{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,13]],"date-time":"2026-10-13T08:49:49Z","timestamp":1791881389876,"version":"4.3.4"},"reference-count":39,"publisher":"MDPI AG","issue":"19","license":[{"start":{"date-parts":[[2026,10,8]],"date-time":"2026-10-08T00:00:00Z","timestamp":1791417600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,10,8]],"date-time":"2026-10-08T00:00:00Z","timestamp":1791417600000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>The simultaneous separation of therapeutic insulin analogues by capillary electrophoresis (CE) remains challenging because of their high structural similarity and their tendency to adsorb onto the capillary surface. This study investigated the applicability of cyclodextrin (CD) derivatives as selectivity-modifying additives for the CE separation of human insulin and five therapeutic insulin analogues using five-layer poly(diallyldimethylammonium chloride)\/poly(sodium 4-styrenesulfonate) (PDADMAC\/PSS)2.5 and four-layer (PDADMAC\/PSS)2 successive multiple ionic-polymer layer (SMIL)-coated capillaries under acidic and alkaline conditions, respectively. A broad range of CD derivatives was systematically evaluated under optimized background electrolyte conditions. Under acidic conditions, neutral and predominantly uncharged CD derivatives improved separation selectivity, with carboxyethyl-\u03b2-CD providing the most favorable discrimination within the critical insulin lispro\u2013human insulin\u2013insulin aspart migration group. In contrast, highly charged anionic CD derivatives disrupted the PDADMAC-terminated coating and could not be applied. The PSS-terminated coating remained stable under alkaline conditions, enabling the evaluation of both neutral and anionic CD derivatives. Under these conditions, the migration order of the insulin species was completely reversed, while several CD derivatives further modulated the relative spacing of neighboring peaks. The use of organic modifiers produced system-dependent effects on separation selectivity and peak shape. These findings demonstrate that the appropriate selection of CD derivatives and SMIL coating strategy provides an effective approach for modulating selectivity in the CE separation of structurally related therapeutic peptides.<\/jats:p>","DOI":"10.3390\/molecules31193578","type":"journal-article","created":{"date-parts":[[2026,10,8]],"date-time":"2026-10-08T15:16:52Z","timestamp":1791472612000},"page":"3578","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Effect of Cyclodextrin Complexation on the Separation of Insulin Analogues in Capillary Electrophoresis Using Dynamically Coated Capillaries"],"prefix":"10.3390","volume":"31","author":[{"given":"Csenge","family":"Kis","sequence":"first","affiliation":[{"name":"Department of Physical Chemistry, \u201cGeorge Emil Palade\u201d University of Medicine, Pharmacy, Science, and Technology of Targu Mures, 540142 T\u00e2rgu Mure\u015f, Romania"},{"name":"Doctoral School of Medicine and Pharmacy, \u201cGeorge Emil Palade\u201d University of Medicine, Pharmacy, Science, and Technology of Targu Mures, 540142 T\u00e2rgu Mure\u015f, Romania"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"software","vocabulary":"credit"},{"role":"formal-analysis","vocabulary":"credit"},{"role":"writing-original-draft","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"visualization","vocabulary":"credit"}]},{"given":"Emese","family":"Sipos","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Industry and Biotechnology, \u201cGeorge Emil Palade\u201d University of Medicine, Pharmacy, Science, and Technology of Targu Mures, 540142 T\u00e2rgu Mure\u015f, Romania"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"conceptualization","vocabulary":"credit"},{"role":"resources","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"funding-acquisition","vocabulary":"credit"},{"role":"project-administration","vocabulary":"credit"},{"role":"supervision","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5341-319X","authenticated-orcid":false,"given":"Gerg\u0151","family":"T\u00f3th","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Chemistry, Semmelweis University, H\u0151gyes E. u. 9, 1092 Budapest, Hungary"},{"name":"Center for Pharmacology and Drug Research & Development, Semmelweis University, 1092 Budapest, Hungary"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"supervision","vocabulary":"credit"},{"role":"project-administration","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8740-0212","authenticated-orcid":false,"given":"Zolt\u00e1n-Istv\u00e1n","family":"Szab\u00f3","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Industry and Biotechnology, \u201cGeorge Emil Palade\u201d University of Medicine, Pharmacy, Science, and Technology of Targu Mures, 540142 T\u00e2rgu Mure\u015f, Romania"},{"name":"Sz-imfidum Ltd., 525401 Lunga, Romania"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,10,8]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"687","DOI":"10.1002\/bip.20734","article-title":"Insulin Structure and Function","volume":"88","author":"Mayer","year":"2007","journal-title":"Biopolym. Pept. Sci."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"799","DOI":"10.1038\/414799a","article-title":"Insulin Signalling and the Regulation of Glucose and Lipid Metabolism","volume":"414","author":"Saltiel","year":"2001","journal-title":"Nature"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"349","DOI":"10.1038\/s41574-020-0355-7","article-title":"Pancreatic \u03b2-Cells in Type 1 and Type 2 Diabetes Mellitus: Different Pathways to Failure","volume":"16","author":"Eizirik","year":"2020","journal-title":"Nat. Rev. Endocrinol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"S9","DOI":"10.1016\/j.amjmed.2008.03.022","article-title":"Pharmacokinetic and Pharmacodynamic Advantages of Insulin Analogues and Premixed Insulin Analogues Over Human Insulins: Impact on Efficacy and Safety","volume":"121","author":"Rolla","year":"2008","journal-title":"Am. J. Med."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"344","DOI":"10.1016\/j.jpba.2014.12.038","article-title":"Simultaneous Determination of Insulin and Its Analogues in Pharmaceutical Formulations by Micellar Electrokinetic Chromatography","volume":"111","author":"Lamalle","year":"2015","journal-title":"J. Pharm. Biomed. Anal."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"4139","DOI":"10.1039\/C9AN00112C","article-title":"Insulin: A Review of Analytical Methods","volume":"144","author":"Shen","year":"2019","journal-title":"Analyst"},{"key":"ref_7","unstructured":"European Directorate for the Quality of Medicines & HealthCare (2025, December 01). Insulin Lispro\u2014European Pharmacopoeia 11.8. Available online: https:\/\/pheur.edqm.eu\/app\/11-8\/content\/11-8\/2085E.htm."},{"key":"ref_8","unstructured":"European Directorate for the Quality of Medicines & HealthCare (2025, December 01). Insulin, Human\u2014European Pharmacopoeia 11.8. Available online: https:\/\/pheur.edqm.eu\/app\/11-8\/content\/11-8\/0838E.htm."},{"key":"ref_9","unstructured":"European Directorate for the Quality of Medicines & HealthCare (2025, December 01). Insulin Glargine\u2014European Pharmacopoeia 11.8. Available online: https:\/\/pheur.edqm.eu\/app\/11-8\/content\/11-8\/2571E.htm."},{"key":"ref_10","unstructured":"European Directorate for the Quality of Medicines & HealthCare (2025, December 01). Insulin Aspart\u2014European Pharmacopoeia 11.8. Available online: https:\/\/pheur.edqm.eu\/app\/11-8\/content\/11-8\/2084E.htm."},{"key":"ref_11","first-page":"00195","article-title":"Development of a New HPLC Method for the Determination of Lispro and Glargine Insulin Analogues in Pharmaceutical Preparations","volume":"7","year":"2018","journal-title":"J. Anal. Pharm. Res."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"122557","DOI":"10.1016\/j.jchromb.2021.122557","article-title":"Simultaneous Separation of Insulin and Six Therapeutic Analogues on a Mixed Mode Column: HPLC-UV Method Development and Application","volume":"1171","author":"Kristl","year":"2021","journal-title":"J. Chromatogr. B Anal. Technol. Biomed. Life Sci."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"71","DOI":"10.1016\/j.jpba.2018.12.034","article-title":"Development and Validation of a New UHPLC Method for Related Proteins in Insulin and Insulin Analogues as an Alternative to the European Pharmacopoeia RP-HPLC Method","volume":"166","author":"Wahl","year":"2019","journal-title":"J. Pharm. Biomed. Anal."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"219","DOI":"10.1002\/dta.35","article-title":"Sensitive and Fast Identification of Urinary Human, Synthetic and Animal Insulin by Means of Nano-UPLC Coupled with High-Resolution\/High-Accuracy Mass Spectrometry","volume":"1","author":"Thomas","year":"2009","journal-title":"Drug Test. Anal."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1125","DOI":"10.1002\/dta.1710","article-title":"Determination of Human Insulin and Its Analogues in Human Blood Using Liquid Chromatography Coupled to Ion Mobility Mass Spectrometry (LC-IM-MS)","volume":"6","author":"Thomas","year":"2014","journal-title":"Drug Test. Anal."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1761","DOI":"10.1002\/dta.2518","article-title":"Analysis of Insulin and Insulin Analogs from Dried Blood Spots by Means of Liquid Chromatography\u2013High Resolution Mass Spectrometry","volume":"10","author":"Thomas","year":"2018","journal-title":"Drug Test. Anal."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1560","DOI":"10.1002\/elps.200900547","article-title":"Investigations on the Migration Behavior of Insulin and Related Synthetic Analogues in CZE, MEKC and MEEKC Employing Different Surfactants","volume":"31","author":"Haunschmidt","year":"2010","journal-title":"Electrophoresis"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"167","DOI":"10.31083\/ph.2020.9188","article-title":"Application of the Capillary Zone Electrophoresis (CZE) and Capillary Gel Electrophoresis (CGE) for the Separation of Human Insulin, Insulin Lispro and Their Degradation Products","volume":"75","author":"Pajaziti","year":"2020","journal-title":"Pharmazie"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"145","DOI":"10.1016\/j.jpba.2010.02.012","article-title":"Simultaneous Determination of Regular Insulin and Insulin Aspart by Capillary Zone Electrophoresis and Application in Drug Formulations","volume":"53","author":"Yeh","year":"2010","journal-title":"J. Pharm. Biomed. Anal."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"461344","DOI":"10.1016\/j.chroma.2020.461344","article-title":"Determination of Deamidated Isoforms of Human Insulin Using Capillary Electrophoresis","volume":"1626","author":"Andrasi","year":"2020","journal-title":"J. Chromatogr. A"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1651","DOI":"10.1002\/elps.70040","article-title":"A Simple and Affordable CZE\u2013UV Method for Quality Control of Insulin in Active Pharmaceutical Ingredient and Formulations Using Quadruple Polymer-Coated Capillary","volume":"46","author":"Kidanemariam","year":"2025","journal-title":"Electrophoresis"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"464286","DOI":"10.1016\/j.chroma.2023.464286","article-title":"Comparative Study on the Deamidation of Three Recombinant Human Insulins Using Capillary Electrophoresis","volume":"1706","author":"Andrasi","year":"2023","journal-title":"J. Chromatogr. A"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"464802","DOI":"10.1016\/j.chroma.2024.464802","article-title":"Investigating Cationic and Zwitterionic Successive Multiple Ionic-Polymer Layer Coatings for Protein Separation by Capillary Electrophoresis","volume":"1720","author":"Dhellemmes","year":"2024","journal-title":"J. Chromatogr. A"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"e202200028","DOI":"10.1002\/cplu.202200028","article-title":"Polyelectrolyte multilayers in capillary electrophoresis","volume":"87","author":"Roca","year":"2022","journal-title":"ChemPlusChem"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"463351","DOI":"10.1016\/j.chroma.2022.463351","article-title":"Determination of Human Insulin and Its Six Therapeutic Analogues by Capillary Electrophoresis\u2013Mass Spectrometry","volume":"1678","author":"Hamidli","year":"2022","journal-title":"J. Chromatogr. A"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"2953","DOI":"10.1016\/j.chroma.2008.11.008","article-title":"Capillary Electrokinetic Chromatography of Insulin and Related Synthetic Analogues","volume":"1216","author":"Ortner","year":"2009","journal-title":"J. Chromatogr. A"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"371","DOI":"10.1007\/978-1-0716-5023-3_19","article-title":"Cyclodextrins as Chiral Selectors in Capillary Electromigration Techniques","volume":"Volume 2994","author":"Scriba","year":"2026","journal-title":"Methods in Molecular Biology"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"696","DOI":"10.1002\/elps.70116","article-title":"Cyclodextrins as Chiral Selectors in Capillary Electrophoresis\u2014Recent Developments in Understanding Analyte Complexation and Applications in Pharmaceutical Analysis","volume":"47","author":"Scriba","year":"2026","journal-title":"Electrophoresis"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"367","DOI":"10.1016\/S0021-9673(97)00994-1","article-title":"Cyclodextrin Aided Separation of Peptides and Proteins by Capillary Zone Electrophoresis","volume":"796","author":"Rathore","year":"1998","journal-title":"J. Chromatogr. A"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"2285","DOI":"10.1002\/elps.1150191306","article-title":"Cyclodextrins as Selectivity Enhancers in Capillary Zone Electrophoresis of Proteins","volume":"19","author":"Rathore","year":"1998","journal-title":"Electrophoresis"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"824","DOI":"10.1002\/elps.200390103","article-title":"Separation of Peptides and Proteins by Capillary Electrophoresis Using Acidic Buffers Containing Tetraalkylammonium Cations and Cyclodextrins","volume":"24","author":"Quang","year":"2003","journal-title":"Electrophoresis"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"4835","DOI":"10.1002\/jssc.201700610","article-title":"A Cationic \u03b2-Cyclodextrin as a Dynamic Coating for the Separation of Proteins in Capillary Electrophoresis","volume":"40","author":"Quirino","year":"2017","journal-title":"J. Sep. Sci."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"100011","DOI":"10.1016\/j.jcoa.2021.100011","article-title":"Cyclodextrins as Modulators for Separation of Charged Variants of mAbs by Capillary Zone Electrophoresis","volume":"1","author":"Kumar","year":"2021","journal-title":"J. Chromatogr. Open"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"101","DOI":"10.1016\/S0169-409X(98)00057-X","article-title":"Cyclodextrins in Peptide and Protein Delivery","volume":"36","author":"Irie","year":"1999","journal-title":"Adv. Drug Deliv. Rev."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"229","DOI":"10.1016\/j.ijpharm.2015.05.037","article-title":"Insulin Complexation with Hydroxypropyl-\u03b2-Cyclodextrin: Spectroscopic Evaluation of Molecular Inclusion and Use of the Complex in Gel for Healing of Pressure Ulcers","volume":"490","author":"Valentini","year":"2015","journal-title":"Int. J. Pharm."},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Bucur, P., F\u00fcl\u00f6p, I., and Sipos, E. (2022). Insulin Complexation with Cyclodextrins\u2014A Molecular Modeling Approach. Molecules, 27.","DOI":"10.3390\/molecules27020465"},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Hancu, G., Papp, L.A., T\u00f3th, G., and Kelemen, H. (2021). The Use of Dual Cyclodextrin Chiral Selector Systems in the Enantioseparation of Pharmaceuticals by Capillary Electrophoresis: An Overview. Molecules, 26.","DOI":"10.3390\/molecules26082261"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"2799","DOI":"10.1002\/elps.201900107","article-title":"Chiral Separation of Lansoprazole and Rabeprazole by Capillary Electrophoresis Using Dual Cyclodextrin Systems","volume":"40","author":"Papp","year":"2019","journal-title":"Electrophoresis"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1377","DOI":"10.1007\/s10337-015-2967-y","article-title":"Simultaneous Chiral Separation of Four H1-Antihistamines by Capillary Zone Electrophoresis Using a Dual Cyclodextrin System","volume":"78","author":"Hancu","year":"2015","journal-title":"Chromatographia"}],"container-title":["Molecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1420-3049\/31\/19\/3578\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,10,13]],"date-time":"2026-10-13T08:15:51Z","timestamp":1791879351000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1420-3049\/31\/19\/3578"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,10,8]]},"references-count":39,"journal-issue":{"issue":"19","published-online":{"date-parts":[[2026,10]]}},"alternative-id":["molecules31193578"],"URL":"https:\/\/doi.org\/10.3390\/molecules31193578","relation":{},"ISSN":["1420-3049"],"issn-type":[{"value":"1420-3049","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,10,8]]}}}