Claudius Rapp

Comparative investigation of human stratum corneum ceramides

The stratum corneum (SC) requires ceramides, cholesterol, and fatty acids to provide the cutaneous permeability barrier. SC lipids can be analyzed by normal-phase high-performance thin-layer chromatography (HPTLC). However, without further analysis, some uncertainty remains about the molecular composition of lipids represented by every TLC band of an unknown sample. We therefore analyzed each ceramide band further by subjecting the isolated lipids to a direct coupling of reversed-phase high-performance liquid chromatography and electrospray ionization-mass spectrometry (HPLC/ESI-MS, or IC/MS). IC/MS analysis and ESI-MS/MS negative ion and collision-induced dissociation experiments revealed that ceramide band 4 contained not only N-(ω-OH-acyl)acyl-6-OH-sphingosine, Cer(EOH), but also N-(α-OH-acyl)-sphingosine. Band 5 exclusively contained N-acyl-6-OH-sphingosine. Our results demonstrate the benefit of LC/MS analysis for selective identification of human SC ceramides. Moreover, the combination of HPTLC for pre-separation and LC/MS for identification of lipids is an even more powerful tool for detailed ceramide analysis.

Determination of penetration profiles of topically applied substances by means of tape stripping and optical spectroscopy: UV filter substance in sunscreens

Penetration profiles of topically applied drugs and cosmetic products provide important information on their efficacy. The application of tape stripping in combination with UV/VIS spectroscopy is checked to determine the local position of topically applied substances inside the stratum corneum, the penetration profile. The amount of corneocytes removed with each tape strip is quantified via the particle-dependent absorption, the pseudoabsorption, in the visible spectral range. The concentration of a typical UV filter substance, 4-methylbenzylidene camphor, is determined by optical spectroscopy using the tape strips removed originally. In this case, a time-dependent increase in the absorbance must be taken into account. Laser scanning microscopic investigations confirm that the nonhomogeneous distribution of the filter substance, on the strips, can explain this spectroscopic behavior. When reaching a homogeneous distribution, the UV spectroscopic signal reflects the correct concentration. These spectroscopic values are compared with high performance liquid chromatography (HPLC) data. The values obtained with both methods for the concentrations of 4-methylbenzylidene camphor are in good agreement. The data obtained are used to illustrate the determination of a penetration profile of a UV filter substance. The results demonstrate that the described protocol is well suited to characterize, in a simple manner, topically applied substances that have a characteristic UV/VIS absorption band.

MALDI imaging in human skin tissue sections: focus on various matrices and enzymes.

AbstractMatrix-assisted laser/desorption ionization (MALDI) mass-spectrometric imaging (MSI), also known as MALDI imaging, is a powerful technique for mapping biological molecules such as endogenous proteins and peptides in human skin tissue sections. A few groups have endeavored to apply MALDI-MSI to the field of skin research; however, a comprehensive article dealing with skin tissue sections and the application of various matrices and enzymes is not available. Our aim is to present a multiplex method, based on MALDI-MSI, to obtain the maximum information from skin tissue sections. Various matrices were applied to skin tissue sections: (1) 9-aminoacridine for imaging metabolites in negative ion mode; (2) sinapinic acid to obtain protein distributions; (3) α-cyano-4-hydroxycinnamic acid subsequent to on-tissue enzymatic digestion by trypsin, elastase, and pepsin, respectively, to localize the resulting peptides. Notably, substantial amounts of data were generated from the distributions retrieved for all matrices applied. Several primary metabolites, e.g. ATP, were localized and subsequently identified by on-tissue postsource decay measurements. Furthermore, maps of proteins and peptides derived from on-tissue digests were generated. Identification of peptides was achieved by elution with different solvents, mixing with α-cyano-4-hydroxycinnamic acid, and subsequent tandem mass spectrometry (MS/MS) measurements, thereby avoiding on-tissue MS/MS measurements. Highly abundant peptides were identified, allowing their use as internal calibrants in future MALDI-MSI analyses of human skin tissue sections. Elastin as an endogenous skin protein was identified only by use of elastase, showing the high potential of alternative enzymes. The results show the versatility of MALDI-MSI in the field of skin research. This article containing a methodological perspective depicts the basics for a comprehensive comparison of various skin states. FigureMatrix-assisted laser/desorption ionization (MALDI) mass-spectrometric imaging (MSI), also known as MALDI imaging, is a powerful technique for mapping biological molecules in human skin tissue sections. In this body of work, a multiplex method, based on MALDI-MSI, is presented to obtain maximum information from skin tissue sections. Therefore, various matrices were applied to skin tissue sections: (1) 9-aminoacridine (9-AA) for imaging small molecules in negative ion mode; (2) sinapinic acid (SA) to obtain protein distributions; (3) α-cyano-4-hydroxycinnamic acid (α-HCHA) subsequent to on-tissue enzymatic digestion by trypsin, elastase, and pepsin, respectively, to localize the resulting peptides. Of note, identification of metabolites was achieved by post-source decay (PSD) MALDI, and proteins were identified subsequent to enzymatic digestion via the resulting peptides which were eluted from the skin tissue section and afterwards analyzed with use of a tandem time-of-flight (ToF) mass spectrometer. The application of alternative enzymes, such as pepsin and elastase, is highlighted within this article

Qualitative Analysis of Tackifier Resins in Pressure Sensitive Adhesives Using Direct Analysis in Real Time Time-of-Flight Mass Spectrometry

Tackifier resins play an important role as additives in pressure sensitive adhesives (PSAs) to modulate their desired properties. With dependence on their origin and processing, tackifier resins can be multicomponent mixtures. Once they have been incorporated in a polymer matrix, conventional chemical analysis of tackifiers usually tends to be challenging because a suitable sample pretreatment and/or separation is necessary and all characteristic components have to be detected for an unequivocal identification of the resin additive. Nevertheless, a reliable analysis of tackifiers is essential for product quality and safety reasons. A promising approach for the examination of tackifier resins in PSAs is the novel direct analysis in real time mass spectrometry (DART-MS) technique, which enables screening analysis without time-consuming sample preparation. In the present work, four key classes of tackifier resins were studied (rosin, terpene phenolic, polyterpene, and hydrocarbon resins). Their corresponding complex mass spectra were interpreted and used as reference spectra for subsequent analyses. These data were used to analyze tackifier additives in synthetic rubber and acrylic adhesive matrixes. To prove the efficiency of the developed method, complete PSA products containing two or three different tackifiers were analyzed. The tackifier resins were successfully identified, while measurement time and interpretation took less than 10 mins per sample. Determination of resin additives in PSAs can be performed down to 0.1% (w/w, limit of detection) using the three most abundant signals for each tackifier. In summary, DART-MS is a rapid and efficient screening method for the analysis of various tackifiers in PSAs.

Determination of green-tea catechins in cosmetic formulations and in in-vitro skin extracts by high-performance liquid chromatography coupled with electrospray ionization mass spectrometry

SummaryA simple, rapid and reproducible method is presented for the analysis of green-tea extracts in different cosmetic formulations and in in-vitro skin extracts. Cosmetically active principal components were used for determination of complex assembled green-tea extracts. The catechins selected were catechin, epicatechin, epigallocatechin, epigallocatechin gallate, and epicatechin gallate, because of their efficacy and their concentrations in green-tea extracts. The determination was performed by high-performance liquid chromatography (HPLC), on a reversed-phase (RP) column, coupled to a single-quadrupole mass spectrometer (MS) via an electrospray ionization (ESI) interface. Detection was performed in the negative selected-ion-monitoring (SIM) mode. A detection limit between 5 and 15 ng g−1 was achieved in methanol-water-ascorbic acid extracts from different emulsions. A routine analytical procedure could be established with good quantitative reliability. During validation, the repeatabilities (relative standard deviation) for catechin standard solutions were found to be 1.1–2.7% (within one day) and 2.2–4.3% (day-to-day). Recoveries from spiked placebos were 98–105%. The method was successfully used to determine the storage stability of green-tea extract in cosmetic formulations and the in-vitro penetration of green-tea extract into the skin.

A novel sampling method for identification of endogenous skin surface compounds by use of DART-MS and MALDI-MS

Identification of endogenous skin surface compounds is an intriguing challenge in comparative skin investigations. Notably, this short communication is focused on the analysis of small molecules, e.g. natural moisturizing factor (NMF) components and lipids, using a novel sampling method with DIP-it samplers for non-invasive examination of the human skin surface. As a result, extraction of analytes directly from the skin surface by use of various solvents can be replaced with the mentioned procedure. Screening of measureable compounds is achieved by direct analysis in real time mass spectrometry (DART-MS) without further sample preparation. Results are supplemented by dissolving analytes from the DIP-it samplers by use of different solvents, and subsequent matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) measurements. An interesting comparison of the mentioned MS techniques for determination of skin surface compounds in the mass range of 50-1000 Da is presented.

Observation of a Penetration Depth Gradient in Attenuated Total Reflection Fourier Transform Infrared Spectroscopic Imaging Applications

Fourier transform infrared (FT-IR) spectroscopic imaging offers the possibility of combining spectral and spatial information, thereby enabling a spatial chemical visualization of samples. Thus, sample areas can be analyzed by infrared spectroscopy with high lateral resolution (up to 3–4 lm). The performance of FT-IR imaging equipment has been discussed by many authors, and numerous applications have been described. An important application area is the analysis of polymers, including diffusion and dissolution processes. Another field of research is the analysis of skin sections, biological tissues, and biomaterials. Pharmaceutical issues such as drug release or dissolution of tablets have also been analyzed by FT-IR imaging. Besides transmission measurements, attenuated total reflection (ATR) imaging is often applied. In this note we report on investigations of homogeneous samples by ATR imaging measurements with the FastIR accessory. In order to check the illumination uniformity of the ATR crystal, we chose a homogeneous liquid sample (paraffin oil) because of its perfect contact to the ATR crystal. However, despite this perfect contact, the integrated absorbance for a particular band is not constant for the imaged area. Instead, an absorbance gradient in the Z direction of the image (direction of the IR beam from the source to the detector) is revealed. These findings were also analyzed for two solid samples (adhesive tapes) and an emulsion. The influence of this absorbance gradient on ATR imaging results will be discussed.

Rapid quantification of iodopropynyl butylcarbamate as the preservative in cosmetic formulations using high-performance liquid chromatography-electrospray mass spectrometry

A simple, rapid and reproducible method for identification and quantification of iodopropynyl butylcarbamate (IPBC) in different cosmetic formulations is presented. The determination was carried out using a high-performance liquid chromatography (HPLC) procedure on a reversed phase column coupled to a single quadrupole mass spectrometer (MS) via an electrospray ionization (ESI) interface. Detection was performed in the positive selected ion-monitoring mode. In methanol/water extracts from different cosmetic formulations a detection limit between 50 and 100 ng/g could be achieved. A routine analytical procedure could be set up with good quantification reliability (relative standard deviation between 0.9 and 2.9%).

Influence of various on-tissue washing procedures on the entire protein quantity and the quality of matrix-assisted laser desorption/ionization spectra

RATIONALE For the matrix-assisted laser desorption/ionization (MALDI) imaging of proteins and tryptic peptides it is recommendable to remove salts, lipids, and phospholipids prior to analysis. However, thorough investigations of the influence of commonly used washing protocols on the entire protein content and the spectral quality have not been carried out. METHODS After the application of various on-tissue washing protocols, proteins and peptides were eluted by use of different solvents. Subsequently, protein quantities of the eluates were determined by a bicinchoninic acid assay. The spectral quality of the tryptic peptide eluates was investigated based upon peak picking. A MALDI time-of-flight (TOF) mass spectrometer was used to generate mass spectra. Skin tissue samples were prepared by embedding them either in carboxymethyl cellulose or in a cutting medium containing polyethylene glycol. RESULTS Our work shows the numerical decrease in protein content after applying different on-tissue washing protocols. Protein losses in a range of 17-38% were observed. From evaluating the spectral quality, two washing protocols were shown to be beneficial, enabling the detection of a high number of tryptic peptides. Procedures to thoroughly remove polyethylene glycol (deriving from special embedding media) were determined. Critically, aqueous washing steps conducted as short dips in two different jars were beneficial in achieving complete removal. CONCLUSIONS Washing steps have a strong impact on improving the spectral quality, but they may lead to a high decrease in the protein content. Our results show that there is a balancing act between avoiding protein loss and obtaining high spectra quality.

Quantitative determination of non-ionic surfactants by high-performance liquid-chromatography-ion trap mass-spectrometry

SummaryThis work presents a method for the qualitative and quantitative analysis of non-ionic surfactant of the fatty alcohol ethoxylate and nonylphenyl ethoxylate types in lanolin.The lanolin dissolved in a mixture of methanol and methyl tert-butyl ether was analyzed with no time-consuming sample preparation. Coupling high-performance liquid-chromatography with electrospray ion trap mass-spectrometry gives an extracted ion chromatogram of every individual substance in the surfactant used. Sensitivity factors were determined as a function of fatty alcohol chain length and the degree of ethoxylation. The relationships found allow calculation of calibration curves for each single substance in the surfactant. In this way the content and distribution of each substance can be determined regardless of the surfactant contained in the sample. In contrast to calibration using reference surfactants or principal substances, even surfactants and surfactant mixtures with an unknown single substance distribution can be quantified.With this method of analysis the surfactants used for wool washing could be identified and quantified on the basis of reference samples. In addition, changes in the surfactant composition during the production of lanolin and its processing to Eucerit® could be followed.