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The number and position of phosphoryl and acyl groups in lipid A molecules are key structural determinants in their bioactivities. In this study, a NACE\u2013ESI\u2010MS\/MS method was developed for the simultaneous analysis of lipid A isomers possessing a different degree of phosphorylation and acylation. Various C4\u2019\u2010 and C1\u2010monophosphorylated lipid A isobars, as well as acylation isomers, were baseline separated within 43\u00a0min in a separation medium of methanol\/dichloromethane\/triethylamine\/acetic acid 60:40:1.08:0.36 (v\/v\/v\/v). Both normal and reverse CE polarities could be applied for proper detection of the analytes owing to the combination of a suction effect caused by the nebulizer gas at the outlet end of the capillary and external pressure applied on the inlet vial. The separated lipid A species could be identified unequivocally by their characteristic fragmentation patterns through CID performed in both negative\u2010 and positive\u2010ionization modes. The uniqueness of the NACE\u2013ESI\u2010MS\/MS method lies in its simplicity and reliability for proving the phosphorylation isomerism (C1 or C4\u2019) and acylation pattern of native lipid A species or those designed for therapeutic applications.<\/jats:p>","DOI":"10.1002\/elps.201900251","type":"journal-article","created":{"date-parts":[[2020,4,26]],"date-time":"2020-04-26T08:39:26Z","timestamp":1587890366000},"page":"1178-1188","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":14,"title":["NACE\u2013ESI\u2010MS\/MS method for separation and characterization of phosphorylation and acylation isomers of lipid A"],"prefix":"10.1002","volume":"41","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8839-2648","authenticated-orcid":false,"given":"Viktor","family":"S\u00e1ndor","sequence":"first","affiliation":[{"name":"Institute of Bioanalysis Medical School and Szent\u00e1gothai Research Centre University of P\u00e9cs  P\u00e9cs Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Bal\u00e1zs Viktor","family":"Berkics","sequence":"additional","affiliation":[{"name":"Institute of Bioanalysis Medical School and Szent\u00e1gothai Research Centre University of P\u00e9cs  P\u00e9cs Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Anik\u00f3","family":"Kil\u00e1r","sequence":"additional","affiliation":[{"name":"Institute of Bioanalysis Medical School and Szent\u00e1gothai Research Centre University of P\u00e9cs  P\u00e9cs Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"B\u00e9la","family":"Kocsis","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, Medical School University of P\u00e9cs  P\u00e9cs Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5737-5514","authenticated-orcid":false,"given":"Ferenc","family":"Kil\u00e1r","sequence":"additional","affiliation":[{"name":"Institute of Bioanalysis Medical School and Szent\u00e1gothai Research Centre University of P\u00e9cs  P\u00e9cs Hungary"},{"name":"Department of Bioengineering Sapientia Hungarian University of Transylvania  Miercurea Ciuc Romania"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"\u00c1gnes","family":"D\u00f6rnyei","sequence":"additional","affiliation":[{"name":"Department of Analytical and Environmental Chemistry, Faculty of Science University of P\u00e9cs  P\u00e9cs Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2020,5,12]]},"reference":[{"key":"e_1_2_8_2_1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.biochem.71.110601.135414"},{"key":"e_1_2_8_3_1","doi-asserted-by":"publisher","DOI":"10.1038\/nature07830"},{"key":"e_1_2_8_4_1","doi-asserted-by":"publisher","DOI":"10.1128\/IAI.00939-07"},{"key":"e_1_2_8_5_1","doi-asserted-by":"publisher","DOI":"10.1002\/chem.201403923"},{"key":"e_1_2_8_6_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M109.004887"},{"key":"e_1_2_8_7_1","doi-asserted-by":"publisher","DOI":"10.4049\/jimmunol.1000953"},{"key":"e_1_2_8_8_1","doi-asserted-by":"publisher","DOI":"10.1177\/09680519060120040201"},{"key":"e_1_2_8_9_1","doi-asserted-by":"publisher","DOI":"10.1177\/09680519010070030101"},{"key":"e_1_2_8_10_1","doi-asserted-by":"publisher","DOI":"10.1111\/j.1462-5822.2009.01349.x"},{"key":"e_1_2_8_11_1","doi-asserted-by":"publisher","DOI":"10.1128\/IAI.00937-10"},{"key":"e_1_2_8_12_1","doi-asserted-by":"crossref","first-page":"3229","DOI":"10.4049\/jimmunol.142.9.3229","volume":"142","author":"Loppnow H.","year":"1989","journal-title":"J. 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