Oxysterols in cosmetics-Determination by planar solid phase extraction and gas chromatography-mass spectrometry.

Sterol oxidation products (SOPs) are linked to several toxicological effects. Therefore, investigation of potential dietary uptake sources particularly food of animal origin has been a key issue for these compounds. For the simultaneous determination of oxysterols from cholesterol, phytosterols, dihydrolanosterol and lanosterol in complex cosmetic matrices, planar solid phase extraction (pSPE) was applied as clean-up tool. SOPs were first separated from more non-polar and polar matrix constituents by normal phase thin-layer chromatography and then focussed into one target zone. Zone extraction was performed with the TLC-MS interface, followed by gas chromatography-mass spectrometry analysis. pSPE showed to be effective for cleaning up cosmetic samples as sample extracts were free of interferences, and gas chromatographic columns did not show any signs of overloading. Recoveries were between 86 and 113% with relative standard deviations of below 10% (n=6). Results of our market survey in 2016 showed that some cosmetics with ingredients of plant origin contained phytosterol oxidation products (POPs) in the low ppm range and therefore in line with levels reported for food. In lanolin containing products, total SOPs levels (cholesterol oxidation products (COPs), lanosterol oxidation products (LOPs), dihydrolanosterol oxidation products (DOPs)) being in the low percent range exceeded reported levels for food by several orders of magnitudes.

Determination of cholesterol oxides by gas chromatography-flame ionization detection/mass selective detection and their occurence in lanolin-containing cosmetics and ointments.

OBJECTIVE: Cholesterol oxides (COPs) are thought to be of toxicological relevance in cholesterol-containing foods. For cholesterol-containing cosmetics and the like, no information is available up to this date. Therefore, the first of two main aims of this study was to develop and validate a method for determining COPs in lanolin-containing cosmetics such as lipsticks and fatty creams as well as in nipple ointments. The second aim was to study the occurrence of COPs and their concentration levels in the respective product classes. METHODS: The procedure is based on a published method for food comprising some necessary modifications. Sample preparation consisted of transesterfication, solid-phase extraction and silylation of target compounds. Separation of the derivatized COPs and their quantification were performed with gas chromatography (GC) using a flame ionization detector (FID) or a mass spectrometer (MS). RESULTS: The successful validation and the trouble-free application during the market survey showed that the method was fit for purpose. Total COP levels found were in the low per cent range (up to 3%) and surprisingly high, being many orders of magnitude higher than those published for foods. CONCLUSION: To our knowledge, we present for the first time a method for the determination of COPs in non-food consumer products. Furthermore, our study demonstrates that lanolin-containing cosmetics may be an additional exogenous source of COPs. We further show evidence, that at least part of the COPs are already formed on the sheep's wool.

Photoprotection in changing times - UV filter efficacy and safety, sensitization processes and regulatory aspects.

As excessive sun exposure is tightly associated with different pathological changes of the skin, for example premature skin ageing or the development of skin cancer, an appropriate protection of the skin against UV radiation is of particular importance. Sun protection products and UV filter substances have evolved continuously in the past few decades. New developments and improved technical conditions of production have led to increasingly effective and efficient products with broadband protection ability. Accordingly, legal requirements have also changed and expanded. Although certain trends exist to harmonize the regulation of sunscreens at a global level, there are still large differences how UV absorbers are approved, which testing methods are prescribed, and which general requirements sun protection products must fulfil. Modern UV filters provide efficient protection against UVA and UVB radiation, are heat and photostable, user-friendly, cost-effective, water resistant and non-toxic. As inorganic and organic UV filters are topically applied to the skin in relatively high concentrations (up to 25%), especially the assessment of their (photo)sensitization potential is of particular importance. Accordingly, skin sensitization is a key endpoint for the legally required safety assessment of cosmetic ingredients in Europe and many other countries. This review will summarize the current regulatory status of different approved UV filters, will describe their beneficial and adverse properties and will give an overview of how the efficacy of sunscreens can be evaluated. Finally, an insight into the basic mechanism of (photo)allergic reactions and existing skin sensitization test methods will be provided.

Reactivity of cosmetic UV filters towards skin proteins: model studies with Boc-lysine, Boc-Gly-Phe-Gly-Lys-OH, BSA and gelatin.

OBJECTIVE: Organic UV filters are used as active ingredients in most sunscreens and also in a variety of daily care products. Their good (photo) stability is of special interest to guarantee protective function and to prevent interactions with the human skin. Due to the mostly electrophilic character of the UV filters, reactions with nucleophilic protein moieties like lysine side chains are conceivable. Prior studies showed that the UV filters octocrylene (OCR), butyl methoxydibenzoylmethane (BM-DBM), ethylhexyl salicylate (EHS), ethylhexyl methoxycinnamate (EHMC), benzophenone-3 (BP-3), ethylhexyl triazone (EHT) and dibenzoylmethane (DBM) were able to covalently bind to an HPTLC amino phase and the amino acid models ethanolamine and butylamine after slightly heating and/or radiation. METHODS: Boc-protected lysine, the tetrapeptide Boc-Gly-Phe-Gly-Lys-OH, bovine serum albumin (BSA) and porcine gelatin were used as more complex models to determine the reactivity of the mentioned UV filters towards skin proteins under thermal or UV irradiation conditions. RESULTS: After gentle heating at 37°C, benzophenone imines were identified as reaction products of BP-3 and OCR with Boc-lysine and the tetrapeptide, whereas DBM and BM-DBM yielded enamines. For EHMC, a Michael-type reaction occurred, which resulted in addition of Boc-lysine or the tetrapeptide to the conjugated double bond. Ester aminolysis of EHS and EHT mainly afforded the corresponding amides. Reactions of the UV filters with BSA changed the UV spectrum of BSA, generally associated with an increase of the absorption strength in the UVA or UVB range. For all protein models, the UV filters showed an increasing reactivity in the order EHT < EHMC < EHS < BP-3 < OCR < DBM < BM-DBM. CONCLUSION: Especially the UV absorbers BM-DBM, OCR and BP-3, which are seen as common allergens or photoallergens, showed a high reactivity towards the different skin protein models. As the formation of protein adducts is recognized as important key element in the induction of skin sensitization, the results of this study can contribute to a better understanding of the underlying chemical mechanisms of such reactions.

Rapid screening method to study the reactivity of UV filter substances towards skin proteins by high-performance thin-layer chromatography.

OBJECTIVE: Most UV filters used in sunscreens and other cosmetic products contain carbonyl groups, which generally are able to react with peptides or free amino acids of the human skin. To estimate their reactivity, we studied different prominent UV filter substances, octocrylene, ethylhexyl salicylate, 4-t-butyl-4'-methoxydibenzoylmethane, ethylhexyl methoxycinnamate, benzophenone-3, hydroxymethylbenzoyl sulphonic acid, octyldimethyl p-aminobenzoic acid, 3-benzylidene camphor, 4-methylbenzylidene camphor, diethylhexyl butamido triazone and ethylhexyl triazone. METHODS: A simple screening method using an amino HPTLC plate as protein model was established. The influence of different reaction conditions like heating and irradiation was determined. RESULTS: The ketones BP-3, HMBS and BM-DBM revealed the highest binding rates after both irradiation and heating. After 1 h of irradiation, 82%, 28% and 96%, respectively, were bonded to the amino phase, while heating resulted in values of 52%, 36% and 16%. For BP-3 and HMBS, even storage in the dark at room temperature resulted in a low binding. Contrarily, for the two camphor derivatives 3-BC and 4-MBC, only irradiation led to a slightly turnover. UV filters with ester groups also showed a different behaviour depending on their main skeleton. While OCR especially reacted under heating with the amino phase, resulting in 36% of bound species after one hour, UV irradiation particularly encouraged a reaction of the other esters. After 1 h irradiation, 15% of EHMC, 38% of EHS and 48% of OD-PABA were bonded to the amino groups of the HPTLC plate, whereas the reactivity of the two triazones, EHT and DEBT, was comparatively low. CONCLUSION: Especially the UV filters BP-3, BM-DBM, HMBS, EHMC or OCR, which are commonly known to cause contact dermatitis, showed a high tendency to form adducts with the amino layer. Thus, the amino plate seems to be a proper tool to screen for skin sensitizers.

Engineered anisotropic microstructures for ultrathin-layer chromatography.

The strong dependence of separation behavior on ultrathin-layer chromatography (UTLC) stationary phase microstructure motivates continued UTLC plate design optimization efforts. We fabricated 4.6-5.3 mum thick normal phase silica UTLC stationary phases with several types of in-plane macropore anisotropies using the glancing angle deposition (GLAD) approach to engineering nanostructured thin films. The separation behaviors of two new media, isotropic vertical posts and anisotropic bladelike films, were compared to that of anisotropic chevron media. Channel-like structures within the anisotropic media introduced preferential mobile phase flow directions that could be exploited to give separation tracks diagonal to the development direction. Extraction of chromatograms from these angled tracks required the development of a new analytical approach that involved a commercial flatbed film scanner and custom numerical image analysis software. GLAD stationary phase performance was quantified using the Dimethyl Yellow dye separated from a lipophilic dye mixture over migration distances less than approximately 10 mm. The limits of detection were 10 +/- 4 ng for the vertical posts and 11 +/- 3 ng for the bladelike media. We obtained theoretical plate heights that varied with film microstructure between 12 and 28 mum. Unoptimized separation performance was comparable to that of other planar chromatography media. Macropore anisotropies engineered by GLAD may expand the capabilities of future UTLC stationary phases.

Determination of cutin-bound residues of chlorothalonil by immunoassay.

An indirect competitive enzyme-linked immunosorbent assay (ELISA) was used to determine photochemically cutin-bound residues of chlorothalonil in enzymatically isolated tomato and apple fruit cuticles. The samples were spiked, irradiated, exhaustively extracted, and depolymerized with boron trifluoride complex resulting in a soluble depolymerisate. With this procedure, the ELISA could be calibrated with free target molecules for the quantification of originally bound chlorothalonil residues. In fruit cuticles that were irradiated for 8 h by simulated sunlight, 0.030 and 0.068 mg/g photoinduced cutin-bound residues of wax-free cuticles (calculated as chlorothalonil) were determined for tomatoes and apples, respectively. For the used antibody mAb chl. 4/11, cross-reactivities with derivatives of chlorothalonil simulating different types of cuticle-bound residues are given and discussed.

Photoinduced addition of the fungicide anilazine to cyclohexene and methyl oleate as model compounds of plant cuticle constituents.

Photoreactions, initiated by sunlight irradiation, between organochlorine pesticides and olefinic compounds of plant cuticles have been postulated. Concerning the formation of bound residues, which so far have not been detectable by common analytical techniques, photoaddition reactions are of main interest. In order to study the photochemical behavior of chlorinated fungicides, anilazine was irradiated in cyclohexene and methyl oleate as model compounds for olefinic plant cuticle constituents. Anilazine extensively reacted with the cis-double bond of both model compounds via radical mechanisms. In addition to a dechlorinated photoproduct several addition products were formed showing plausible reaction pathways for the formation of bound residues in plant cuticles. Photoproducts were isolated by preparative HPLC and analyzed by HPLC, MS, 1H-, and 13C-NMR.

Selective trace determination of dithiocarbamate fungicides in fruits and vegetables by reversed-phase ion-pair liquid chromatography with ultraviolet and electrochemical detection.

The predominant methods for determination of dithiocarbamate fungicides (DTC) have been based on quantitation of carbon disulfide released by hot acid digestion. Because the subgroups of the DTC family differ in their chemical properties and toxicological behavior, selective determination is required. To meet the demand for a fast, simple, and sensitive procedure, a new reversed-phase ion-pair chromatographic method was developed, consisting of surface extraction followed by direct injection into a liquid chromatographic system equipped with ultraviolet and electrochemical detectors, connected in series. The procedure is applicable to residues of N-methyldithiocarbamates (metam-sodium), N,N-dimethyldithiocarbamates (e.g., ziram and ferbam), ethylenebisdithiocarbamates (e.g., nabam, maneb, zineb, and mancozeb), and propylenebisdithiocarbamates (e.g., propineb) in fruits and vegetables. Limits of quantitation, calculated according to the procedure of the Deutsche Forschungsgemeinschaft, are 9, 12, 8, and 12 microg CS2/L for N-methyl-DTC, N,N-dimethyl-DTC, ethylenebis-DTC, and propylenebis-DTC, respectively, when electrochemical detection is used.

CS(2) blinds in Brassica crops: false positive results in the dithiocarbamate residue analysis by the acid digestion method.

Various members of the Brassicaceae family (cauliflower, savoy cabbage, red cabbage, turnip-rooted cabbage) grown without any application of pesticides were analyzed according to the acid digestion method commonly used for the determination of dithiocarbamate fungicide residues. Depending on postharvest treatments, high non-anthropogenic CS(2) values up to 4 mg/kg were found in some cases, especially in frozen raw cabbage samples, exceeding maximum residue limits. To explore phytogenic CS(2) occurrences, two model substances (phenylisothiocyanate and methyl tryptaminedithiocarbamate) representing natural mustard oils and brassinines, respectively, were analyzed for their acid hydrolysis decomposition products. In both cases, COS was found generally, but CS(2) was readily formed during acid digestion, too, when sulfides were present. The results obtained clearly demonstrate that CS(2) values determined by using the acid digestion method of crops rich in secondary metabolism sulfur compounds have to be interpreted carefully.

Analysis of carbendazim, benomyl, thiophanate methyl and 2,4-dichlorophenoxyacetic acid in fruits and vegetables after supercritical fluid extraction.

Simple methods for the analysis of carbendazim, benomyl and thiophanate methyl in fruits and vegetables and of 2,4-D in citrus fruits are presented. Sample preparation involves supercritical fluid extraction with carbon dioxide and further analysis is performed without any additional clean-up by GC-MS after derivatisation or directly by HPLC-diode array detection. The SFE methods presented are clearly faster and more cost effective than traditional solvent based approaches. The recoveries, detection limits and repeatabilities achieved, meet the needs of tolerance level monitoring of these compounds in fruits and vegetables.

[Dietary folates--a timely review. Stability, physiological significance, bioavailability, analytical determination methods, effect of food handling]. / Nahrungsfolate--Eine aktuelle Ubersicht. Stabilität, Physiologische Bedeutung, Bioverfügbarkeit, analytische Bestimmungsmethoden, Einfluss der Lebensmittelbehandlung.

Because of the unequal and, in some instances, low stability of different folate vitamers against extreme conditions the analytical determination of folate and the estimation of folate losses in food processing and preparation cause considerable difficulties. HPLC allows determination of the native folate derivative patterns. As the bioavailability of folates is influenced by a variety of factors and different methods were employed for assessing bioavailability there is a considerable inconsistency in the results of these studies. Folates labeled with radioactive or stable isotopes provide new approaches to metabolic and bioavailability studies.

Fungicides and photochemistry: photodegradation of the azole fungicide penconazole.

In order to examine the photostability of the fungicide penconazole (1-(2,4-dichloro-beta-propylphenethyl)-1H-1,2,4-triazole, 1) in the field, model experiments with organic solvents were performed. Photodegradation (lambda > 280 nm) of penconazole was found to be more efficient in isopropanol and cyclohexane solution than in the presence of cyclohexene. Photolysis in isopropanol and cyclohexane resulted in considerable formation of 1-(4-chloro-beta-propylphenethyl)-1H-1,2,4-triazole (2) and 5H,6H-(1,2,4-triazolo)-[5,1-a]-9-chloro-6-propyl-isoquinoline (3). Furthermore, photodehalogenation of 3 yielded traces of 5H,6H-(1,2,4-triazolo)-[5,1-a]-6-propyl-isoquinoline (4) and, in the presence of isopropanol 5H,6H-(1,2,4-triazolo)-[5,1-a]-9-(2-hydroxy-2-methylethyl)-6-propyl- isoquioline (5). Additionally, a lot of polar products were found in high yields which could not be isolated and characterized individually. In the presence of cyclohexene, on the other hand, photodecomposition and photodehalogenation to photoproduct 2 were found to be the main degradation pathways and photoproduct 3 was only detected as a trace component.

Analysis of dithiocarbamate fungicides. Second-derivative UV-spectroscopic determination of CS2, COS, and thiram (TMTD).

The application of second-derivative UV-spectroscopy offers a highly sensitive and selective method for the determination of CS2 and COS, as acid hydrolysis products of dithiocarbamate und thiuram disulphide fungicides, using a methanolic amine absorption reagent (ethylenediamine, piperidine). With standard concentrations of 0.08-1.1 microgram CS2/ml and 0.3 to 2.0 micrograms COS/ml, respectively, calibration curves with good correlation coefficients (r > 0.999) were obtained. In comparison to the official method of the Deutsche Forschungsgemeinschaft (DFG method S15) the proposed alternative is at least 100 times more sensitive to CS2. Using the second derivative method it is possible not only to clearly differentiate between CS2 and COS but also to quantify both gases without resorting to tedious background corrections as compared to the direct photometric methods. Additionally, second derivative spectroscopy allows the direct determination of thiram in the concentration range 1-10 micrograms/ml after its extraction with chloroform. For example, thiram in water (10 micrograms/l) and in thiram/talc standards (10 mg/g) were determined with good precision (+/- 2.0%).

Analysis of dithiocarbamate fungicides. Reaction products of the thiuram disulphide fungicide thiram (TMTD) during acid hydrolysis.

The acid hydrolysis products (CS2, COS, and H2S) of thiram (tetramethylthiram disulphide, TMTD) absorbed in a methanolic amine reagent (ethylenediamine, piperidine) were investigated by second derivative UV spectroscopy. When the hydrolysis temperatures are below the boiling point the formation of the side products COS and H2S at the expense of CS2 is unavoidable. Failure to carefully scrub the liberated gases with a lead acetate solution leads to the absorption of H2S also in the amine reagent and, like COS, causes an absorption at 230 nm that is erroneously attributed to COS. With the second-derivative technique it has been undoubtedly proved that, under the conditions of the Deutsche Forschungsgemeinschaft method S 15, thiram liberates CS2 and COS in a molar ratio of 1.92 to 0.06 (average of eight determinations) but not in a molar ratio of 1.5 to 0.5, as previously reported by some workers. Under these conditions it is impossible, contrary to earlier claims, to differentiate thiram from other dithiocarbamates based on the ratio of the hydrolysis products in residue analysis.

Photodegradation of the carbamate insecticide pirimicarb.

In order to study the photoreactivity of pirimicarb (2-(dimethylamino)-5,6-dimethyl-pyrimidin-4-yl-N-dimethylcarbamate ) on plant surfaces, model experiments with organic solvents were performed. Pirimicarb decomposed readily when irradiated (lambda > 280 nm) in the presence of the selected model solvents to form a number of products. Half-lives were in the order isopropanol < cyclohexane < cyclohexene (up to a maximum of 140 min). In the presence of all three solvents photooxidation predominated leading to N-formylpirimicarb and, in a second step, to N-desmethylpirimicarb as well as to further oxygenated products with the carbamate moiety intact. Photolysis in cyclohexene resulted in photomineralisation as the main degradation pathway. Irradiation of pirimicarb in the presence of isopropanol also yielded an addition product of the parent compound with the secondary alcohol.