Magdalena Ligor

Determination of volatile organic compounds in exhaled breath of patients with lung cancer using solid phase microextraction and gas chromatography mass spectrometry

Abstract Background: Analysis of exhaled breath is a promising diagnostic method. Sampling of exhaled breath is non-invasive and can be performed as often as considered desirable. There are indications that the concentration and presence of certain of volatile compounds in exhaled breath of lung cancer patients is different from concentrations in healthy volunteers. This might lead to a future diagnostic test for lung cancer. Methods: Exhaled breath samples from 65 patients with different stages of lung cancer and undergoing different treatment regimes were analysed. Mixed expiratory and indoor air samples were collected. Solid phase microextraction (SPME) with carboxen/polydimethylsiloxane (CAR/PDMS) sorbent was applied. Compounds were analysed by means of gas chromatography (GC) and mass spectrometry (MS). Results: The method we used allowed identification with the spectral library of 103 compounds showing at least 15% higher concentration in exhaled breath than in inhaled air. Among those 103 compounds, 84 were confirmed by determination of the retention time using standards based on the respective pure compound. Approximately, one third of the compounds detected were hydrocarbons. We found aromatic hydrocarbons, alcohols, aldehydes, ketones, esters, ethers, sulfur compounds, nitrogen-containing compounds and halogenated compounds. Acetonitrile and benzene were two of 10 compounds which correlated with smoking behaviour. A comparison of results from cancer patients with those of 31 healthy volunteers revealed differences in the concentration and presence of certain compounds. The sensitivity for detection of lung cancer patients based on eight different compounds not seen in exhaled breath of healthy volunteers was 51% and the specificity was 100%. These eight potential markers for detection of lung cancer are 1-propanol, 2-butanone, 3-butyn-2-ol, benzaldehyde, 2-methyl-pentane, 3-methyl-pentane, n-pentane and n-hexane. Conclusions: SPME is a relatively insensitive method and compounds not observed in exhaled breath may be present at a concentration lower than LOD. The main achievement of the present work is the validated identification of compounds observed in exhaled breath of lung cancer patients. This identification is indispensible for future work on the biochemical sources of these compounds and their metabolic pathways. Clin Chem Lab Med 2009;47:550–60.

The analysis of healthy volunteers' exhaled breath by the use of solid-phase microextraction and GC-MS

We analysed breath and inhaled room air samples from 39 healthy volunteers (28 non-smokers, 8 smokers and 3 ex-smokers) by SPME-GC-MS. Mixed expiratory and indoor air samples were collected in freshly cleaned Tedlar bags. Eighteen millilitres of each sample were transferred into sealed, evacuated glass vials, preconcentrated by solid-phase microextraction (SPME, carboxen/polydimethylsiloxane) and investigated by gas chromatography with mass spectrometric detection (GC-MS). For the unequivocal identification of potential marker compounds, pure calibration mixtures of reference compounds (depending on commercial availability) were prepared to determine the retention time and mass spectra with respect to our analytical setting. Applying the adapted SPME-GC/MS method with limit of detection in the high ppb range (0.05-15.00 ppb), we succeeded in identifying altogether 38 compounds with concentrations in exhaled breath being at least 50% higher than concentration in inhaled air. From these 38 compounds, 31 were identified not only by the spectral library match but also by retention time of standards. A comparison of retention times and spectrum obtained for standards and determined compounds was performed. We found hydrocarbons (isoprene, 2-pentene, 2-methyl-1-pentene, benzene, toluene, p-cymene, limonene, 2,4-dimethylheptane, n-butane), ketones (acetone, hydroxypropanone, methylvinyl ketone), ethers (dimethyl ether, 1,3-dioxolane), esters (ethyl acetate), aldehydes (propanal, hexanal, heptanal, acrolein) and alcohols (ethanol, 2-metoxyethanol, isopropyl alcohol, 2,2,3,3- tetramethylcyclopropanemethanol, 3,4-dimethylcyclohexanol). Proper identification of compounds in different cohorts of patients and volunteers is the base for further investigation of origin, biochemical background and distribution of potential breath biomarkers.

Determination of menthol and menthone in food and pharmaceutical products by solid-phase microextraction-gas chromatography

Abstract In the current contribution the optimization of menthol and menthone isolated from food and pharmaceutical samples by solid-phase microextraction (SPME) will be presented. Extraction efficiencies for commercial (polydimethylsiloxane) and laboratory-made (ethoxypolydimethylsiloxane) coated quartz fibers were compared. The results show, that SPME coupled with GC–flame ionisation detection is a reproducible method for isolation and for qualitative and quantitative determination of menthol and menthone at ppm as well as ppb levels. The described procedure can be recommended for routine food and pharmaceutical analyses.

Preparation and characterization of microporous fibers for sample preparation and LC‐MS determination of drugs

The aim of this study was the preparation of polypyrrole (PPy) fibers for solid phase microextraction (SPME). PPy coatings were obtained during the electrochemical polymerization process. The utility of various metal wires (Fe, Cu, Ag, Cu/Ag, kanthal and medical stainless steel) as a support for polymers was compared. Various experimental conditions of the synthesis process such as scan rate, voltage limits and number of scans and deposition time were applied. The average polymer thickness was in the range of 7-125 microm and its weight was in the scope of 0.65-5.6 mg. Different techniques, mainly elemental analysis, Fourier transform infrared spectroscopy, microscopy, and chromatography were performed for the characterization of obtained fibers with microporous structure. The extraction efficiency of cardiovascular drugs (metoprolol, propranolol, oxprenolol, propafenone and mexiletine) by means of fibers was tested. The concentration of mentioned compounds in standard solution was in the span of 10-150 ng/mL. LC-MS was employed for determination of drugs in desorption solution. LODs varied from 0.013 to 1.51 ng/mL for metoprolol and mexiletine respectively. The repeatability of extraction was obtained with the RSD values lower than 10%.

Corona-Charged Aerosol Detection: An Analytical Approach

An alternative to other universal detection methods like refractive index (RI), low wavelength or ultraviolet (UV), and evaporative light scattering detection (ELSD), chemiluminescent nitrogen detection is a relatively new method based upon aerosol charging and involves Corona-charged aerosol detection (Corona-CAD). Some reports show that the sensitivity of this method is lower than that of fluorescence detection. Others confirm approximately five times higher sensitivity than conventional UV absorbance detection for selected analytes. This is the reason that Corona-CAD can be widely used for the determination of nonvolatile or semivolatile compounds, including: lipids, oligosaccharides, carbohydrates, proteins, steroids, surfactants, polymers, peptides, and others. The response of Corona-CAD is independent of chemical properties of the analyte. This article reviews applications of Corona-CAD and principles of the aerosol charging method as well as advantages and disadvantages of the method. The modern solution using an ultra detector, which brings charged aerosol detection to UHPLC, is also discussed.

Determination of volatile and non-volatile products of milk fermentation processes using capillary zone electrophoresis and solid phase microextraction coupled to gas chromatography

The aim of the investigations was to develop analytical methods for the determination of selected volatile and non-volatile organic compounds numbering among the final products of milk fermentation. The analyzed compounds were as follows: biacetyl and carboxylic acids (formic, acetic, citric, and lactic). The model yogurt was prepared under controlled conditions in our laboratory by addition of the selected bacteria (Lactobacillus bulgaricus and Streptococcus thermophilus) to the milk sample. The temperature, time, and stirring were controlled during the fermentation process. Factors considered in SPMPE-GC-FID method development included fiber exposure time, salt addition, temperature of extraction, and temperature of desorption. Various SPME fibers, for example with PDMS, CAR/PDMS, PA, and PDMS/DVB coatings, were tested to obtain the highest recovery of the investigated compounds extracted from yogurt samples. Based on these preliminary experiments, qualitative and quantitative analyses for the determination of biacetyl were performed by SPME-GC-FID. Moreover, a capillary zone electrophoresis method was developed for the determination of carboxylic acids in the yogurt samples. The buffer composition as well as deproteinization by acetonitrile were found to have a crucial effect on the analysis.

The comparison of solid phase microextraction-GC and static headspace-GC for determination of solvent residues in vegetable oils

The objective of these investigations has been the determination of volatile organic compounds including residue solvents present in vegetable oil samples. Some olive oil, rape oil, sunflower oil, soy-bean oil, pumpkin oil, grape oil, rice oil as well as hazel-nut oil samples were analysed. Among residue solvents the following compounds have been mentioned: acetone, n-hexane, benzene, and toluene. Some experiments for the solid phase microextraction (SPME)-GC-flame ionisation detection (FID) were performed to examine extraction conditions such as fiber exposure time, temperature of extraction, and temperature of desorption. Various SPME fibers such as polydimethylsiloxane, Carboxen/polydimethylsiloxane and polydimethylsiloxane/divinylbenzene coatings were used for the isolation of tested compounds from vegetable oil samples. After optimisation of SPME, real vegetable oil samples were examined using SPME-GC/MS. Based on preliminary experiments the qualitative and quantitative analyses for the determination of acetone, n-hexane, benzene and toluene were performed by SPME-GC-FID and static head-space (SHS)-GC-FID methods. The regression coefficients for calibration curves for the examined compounds were R(2) > or = 0.992. This shows that the used method is linear in the examined concentration range (0.005-0.119 mg/kg for SPME-GC-FID and 0.003-0.728 mg/kg for SHS-GC-FID). Chemical properties of analysed vegetable oils have been characterised by chemometric procedure (cluster analysis).

Determination of Flavonoids in Tea and Rooibos Extracts by TLC and HPLC

In this study thin-layer chromatography (TLC) was used for analysis of the constituents of extracts of tea (Camellia sinensis L.) and Rooibos (Aspalathus linearis) leaves. In particular, flavonoids, a group of phenolic compounds, for example myrecetin, rutin, catechin, quercetin, and kaempferol were analyzed. Extracts of various types of tea, for example black, green, and Rooibos herbal tea (called red tea) were analyzed. The efficiency of extraction of flavonoids from plant material by classical liquid extraction (LE) and supercritical-fluid extraction (SFE) was also compared. Recoveries of individual flavonoids were approximately ten times higher after use of solvent extraction. Separation of flavonoids from tea extracts on the basis of their polarities was optimized by selection of solvents, stationary phases, and chromatographic conditions. For final analyses glass-backed silica gel 60 F254 plates and the mobile phase acetone-chloroform-water 80:20:10 (v/v) were used. UV detection of TLC chromatograms (254 and 366 nm) was used. Better detection of the flavonoids was achieved at 366 nm. The concentration of some important flavonoids in real tea samples was obtained by high-performance liquid chromatography (HPLC).

Thin Layer Chromatographic Techniques (TLC, OP TLC) for Determination of Biological Activated Compounds from Herb Extracts

Abstract In the current contribution the comparison of various chromatographic techniques (TLC, OP TLC) for the determination of volatile organic compounds including monoterpenes from peppermint (Folium Mentha piperita) is presented. Flavonoids from hawthorn (Crategus oxyacantha), Passiflora incarnata, hop (Humulus lupulus), cacao (Theobroma cacao), as well as tea (Thea sinensis) extracts were also determined by TLC and OP TLC. Planar chromatographic techniques, thin‐layer chromatography (TLC) and over pressure thin‐layer chromatography (OP TLC) have been developed for the quantitative determination of constituent menthol and menthone in extracts of peppermint leaves. The optimisation of separation of monoterpenes from peppermint based on their principles of polarity including the selection of solvents, stationary phases, and chromatographic conditions, was performed. For separation of determined compounds from plant material, different extraction methods such as supercritical fluid extraction (SFE) and solvent extraction were used.

Design of the extraction process for terpenes and other volatiles from allspice by solid‐phase microextraction and hydrodistillation

Methods for the separation and determination of terpenes (mono- and sesqui-) and phenylpropanoids such as eugenol and methyleugenol from samples of allspice berries have been developed. Chromatographic analyses of isolated groups of compounds were carried out by means of gas chromatography coupled with mass spectrometry. A comparison of various types of solid-phase microextraction fibers was performed. The highest yields of terpenes were extracted by polydimethylsiloxane and divinylbenzene/Carboxen/polydimethylsiloxane fibers (almost the same for these two fibers), approximately twice as much as by Carbowax/divinylbenzene fiber. The highest amounts of monoterpenes were extracted by divinylbenzene/Carboxen/polydimethylsiloxane fiber, and the highest amounts of sesquiterpenes were extracted by polydimethylsiloxane fiber. Moreover, the effect of water addition on extraction yields as well as time and temperature of extraction were tested. Aroma profiles of extracts obtained by solid-phase microextraction and essential oil obtained by hydrodistillation of allspice berries were compared. The aroma profile of the divinylbenzene/Carboxen/polydimethylsiloxane fiber extract was similar to the aroma profile of essential oil. Particular characteristics of volatile allspice matters were presented. The linear retention indices for each compound were calculated.