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Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) has seen a growing interest as a leading technique in the pharmaceutical industry for mapping label-free exogenous and endogenous species in biological tissues. However, the use of MALDI-MSI to perform spatially resolved absolute quantitation of species directly in tissues is still challenging, and robust quantitative mass spectrometry imaging (QMSI) methods need to be developed. In this study, we describe the microspotting technique for analytical and internal standard deposition, matrix sublimation, powerful QMSI software, and mass spectrometry imaging setup to obtain absolute quantitation of drug distribution in 3D skin models., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

In skin penetration studies, HPLC-MS/MS analysis on extracts of heat-separated epidermis and dermis provides an estimate of the amount of drug penetrated. In this study, MALDI-MSI enabled qualitative skin distribution analysis of endogenous molecules and the drug molecule, tofacitinib and quantitative analysis of the amount of tofacitinib in the epidermis. The delivery of tofacitinib to the skin was investigated in a Franz diffusion cell using three different formulations (two oil-in-water creams, C1 and C2 and an aqueous gel). Further, in vitro release testing (IVRT) was performed and resulted in the fastest release of tofacitinib from the aqueous gel and the lowest from C2. In the ex vivo skin penetration and permeation study, C1 showed the largest skin retention of tofacitinib, whereas, lower retention and higher permeation were observed for the gel and C2. The quantitative MALDI-MSI analysis showed that the content of tofacitinib in the epidermis for the C1 treated samples was comparable to HPLC-MS/MS analysis, whereas, the samples treated with C2 and the aqueous gel were below LOQ. The study demonstrates that MALDI-MSI can be used for the quantitative determination of drug penetration in epidermis, as well as, to provide valuable information on qualitative skin distribution of tofacitinib., (Copyright © 2020 Elsevier B.V. All rights reserved.)

We report for the first time label-free quantification of xenobiotic metabolizing enzymes (XME), transporters, redox enzymes, proteases, and nucleases in six human skin explants and a three-dimensional living skin equivalent model from LabSkin. We aimed to evaluate the suitability of LabSkin as an alternative to animal testing for the development of topical formulations. More than 2000 proteins were identified and quantified from total cellular protein. Alcohol dehydrogenase 1C, the most abundant phase I XME in human skin, and glutathione S-transferase pi 1, the most abundant phase II XME in human skin, were present in similar abundance in LabSkin. Several esterases were quantified and esterase activity was confirmed in LabSkin using substrate-based mass spectrometry imaging. No cytochrome P450 (P450) activity was observed for the substrates tested, in agreement with the proteomics data, where the cognate P450s were absent in both human skin and LabSkin. Label-free protein quantification allowed insights into other related processes such as redox homeostasis and proteolysis. For example, the most abundant antioxidant enzymes were thioredoxin and peroxiredoxin-1. This systematic determination of functional equivalence between human skin and LabSkin is a key step toward the construction of a representative human in vitro skin model, which can be used as an alternative to current animal-based tests for chemical safety and for predicting dosage of topically administered drugs. SIGNIFICANCE STATEMENT: The use of label-free quantitative mass spectrometry to elucidate the abundance of xenobiotic metabolizing enzymes, transporters, redox enzymes, proteases, and nucleases in human skin enhance our understanding of the skin physiology and biotransformation of topical drugs and cosmetics. This will help to develop mathematical models to predict drug metabolism in human skin and to develop more robust in vitro engineered human skin tissue as alternatives to animal testing., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

The combination of microspotting of analytical and internal standards, matrix sublimation, and recently developed software for quantitative mass spectrometry imaging has been used to develop a high-resolution method for the determination of terbinafine hydrochloride in the epidermal region of a full thickness living skin equivalent model. A quantitative assessment of the effect of the addition of the penetration enhancer (dimethyl isosorbide (DMI)) to the delivery vehicle has also been performed, and data have been compared to those obtained from LC-MS/MS measurements of homogenates of isolated epidermal tissue. At 10% DMI, the levels of signal detected for the drug in the epidermis were 0.20 ± 0.072 mg/g tissue for QMSI and 0.28 ± 0.040 mg/g tissue for LC-MS/MS at 50% DMI 0.69 ± 0.23 mg/g tissue for QMSI and 0.66 ± 0.057 mg/g tissue for LC-MS/MS. Comparison of means and standard deviations indicates no significant difference between the values obtained by the two methods.

Objective: Examination of the skin barrier repair/wound healing process using a living skin equivalent (LSE) model and matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) to identify lipids directly involved as potential biomarkers. These biomarkers may be used to determine whether an in vivo wound is going to heal for example if infected., Methods: An in vitro LSE model was wounded with a scalpel blade and assessed at day 4 post-wounding by histology and MALDI-MSI. Samples were sectioned at wound site and were either formalin-fixed paraffin-embedded (FFPE) for histology or snapped frozen (FF) for MSI analysis., Results: The combination of using an in vitro wounded skin model with MSI allowed the identification of lipids involved in the skin barrier repair/wound healing process. The technique was able to highlight lipids directly in the wound site and distinguish differences in lipid distribution between the epidermis and wound site., Conclusion: This novel method of coupling an in vitro LSE with MSI allowed in-depth molecular analysis of the skin barrier repair/wound healing process. The technique allowed the identification of lipids directly involved in the skin barrier repair/wound healing process, indicating these biomarkers may be potentially be used within the clinic. These biomarkers will help to determine, which stage of the skin barrier repair/wound healing process the wound is in to provide the best treatment., (© 2018 Society of Cosmetic Scientists and the Société Française de Cosmétologie.)

Matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) is now a well-established technique for imaging analysis of sectioned biological tissues. One of the growing areas of interest is in the analysis of skin. MALDI-MSI can provide a wealth of information from within sections of skin. This includes information on the distribution of pharmaceuticals following topical treatments, through to the examination of the composition of different skin layers and studies of proteomic, lipidomic, and metabolomic responses to disease, wounds, and external stimuli. Here, we describe the handling procedures, preparatory treatment, and mass spectrometry setup required for the MALDI MSI analysis of lipids within human skin samples.

Mass spectrometry imaging (MSI) is a powerful tool for the study of intact tissue sections. The use of matrix-assisted laser desorption/ionisation (MALDI) MSI for the study of the distribution and effect of emollient treatment on sections of reconstructed living skin equivalents during their development and maturation is described. Living skin equivalent (LSE) samples were obtained at 14days development, re-suspended in maintenance medium and incubated for 24h after delivery. The medium was changed, the LSE treated with either Physiogel A.I.® or Oilatum Junior® emollients and then re-incubated and samples taken at 4, 6 and 24h time points. Mass spectra and mass spectral images were recorded from 12μm sections of the LSE taken at each time point for comparison using MALDI mass spectrometry (MS). It was possible to detect ions characteristic of each emollient in the LSE. In addition a number of lipid species previously reported as being significant in the maturation of the LSE were observable. At the 24h time point, the images revealed what appeared to be differences in the organisation of the skin cells observed across the Physiogel A.I.® treatment group tissue sections when directly compared to the untreated tissue group., (Copyright © 2016 Elsevier Inc. All rights reserved.)

MALDI-MS Imaging is a novel label-free technique that can be used to visualize the changes in multiple mass responses following treatment. Following treatment with proinflammatory cytokine interleukin-22 (IL-22), the epidermal differentiation of Labskin, a living skin equivalent (LSE), successfully modeled psoriasis in vitro. Masson's trichrome staining enabled visualization and quantification of epidermal differentiation between the untreated and IL-22 treated psoriatic LSEs. Matrix-assisted laser desorption ionization mass spectrometry imaging was used to observe the spatial location of the psoriatic therapy drug acetretin following 48 h treatments within both psoriatic and normal LSEs. After 24 h, the drug was primarily located in the epidermal regions of both the psoriatic and nonpsoriatic LSE models whereas after 48 h it was detectible in the dermis., (© 2016 The Authors. Proteomics Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Lipidomics is a rapidly expanding area of scientific research and there are a number of analytical techniques that are employed to facilitate investigations. One such technique is matrix-assisted laser desorption ionisation (MALDI) mass spectrometry (MS). Previous MALDI-MS studies involving lipidomic investigation have included the analysis of a number of different ex vivo tissues, most of which were obtained from animal models, with only a few being of human origin. In this study, we describe the use of MALDI-MS, MS/MS and MS imaging methods for analysing lipids within cross-sections of ex vivo human skin. It has been possible to tentatively identify lipid species via accurate mass measurement MALDI-MS and also to confirm the identity of a number of these species via MALDI-MS/MS, in experiments carried out directly on tissue. The main lipid species detected include glycerophospholipids and sphingolipids. MALDI images have been generated at a spatial resolution of 150 and 30 μm, using a MALDI quadrupole time-of-flight Q-Star Pulsar-i (TM) (Applied Biosystems/MDS Sciex, Concord, ON, Canada) and a MALDI high-definition MS (HDMS) SYNAPT G2-HDMS(TM) system (Waters, Manchester, UK), respectively. These images show the normal distribution of lipids within human skin, which will provide the basis for assessing alterations in lipid profiles linked to specific skin conditions e.g. sensitisation, in future investigations.

Matrix-assisted laser desorption/ionisation (MALDI) quadrupole time-of-flight mass spectrometry (Q-TOFMS) has been used to detect and image the distribution of a xenobiotic substance in skin. Porcine epidermal tissue was treated with 'Nizoral', a medicated shampoo containing ketoconazole (+/-)-1-acetyl-4-[p-[[(2R,4S)-2-(2,4-dichlorophenyl)-2-(imidazol-1-ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl]piperazine) as active ingredient. Following incubation for 1 h at 37 degrees C all excess formulation was washed from the surface. A cross-section of the drug-treated tissue was then blotted onto a cellulose membrane, precoated in matrix (alpha-cyano-4-hydroxycinnamic acid (CHCA)), by airspray deposition. In separate experiments the tissue surface was treated with Nizoral within a triangular former, and subsequently blotted onto a matrix-coated membrane. Sample membranes were then mounted into the recess of specialised MALDI targets with adhesive tape. All samples were analysed by MALDI-TOFMS using an Applied Biosystem 'Q-star Pulsar i' hybrid Q-TOF mass spectrometer fitted with an orthagonal MALDI ion source and imaging software. Detection of the protonated molecule was readily achievable by this technique. Treatment of the tissue within a template gave rise to images depicting the expected distribution of the drug, demonstrating that this technique is capable of producing spatially useful data. Ion images demonstrating the permeation of the applied compound into the skin were achieved by imaging a cross-sectional imprint of treated tissue. A calibration graph for the determination of ketoconazole was prepared using the sodium adduct of the matrix ion as an internal standard. This enabled construction of a quantitative profile of drug in skin. Conventional haematoxylin and eosin staining and microscopy methods were employed to obtain a histological image of the porcine epidermal tissue. Superimposing the mass spectrometric and histological images appeared to indicate drug permeation into the dermal tissue layer., (Copyright 2004 John Wiley & Sons, Ltd.)