Identifikacija onečišćenja daptomicina primjenom dvodimenzionalne tekućinske kromatografije spregnute s masenim spektrometrom

professional thesis

• Author: Vedrana Bukal
• Mentor: Ana Mornar Turk (mentor)
• Committee member: Biljana Nigović (predsjednik povjerenstva)
• Committee member: Ana Mornar Turk (član povjerenstva)
• Committee member: Maja Lusina Kregar (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of pharmaceutical analysis); Zagreb
• Defense date and country: 2021-09-29, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Pharmacy

Abstract

Cilj istraživanja
U ovom radu dan je pregled tehnike dvodimenzionalna tekućinska kromatografija.
Opisana je primjena dvodimenzionalne tekućinske kromatografije u identifikaciji onečišćenja u ljekovitim oblicima. Cilj rada bio je razviti metodu primjenom navedene tehnike za identifikaciju onečišćenja daptomicina u ljekovitim oblicima, a ista može biti primijenjena i za identifikaciju onečišćenja u aktivnoj farmaceutskoj tvari.
Materijali/metode
Za identifikaciju onečišćenja daptomicina korišten je dvodimenzionalni tekućinski kromatograf visoke djelotvornosti s analizatorom masa Agilent 1290 Infinity II povezanim s detektorom masa Q-TOF MS, model 6545 (Agilent Technologies, Santa Clara, CA, SAD). Analitička metoda korištena u prvoj dimenziji modificirana je prema literaturno dostupnim podacima kako bi se postiglo bolje razdvajanje kromatografskih pikova daptomicina i prisutnih onečišćenja. U drugoj dimenziji dužina i vrsta kolone, protok i gradijentni program su mijenjani sa svrhom poboljšanja selektivnosti metode. Parametri masenog spektrometra su optimirani direktnim unošenjem radnog standarda daptomicina koncentracije 0,2 mg/mL u instrument.
Teorijska pozadina s naglaskom na aktivnu modulaciju i obrnutofaznu kromatografiju istražena je koristeći znanstvene izvore: PubMed, Science Direct.
Rezultati
2D-LC/MS metoda razvijena je za identifikaciju onečišćenja daptomicina: lakton daptomicina, β-izomer daptomicina i anhidro-daptomicina. Kako bi se postigla prikladna selektivnost metode razvoj je započeo modifikacijom poznate metode koja je korištena u prvoj dimenziji. Razdvajanje analita provedeno je primjenom obrnutofazne kromatografske kolone (C8). Kromatografskim postupkom u prvoj dimenziji postignuto je zadovoljavajuće razdvajanje ispitanih onečišćenja te je druga dimenzija primijenjena za provjeru čistoće pikova onečišćenja. Za optimizaciju kromatografskog postupka u drugoj dimenziji korištene su također obrnutofazne kromatografske kolone (C18). Za „povezivanje“ dviju dimenzija korišten je prvo engl. multi heart cutting način rada. Međutim, utvrđeno je kako su svi „rezovi“ iz prve dimenzije bili „čisti“ odnosno nije došlo do preklapanja kromatografskih pikova osim u uzorku XEL540/02-17/032.
Kako bi se utvrdila čistoća navedenog kromatografskog pika primijenjeno je uzorkovanje u visokoj rezoluciji (engl. High Resolution Sampling) odnosno engl. selective comprehensive 2D-LC način rada. Primjenom ovog načina rada potvrđeno je preklapanje pikova onečišćenja β-izomera daptomicina i drugih strukturno sličnih onečišćenja u uzorku XEL540/02-17/032. Masenom spektrometrijom potvrđen je identitet onečišćenja daptomicina: lakton daptomicina, β-izomer daptomicina i anhidro-daptomicin u tri ispitana ljekovita oblika.
Zaključak
Dvodimenzionalna tekućinska kromatografija je napredna instrumentalna tehnika kojom je moguće razdvojiti sastavnice složenih uzoraka, pogotovo kada je moguće u potpunosti primijeniti ortogonalnost u separacijskim koracima i spregnuti je s masenim detektorom kako bi dodatno identificirali analit ili dobili više informacija o strukturi same molekule.

Abstract (english)

Objectives
The objective of the research was to give the overview of two-dimensional liquid chromatography. This paper will present application of the two-dimensional liquid chromatography in identification of impurities in final dosage form containing daptomycin as active ingredient. The aim of the study was to develop a method using the above technique to identify daptomycin impurities in final dosage form, the same can be applied for the identification of impurities in active pharmaceutical ingredient.
Material and methods
For experimental work two-dimensional liquid chromatograph Agilent, 1290 Infinity II hyphenated with mass spectrometer Q-TOF MS, model 6545 was used.
Instrumental conditions for separation in first dimension was taken over from literature and optimized additionally with change of flow and gradient program introduction for better peak separation. In second dimension column length, flow and gradient program were investigated with the optimization purpose. Mass spectrometer instrumental parameters were optimized by injecting directly daptomycin working standard in concentration 0,2 mg/mL.
Theoretical background with accent on active modulation and reverse phase chromatography was studied using scientific sources: PubMed, Science Direct.
Results
2D-LC/MS method was developed for identification of daptomycin impurities: lactone daptomycin, β-isomer of daptomycin and anhydro-daptomycin. For development of first dimension chromatographic method previously published method was modified. Separation of selected analytes was performed using reversed phase column (C8). Satisfactory separation of all analytes was obtained using developed method. Therefore, the peak purity was evaluated using second dimensional chromatography. For optimization of chromatographic procedure reversed phase chromatography was (C18) used. Firstly, the multi heart cutting mode was used for connection of two dimensions. It was found that all cuts from first dimension were pure except those found in the sample XEL540/02-17/032.
Therefore, High Resolution Sampling or selective comprehensive 2D-LC mode was applied. Using this approach, co-elution of impurity daptomycin β-isomer and other daptomycin related impurities in the sample XEL540/02-17/032 was detected. Mass spectrometry was used for daptomycin impurity identification (lactone daptomycin, daptomycin β-isomer and anhydro-daptomycin) in investigated samples.
Conclusion
Two-dimensional liquid chromatography is a powerful technique for separation of components of interest, especially when it is possible to apply completely orthogonal approach in separation steps and hyphenated 2D-LC with mass spectrometer for further identification of analyte and for gathering the knowledge and more information about the molecule structure.

• Keywords: dvodimenzionalna tekućinska kromatografija; identifikacija onečišćenja; daptomicin
• Keywords (english): two-dimensional liquid chromatography; identification of impurities; daptomycin
• Language: croatian
• URN:NBN: urn:nbn:hr:163:081310
• Study programme: Title: Drug development; Study programme type: university; Study level: postgraduate specialist; Academic / professional title: sveučilišni magistar/sveučilišna magistra razvoja lijekova (sveučilišni magistar/sveučilišna magistra razvoja lijekova)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2022-03-23 15:29:58