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The use of MALDI-MSI for the analysis of bone tissue has been limited due to the insulating properties of the material but more importantly the absence of a proper sample preparation protocol for undecalcified bone tissue. Undecalcified sections are preferred to retain sample integrity as much as possible or to study the tissue-bone bio interface in particular. Here, we optimized the sample preparation protocol of undecalcified bone samples, aimed at both targeted and untargeted applications for forensic and preclinical applications, respectively. Different concentrations of gelatin and carboxymethyl cellulose (CMC) were tested as embedding materials. The composition of 20% gelatin and 7.5% CMC showed to support the tissue best while sectioning. Bone tissue has to be sectioned with a tungsten carbide knife in a longitudinal fashion, while the sections need to be supported with double-sided tapes to maintain the morphology of the tissue. The developed sectioning method was shown to be applicable on rat and mouse as well as human bone samples. Targeted (methadone and EDDP) as well as untargeted (unknown lipids) detection was demonstrated. DHB proved to be the most suitable matrix for the detection of methadone and EDDP in positive ion mode. The limit of detection (LOD) is estimated to approximately 50\u00a0pg\/spot on bone tissue. The protocol was successfully applied to detect the presence of methadone and EDDP in a dosed rat femur and a dosed human clavicle. The best matrices for the untargeted detection of unknown lipids in mouse hind legs in positive ion mode were CHCA and DHB based on the number of tissue-specific peaks and signal-to-noise ratios. The developed and optimized sample preparation method, applicable on animal and human bones, opens the door for future forensic and (pre)clinical investigations.<\/jats:p>","DOI":"10.1007\/s00216-020-02920-1","type":"journal-article","created":{"date-parts":[[2020,9,15]],"date-time":"2020-09-15T08:03:41Z","timestamp":1600157021000},"page":"2683-2694","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":46,"title":["Sample preparation of bone tissue for MALDI-MSI for forensic and (pre)clinical applications"],"prefix":"10.1007","volume":"413","author":[{"given":"Michiel","family":"Vandenbosch","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sylvia P.","family":"Nauta","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anastasiya","family":"Svirkova","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Martijn","family":"Poeze","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ron M.A.","family":"Heeren","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tiffany Porta","family":"Siegel","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eva","family":"Cuypers","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8060-7851","authenticated-orcid":false,"given":"Martina","family":"Marchetti-Deschmann","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2020,9,15]]},"reference":[{"issue":"6","key":"2920_CR1","doi-asserted-by":"publisher","first-page":"2283","DOI":"10.1007\/s00204-016-1905-6","volume":"91","author":"O Karlsson","year":"2017","unstructured":"Karlsson O, Hanrieder J. Imaging mass spectrometry in drug development and toxicology. Arch Toxicol. 2017;91(6):2283\u201394.","journal-title":"Arch Toxicol"},{"key":"2920_CR2","doi-asserted-by":"crossref","unstructured":"Chughtai S, Chughtai K, Cillero-Pastor B, Kiss A, Agrawal P, MacAleese L, et al. A multimodal mass spectrometry imaging approach for the study of musculoskeletal tissues. Int J Mass Spectrom. 2012;325\u2013327:150\u201360 Available from: http:\/\/www.sciencedirect.com\/science\/article\/pii\/S1387380612002308. Accessed 11 Sept 2020.","DOI":"10.1016\/j.ijms.2012.07.008"},{"issue":"15","key":"2920_CR3","doi-asserted-by":"publisher","first-page":"2690","DOI":"10.1039\/C7AN00565B","volume":"142","author":"PM Vaysse","year":"2017","unstructured":"Vaysse PM, Heeren RMA, Porta T, Balluff B. Mass spectrometry imaging for clinical research-latest developments, applications, and current limitations. Analyst. 2017;142(15):2690\u2013712.","journal-title":"Analyst."},{"issue":"10","key":"2920_CR4","doi-asserted-by":"publisher","first-page":"6051","DOI":"10.1021\/acs.analchem.7b05239","volume":"90","author":"JG Swales","year":"2018","unstructured":"Swales JG, Dexter A, Hamm G, Nilsson A, Strittmatter N, Michopoulos F, et al. Quantitation of endogenous metabolites in mouse tumors using mass-spectrometry imaging. Anal Chem. 2018;90(10):6051\u20138.","journal-title":"Anal Chem"},{"key":"2920_CR5","doi-asserted-by":"crossref","unstructured":"Cohen L, Go E, Siuzdak G. Small molecule desorption-ionization mass analysis. MALDI MS. A practical guide to instrumentation, methods and applications. 2007. p. 299\u2013337.","DOI":"10.1002\/9783527610464.ch9"},{"issue":"5","key":"2920_CR6","doi-asserted-by":"publisher","first-page":"1355","DOI":"10.1007\/s00216-013-7075-y","volume":"406","author":"H Hirano","year":"2014","unstructured":"Hirano H, Masaki N, Hayasaka T, Watanabe Y, Masumoto K, Nagata T, et al. Matrix-assisted laser desorption\/ionization imaging mass spectrometry revealed traces of dental problem associated with dental structure. Anal Bioanal Chem. 2014 Feb;406(5):1355\u201363.","journal-title":"Anal Bioanal Chem"},{"key":"2920_CR7","doi-asserted-by":"publisher","unstructured":"Schaepe K, Bhandari DR, Werner J, Henss A, Pirkl A, Kleine-Boymann M, et al. Imaging of lipids in native human bone sections using TOF\u2013secondary ion mass spectrometry, atmospheric pressure scanning microprobe matrix-assisted laser desorption\/ionization Orbitrap mass spectrometry, and Orbitrap\u2013secondary ion mass spectrometry. Anal Chem. 2018;90(15):8856\u201364. Available from: https:\/\/doi.org\/10.1021\/acs.analchem.8b00892.","DOI":"10.1021\/acs.analchem.8b00892"},{"key":"2920_CR8","doi-asserted-by":"crossref","unstructured":"Seeley EH, Wilson KJ, Yankeelov TE, Johnson RW, Gore JC, Caprioli RM, et al. Co-registration of multi-modality imaging allows for comprehensive analysis of tumor-induced bone disease. Bone. 2014\/01\/30. 2014;61:208\u201316. Available from: https:\/\/www.ncbi.nlm.nih.gov\/pubmed\/24487126. Accessed 11 Sept 2020.","DOI":"10.1016\/j.bone.2014.01.017"},{"key":"2920_CR9","doi-asserted-by":"publisher","unstructured":"Fujino Y, Minamizaki T, Yoshioka H, Okada M, Yoshiko Y. Imaging and mapping of mouse bone using MALDI-imaging mass spectrometry. Bone Rep. 2016;5:280\u20135. Available from: https:\/\/doi.org\/10.1016\/j.bonr.2016.09.004.","DOI":"10.1016\/j.bonr.2016.09.004"},{"key":"2920_CR10","doi-asserted-by":"publisher","unstructured":"Svirkova A, Turyanskaya A, Perneczky L, Streli C, Marchetti-Deschmann M. Multimodal imaging of undecalcified tissue sections by MALDI MS and \u03bcXRF. Analyst. 2018;143(11):2587\u201395. Available from: https:\/\/doi.org\/10.1039\/C8AN00313K.","DOI":"10.1039\/C8AN00313K"},{"issue":"46","key":"2920_CR11","doi-asserted-by":"publisher","first-page":"5929","DOI":"10.1039\/C9AY01886G","volume":"11","author":"DC Muddiman","year":"2019","unstructured":"Muddiman DC, Khodjaniyazova S, Hanne NJ, Cole JH. Mass spectrometry imaging (MSI) of fresh bones using infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI). 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Forensic Sci Int. 2020;309.","DOI":"10.1016\/j.forsciint.2020.110225"},{"key":"2920_CR26","doi-asserted-by":"publisher","first-page":"275","DOI":"10.1016\/j.foodchem.2018.12.126","volume":"283","author":"D Resetar","year":"2019","unstructured":"Resetar D, Svirkova A, Allmaier G, Marchetti-deschmann M, Kraljevi S. Optimization of MALDI-TOF mass spectrometry imaging for the visualization and comparison of peptide distributions in dry-cured ham muscle fibers. Food Chem. 2019;283:275\u201386.","journal-title":"Food Chem"},{"key":"2920_CR27","doi-asserted-by":"publisher","unstructured":"Goodwin RJA. Sample preparation for mass spectrometry imaging : small mistakes can lead to big consequences. J Proteome. 2012;75(16):4893\u2013911. Available from: https:\/\/doi.org\/10.1016\/j.jprot.2012.04.012.","DOI":"10.1016\/j.jprot.2012.04.012"},{"key":"2920_CR28","doi-asserted-by":"crossref","unstructured":"Boesl U. Time-of-flight mass spectrometry: introduction to the basics. 2017;86\u2013109.","DOI":"10.1002\/mas.21520"},{"issue":"12","key":"2920_CR29","doi-asserted-by":"publisher","first-page":"1156","DOI":"10.1038\/nmeth.4433","volume":"14","author":"M Kompauer","year":"2017","unstructured":"Kompauer M, Heiles S, Spengler B. Autofocusing MALDI mass spectrometry imaging of tissue sections and 3D chemical topography of nonflat surfaces. Nat Methods. 2017;14(12):1156\u20138.","journal-title":"Nat Methods"},{"key":"2920_CR30","doi-asserted-by":"publisher","first-page":"29","DOI":"10.1007\/s00216-011-4696-x","volume":"401","author":"J Fernandez","year":"2011","unstructured":"Fernandez J, Ochoa B, Fresnedo O, Giralt MT. Matrix-assisted laser desorption ionization imaging mass spectrometry in lipidomics. 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Available from: https:\/\/doi.org\/10.1016\/j.jprot.2012.07.006.","DOI":"10.1016\/j.jprot.2012.07.006"}],"container-title":["Analytical and Bioanalytical Chemistry"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1007\/s00216-020-02920-1.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/article\/10.1007\/s00216-020-02920-1\/fulltext.html","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1007\/s00216-020-02920-1.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,11,18]],"date-time":"2022-11-18T11:03:18Z","timestamp":1668769398000},"score":1,"resource":{"primary":{"URL":"https:\/\/link.springer.com\/10.1007\/s00216-020-02920-1"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,9,15]]},"references-count":31,"journal-issue":{"issue":"10","published-print":{"date-parts":[[2021,4]]}},"alternative-id":["2920"],"URL":"https:\/\/doi.org\/10.1007\/s00216-020-02920-1","relation":{},"ISSN":["1618-2642","1618-2650"],"issn-type":[{"value":"1618-2642","type":"print"},{"value":"1618-2650","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,9,15]]},"assertion":[{"value":"26 May 2020","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"23 July 2020","order":2,"name":"revised","label":"Revised","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"27 August 2020","order":3,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"15 September 2020","order":4,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"All applicable international, national, and institutional guidelines for the care and use of the animals were followed. All experiments were in accordance with ethical standards as approved by the Ethical Committee for Animal Experimentation of the University of Leuven (P 113\/2011). Approval for this study was received from the Medical Ethics Committee of the faculty of Medicine of the University Hospital of Leuven, Belgium, and the Animal Experiments Committee of Maastricht University (2014-030).","order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Compliance with ethical standards"}},{"value":"The authors declare that they have no conflict of interest.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Conflict of interest"}}]}}