{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,8]],"date-time":"2026-06-08T22:59:23Z","timestamp":1780959563452,"version":"3.54.1"},"reference-count":26,"publisher":"MDPI AG","issue":"13","license":[{"start":{"date-parts":[[2025,6,25]],"date-time":"2025-06-25T00:00:00Z","timestamp":1750809600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003196","name":"Italian Ministry of Health granted to IRCCS Mondino Foundation","doi-asserted-by":"publisher","award":["GR-2018-12366771"],"award-info":[{"award-number":["GR-2018-12366771"]}],"id":[{"id":"10.13039\/501100003196","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Heterozygous mutations in the GBA1 gene, encoding the enzyme glucocerebrosidase (GCase), are major risk factors for Parkinson\u2019s Disease (PD). Ambroxol, a small chaperone originally used as a mucolytic agent, has been shown to cross the blood\u2013brain barrier, enhance GCase activity, and reduce \u03b1-synuclein levels, making it a promising therapeutic candidate for disease-modifying effects in GBA1-associated PD (GBA1-PD). This study aimed to develop a method to quantify ambroxol levels in human plasma and cerebrospinal fluid (CSF) using liquid chromatography\u2013tandem mass spectrometry (LC-MS\/MS). Ambroxol was determined by online solid-phase extraction (SPE), coupled with LC-MS\/MS, by gradient elution on a monolithic column. Detection employed a 3200 QTRAP tandem mass spectrometer in the positive electrospray ionization mode. Calibration curves exhibited linearity across the analyzed ranges in both plasma and CSF. The recovery rate ranged from 106.7% to 113.5% in plasma and from 99.0% to 103.0% in CSF. No significant matrix effect was observed. Intra-day and inter-day precisions were below 11.8% in both matrices, and accuracy ranged from 89.9% to 103.1% in plasma and from 96.3% to 107.8% in CSF. We evaluated and confirmed the stability of the analyte in plasma and CSF across various storage conditions. The method was successfully validated according to European Medicine Agency (EMA) guidelines and its applicability was confirmed in the context of a multicenter, randomized, double-blind, placebo-controlled, phase II study, designed to monitor the ambroxol levels in the plasma and CSF of GBA1-PD.<\/jats:p>","DOI":"10.3390\/ijms26136094","type":"journal-article","created":{"date-parts":[[2025,6,26]],"date-time":"2025-06-26T05:53:13Z","timestamp":1750917193000},"page":"6094","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["LC-MS\/MS-Based Determination of Ambroxol in Human Plasma and Cerebrospinal Fluid: Validation and Applicability in a Phase II Study on GBA-Associated Parkinson\u2019s Disease Patients"],"prefix":"10.3390","volume":"26","author":[{"given":"Valentina","family":"Franco","sequence":"first","affiliation":[{"name":"Clinical and Experimental Pharmacology Unit, Department of Internal Medicine and Therapeutics, University of Pavia, 27100 Pavia, Italy"},{"name":"IRCCS Mondino Foundation, 27100 Pavia, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0005-7155-9447","authenticated-orcid":false,"given":"Michela","family":"Palmisani","sequence":"additional","affiliation":[{"name":"Clinical and Experimental Pharmacology Unit, Department of Internal Medicine and Therapeutics, University of Pavia, 27100 Pavia, Italy"},{"name":"IRCCS Mondino Foundation, 27100 Pavia, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1945-8153","authenticated-orcid":false,"given":"Fabiana","family":"Colucci","sequence":"additional","affiliation":[{"name":"Parkinson and Movement Disorders Unit, Department of Clinical Neurosciences, Fondazione IRCCS Istituto Neurologico Carlo Besta, 20133 Milano, Italy"},{"name":"Department of Neuroscience and Rehabilitation, University of Ferrara, 44121 Ferrara, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rosa","family":"De Micco","sequence":"additional","affiliation":[{"name":"Department of Advanced Medical and Surgical Sciences, University of Campania \u201cLuigi Vanvitelli\u201d, 80138 Naples, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-4675-6059","authenticated-orcid":false,"given":"Simone","family":"Aloisio","sequence":"additional","affiliation":[{"name":"Department of Advanced Medical and Surgical Sciences, University of Campania \u201cLuigi Vanvitelli\u201d, 80138 Naples, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Federico","family":"Cazzaniga","sequence":"additional","affiliation":[{"name":"Laboratory of Clinical Pathology, Unit of Laboratory Medicine, Fondazione IRCCS Istituto Neurologico Carlo Besta, 20133 Milano, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6466-5381","authenticated-orcid":false,"given":"Silvia","family":"Cerri","sequence":"additional","affiliation":[{"name":"IRCCS Mondino Foundation, 27100 Pavia, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Francesca","family":"Crema","sequence":"additional","affiliation":[{"name":"Clinical and Experimental Pharmacology Unit, Department of Internal Medicine and Therapeutics, University of Pavia, 27100 Pavia, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-3794-1363","authenticated-orcid":false,"given":"Francesca","family":"Dattrino","sequence":"additional","affiliation":[{"name":"Clinical and Experimental Pharmacology Unit, Department of Internal Medicine and Therapeutics, University of Pavia, 27100 Pavia, Italy"},{"name":"IRCCS Mondino Foundation, 27100 Pavia, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4323-9145","authenticated-orcid":false,"given":"Barbara","family":"Garavaglia","sequence":"additional","affiliation":[{"name":"Unit of Medical Genetics and Neurogenetics, Fondazione IRCCS Istituto Neurologico Carlo Besta, 20133 Milano, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Matteo","family":"Gastaldi","sequence":"additional","affiliation":[{"name":"IRCCS Mondino Foundation, 27100 Pavia, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3574-1290","authenticated-orcid":false,"given":"Pierfrancesco","family":"Mitrotti","sequence":"additional","affiliation":[{"name":"IRCCS Mondino Foundation, 27100 Pavia, Italy"},{"name":"Department of Brain and Behavioral Sciences, University of Pavia, 27100 Pavia, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2820-9880","authenticated-orcid":false,"given":"Fabio","family":"Moda","sequence":"additional","affiliation":[{"name":"Laboratory of Clinical Pathology, Unit of Laboratory Medicine, Fondazione IRCCS Istituto Neurologico Carlo Besta, 20133 Milano, Italy"},{"name":"Department of Medical Biotechnology and Translational Medicine, Universit\u00e0 degli Studi di Milano, 20126 Milan, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5795-4989","authenticated-orcid":false,"given":"Paola","family":"Rota","sequence":"additional","affiliation":[{"name":"Department of Biomedical, Surgical and Dental Sciences, University of Milan, 20133 Milan, Italy"},{"name":"Institute for Molecular and Translational Cardiology (IMTC), San Donato Milanese, 20097 Milan, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-5595-9066","authenticated-orcid":false,"given":"Rita","family":"Stiuso","sequence":"additional","affiliation":[{"name":"IRCCS Mondino Foundation, 27100 Pavia, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1513-2113","authenticated-orcid":false,"given":"Cristina","family":"Tassorelli","sequence":"additional","affiliation":[{"name":"IRCCS Mondino Foundation, 27100 Pavia, Italy"},{"name":"Department of Brain and Behavioral Sciences, University of Pavia, 27100 Pavia, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6035-9715","authenticated-orcid":false,"given":"Roberto","family":"Eleopra","sequence":"additional","affiliation":[{"name":"Parkinson and Movement Disorders Unit, Department of Clinical Neurosciences, Fondazione IRCCS Istituto Neurologico Carlo Besta, 20133 Milano, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alessandro","family":"Tessitore","sequence":"additional","affiliation":[{"name":"Department of Advanced Medical and Surgical Sciences, University of Campania \u201cLuigi Vanvitelli\u201d, 80138 Naples, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0600-6820","authenticated-orcid":false,"given":"Enza Maria","family":"Valente","sequence":"additional","affiliation":[{"name":"IRCCS Mondino Foundation, 27100 Pavia, Italy"},{"name":"Department of Molecular Medicine, University of Pavia, 27100 Pavia, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7880-9921","authenticated-orcid":false,"given":"Micol","family":"Avenali","sequence":"additional","affiliation":[{"name":"IRCCS Mondino Foundation, 27100 Pavia, Italy"},{"name":"Department of Brain and Behavioral Sciences, University of Pavia, 27100 Pavia, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Roberto","family":"Cilia","sequence":"additional","affiliation":[{"name":"Parkinson and Movement Disorders Unit, Department of Clinical Neurosciences, Fondazione IRCCS Istituto Neurologico Carlo Besta, 20133 Milano, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,6,25]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"567","DOI":"10.1002\/humu.20676","article-title":"Gaucher disease: Mutation and polymorphism spectrum in the glucocerebrosidase gene (GBA)","volume":"29","author":"Hruska","year":"2008","journal-title":"Hum. Mutat."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"44","DOI":"10.1007\/s11910-018-0860-4","article-title":"GBA-Associated Parkinson\u2019s Disease and Other Synucleinopathies","volume":"18","author":"Liong","year":"2018","journal-title":"Curr. Neurol. Neurosci. Rep."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"2648","DOI":"10.1093\/brain\/awv179","article-title":"Glucocerebrosidase activity in Parkinson\u2019s disease with and without GBA mutations","volume":"138","author":"Alcalay","year":"2015","journal-title":"Brain"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"9617","DOI":"10.1523\/JNEUROSCI.1525-17.2017","article-title":"Glucosylsphingosine Promotes alpha-Synuclein Pathology in Mutant GBA-Associated Parkinson\u2019s Disease","volume":"37","author":"Taguchi","year":"2017","journal-title":"J. Neurosci."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"e118","DOI":"10.1182\/blood-2011-05-352971","article-title":"Elevated plasma glucosylsphingosine in Gaucher disease: Relation to phenotype, storage cell markers, and therapeutic response","volume":"118","author":"Dekker","year":"2011","journal-title":"Blood"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1016\/j.nbd.2015.06.008","article-title":"Ambroxol-induced rescue of defective glucocerebrosidase is associated with increased LIMP-2 and saposin C levels in GBA1 mutant Parkinson\u2019s disease cells","volume":"82","author":"Ambrosi","year":"2015","journal-title":"Neurobiol. Dis."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"427","DOI":"10.1001\/jamaneurol.2019.4611","article-title":"Ambroxol for the Treatment of Patients with Parkinson Disease with and Without Glucocerebrosidase Gene Mutations: A Nonrandomized, Noncontrolled Trial","volume":"77","author":"Mullin","year":"2020","journal-title":"JAMA Neurol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1099","DOI":"10.2116\/analsci.33.1099","article-title":"Quantitative Detection of Ambroxol in Human Plasma Using HPLC-APCI-MS\/MS: Application to a Pharmacokinetic Study","volume":"33","author":"Mao","year":"2017","journal-title":"Anal. Sci."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1134","DOI":"10.1016\/j.jpba.2009.11.024","article-title":"Quantification of the major metabolites of bromhexine in human plasma using RRLC-MS\/MS and its application to pharmacokinetics","volume":"51","author":"Liu","year":"2010","journal-title":"J. Pharm. Biomed. Anal."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1108","DOI":"10.1002\/bmc.1032","article-title":"Rapid and sensitive liquid chromatography tandem mass spectrometry method for the quantification of ambroxol in human plasma","volume":"22","author":"Hu","year":"2008","journal-title":"Biomed. Chromatogr."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"597","DOI":"10.5414\/CP203212","article-title":"Pharmacokinetics and safety of salbutamol\/ambroxol fixed-dose combination granules in healthy Chinese subjects","volume":"56","author":"Wang","year":"2018","journal-title":"Int. J. Clin. Pharmacol. Ther."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"247","DOI":"10.5414\/CP203188","article-title":"Investigation of a potential drug-drug interaction between salbutamol and ambroxol and bioequivalence of a new fixed-dose combination containing these two drugs in healthy Chinese subjects","volume":"56","author":"Wang","year":"2018","journal-title":"Int. J. Clin. Pharmacol. Ther."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"399","DOI":"10.5414\/CP202527","article-title":"Bioequivalence assessment of ambroxol orally-disintegrating tablet after a single oral-dose administration to healthy volunteers","volume":"54","author":"Ni","year":"2016","journal-title":"Int. J. Clin. Pharmacol. Ther."},{"key":"ref_14","first-page":"18744","article-title":"Pharmacokinetics of ambroxol and clenbuterol tablets in healthy Chinese volunteers","volume":"8","author":"Yang","year":"2015","journal-title":"Int. J. Clin. Exp. Med."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1789","DOI":"10.1002\/bmc.3754","article-title":"Simultaneous determination of ambroxol and salbutamol in human plasma by ultra-performance liquid chromatography-tandem mass spectrometry and its application to a pharmacokinetic study","volume":"30","author":"Guo","year":"2016","journal-title":"Biomed. Chromatogr."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"829","DOI":"10.1016\/j.jpba.2008.05.032","article-title":"Simultaneous determination of amoxicillin and ambroxol in human plasma by LC-MS\/MS: Validation and application to pharmacokinetic study","volume":"48","author":"Wen","year":"2008","journal-title":"J. Pharm. Biomed. Anal."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"20","DOI":"10.1016\/j.cca.2007.03.015","article-title":"Simultaneous determination and pharmacokinetic study of roxithromycin and ambroxol hydrochloride in human plasma by LC-MS\/MS","volume":"382","author":"Hang","year":"2007","journal-title":"Clin. Chim. Acta"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"209","DOI":"10.1016\/S0731-7085(03)00059-1","article-title":"Determination of ambroxol in human plasma using LC-MS\/MS","volume":"32","author":"Kim","year":"2003","journal-title":"J. Pharm. Biomed. Anal."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"520","DOI":"10.1002\/bmc.2824","article-title":"A sensitive LC-MS\/MS method for the simultaneous determination of amoxicillin and ambroxol in human plasma with segmental monitoring","volume":"27","author":"Dong","year":"2013","journal-title":"Biomed. Chromatogr."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Zhou, H., Zhang, M., Sun, L., Li, W., and Liu, Q. (2021). A simple LC-MS\/MS method for simultaneous determination of cilostazol and ambroxol in Sprague-Dawley rat plasma and its application to drug-drug pharmacokinetic interaction study following oral delivery in rats. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci., 1179.","DOI":"10.1016\/j.jchromb.2021.122766"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"364","DOI":"10.1016\/j.jchromb.2007.03.015","article-title":"Determination of ambroxol in human plasma by high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-MS\/ESI)","volume":"853","author":"Su","year":"2007","journal-title":"J. Chromatogr. B Analyt. Technol. Biomed. Life Sci."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"e000535","DOI":"10.1136\/bmjno-2023-000535","article-title":"Ambroxol as a disease-modifying treatment to reduce the risk of cognitive impairment in GBA-associated Parkinson\u2019s disease: A multicentre, randomised, double-blind, placebo-controlled, phase II trial. The AMBITIOUS study protocol","volume":"5","author":"Colucci","year":"2023","journal-title":"BMJ Neurol. Open"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"EMA (2024, April 26). ICH Guideline M10 on Bioanalytical Method Validation and Study Sample Analysis. Available online: https:\/\/www.ema.europa.eu\/en\/documents\/scientific-guideline\/ich-guideline-m10-bioanalytical-method-validation-step-5_en.pdf.","DOI":"10.1208\/s12248-024-00974-y"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1591","DOI":"10.1002\/mds.26424","article-title":"MDS clinical diagnostic criteria for Parkinson\u2019s disease","volume":"30","author":"Postuma","year":"2015","journal-title":"Mov. Disord."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"248","DOI":"10.1002\/mds.24059","article-title":"Clinical validation of Movement Disorder Society-recommended diagnostic criteria for Parkinson\u2019s disease with dementia","volume":"27","author":"Barton","year":"2012","journal-title":"Mov. Disord."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1689","DOI":"10.1002\/mds.21507","article-title":"Clinical diagnostic criteria for dementia associated with Parkinson\u2019s disease","volume":"22","author":"Emre","year":"2007","journal-title":"Mov. Disord."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/13\/6094\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T17:58:20Z","timestamp":1760032700000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/13\/6094"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,6,25]]},"references-count":26,"journal-issue":{"issue":"13","published-online":{"date-parts":[[2025,7]]}},"alternative-id":["ijms26136094"],"URL":"https:\/\/doi.org\/10.3390\/ijms26136094","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,6,25]]}}}