high-resolution (HR) dependent and independent acquisition schemes on a QqTOF MS (with resolving power 20,000–40,000) were investigated for the analysis of pharmaceutical compounds in biological fluids. High-resolution selected reaction monitoring (HR-SRM) was found to be linear over three orders of magnitude for quantitative analysis of paracetamol in human plasma, offering a real alternative to triple quadrupole LC–SRM/MS. Metabolic stability of talinolol in microsomes was characterized by use of three different acquisition schemes: (i) information-dependent acquisition (IDA) with a TOF MS experiment as survey scan and product-ion scan as dependent scan; (ii) MS^ALL by collecting TOF mass spectra with and without fragmentation by alternating the collision energy of the collision cell between a low (i.e., 10 eV) and high setting (i.e., 40 eV); and (iii) a novel independent acquisition mode referred to as “sequential window acquisition of all theoretical fragment-ion spectra” (SWATH) or “global precursor ions scan mode” (GPS) in which sequential precursor ions windows (typically 20 u) are used to collect the same spectrum precursor and fragment ions using a collision energy range. SWATH or GPS was found to be superior to IDA or MS^ALL in combination with UHPLC for qualitative analysis but requires a rapidly acquiring mass spectrometer. Finally, the GPS concept was used for QUAL/QUAN analysis (i.e. integration of qualitative and quantitative analysis) of bosentan and its metabolites in urine over a concentration range from 5 to 2,500 ng mL⁻¹.     

Analysis of nitrophenols and other polar nitroaromatic compounds in ammunition wastewater by high-field proton nuclear magnetic resonance (1H-NMR) spectroscopy and chromatographic methods

 It is shown that by using high-field proton nuclear magnetic resonance (¹H-NMR) spectroscopy it is possible, without prior separation, to analyse nitrophenols and other acidic nitroaromatic compounds in the “pH 2 extract” of ammunition wastewater. The ¹H-NMR chemical shifts data of a variety of reference compounds are presented. Two groundwater samples from the former ammunition plant in Elsnig (Saxony) were analysed by ¹H-NMR and also by chromatographic methods (GC/MS, HPLC). The results are compared and discussed. Received: 17 January 1996/Revised: 15 May 1996/Accepted: 17 May 1996     

Analysis of nitrophenols and other polar nitroaromatic compounds in ammunition wastewater by high-field proton nuclear magnetic resonance (1H-NMR) spectroscopy and chromatographic methods

 It is shown that by using high-field proton nuclear magnetic resonance (¹H-NMR) spectroscopy it is possible, without prior separation, to analyse nitrophenols and other acidic nitroaromatic compounds in the “pH 2 extract” of ammunition wastewater. The ¹H-NMR chemical shifts data of a variety of reference compounds are presented. Two groundwater samples from the former ammunition plant in Elsnig (Saxony) were analysed by ¹H-NMR and also by chromatographic methods (GC/MS, HPLC). The results are compared and discussed. Received: 17 January 1996/Revised: 15 May 1996/Accepted: 17 May 1996     

Isomeric composition of the ester fraction from epicuticular waxes of Festuca arundinacea Schreb

The ester portion of epicuticular waxes of tall fescue (Festuca arundinacea Schreb.) is composed of mixtures of homologous series of fatty acids esterfied with fatty alcohols. GC analyses of natural esters gave rise to the separation of several compounds of different chain length. To identify and quantify individual components in the mixture, GC/MS analyses were carried out, and the mass spectra of each peak were recorded and analyzed for characteristic ions. This approach permitted a comparison with data obtained from classical methods using GC analysis of acid and alcohol moieties obtained by hydrolysis.     

气相色谱/离子阱质谱中组分图谱提取的主成分分析方法

由于离子阱质谱中容易发生分子-离子反应, 致使质谱图发生改变, 影响与主要由四极质谱建立的参照图谱的匹配效果. 本文采用主成分分析方法对气相色谱/离子阱质谱中组成色谱峰的所有全扫描质谱进行提取, 提取后的质谱图能够有效克服分子-离子反应和基线干扰的影响, 应用不同的匹配算法均能很好地和参照图谱匹配, 和传统方法相比匹配度显著提高, 对目标化合物定性准确.     

A study on mass spectrometry of methylated [60] fullerenes using the “in-beam” electron impact technique

Mass spectra of the methylated [60]fullerenes were obtained by EI mass spectrometry using “desorption” or “in-beam” technique. The mass spectra of the methylated fullerenes, C₆₀Me_n, have the molecular ion peak M⁺ indicating that the product is stable under the MS (EI) conditions. The appearance of an intense peak at m/z 360 was assigned to the formation of fullerene dication C₆₀⁺⁺. The remaining peaks were assigned to successive loss of methyl groups from molecular monocation and dication.     

Identification of Abnormal Urinary Organic Acids in Inherited Metabolic Diseases by Gas Chromatography-Mass Spectrometry

A gas chromatography/mass spectrometry (GC/MS) coupled system has been established for the confirmatory identification of abnormal urinary organic acids in inherited metabolic diseases. Samples of patient urines were extracted with an organic solvent and trimethylsilylated (TMS). A mass spectra of gas chromatographically separated TMS derivatives can be obtained using the GC/MS coupled system with a single analytical run. Those compounds with close methylene units (e.g., 4-hydroxyphenylacetaic acid and phenylpyruvic acid) in the gas chromatograph can be identified by their specific mass spectra. The results indicate that this GC/MS system is a powerful method for identifying abnormal urinary organic acids. These acids can be identified by comparison with authentic mass spectra established in our laboratories or with mass spectra files from other sources or they can be directly identified by analysis of the mass spectrum. By using this system, we were able to make positive identification of several inherited metabolic diseases found in Chinese patients, including phenylketonuria, propionic acidemia, and methylmalonic aciduria. This GC/MS system is a powerful tool for the diagnosis of inherited metabolic diseases.     

Rapid detection of <Emphasis Type="Italic">Aspergillus flavus</Emphasis> in rice using biofunctionalized carbon nanotube field effect transistors

Popularity of new psychoactive substances, known as legal highs or herbal highs, is continuously growing. These products are typically sold via internet and in so-called head shops. The aim of this study was to identify active ingredients of herbal highs and to compare their chemical composition. Twenty-nine various products seized by the police in one of the “head shops” were analysed. Herbal mixtures (0.2 g) were prepared by ultrasonic-assisted extraction with 2.0 ml of ethanol for 2 h. The extracts were analysed by gas chromatography coupled to mass spectrometry (GC/MS). The main active compounds of the herbal mixtures were synthetic cannabinoids: JWH-018, JWH-073 and cannabicyclohexanol (CP-47,497-C8-homolog). Their content differed between the products; some contained only one cannabinoid whereas the others contained two or more. Cluster analysis and principal component analysis revealed that chemical composition of many products was very similar. The similarity was connected with their flavour and not the common name. This statement was true for the synthetic cannabinoids, other potential agonists of cannabinoid receptors (amides of fatty acids) and ingredients of natural origin and confirms that herbal highs are a threat to human health because the purchaser has no information on their real composition.     

An activated carbon substrate surface for laser desorption mass spectrometry

A method to obtain laser desorption/ionization mass spectra of organic compounds by depositing sample solutions onto a carbon substrate surface is demonstrated. The substrate consists of a thin layer of activated carbon particles immobilized on an aluminum support. In common with the porous carbon suspension samples used in previous “surface-assisted laser desorption/ionization” (SALDI) work, the mass spectra contain only a few “matrix” background ion peaks, minimizing interference with analyte ion peaks. The presence of glycerol ensured that the ion signals were stable over hundreds of laser shots. In addition, the carbon substrate surface has several advantages over the suspension samples. The use of a very thin layer of carbon significantly improves the sensitivity. Detection limits range from attomoles for crystal violet to femtomoles for bradykinin. Very little sample preparation is required as the analyte solution is simply pipetted onto the substrate surface and glycerol added. When using an alternate sample deposition method, a mass resolution for bradykinin of 1800 is achieved in linear time-of-flight mode. This is close to the resolution limit set by the detector system and above instrument specification for matrix-assisted laser desorption/ionization mass spectra.     

Identification of essential components of Houttuynia cordata by gas chromatography/mass spectrometry and the integrated chemometric approach

Starting with Biller-Biemann's work [J.E. Biller, K. Biemann, Anal. Lett. 7 (1974) 515], various kinds of approaches have been proposed to extract GC/MS data to obtain pure components responses. In this paper, an integrated chemometric approach is proposed, which combine four sequential steps, data pretreatment, component perception, resolution and component identification, and then the proposed approach is manipulated to analyze the essential oils of a herbal medicine named Houttuynia cordata (HC). On the basis of the selective information obtained from both chromatograms and mass spectra, the proposed integrated chemometric approach can resolve the two-way GC/MS responses matrix into pure chromatograms and mass spectra without any model assumption on the peak shape. The resolution results obtained from HC samples demonstrate the performance of the proposed approach and indicate that it may be a promising one for analyzing complex chromatograms.     

Tunable fragmentation of organic molecules in laser ablation glow discharge time-of-flight mass spectrometry

A DC-pulsed glow discharge (GD) has distinct temporal regimes which are characterized by “softer” or “harder” ionization of analytes introduced into the discharge. It is thus possible to obtain both molecular weight and structural fragment information from the same spectra. In order to extend the capabilities of this technique a laser ablation (LA) sampling system was coupled to a DC-pulsed GD and to a time-of-flight (TOF) mass spectrometer (MS) for characterizing organic samples such as oleic acid, reserpine, two different peptides, and a polymer. Both hard and soft ionization regimes were studied. These LAGD-TOFMS results were compared to matrix-assisted laser desorption ionization (MALDI) spectra using the same compounds (i.e., analytes, concentration, and matrix). It was found that LAGD offers tunable ionization and provides a reduced matrix dependence. However, the sensitivity achieved by the prototype LAGD-TOFMS was significantly lower when compared with commercially available MALDI-TOFMS instrumentation. Since LAGD-TOFMS is rather new, some technical details to increase its sensitivity are discussed.     

Multivariate statistical analysis of coastal sediment data

Multivariate statistical analysis of sediment data (information matrix 123 × 16) from the Gulf of Mexico, USA shows that the data structure is defined by four latent factors conditionally called “inorganic natural”, “inorganic anthropogenic”, “bioorganic” and “organic anthropogenic” explaining 39.24%, 23.17%, 10.77% and 10.67% of the total variance of the data system, respectively. The receptor model obtained by the application of the PCR approach makes it possible to apportion the contribution of each chemical component for the latent factor formation. A separation of the contribution of each chemical parameter is achieved within the frames of “natural” and “anthropogenic” origin of the respective heavy metal or organic matter to the sediment formation process. This is a new approach as compared to the traditional “one dimensional” search with a limited number of preliminary selected tracer components. The model suggested divides natural from anthropogenic influences and allows in this way each participant in the sediment formation process to be used as marker of either natural or anthropogenic effects. Received: 20 March 1999 / Revised: 1 June 1999 / Accepted: 3 June 1999     

Multivariate statistical analysis of coastal sediment data

Multivariate statistical analysis of sediment data (information matrix 123 × 16) from the Gulf of Mexico, USA shows that the data structure is defined by four latent factors conditionally called “inorganic natural”, “inorganic anthropogenic”, “bioorganic” and “organic anthropogenic” explaining 39.24%, 23.17%, 10.77% and 10.67% of the total variance of the data system, respectively. The receptor model obtained by the application of the PCR approach makes it possible to apportion the contribution of each chemical component for the latent factor formation. A separation of the contribution of each chemical parameter is achieved within the frames of “natural” and “anthropogenic” origin of the respective heavy metal or organic matter to the sediment formation process. This is a new approach as compared to the traditional “one dimensional” search with a limited number of preliminary selected tracer components. The model suggested divides natural from anthropogenic influences and allows in this way each participant in the sediment formation process to be used as marker of either natural or anthropogenic effects. Received: 20 March 1999 / Revised: 1 June 1999 / Accepted: 3 June 1999     

Comparison of differential mobility spectrometry and mass spectrometry for gas chromatographic detection of ignitable liquids from fire debris using projected difference resolution

The significance of forensic arson analysis accelerates the applications of new technologies in this area. Based on the previously reported application of differential mobility spectrometry (DMS) as a detection method for gas chromatography (GC) in arson analysis, the performances of DMS and mass spectrometry (MS) were compared using a novel chemometric tool, projected difference resolutions (PDRs). The PDR results show that one-way mass spectra data exhibit higher resolution than DMS data, while total ion chromatograms from GC–DMS show higher resolution than that from GC/MS for differentiating seven kinds of ignitable liquids. Combining the information from both chromatography and spectra, two-way data always have higher resolution than one-way data for these two detection methods, and GC/MS would exhibit better performance than GC–DMS according to the minimum resolution value. To verify the PDR results, a fuzzy rule-building expert system was applied for classifying these seven kinds of ignitable liquids from fire debris based on GC–DMS and GC/MS data, respectively. The prediction accuracies were consistent with PDR results, which proved that PDR is a powerful tool in comparing the performances of different analysis methods for pattern recognition. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Determination of ion structures in structurally related compounds using precursor ion fingerprinting

Structurally-related alkaloids were analyzed by electrospray ionization/multiple stage mass spectrometry (ESI/MS ⁿ ) at varying collision energies to demonstrate a conceptual algorithm, precursor ion fingerprinting (PIF). PIF is a new approach for interpreting and library-searching ESI mass spectra predicated on the precursor ions of structurally-related compounds and their matching product ion spectra. Multiple-stage mass spectra were compiled and constructed into “spectral trees” that illustrated the compounds’ product ion spectra in their respective mass spectral stages. The precursor ions of these alkaloids were characterized and their spectral trees incorporated into an MS ⁿ library. These data will be used to construct a universal, searchable, and transferable library of MS ⁿ spectra. In addition, PIF will generate a proposed structural arrangement utilizing previously characterized ion structures, which will assist in the identification of unknown compounds.     

Comparison of electron ionization mass spectra of some organic compounds (MW < 200 Da) recorded on mass spectrometers of various types

Low-resolution electron ionization mass spectra recorded on various types of mass spectrometers (time-of-flight, quadrupole, and three-dimensional ion trap) were compared. A model mixture of 10 organic compounds (MW < 200 Da) was analyzed by gas chromatography-mass spectrometry. Pure mass spectra of analytes were extracted using the AMDIS software. The best repeatability was achieved for the time-of-flight mass spectrometer. The mass spectra recorded by a quadrupole and a time-of-flight mass spectrometer were quite similar. In the case of these instruments, library search using a commercial mass spectral data base (NIST’05) gave satisfactory result for each analyte (rank 1 or 2 in the “hit list”; Match > 900). In some cases, the mass spectra of model compounds recorded by the ion trap mass spectrometer differed in intensity of certain mass spectral peaks (but not in the set of peaks) from the mass spectra presented in the library and from the experimental mass spectra recorded by the time-of-flight and quadrupole instruments.