Rapid Identification of Unknown Impurities in 3-Bromo-5-(trifluoromethyl)aniline by LC-SPE/NMR

Identification of unknown pharmaceutical impurities is an essential part in the drug development process. In the present study, we developed and applied liquid chromatography (LC) – solid-phase extraction (SPE) / nuclear magnetic resonance (NMR) methodology with cryoprobe for identification and structural characterization of unknown impurities in 3-bromo-5-(trifluoromethyl)aniline. The three main impurities were separated and isolated by LC-SPE system. After multiple trapping, isolated impurities were eluted from the SPE cartridges with deuterated acetonitrile and one- and two-dimensional homo- and heteronuclear NMR spectra were recorded. The structures of the unknown impurities were determined by detailed inspection of NMR spectra and by mass spectrometric (MS) analysis. The results of the present preliminary study demonstrated that LC-SPE/NMR can be used for rapid impurity profiling of 3-bromo-5-(trifluoromethyl)aniline. This work is licensed under a Creative Commons Attribution 4.0 International License

Isolation and structural characterization of process impurities of 3-bromo-5-(trifluoromethyl)aniline using LC-SPE/NMR

U sklopu ove disertacije provedena je izolacija i strukturna karakterizacija nepoznatih procesnih onečišćenja 3-brom-5-(trifluormetil)anilina, polaznog materijala u sintezi djelatnih tvari. Sustav tekućinske kromatografije ultra visoke djelotvornosti (UHPLC) primijenjen je za prvotne analize istraživanih uzoraka te detekciju i određivanje količina pojedinih onečišćenja. Tehnika semi-preparativne tekućinske kromatografije korištena je kako bi se na stvarnom primjeru istražila primjenjivost tradicionalnog pristupa izolaciji i identifikaciji niskih razina onečišćenja, a izolirani spojevi su analizirani uz pomoć spektroskopije nuklearne magnetske rezonancije (NMR) i tehnike spektrometrije masa (MS). Veći dio istraživanja proveden je vezanim sustavom tekućinske kromatografije, ekstrakcije na čvrstoj fazi i spektroskopije NMR s krio-sondom (LC-SPE/NMR) koji je korišten za odjeljivanje, izolaciju, pohranu i ukoncentriravanje više od 15 analita na SPE nosačima te strukturnu karakterizaciju deset uspješno izoliranih onečišćenja 3-brom-5-(trifluormetil)anilina. Na temelju analize jednodimenzijskih 1H i 19F te dvodimenzijskih homo- i heteronuklearnih spektara NMR (COSY, 1H-13C HSQC i 1H-13C HMBC) te evaluacije rezultata MS predložene su strukture izoliranih onečišćenja.Within this thesis, an isolation and structural characterization of the process impurities of 3-bromo-5-(trifluoromethyl)aniline, a starting material in the synthesis of active pharmaceutical ingredients (APIs), was performed. Ultra-high performance liquid chromatography (UHPLC) was used for initial analysis of the investigated samples in order to detect and quantify individual impurities. Semi-preparative liquid chromatography was employed to evaluate the applicability of the traditional approach to isolation and identification of low level impurities, and the isolated compounds were analysed by nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). Most of the research was carried out by hyphenated system comprising liquid chromatography, solid phase extraction and NMR spectroscopy with cryoprobe (LC-SPE/NMR) used for separation, isolation, trapping and structural characterization. More than 15 analytes were separated and isolated, and structural characterization was performed on ten successfully isolated 3-bromo- 5-(trifluoromethyl)aniline impurities. Based on the analysis of one-dimensional 1H and 19F and two-dimensional homo- and heteronuclear NMR spectra (COSY, 1H-13C HSQC and 1H- 13C HMBC) and evaluation of MS results the structures of isolated impurities were proposed

Isolation and structural characterization of process impurities of 3-bromo-5-(trifluoromethyl)aniline using LC-SPE/NMR

U sklopu ove disertacije provedena je izolacija i strukturna karakterizacija nepoznatih procesnih onečišćenja 3-brom-5-(trifluormetil)anilina, polaznog materijala u sintezi djelatnih tvari. Sustav tekućinske kromatografije ultra visoke djelotvornosti (UHPLC) primijenjen je za prvotne analize istraživanih uzoraka te detekciju i određivanje količina pojedinih onečišćenja. Tehnika semi-preparativne tekućinske kromatografije korištena je kako bi se na stvarnom primjeru istražila primjenjivost tradicionalnog pristupa izolaciji i identifikaciji niskih razina onečišćenja, a izolirani spojevi su analizirani uz pomoć spektroskopije nuklearne magnetske rezonancije (NMR) i tehnike spektrometrije masa (MS). Veći dio istraživanja proveden je vezanim sustavom tekućinske kromatografije, ekstrakcije na čvrstoj fazi i spektroskopije NMR s krio-sondom (LC-SPE/NMR) koji je korišten za odjeljivanje, izolaciju, pohranu i ukoncentriravanje više od 15 analita na SPE nosačima te strukturnu karakterizaciju deset uspješno izoliranih onečišćenja 3-brom-5-(trifluormetil)anilina. Na temelju analize jednodimenzijskih 1H i 19F te dvodimenzijskih homo- i heteronuklearnih spektara NMR (COSY, 1H-13C HSQC i 1H-13C HMBC) te evaluacije rezultata MS predložene su strukture izoliranih onečišćenja.Within this thesis, an isolation and structural characterization of the process impurities of 3-bromo-5-(trifluoromethyl)aniline, a starting material in the synthesis of active pharmaceutical ingredients (APIs), was performed. Ultra-high performance liquid chromatography (UHPLC) was used for initial analysis of the investigated samples in order to detect and quantify individual impurities. Semi-preparative liquid chromatography was employed to evaluate the applicability of the traditional approach to isolation and identification of low level impurities, and the isolated compounds were analysed by nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). Most of the research was carried out by hyphenated system comprising liquid chromatography, solid phase extraction and NMR spectroscopy with cryoprobe (LC-SPE/NMR) used for separation, isolation, trapping and structural characterization. More than 15 analytes were separated and isolated, and structural characterization was performed on ten successfully isolated 3-bromo- 5-(trifluoromethyl)aniline impurities. Based on the analysis of one-dimensional 1H and 19F and two-dimensional homo- and heteronuclear NMR spectra (COSY, 1H-13C HSQC and 1H- 13C HMBC) and evaluation of MS results the structures of isolated impurities were proposed