Detection of metabolites of trapped humans using ion mobility spectrometry coupled with gas chromatography.

For the first time, ion mobility spectrometry coupled with rapid gas chromatography, using multicapillary columns, was applied for the development of a pattern of signs of life for the localization of entrapped victims after disaster events (e.g., earthquake, terroristic attack). During a simulation experiment with entrapped volunteers, 12 human metabolites could be detected in the air of the void with sufficient sensitivity to enable a valid decision on the presence of a living person. Using a basic normalized summation of the measured concentrations, all volunteers involved in the particular experiments could be recognized only few minutes after they entered the simulation void and after less than 3 min of analysis time. An additional independent validation experiment enabled the recognition of a person in a room of ∼25 m(3) after ∼30 min with sufficiently high sensitivity to detect even a person briefly leaving the room. Undoubtedly, additional work must be done on analysis time and weight of the equipment, as well as on validation during real disaster events. However, the enormous potential of the method as a significantly helpful tool for search-and-rescue operations, in addition to trained canines, could be demonstrated.

Detection of characteristic metabolites of Aspergillus fumigatus and Candida species using ion mobility spectrometry-metabolic profiling by volatile organic compounds.

Volatile metabolites of Aspergillus fumigatus and Candida species can be detected by gas chromatography/mass spectrometry (GC/MS). A multi-capillary column - ion mobility spectrometer (MCC-IMS) was used in this study to assess volatile organic compounds (VOCs) in the headspace above A. fumigatus and the four Candida species Candida albicans, Candida parapsilosis, Candida glabrata and Candida tropicalis in an innovative approach, validated for A. fumigatus and C. albicans by GC/MS analyses. For the detection of VOCs, a special stainless steel measurement chamber for the microbial cultures was used. The gas outlet was either attached to MCC-IMS or to adsorption tubes (Tenax GR) for GC/MS measurements. Isoamyl alcohol, cyclohexanone, 3-octanone and phenethylalcohol can be described as discriminating substances by means of GC/MS. With MCC-IMS, the results for 3-octanone and phenethylalcohol are concordant and additionally to GC/MS, ethanol and two further compounds (p_0642_1/p_683_1 and p_705_3) can be described. Isoamyl alcohol and cyclohexanone were not properly detectable with MCC-IMS. The major advantage of the MCC-IMS system is the feasibility of rapid analysis of complex gas mixtures without pre-concentration or preparation of samples and regardless of water vapour content in an online setup. Discrimination of fungi on genus level of the investigated germs by volatile metabolic profile and therefore detection of VOC is feasible. However, a further discrimination on species level for Candida species was not possible.

Software tool for mining liquid chromatography/multi-stage mass spectrometry data for comprehensive glycerophospholipid profiling.

Electrospray ionization mass spectrometry (ESI-MS) has emerged as an indispensable tool in the field of lipidomics. Despite the growing interest in lipid analysis, there are only a few software tools available for data evaluation, as compared for example to proteomics applications. This makes comprehensive lipid analysis a complex challenge. Thus, a computational tool for harnessing the raw data from liquid chromatography/mass spectrometry (LC/MS) experiments was developed in this study and is available from the authors on request. The Profiler-Merger-Viewer tool is a software package for automatic processing of raw-data from data-dependent experiments, measured by high-performance liquid chromatography hyphenated to electrospray ionization hybrid linear ion trap Fourier transform mass spectrometry (FTICR-MS and Orbitrap) in single and multi-stage mode. The software contains three parts: processing of the raw data by Profiler for lipid identification, summarizing of replicate measurements by Merger and visualization of all relevant data (chromatograms as well as mass spectra) for validation of the results by Viewer. The tool is easily accessible, since it is implemented in Java and uses Microsoft Excel (XLS) as output format. The motivation was to develop a tool which supports and accelerates the manual data evaluation (identification and relative quantification) significantly but does not make a complete data analysis within a black-box system. The software's mode of operation, usage and options will be demonstrated on the basis of a lipid extract of baker's yeast (S. cerevisiae). In this study, we focused on three important representatives of lipids: glycerophospholipids, lyso-glycerophospholipids and free fatty acids.

Alignment of retention time obtained from multicapillary column gas chromatography used for VOC analysis with ion mobility spectrometry.

Multicapillary column (MCC) ion mobility spectrometers (IMS) are increasingly in demand for medical diagnosis, biological applications and process control. In a MCC-IMS, volatile compounds are differentiated by specific retention time and ion mobility when rapid preseparation techniques are applied, e.g. for the analysis of complex and humid samples. Therefore, high accuracy in the determination of both parameters is required for reliable identification of the signals. The retention time in the MCC is the subject of the present investigation because, for such columns, small deviations in temperature and flow velocity may cause significant changes in retention time. Therefore, a universal correction procedure would be a helpful tool to increase the accuracy of the data obtained from a gas-chromatographic preseparation. Although the effect of the carrier gas flow velocity and temperature on retention time is not linear, it could be demonstrated that a linear alignment can compensate for the changes in retention time due to common minor deviations of both the carrier gas flow velocity and the column temperature around the MCC-IMS standard operation conditions. Therefore, an effective linear alignment procedure for the correction of those deviations has been developed from the analyses of defined gas mixtures under various experimental conditions. This procedure was then applied to data sets generated from real breath analyses obtained in clinical studies using different instruments at different measuring sites for validation. The variation in the retention time of known signals, especially for compounds with higher retention times, was significantly improved. The alignment of the retention time--an indispensable procedure to achieve a more precise identification of analytes--using the proposed method reduces the random error caused by small accidental deviations in column temperature and flow velocity significantly.

Comparison of metabolites in exhaled breath and bronchoalveolar lavage fluid samples in a mouse model of asthma.

BACKGROUND: A multi-capillary column ion mobility spectrometer (MCC/IMS) was developed to provide a method for the noninvasive diagnosis of lung diseases. The possibility of measuring the exhaled breath of mice was evaluated previously. The aim of the present study was to reveal whether mice affected by airway inflammation can be identified via MCC/IMS. METHODS: Ten mice were sensitized and challenged with ovalbumin to induce allergic airway inflammation. The breath and volatile compounds of bronchoalveolar lavage fluid (BALF) were measured by MCC/IMS. Furthermore, histamine, nitric oxide, and arachidonic acid were determined as inflammatory markers in vitro. RESULTS: Six volatile molecules were found in the BALF headspace at a significantly higher concentration in mice with airway inflammation compared with healthy animals. The concentration of substances correlated with the numbers of infiltrating eosinophilic granulocytes. However, substances showing a significantly different concentration in the BALF headspace were not found to be different in exhaled breath. Histamine and nitric oxide were identified by MCC/IMS in vitro but not in the BALF headspace or exhaled breath. CONCLUSION: Airway inflammation in mice is detectable by the analysis of the BALF headspace via MCC/IMS. Molecules detected in the BALF headspace of asthmatic mice at a higher concentration than in healthy animals may originate from oxidative stress induced by airway inflammation. As already described for humans, we found no correlation between the biomarker concentration in the BALF and the breath of mice. We suggest using the model described here to gain deeper insights into this discrepancy.

Analyses of mouse breath with ion mobility spectrometry: a feasibility study.

Exhaled breath can provide comprehensive information about the metabolic state of the subject. Breath analysis carried out during animal experiments promises to increase the information obtained from a particular experiment significantly. This feasibility study should demonstrate the potential of ion mobility spectrometry for animal breath analysis, even for mice. In the framework of the feasibility study, an ion mobility spectrometer coupled with a multicapillary column for rapid preseparation was used to analyze the breath of orotracheally intubated spontaneously breathing mice during anesthesia for the very first time. The sampling procedure was validated successfully. Furthermore, the breath of four mice (2 healthy control mice, 2 with allergic airway inflammation) was analyzed. Twelve peaks were identified directly by comparison with a database. Additional mass spectrometric analyses were carried out for validation and for identification of unknown signals. Significantly different patterns of metabolites were detected in healthy mice compared with asthmatic mice, thus demonstrating the feasibility of analyzing mouse breath with ion mobility spectrometry. However, further investigations including a higher animal number for validation and identification of unknown signals are needed. Nevertheless, the results of the study demonstrate that the method is capable of rapid analyses of the breath of mice, thus significantly increasing the information obtained from each particular animal experiment.

Breath analysis-performance and potential of ion mobility spectrometry.

Ion mobility spectrometry is a fast and sensitive analytical method for the detection of gas phase analytes in the ppb(v)-ppt(v) range under ambient conditions (pressure and temperature). Ion mobility spectrometers coupled with rapid pre-separation like multi-capillary columns (MCC/IMS) are suitable for the selective characterization of complex and humid mixtures. Recently, MCC/IMS have been applied to analyses of human breath for early diagnosis as well as medication and therapy control. The complete procedure of breath analyses including evaluation and interpretation of the data obtained is demonstrated for the first time on exhaled breath after the consumption of a particular candy as an example. An MCC/IMS equipped with a ß-radiation source ((63)Ni) requires 5 to 10 min for a complete analysis of exhaled breath. Retention time and reduced ion mobility of the detected signals are compared to an analyte database for the identification of the related analytes. These findings were successfully validated by gas-chromatographic mass spectroscopy of the headspace of the candy via solid-phase micro-extraction and of breath samples on Tenax adsorption tubes. Furthermore, signal height of particular analyte signals as a measure for their concentration was used to monitor the concentration development with time. This exemplary investigation demonstrates that MCC/IMS is a powerful and rapid non-invasive tool for human breath analyses. The method can be used for medical applications (diagnosis, therapy control, metabolic profiling) as well as for a general determination of the metabolic state of a subject (medication, nutrition, fasting). The demonstrated procedure is independent of whether the analytes detected in breath are caused by nutrition or medication or whether they are metabolite characteristics for a particular disease. Therefore, it can directly be transferred to any relevant peak pattern.

Changes of the metabolism of the colon cancer cell line SW-480 under serum-free and serum-reduced growth conditions.

Serum of animal origin, like foetal calf serum (FCS), is used as a standard supplement for media to cultivate mammalian cells, mostly due to its growth-supporting properties. Unfortunately, animal serum has many disadvantages like the risk of contamination, high costs, fluctuations within the composition of different batches and the high amount of foetuses, which have to be harvested. To avoid all this, it is necessary to provide alternatives, which combine as many positive properties of the animal serum as possible but do not influence the cellular metabolism negatively. Today, several serum-free complete media as well as serum substitutes are commercially available. In the present study, a serum substitute, a serum-reduced medium and a serum-free medium were evaluated concerning their influence on the metabolism on the colon cancer cell line SW-480. The evaluation of morphological changes of the cells was done by microscopic analysis whereas differences in the volatile metabolome were analysed by solid phase micro extraction (SPME) followed by gas chromatography/mass spectrometry (GC/MS).

Ultrafiltration behavior of selected pharmaceuticals on natural and synthetic membranes in the presence of humic-rich hydrocolloids.

The separation behavior of the frequently administered pharmaceuticals sulfamethoxazol (Sulfa), carbamazepine (Carba), diclofenac (Diclo), and ibuprofen (Ibu) on different natural and synthetic ultrafiltration membranes was studied. Commercially available cattle intestine natural membranes (NM), polyethersulfone (PES), and regenerated cellulose-based (RC) flat membranes (nominal cut-off 1 kDa) have been investigated as ultrafiltration membranes in a small tangential-flow ultrafiltration unit (TF-UF). First, the nominal cut-off of the NM membranes under study was assessed at approximately 5 kDa, by using polystyrenesulfonate standards for pore-size classification at low TF-UF pressure (0.25 x 10(5) Pa). Working pressures of >1.5 x 10(5) Pa strongly increased the cut-off of NM, in contrast with that of PES and RC membranes. Sulfa, Carba, Diclo and Ibu (1 mg L(-1) each) in colloid-free aqueous solutions (400 mg L(-1) NaCl) completely permeated through NM membranes, but less through PES and RC, which had particular sorption capability towards Diclo. The drugs were routinely determined by using high-performance liquid chromatography (HPLC). Detailed TF-UF investigation of drug retention on NM in the presence of humic hydrocolloids revealed strong interactions between aquatic humic substances (HS) and Diclo and Ibu (but not with Sulfa and Carba) causing retention of up to 80% of Diclo and Ibu, probably because of their binding to macromolecular HS. The standard deviation (SD) of both drug and HS permeation through a single NM was between 2.5 (Sulfa) and 4.0% (Diclo), in contrast with the SD of permeation through separate membranes taken from different lots [SD up to 14.0% (Diclo)], presumably caused by natural variation of the studied NM. Accordingly, membrane filtration of drug-containing water samples on cattle intestines enables both analyte/matrix separations for Carba and Sulfa in the presence of humic colloids and analytical discrimination between free and colloid-bound Diclo and Ibu fractions.

Separation of drug traces from water with particular membrane systems.

The purpose of the present project is to examine the applicability of certain natural biological and liquid membranes for the separation of drugs of environmental concern such as ibuprofen, diclofenac, carbamazepine, and sulfamethoxazol from dilute aqueous solutions. Different types of intestine parts of cattle, sheep, and pig were applied as biological flat sheet membranes after different modes of pretreatment. Best results were obtained with special parts of cattle appendix. The concentration of each drug in the aqueous feed phase was in the range of 0.1-10 mg (cm3)(-1), the pH-values adjusted between 8 and 10. Pure water was used as the permeation phase. The influence of experimental parameters such as stirring velocity, temperature, pH-value, salt concentration, and the presence of surfactants as well as humic substances was studied. Under all conditions chosen the combined drugs permeate simultaneously through the natural membranes as the permeation kinetics of the individual compounds are very similar, while humic compounds were retained. Additional treatment of the permeate with liquid or solid phase extraction techniques increases crucially the depletion of the drugs from the feed. The mass transfer of the pharmaceuticals through the liquid membranes was carried out in three-compartment transport cells and supported liquid membrane-chambers. The three-phase liquid bulk membrane systems consisted of an aqueous feed solution, an organic solvent (dihexyl ether, decane, undecane, or decanol) with and without a dissolved sulfonic acid, tertiary amine or Cu(II)-chelate compound as a liquid bulk membrane and an aqueous stripping solutions containing dilute solutiuons of Na2CO3, NaOH, HCl, or HClO4. The transport of the drugs shows some differences, which can be attributed to their acid/base-behavior and partition coefficients log Kow. High extraction yields were obtained for sulfamethoxazol and carbamazepine by using polar organic solvents. Maximum transport efficiencies were obtained for the acidic compounds ibuprofen and diclofenac. They were completely extracted by using dihexyl ether loaded with octane sulfonic acid. A pH-gradient between feed and strip increases the efficiency of the transport. Certain three-phase compositions were successfully utilized in supported liquid membrane systems (SLM) so that high enrichment factors (approximately 75) were achieved for traces of diclofenac and ibuprofen. The solid and liquid membrane systems employed aim for technical as well as analytical purposes, such as sample pretreatment prior to HPLC-UV or LC-MS analysis of drug traces.