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However, challenges associated with precise liquid confinement and sample targeting persist. In this paper, we present a laser micromachining-based approach to prepare DMSs on hydrophobic paper substrates that include visual recognition elements, or reticles, around surface energy traps (SETs). This targeted DMS substrate is combined with direct mass spectrometric analyses, namely liquid microjunction\u2013surface sampling probe\u2013mass spectrometry (LMJ-SSP-MS) and flowing atmospheric-pressure afterglow\u2013mass spectrometry (FAPA-MS). With the laser-based micromachining approach, DMSs flanked by crosshairs for enhanced visualization are prepared on SETs as small as 0.55 mm in diameter, which offers an approximately 12-fold reduction in size compared to traditional DMS preparations. The DMSs prepared on these targeting SETs are demonstrated with the detection of caffeine in model aqueous and artificial urine solutions using LMJ-SSP-MS and FAPA-MS, respectively. With further refinement, this approach could be automated using computer vision and robotics to broaden the scope of DMSs and improve the analytical workflow.<\/jats:p>","DOI":"10.3390\/mi17050559","type":"journal-article","created":{"date-parts":[[2026,4,30]],"date-time":"2026-04-30T14:35:53Z","timestamp":1777559753000},"page":"559","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Spot on: A Laser Micromachining-Based Approach to Improve Dried Matrix Spot Preparation with Proof-of-Principle Analytical Demonstrations Using Ambient Ionization Mass Spectrometry"],"prefix":"10.3390","volume":"17","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2496-4520","authenticated-orcid":false,"given":"Daniel O.","family":"Reddy","sequence":"first","affiliation":[{"name":"Department of Chemistry, Queen\u2019s University, Kingston, ON K7L 3N6, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9715-8087","authenticated-orcid":false,"given":"Malek","family":"Hassan","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Queen\u2019s University, Kingston, ON K7L 3N6, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jonathan O.","family":"Graham","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, NY 12180, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jared","family":"Viggers","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, NY 12180, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Katherine E.","family":"Williams","sequence":"additional","affiliation":[{"name":"School of Computing, Queen\u2019s University, Kingston, ON K7L 2N8, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Randy E.","family":"Ellis","sequence":"additional","affiliation":[{"name":"School of Computing, Queen\u2019s University, Kingston, ON K7L 2N8, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thomas R.","family":"Covey","sequence":"additional","affiliation":[{"name":"SCIEX, Concord, ON L4K 4V8, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8470-1338","authenticated-orcid":false,"given":"Jacob T.","family":"Shelley","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, NY 12180, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Richard D.","family":"Oleschuk","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Queen\u2019s University, Kingston, ON K7L 3N6, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,4,30]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"2769","DOI":"10.4155\/bio.11.270","article-title":"Dried Matrix Spot Direct Analysis: Evaluating the Robustness of a Direct Elution Technique for Use in Quantitative Bioanalysis","volume":"3","author":"Spooner","year":"2011","journal-title":"Bioanalysis"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"2375","DOI":"10.4155\/bio.15.166","article-title":"Potential Missing Steps for a Wide Use of Dried Matrix Spots in Biomedical Analysis","volume":"7","author":"Leuthold","year":"2015","journal-title":"Bioanalysis"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1441","DOI":"10.4155\/bio-2021-0135","article-title":"Dried Matrix Spots in Forensic Toxicology","volume":"13","author":"Fabris","year":"2021","journal-title":"Bioanalysis"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"2581","DOI":"10.4155\/bio.13.214","article-title":"Recent Advances in the Bioanalytical Applications of Dried Matrix Spotting for the Analysis of Drugs and Their Metabolites","volume":"5","author":"Zimmer","year":"2013","journal-title":"Bioanalysis"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"101","DOI":"10.1002\/xrs.710","article-title":"Nanodroplets: A New Method for Dried Spot Preparation and Analysis","volume":"33","author":"Miller","year":"2004","journal-title":"X-Ray Spectrom."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"5723","DOI":"10.1021\/acs.analchem.5b00893","article-title":"Enhanced Stability of Blood Matrices Using a Dried Sample Spot Assay To Measure Human Butyrylcholinesterase Activity and Nerve Agent Adducts","volume":"87","author":"Perez","year":"2015","journal-title":"Anal. 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