Institut für Lebensmittelchemie

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Determination of potentially hazardous oxidation products in cosmetics containing lanolin or 1-(1,2,3,4,5,6,7,8-octahydro-2,3,8,8-tetramethyl-2-naphthalenyl)ethanone (OTNE)
(2019) Schrack-Belschner, Sonja Miriam Irmgard; Schwack, Wolfgang
Cosmetic products are important consumer goods in the "non-food" sector, which should not have negative effects of the human health. Critical compounds, however, can be formed by the oxidation of an unsaturated organic compound.Thereby formed oxidation products with potentially adverse properties are well known from the food sector. As the oxidation of cosmetic ingredients, however, has less been studied, the oxidation of selected cosmetic ingredients with respect to the formation of potentially critical compounds was investigated within the framework of this thesis.The oxidation of cholesterol to various cholesterol oxidation products (COPs) was investigated in a first step.COPs are known from the food sector and are suspected of causing certain diseases such as arteriosclerosis.Cosmetic products have not yet been tested for COPs, although a versatile ingredient used only in cosmetic products, lanolin, contains above-average levels of the cholesterol, which is the precursor.Total COPs contents in cosmetics containing lanolin, namely lip care products, fat creams and ointments for nursing women were in the low percent range (up to 3 %) and were thus several orders of magnitudes higher than the contents found in food.The oxidation of fragrances was studied in the second part of this work.The subject is not new as the oxidation of terpenes to contact allergens has been studied in earlier studies. The oxidation of other fragrances was hardly investigated. In order to extend our knowledge in this field, the oxidation of a synthetic fragrance frequently used in perfumes, octahydro tetramethyl naphthalenyl ethanone (OTNE) was studied. Obtained results indicated that peroxides of OTNE were formed during oxidation.It was found out that the OTNE oxidation even occurs, when perfumes are stored indoors under normal temperature and light conditions. An in-vivo test showed that OTNE oxidation can be expected on the skin after application of a perfume.
Development of a planar yeast estrogen screen as screening tool for estrogen active compounds
(2018) Schick, Dinah; Schwack, Wolfgang
Substances that disrupt or impair the hormone system (endocrine system) or that show an irreversible influence on it are referred to as endocrine disruptors or xenohormones. Concerning this, also estrogen active compounds (EAC) are endocrine disruptors, that are under suspicion of being involved in the formation of tumors or to induce disruption during development and reproduction, and are, for example, blamed for being responsible for the feminization of fish. At this, EAC can be natural (human, phytoestrogens) but also synthetic substances, which are discharged to the environment by humans (e.g. pharmaceuticals, pesticides, additives). Regarding the ubiquitous presence of EAC, suitable methods for the analysis of EAC are required. An in vitro method for the determination of EAC is the YES assay (yeast estrogen screen) that is executed in liquid solutions in microtiter plates and that works with genetically modified yeasts, which contain the human estrogen receptor (hER) and a reporter gene encoding for the enzyme beta-galactosidase. In presence of EAC, the enzyme is produced and subsequently cleaves a substrate that is used to measure the receptor activity and thus the estrogenic activity. The transfer of the YES assay to high-performance thin-layer chromatography (HPTLC) was successfully demonstrated and advanced, thus resulting in the combination of a chromatographic separation of analytes and the detection of EAC using genetically modified yeast cells directly on the HPTLC plate (HPTLC planar yeast estrogen screen, HPTLC-pYES). Usually, the substrate 4-methylumbelliferyl-beta-D-galactopyranoside is used for pYES, releasing blue fluorescing 4-methylumbelliferone (MU) after enzymatic cleavage. Various matrices, however, often contain a plenty of different components, partly showing native fluorescences (blue, red), why the detection of the blue fluorescing MU can be interfered. By applying the substrate resorufin-beta-D-galactopyranoside (RGP) and by using automated devices, the RGP-pYES as fast screening tool for EAC was developed and successfully applied to waste water samples and extracts of hops pellet samples. A screening method using HPTLC simultaneously represents a planar clean-up, why samples do not have to undergo complex steps of sample preparation or purification. The chromatographic separation in combination with the detection of estrogenic activity using genetically modified yeasts directly on the plate allowed the detection, the determination and the identification of single EAC. Using RGP, which releases orange fluorescing resorufin after enzymatic cleavage as positive signal of estrogenic activity, enabled a clear differentiation between fluorescences due to estrogenicity and the native fluorescence of sample components. Application of the RGP-pYES to spiked water samples and sewage samples showed high recovery rates and a good precision, and thus the applicability of the method as screening tool for environmental samples. By means of suitable evaluation methods, additionally the generation of dose-response curves of known and unknown EAC and thus the generation of so-called logit-log plots was possible. This enabled the determination of estradiol equivalent factors of known EAC as well as the determination of estradiol equivalent concentrations and amounts, respectively, of known and unknown EAC in liquid and solid samples. Thus, the possibility to estimate the estrogenic potential of a sample or single sample components was given. The coupling of pYES to mass spectrometry additionally allowed the identification of unknown EAC, demonstrated exemplarily by investigation of extracts of hops pellet samples, in which the only detected EAC in the hops extracts was identified as prenylnaringenin. Since the method uses a planar system, the pYES advantageously reveals all chromatographically separated sample components at one look and, as bioassay, additionally detects a possible estrogenic activity (activity at the hER) of single substances, while a differentiation between native occurring fluorescences of sample contaminants and the fluorescence as positive signal for estrogenicity of a substance is granted.
Development of strategies for the prioritization of organic trace substances in water by effect-directed analysis
(2020) Stütz, Lena; Schwack, Wolfgang
The protection of the aquatic environment and the supply of clean drinking water to people all over the world are central challenges of our time. Monitoring of the aquatic environment and the input of anthropogenic trace substances into it is therefore very important. However, since aquatic environmental samples often consist of complex substance mixtures, their characterization and evaluation is very demanding. By using generic target analysis methods, selected known anthropogenic trace substances can be detected and quantified very sensitively. For the detection of previously unknown substances, non-target analysis methods have been increasingly used in recent years. However, these methods do not provide information on the relevance of the anthropogenic trace substances occurring in water. In this context, especially all those trace substances are regarded as relevant from which a harmful effect on humans or water organisms is to be expected. For the detection of such effective substances, effect-directed analysis (EDA) can be used. In EDA, a bioassay is combined with a fractionation method and subsequent chemical analysis, the aim being to identify the bioactive substance. The separation method used in this work is high-performance thin-layer chromatography (HPTLC). After chromatography, the bioassay is performed directly on the HPTLC plate. If an effective zone appears in the bioassay, a prioritization strategy is used to clarify the identity of the substance. Due to the complex aquatic samples, a large number of different substances in a zone must still be expected despite the applied HPTLC separation, which makes it difficult to identify the effective substance. Therefore, a strategy to simplify the identification of effective substances should be developed. The aim was to reduce the complexity by multidimensional separation in such a way that chemical analysis can be used to prioritize to a few candidates in the effective fraction. In the first part of the work, a selective two-dimensional HPTLC separation was developed to reduce the number of substances in a bioactive zone. After the first separation dimension (1D) the acetylcholinesterase inhibition assay (AChE assay) was performed and afterwards only the effective zones were extracted from the HPTLC plate. The selected effective zones were separated in a second separation dimension (2D) and the bioassay was performed again. With this 2D separation, the peak capacity could be increased by a factor of 7 compared to a 1D HPTLC gradient development. If real water samples are examined for their effects, an additional structural elucidation must be carried out to clearly identify the unknown bioactive substances. In this work, the developed 2D EDA was therefore connected to a high-performance liquid chromatography (HPLC) with high-resolution mass spectrometry (HRMS) and a non-target screening (NTS) was performed. Using three water samples(drinking water, surface water and purified sewage water) spiked with six effective substances, it was shown that the developed strategy is suitable for the identification of effective substances and that these can be recovered despite repeated extraction. When applying the developed methodology to real samples, it was also possible to assign and quantify the detected effect in several waters to the substance lumichrome and to linear alkylbenzene sulfonates. Genotoxicity is a crucial endpoint for the effect assessment of water samples. However, this endpoint with metabolic activation cannot yet be performed directly on the HPTLC plate. Since many of the genotoxic substances have an indirect genotoxic effect, i.e. they only acquire their activity after metabolic activation; this endpoint was investigated in the present work with the umu assay in the microtiter plate. However, separation with HPTLC, subsequent extraction of effective zones and non-target analysis of the extracts, should also be performed for this assay. Therefore the umu assay in the microtiter plate was integrated into the existing EDA-with-HPTLC concept. In laboratory experiments, sodium hypochlorite was added to the drug metformin in order to simulate the behavior of the substance during water treatment (chlorination). The metformin sample treated with hypochlorite was examined with the umu assay and a genotoxic effect was detected. After HPTLC separation of the chlorinated sample, zones were extracted over the entire retardation range. When the extracted zones were examined with the umu assay, the genotoxic effect could be clearly assigned to one fraction. Using high-resolution mass spectrometry, the genotoxic effect could be assigned to an already known transformation product of metformin. The HPTLC separation and extraction of the zones from the plate led to a reduction of the possible effective candidate masses by a factor of 10 and thus to a clear prioritization in HRMS analysis.
A fast gas chromatography coupled with electron capture negative ion mass spectrometry in selected ion monitoring mode screening method for short‐chain and medium‐chain chlorinated paraffins
(2022) Schweizer, Sina; Schulz, Tobias; Vetter, Walter
Rationale Chlorinated paraffins (CPs) are a group of anthropogenic pollutants that consist of complex mixtures of polychlorinated n-alkanes of different chain lengths (~C10 to C30). Persistence, bioaccumulation, toxicity, and long-range transport of short-chain chlorinated paraffins (SCCPs, C10- to C13-CPs) have prompted their classification as persistent organic pollutants (POPs) by the Stockholm Convention in 2017. Due to the varying chain lengths and chlorination degrees, quantification of SCCPs and medium-chain chlorinated paraffins (MCCPs, C14- to C17) using gas chromatography coupled with electron capture negative ion mass spectrometry in selected ion monitoring mode (GC/ECNI-MS-SIM) is not only challenging but also very time consuming. In particular, up to eight GC runs per sample are required for the comprehensive GC/ECNI-MS-SIM quantification of SCCPs and MCCPs. These efforts are high especially if the samples do not contain CPs above the limit of detection (LOD), subsequently. Methods We developed a semi-quantitative and sensitive method for the examination of SCCPs and MCCPs in one GC run. This GC/ECNI-MS-SIM screening method was based on the recording of Cl− (m/z 35 and 37), Cl2− (m/z 70 and 72), and HCl2− (m/z 71 and 73) isotope ions and evaluation of the ratios between them. Results Correctness of the results of the screening method was verified by analysis of edible oils with and without CPs, CP standards, as well as a technical CP mixture. Polychlorinated biphenyls (PCBs) and other polyhalogenated aromatic compounds, as well as brominated flame retardants, do not form all of the fragment ions analyzed by the screening method. Conclusions After the screening, only CP-positive samples may need to be measured in detail. Measurement time will already be gained in the case of ~10% samples without CPs.
Geometrical and positional isomers of unsaturated furan fatty acids in food
(2022) Müller, Franziska; Hammerschick, Tim; Vetter, Walter
Furan fatty acids (FuFA) are important antioxidants found in low concentrations in many types of food. In addition to conventional FuFA which normally feature saturated carboxyalkyl and alkyl chains, a few previous studies indicated the FuFA co‐occurrence of low shares of unsaturated furan fatty acids (uFuFA). For their detailed analysis, the potential uFuFA were enriched by centrifugal partition chromatography (CPC) or countercurrent chromatography (CCC) followed by silver ion chromatography from a 4,7,10,13,16,19‐docosahexaenoic acid ethyl ester oil, a 5,8,11,14,17‐eicosapentaenoic acid ethyl ester oil and a latex glove extract. Subsequent gas chromatography with mass spectrometry (GC/MS) analysis enabled the detection of 16 individual uFuFA isomers with a double bond in conjugation with the central furan moiety. In either case, four instead of two uFuFA isomers previously reported in food, respectively, were detected by GC/MS. These isomers showed characteristic elution and abundance patterns in GC/MS chromatograms which indicated the presence of two pairs of cis/trans‐isomers (geometrical isomers).
High-throughput planar solid phase extraction : a new clean-up concept in multi-residue analysis of pesticides
(2014) Oellig, Claudia; Schwack, Wolfgang
Currently, the most serious problems in pesticide residue analysis by liquid chromatography (LC) or gas chromatography (GC) coupled to mass spectrometry (MS) concern the so-called “matrix effects”. The most common way to avoid these effects is the application of matrix-matched calibration standards. Nevertheless, an efficient clean-up undoubtedly is the best way to prevent matrix effects in multi-residue analysis of pesticides in food by LC–MS or GC–MS. For a totally new powerful clean-up method, called high-throughput planar solid phase extraction (HTpSPE), highly automated planar chromatographic tools were applied to remove co-extracted matrix substances entirely and to eliminate any kind of matrix related effects. For sample extraction, the quick, easy, cheap, effective, rugged, and safe (QuEChERS) method was used to initially collect pesticides from fruits and vegetables. The received acetonitrile extracts were applied directly for the development of the novel HTpSPE clean-up. Thin-layer chromatography (TLC) was used to completely separate pesticides from matrix compounds and to focus them into a sharp zone. A two-fold development on amino-modified silica gel thin-layers with acetonitrile for the first development, and acetone for the second development in the backwards direction, was evaluated to perform the best clean-up result and collect the pesticides in a sharp, single target zone. To easily locate the pesticide zone, the Sudan II dye was added to the extracts. Following this clean-up, the target zones (pesticides) were eluted by the TLC–MS interface into vials for the LC–MS determination. HTpSPE resulted in extracts which were nearly free of co-extracted matrix and matrix effects, as shown for seven chemically representative pesticides (acetamiprid, azoxystrobin, chlorpyrifos, fenarimol, mepanipyrim, penconazole, and pirimicarb) in four different fruit and vegetable matrices (apples, cucumbers, red grapes, and tomatoes). Thanks to the very clean HTpSPE extracts, calibration can simply be performed with pure solvent standards and the quantitation by LC–MS provided excellent mean recoveries and relative standard deviations. In addition, tea samples as rather challenging matrices were chosen to apply for HTpSPE. The matrix load of tea extracts generally was too high for the available thin-layer capacity and the selectivity of the amino-modified phase was not suitable for the separation of caffeine and further matrix compounds from the target analytes (pesticides). By modifying the sample extraction, adding a pre-cleaning by dispersive solid phase extraction (dSPE) and changing the thin-layer phase to normal phase silica gel, the complete separation of pesticides and tea matrix components was possible, when again a two-fold development was applied. Caffeine and other alkaloids were completely removed. The effectiveness of HTpSPE was demonstrated by LC–MS/MS calibration curves from matrix-matched and solvent standards, which were nearly identical and by very good mean recoveries, calculated against pure solvent standards. Concerning all validation parameters, the new acetonitrile-HTpSPE procedure for tea samples was superior to the QuEChERS-dSPE method and offered highly successful results. In recent years, large-scale screening in pesticide residue analysis has gained more and more importance. Keeping this in mind, a screening strategy for HTpSPE extracts, using a high-resolution MS, was developed to analyze the cleaned extracts directly for pesticide residues without a liquid chromatographic separation. By this hyphenation, a completely new microliter-flow injection analysis–time-of-flight mass spectrometry (µL-FIA–TOFMS) screening was introduced. The novel HTpSPE–µL-FIA–TOFMS approach enabled the detection of all pesticides simultaneously in a single mass spectrum within a few minutes. The obtained mass spectra were nearly free of matrix compounds, which is especially the great benefit of the effective HTpSPE clean-up. Recovery studies by HTpSPE–µL-FIA–TOFMS against solvent standards for the matrices and pesticides under study provided excellent results, using the mass signal intensities under the entire FIA sample peak. HTpSPE clearly showed superior results concerning every tested parameter than dSPE. With the help of a self-constructed mass database searching tool, all spiked pesticides were detected and correctly identified, while only very low numbers of false-positive findings occurred. Furthermore, a non-target screening approach was successfully implemented by slightly changing the database searching process, offering a mass list of all substances, which are present in the injected extracts but not included in the mass database. Finally, the new HTpSPE–µL-FIA–TOFMS screening was successfully applied to several real samples, when the identified pesticides were quite identical compared to results of LC–MS/MS analysis of the QuEChERS-dSPE extracts.
HPTLC-bioluminescence detection: methodological improvements and the application of the method to mouthwashes
(2013) Baumgartner, Vera; Schwack, Wolfgang
For the chemical analysis of food, drugs, and environmental samples it becomes more and more important to find substances of a certain (biological) activity. For this, several biological screening assays are available. One of the most versatile is the luminescent bacteria test according to an international norm (DIN EN ISO 11348), a rapid cuvette test on cytotoxicity. The assay employs the naturally bioluminescent bacterium Vibrio fischeri, which emits blue-green light under good living conditions. Because the energy-consuming luminescence metabolism is linked directly to the bacterium?s respiratory chain, a disturbance of the bacterium?s metabolism affects the luminescence, whereas the degree of toxicity is proportional to the luminescence inhibition. Major advantage was achieved by coupling this biotest with previous separation by high-performance thin-layer chromatography (HPTLC), which allowed for a screening for individual components. The workflow consists of sample application onto an HPTLC plate, separation, drying the plate, application of the Vibrio fischeri suspension, and detection with a light-sensitive CCD camera. In the resulting image, dark zones on a brightly luminizing background indicate substances that affect the bacteria?s metabolism. No suitable image evaluation program for the effective correction and quantitative evaluation of the image after Vibrio fischeri detection was available, which was regarded as a great disadvantage. In literature, adaptations of the special corrections based on the cuvette test calculations were described, including horizontal background correction and the recalculation of the sigmoid dose-response-relationship of the bacteria?s reaction. This served as a basis for the development of a new method using existing software which did not only perform the necessary calculations but was easy and convenient enough for use in routine evaluations. Furthermore, the process of applying the aqueous bacteria suspension onto the HPTLC plates was improved. Usually, application was done by dipping with the help of a dipping device. Especially for polar substances, however, it was observed that substances can start to dissolve during this process, leading to blurring and tailing of the zones on the plate. A simple rolling device consisting of commercially available household articles was constructed. To compare rolling with dipping, octhilinone and methylparaben were chosen as test compounds. The results of rolling were far superior to dipping. However, manual rolling depended on the person who did it, and it was not possible to control pressure and velocity. To overcome this problem, a prototype of an automated rolling device was constructed and built. After the successful process optimizations, the applicability of the HPTLC-bioluminescence assay was tested on commercial mouthwashes. Mouthwashes are likely to contain antimicrobial compounds, which are not necessarily indicated on the packaging. HPTLC with biodetection was used as a rapid screening method to detect zones of interest, which were further analyzed by conventional techniques like HPLC and GC. First, the reaction of Vibrio fischeri towards more than 40 standard substances was determined. This database was used for the analysis of six commercially available mouthwashes. It revealed that not only declared preservatives are used in mouthwashes, but also other antimicrobial compounds. These were especially constituents of essential oils having antibacterial properties (anethole, carvone, menthol, thymol), but are summarized as ?aroma?, which is in compliance with legal restrictions. A most interesting question concerns the bacteria?s condition on the HPTLC plate. For the brightly luminizing background, it must be assumed that the bacteria are well alive. But no clear statement can be given for bacteria in the dark zones: they might be dead, inhibited (maybe only temporarily), or absent due to water repelling effects of the zone?s compound. A basic attempt to answer this question was made by applying a combination of classical microbiological techniques. In this dissertation it could be shown that HPTLC coupled with Vibrio fischeri detection can successfully be used in practice and is well suited to complement conventional analytical techniques. This work is meant to serve as a guideline for further research and new applications.
Improvements in the analysis of food contaminations deriving from packaging materials
(2009) Rothenbacher, Thorsten; Schwack, Wolfgang
The dissertation presents in its introduction the sources and process of food contaminations deriving from packaging materials. Subsequent legislative aspects, the analysis of food contact materials and contaminants in food are explained and examples therefore are given. The main part of the dissertation covers the following published papers: 1.T. Rothenbacher, M. Baumann and D. Fuegel. 2-Isopropylthioxanthone (2-ITX) in food and food packaging materials on the German market. Food Additives and Contaminants 2007; 24: pp. 438-444 2.T. Rothenbacher, W. Schwack. Determination of epoxidized soybean oil by gas chromatography/single quadrupole and tandem mass spectrometry stable isotope dilution assay. Rapid Communications in Mass Spectrometry 2007; 21: pp. 1937-1943 3.T. Rothenbacher, W. Schwack. Non-targeted multi-component analytical screening of plastic food contact materials using fast interpretation of deliverables via expert structure-activity relationship software Journal of AOAC INTERNATIONAL 2009; 92 (3): pp. 941-9501 4.T. Rothenbacher, W. Schwack. Rapid and nondestructive analysis of phthalic acid esters in toys of poly(vinyl chloride) by direct analysis in real time?single quadrupole mass spectrometry. Rapid Communications in Mass Spectrometry 2009; 23: pp. 2829?2835 5.T. Rothenbacher, W. Schwack. Rapid identification of additives in poly(vinyl chloride) lid gaskets by direct analysis in real time ionisation and single-quadrupole mass spectrometry. Rapid Communications in Mass Spectrometry 2010; 24: pp. 21-29
Investigations into heat- and light-induced terpene modifications in essential oils
(2023) Bitterling, Hannes; Vetter, Walter
Essential oils belong to secondary plant metabolites, with terpenoids and phenylpropanoids being among the main constituents in terms of quantity. Due to their lipophilic character and high volatility, they are mainly obtained by steam distillation. Citrus essential oils (agrumen oils) are an exception , since they are usually extracted from the peels by means of pressing, whereby less volatile components such as coumarins and furocoumarins are also introduced. Due to their odor and taste-giving properties, essential oils are used in the food, beverage, and cosmetics industries. In addition, due to a wide range of pharmacological properties, they are used in phytotherapy as well as in aromatherapy. However, most essential oils are highly susceptible to oxidation, polymerization, dehydrogenation, and isomerization reactions in the presence of atmospheric oxygen, light, and at high temperatures. The resulting organoleptic changes usually lead to a significant quality reduction. The formation of terpene hydroperoxides is another problem, as these are suspected of causing intolerances such as redness and itching in 1-3% of the European population upon contact with the skin. The detection of these chemical changes forms an integral part of quality control and can be prevented as far as possible by suitable production, transport, and storage strategies. Due to their volatility, essential oils are mainly analyzed by gas chromatography. However, due to their instability, the detection of hydroperoxides places considerable demands on common analytical methods. For this reason, a novel spectrophotometric method for the detection of peroxides and hydroperoxides in terpenes and essential oils was developed (paper 1). The oxidation of N-N-dimethyl-p-phenylenediamine by peroxides yielding an intensely red-colored cation (Wursters red) allowed colorimetric detection and quantitation of even smallest amounts (LOD: 0.5 ppm). The minimal sample amount of only a few milligrams, as well as simple and fast performance predestine this method for daily laboratory routine (paper 1). Among plant terpenoids, the monoterpene R-(+)-limonene is very widespread. Thus, it is not only found in citrus oils but also of in caraway oil, where its proportion amounts to almost 50%. To investigate the storage stability, R-(+)-limonene, S-(+)-carvone, different caraway oils, and the corresponding caraway seeds were stored in desiccators at 25 °C and 40 °C for eighteen months (paper 2). The samples were analyzed monthly by GC/MS and GC/FID, as well as HPLC/DAD-MS/MS. This showed that the comparison of seed, isolated essential oil, and pure substance, whichhad not been considered in storage studies so far, was of extraordinary importance. Here, both the plant matrix and the essential oil had a protective effect on individual terpenes and delayed their degradation (paper 2). Further, a clear difference between photo-oxidation and autoxidation was observed. Light-induced oxidation of terpenes primarily resulted in the formation of hydroperoxides, whereas autoxidation led to a variety of compounds such as alcohols, ketones, and epoxides. Thus, the secondary products can serve as specific markers for conclusions about the preload and quality of essential oils. In the study presented in paper 3, further photo-oxidation experiments were conducted with beta-pinene, R-(+)-limonene, and gamma-terpinene, with added furocoumarins. Furocoumarins can absorb UV-A light in the range of 320 – 380 nm and enter an energetically excited state. This energy difference between the ground state and excited state can be dissipated again by the emission of fluorescent and phosphorescent light. In this process, short-wave energy-rich UV light is converted into lower-energy visible light (bathochromic shift). For this reason, the UV light-induced degradation of the terpenes beta-pinene, R-(+)-limonene, and gamma-terpinene could be significantly reduced by adding 5% each of xanthotoxin, bergapten, bergaptol, and bergamottin. The effect of adding bergaptol was most pronounced in the photooxidation of gamma-terpinene (paper 3). Consequently, in citrus essential oils from which the natural furocoumarins had been previously removed, irradiation with UV light resulted in a strong degradation of the terpenes. This process could be markedly reduced by the re-addition of 5% of the previously removed plant-specific furocoumarins (paper 4). In summary, it can be concluded that not only the plant matrix and the essential oil as a multicomponent mixture but also potential interactions with other substances forming part of the essential oil such as furocoumarins may significantly slow down the oxidation of terpenoids.
Investigations on (photo) reactions of cosmetic UV filters towards skin proteins
(2014) Stiefel, Constanze; Schwack, Wolfgang
Although UV filters are important, widespread used cosmetic ingredients, their reaction potential towards skin proteins has hardly been studied so far. Therefore, the aim of the present thesis was to investigate the reactivity of widespread UV filter substances towards skin proteins using increasingly complex protein and skin model systems and different analytical techniques. At first, the development of a rapid high-performance thin-layer chromatographic (HPTLC) screening method on an amino phase as protein model provided an easy and rapid way to estimate the reactivity of the common UV filters benzophenone-3 (BP-3), hydroxymethoxybenzoyl sulfonic acid (HMBS), butyl methoxydibenzoylmethane (BM-DBM), 3-benzylidene camphor (3 BC), 4 methylbenzylidene camphor (4 BMC), octocrylene (OCR), ethylhexyl methoxycinnamate (EHMC), ethylhexyl salicylate (EHS), diethylhexyl butamido triazone (DEBT), ethylhexyl triazone (EHT), and octyldimethyl p-aminobenzoic acid (OD-PABA) towards amino groups under thermal and irradiation conditions. A direct comparison of the results of the screening with (photo) patch test data of the dermatological practice showed that especially those UV filters, which are known to be common triggers for (photo) allergic reactions, showed the highest tendency to bind to the amino phase. This indicates that the screening may be well suited to identify possible skin sensitizers as part of a multistage testing strategy. The observation that the reactivity of the different UV filters was influenced by both heat and UV irradiation was verified during the subsequent studies with butylamine and ethanolamine. The UV filters showed individual, time- and temperature-dependent reactivities towards the amines. Benzophenone imines, enamines, and amides were identified as typical reaction products by means of electrospray ionization mass spectrometry (ESI-MS), Fourier transform infrared (FTIR), and nuclear magnetic resonance (NMR) spectroscopy. BP-3, HMBS, the dibenzoylmethanes, OCR, and EHS showed by far the highest reactivity what was in good correlation with the previous screening, indicating a different contact-allergic potential of the UV filters. In contrast, the esters EHMC and EHT showed a significantly lower reactivity, and for the UV filters 3 BC, 4-MBC, and OD-PABA no conversion was observable at all. The formation of the reaction products had partly big influence on the respective UV filter spectra. In the case of BP-3, HMBS, and EHS, the conversions led to a strong bathochromic shift and hence to approved UVA protection. In contrary, in the case of DBM and BM-DBM and especially OCR, a breakage of the original molecule structures was observed, resulting in a significant decrease of the respective absorption strength. The same reaction tendencies could also be observed, when using Boc-protected lysine, the tetrapeptide Boc-Gly-Phe-Gly-Lys-OH (Boc-GFGK), and bovine serum albumin (BSA) as increasingly complex protein or skin models. OCR and BM-DBM confirmed to be most reactive towards the lysine side chains of the mentioned model systems, followed by DBM > BP 3 > EHS > EHMC > EHT in decreasing order. To determine the covalent binding of the UV filters to the protein BSA, beside the extraction of the unbound UV filters, the increase of the molar mass of the formed BSA-adducts was additionally exemplarily determined for EHMC and DBM by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). Both methods gave comparable results. Binding to BSA did not affect the UV absorption properties of BM-DBM, EHMC, and EHT, but led to a bathochromic shift in the cases of BP-3 and EHS. For OCR, a strong hypsochromic shift and a nearly complete loss of UVA+B protection was observable. To better reflect the usual application conditions, a thin gelatine layer was chosen as further skin model. The UV filter amounts applied were adapted according to the existing ISO norm for the determination of the SPF. Afterwards, UV irradiation was performed. The binding amounts were determined both by extraction of the unbound UV filters and by isotope-ratio mass spectrometry (IRMS), where two synthetized, stable-isotope labelled UV filter analogues (EHC-d5 and DBM-d5) were used. In contrast to the esters EHMC and EHT, which showed comparatively small binding amounts, for the UV filters OCR, BP-3, EHS, and BM-DBM significant reaction tendencies towards gelatin were observed. Finally, various commercial sunscreens and personal care products with UV protection were applied on either prepared porcine skin or glass plates, followed by UV irradiation. Significant differences were observed for the amounts of UV filters extracted from glass and skin. The lower recoveries in the case of the skin indicated an additional reaction of the UV filters towards the skin samples. BP-3 showed the highest discrepancy between the recoveries from glass and skin after irradiation, followed by EHS > BM DBM > OCR > EHMC > EHT in decreasing order. The present dissertation showed that cosmetic UV filters were able to react with amino structures of different proteins under thermal and irradiation conditions. As the formation of protein adducts is seen as key event in the development of (photo) allergic reactions, the results indicate a specific skin sensitization potential of the UV filters. This is confirmed by the experience of dermatological practice. Since such reactions have partly strong influence on the respective UV filter spectra, the existing in vitro methods using PMMA or quartz glass as substrates have to be questioned, since those methods cannot capture such skin-typical reactions.
Rapid screening of antibiotics in foods by HPTLC-FLD/EDA/MS
(2015) Chen, Yisheng; Schwack, Wolfgang
Nowadays, the usage and partly abuse of veterinary antibiotics resulted in a very pressing need to control residues in foods of animal origin. Particularly, the increasingly demanding MRL issues and the huge number of samples to be monitored raised great challenges in this field. Microbial growth inhibition assays are traditionally employed for screening purposes, while sophisticated HPLC-MS methods are alternatively used or only used for confirmation purposes. To substitute the time consuming growth inhibition assays, HPTLC as a platform hyphenated to multi detection modes was employed in this study for the development of a high throughput, sensitive and cost-efficient screening-oriented methodology for antibiotics residues. The first step was focused on tetracyclines and fluoroquinolones, which are the most problematic antibiotics in the European Union and account for the most of the used veterinary antibiotics. To prevent strong tailing effects, the separation was optimized on normal-phase silica gel plates modified with ethylenediamine tetraacetic acid (EDTA). Besides, selective and sensitive fluorescence densitometry was optimized to achieve best signal/noise ratios. Under these conditions, limits of detection (LODs) and quantitation (LOQs) were in the range 12–25 and 45–95 µg/kg, respectively. Recoveries from milk samples, spiked at 50, 100 and 150 µg/kg and extracted by a modified QuEChERS procedure, ranged from 76 to 105%. To circumvent the ion suppressions due to EDTA, HPTLC-mass spectrometry (HPTLC-MS) was optimized, allowing the selective confirmation of positive findings, also offering high sensitivity of 25 µg/kg, and meeting Commission Regulation (EU) No. 37/2010. In the second step, sulfonamides were targeted, which are the secondly most administered veterinary antibiotics in the European Union. Separation of twelve most important sulfonamides was achieved on HPTLC silica gel plates, followed by fluram derivatization and sensitive and selective quantitation by fluorescent densitometry. LODs and LOQs were determined to 15–40 and 35–70 µg/kg, respectively. Samples of bovine milk, porcine liver and kidney were extracted according to the “QuEChERS” strategy. Additionally, a confirmative detection by HPTLC-MS was optimized, offering straightforward identification of target zones. The method was validated to meet the enforced Commission Regulation (EU) No. 37/2010. Finally, a more universal screening method based on HPTLC-bioautography was developed for most of the first-line veterinary antibiotics. A comprehensive HPTLC plate test revealed that the bio-compatibility of different plate layer materials to the applied bioluminescent bacteria (A. fischeri DSM No. 7151) was surprisingly different. It was then discovered that both bright bioluminescent background and significant inhibition zones of antibiotics can only be achieved on HPTLC amino F254S plates. In this case, HPTLC was not used for the chromatographic separation of individual antibiotics extracted with acetonitrile, but in terms of planar solid phase extraction to separate bioactive matrix compounds and to focus the analytes within two distinct target zones of different polarity. Together with HPTLC-MS for identification and confirmation purposes, the developed procedure enabled the rapid, sensitive and efficient multi-class screening of antibiotic residues (16 species of 5 groups, except sulfonamides and penicillins, which only affect Gram positive bacteria). The multi-sample plate images provided the results within a few hours. Thanks to the high sensitivity and the great matrix tolerance, the established method was successfully applied to bovine milk and porcine kidney samples, each spiked at the EU MLRs.
Trace analysis of acrylamide by high-performance thin-layer chromatography coupled to mass spectrometry
(2011) Alpmann, Alexander; Schwack, Wolfgang
Planar-chromatography (High-Performance Thin-Layer Chromatography, HPTLC) is a rapid and cost-effective offline separation method. Through advances in the automatization of each step the system reproducibility, from application and development to detection, has been improved. This makes planar-chromatography a highly reliable technique. HPTLC shows a couple of features that make it unique. There is great flexibility concerning application, development and detection that distinguishes HPTLC from other techniques. Especially the parallel development of up to 36 tracks per plate, the possibility of pre-chromatographic derivatization on the stationary phase, application volumes from nL up to mL, two-dimensional development, automated single or multiple development, and the multiple detection with different methods (UV, fluorescence, bioluminescence, etc.) have to be emphasized. A further advantage over column- (LC) and gas-chromatography (GC) is the single use of the stationary phase. This leads to a high tolerance towards sample matrix and allows for reducing sample preparation. Because of these aspects, planar-chromatography is an interesting tool for each analyst. However, in the last years hyphenation with mass spectrometry (MS) did not make great advancements in comparison to HPLC and GC: thus, planar-chromatography became less attractive. Therefore an existing universal hyphenation (ChromeXtract by Dr. Luftmann), that was based upon a plunger for elution, was improved (publication 1). The original version of the plunger did not allow any elution from glass backed plates, since they broke easily under the pressure applied during clamping. It was difficult to adjust the pressure depending on the experience of the operator. Furthermore, solvent leakage was possible because of insufficient sealing of the cup-point. For a reproducible contact pressure that was independent from the experience of the operator, a commercial torque wrench was used for clamping of the plates. This guaranteed reproducible contact pressure. The installation of a small plastic buffer into the plunger ensured a slight kind of attenuation. This decreased the frequency of leakage from over 50 % to below 5 %. An important criterion of applicability of this hyphenation is the repeatability of the extractions and thus the measurements. Thus, zones of xanthylethylcarbamat (XEC) and dansylpropanamid (DPA) were extracted after chromatographic development. Their specific masses were detected in positive ESI-mode. The relative standard deviation of the signal in single-ion-monitoring (SIM) mode was 18.6 % for XEC and 8.7 % for DPA. Linearity was given in the range of 10 to 200 ng/zone with a very good correlation coefficient (r > 0.9919). The limit of quantification at an S/N-ratio of 10 was calculated by means of the blank signal and amounted 52 and 160 pg/zone for XEC and DPA, respectively. Additionally, the influence of the elution solvent on the extraction of the HPTLC-plate and signal intensity was demonstrated with tests using different solvents. The second publication addressed the application of planar-chromatography hyphenated with MS by means of the modified ChromeXtractor on the determination of acrylamide in drinking water. The strict limit within the EU of 0.1 mug/L until then was only controlled through costly methods that were almost exclusively based on GC-MS or LC-MS/MS after applying intensive clean-up procedures. Thus it was aimed to develop a low priced and rapid alternative method for routine analysis based on HPTLC. Therefore a pre-chromatographic in-situ derivatization of acrylamide with a fluorescence marker was used. The product was detected densitometrically after chromatographic separation. During development of the method, the mass of the reaction product was determined for analysis of the derivatization step. With the aid of the modified ChromeXtract the product could be directly extracted from the plate and transferred to MS. The exact mass proved that instead of the originally used fluorescence marker dansylhydrazine the dimethylaminonaphthaline(Dan)-sulfinic acid reacts with acrylamide. Consequently, dansulfinic acid was synthesized and used for derivatization. To take advantage of the high tolerance of planar-chromatography towards various sample matrices, an approach was searched in order to skip sample preparation. However the necessity to use excess of reagent led to high background fluorescence. This allowed only a limit of detection of 20 mug/L. Thus, sample preparation and analyt enrichment was necessary to obtain a method able to control the maximum concentration. In accordance with DIN 38413-6 concerning determination of acrylamide in drinking water, activated carbon was used for analyte enrichment by means of solid phase extraction (SPE). An internal standard (dimethylacrylamide) was added prior sample preparation. The final extract was analysed as described. In spiked samples of drinking water, a 1000-fold lower limit of detection of 0.02 mug/L and a very good mean reproducibility across the whole system was shown, which suffices to control the maximum amount. A comparative study with measurements by LC-MS/MS revealed satisfactory correlation. Thus, for the first time a planar-chromatographic method for the determination of acrylamide at ultra-trace levels were presented. The third publication addresses the application of the developed method on a very complex food matrix like coffee. Several publications reported problems during determination of acrylamide in coffee. Therefore the extremely high tolerance of planar-chromatography towards sample matrix effects was used, allowing for a shortened sample preparation. The idea of a rapid method was followed by the extraction of commercial coffee samples by means of accelerated solvent extraction (ASE). This allowed for higher throughput during sample preparation. To remove a part of the co extracted matrix, the whole ASE-extract was cleaned by SPE with activated carbon and evaporated to a defined volume. This represented a simplification of common multistage extraction methods and clean-up steps, that aim for complete removal of co extracted matrix prior injection into LC- or GC-systems. In accordance with determination of acrylamide in drinking water, the extract was derivatized in-situ with the fluorescence marker Dansulfinic acid and detected densitometrically after chromatographic separation. The concentration of acrylamide was quantified by means of parallel preparation of three standard additions. Systematic errors and the influence of the sample were corrected by the calibration within the matrix. The linearity of the calibration (between r = 0.9825 and 0.9995) were acceptable. Good values were reached for the limit of quantification (48 mug/kg) and repeatability (rsd 3 %). After method development the acrylamide concentration of commercial coffee samples was determined, showing results being consistent with literature findings. Thus the applicability of the newly developed method to complex food samples was demonstrated. In summary, the present work shows the applicability of planar-chromatography hyphenated with mass spectrometry for sensitive determination of acrylamide. It was possible to quantify the analyte at ultra-trace levels using less instrumental effort and time than usual. Quantification in complex sample matrices was feasible in spite of a simplified sample preparation. These applications prove the relevance of planar-chromatography to solve current analytical problems.

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