{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,7]],"date-time":"2026-09-07T20:33:32Z","timestamp":1788813212833,"version":"build-2803163510"},"reference-count":31,"publisher":"MDPI AG","issue":"13","license":[{"start":{"date-parts":[[2025,7,1]],"date-time":"2025-07-01T00:00:00Z","timestamp":1751328000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Jichi Medical University Hospital"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Immunosuppressants are essential for preventing allograft rejection; however, they require therapeutic drug monitoring to maintain efficacy and to prevent severe complications such as opportunistic infections. Calcineurin inhibitors (CIs) are primarily distributed in red blood cells, whereas mycophenolic acid (MPA) and its metabolites are found in plasma. These differences necessitate separate analyses for each drug, increasing laboratory workload, analytical complexity, and patient burden. We developed a liquid chromatography\u2013tandem mass spectrometry method for simultaneous quantification of CIs such as tacrolimus (Tac), everolimus (Eve), sirolimus (Sir), cyclosporine A (CycA) and MPA in 2.8-\u00b5L whole-blood samples, with a hematocrit-based correction to estimate plasma-equivalent MPA concentrations. Performance of this method was assessed by comparison with conventional immunoassay results using linear regression and Bland\u2013Altman analyses, demonstrating excellent agreement, with strong linearity (R2 &gt; 0.995) at &lt;2 to 35 ng\/mL for three CIs, 26.0 to 1866 ng\/mL for CycA, and 0.1 to 50 \u03bcg\/mL for MPA. Furthermore, MPA and tacrolimus concentrations closely aligned with routine clinical results (R2 &gt; 0.900), indicating high accuracy and reproducibility. This new approach may be particularly beneficial for hospitalized patients with limited venous access, pediatric populations, and in remote care settings where frequent blood sampling is challenging because of simultaneous quantification and fewer sample volume requirements.<\/jats:p>","DOI":"10.3390\/ijms26136358","type":"journal-article","created":{"date-parts":[[2025,7,1]],"date-time":"2025-07-01T10:45:08Z","timestamp":1751366708000},"page":"6358","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Analytical Validation of an LC-MS\/MS Method for Simultaneous Quantification of Multiple Immunosuppressants in Microvolume Whole Blood"],"prefix":"10.3390","volume":"26","author":[{"given":"Kenichi","family":"Aizawa","sequence":"first","affiliation":[{"name":"Division of Translational Research, Clinical Research Center, Jichi Medical University Hospital, Shimotsuke 329-0498, Tochigi, Japan"},{"name":"Clinical Pharmacology Center, Jichi Medical University Hospital, Shimotsuke 329-0498, Tochigi, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Natsuka","family":"Kimura","sequence":"additional","affiliation":[{"name":"Division of Translational Research, Clinical Research Center, Jichi Medical University Hospital, Shimotsuke 329-0498, Tochigi, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4196-5389","authenticated-orcid":false,"given":"Takahiro","family":"Goda","sequence":"additional","affiliation":[{"name":"Solutions COE, Analytical & Measuring Instruments Division, Shimadzu Corporation, Kawasaki 210-0821, Kanagawa, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sho","family":"Nishida","sequence":"additional","affiliation":[{"name":"Division of Translational Research, Clinical Research Center, Jichi Medical University Hospital, Shimotsuke 329-0498, Tochigi, Japan"},{"name":"Division of Renal Surgery and Transplantation, Department of Urology, Jichi Medical University, Shimotsuke 329-0498, Tochigi, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0961-9622","authenticated-orcid":false,"given":"Yasunaru","family":"Sakuma","sequence":"additional","affiliation":[{"name":"Division of Gastroenterological, General and Transplant Surgery, Department of Surgery, Jichi Medical University, Shimotsuke 329-0498, Tochigi, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8274-848X","authenticated-orcid":false,"given":"Daiki","family":"Iwami","sequence":"additional","affiliation":[{"name":"Division of Renal Surgery and Transplantation, Department of Urology, Jichi Medical University, Shimotsuke 329-0498, Tochigi, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ryozo","family":"Nagai","sequence":"additional","affiliation":[{"name":"Jichi Medical University, Shimotsuke 329-0498, Tochigi, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,7,1]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"133","DOI":"10.1016\/S0003-2670(03)00410-0","article-title":"Liquid chromatography\/mass spectrometry for therapeutic drug monitoring of immunosuppressants","volume":"492","author":"Deters","year":"2003","journal-title":"Anal. 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