Rapid identification of bioactive carbohydrazide reaction products by an LC-DAD-SPE-NMR approach

On-line coupling of high performance liquid chromatography with diode array detection to solid phase extraction combined with nuclear magnetic resonance (LC-DAD-SPE-NMR) was used to monitor carbohydrazide condensation reaction progress. First, a chromatographic method was developed and optimised and individual peak separation was readily achieved by using an isocratic acetonitrile-phosphate buffer mobile phase. Subsequently, separated compounds were trapped on SPE cartridges and dried with nitrogen gas. Peak elution was then performed with deuterated acetonitrile and sent for NMR analysis. Single and multiple trapping options were applied. One- and two-dimensional NMR spectra were recorded using a Prodigy cryoprobe. It was demonstrated that LC-DAD-SPE-NMR setup was proved very useful for rapid and unambiguous identification of the reaction products and for determination of their structure. By using Prodigy cryoprobe in NMR measurements we were able to detect and identify compounds present at microgram level thus proving a high sensitivity of this methodology for monitoring reactions of bioactive molecules and drugs

HPLC Monitoring of Acid Catalyzed Conversion of 7-Ethyltryptophol to Methyl Ester of Etodolac

Small scale experimental model for the preparation of methyl ester of etodolac, the key intermediate in the synthesis of nonsteroidal drug etodolac, is thoroughly investigated in order to define the key parameters needed for its large scale production. Oxa-Pictet-Spengler reaction of 7-ethyltryptophol and methyl 3-oxopentanoate with inorganic acids as catalysts was monitored over time using HPLC method with UV detection. HPLC method for the simultaneous determination of 7-ethyltryptophol and the product was developed first. The conversion of 7-ethyltryptophol to etodolac precursor was performed using different molar equivalents of acid (1-5 with respect to the β-ketoester) and starting 7-ethyltryptophol of different degrees of purity. Kinetic profiles and optimal reaction times were in each case defined and key parameters selected

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

Rapid Structure Determination of Bioactive 4''-tetrahydrofurfuryl Macrozone Reaction Mixture Components by LC/SPE/Cryo NMR and MS

LC-SPE/cryo NMR and MS methodologies have been developed and employed for a rapid structure determination of 4″-tetrahydrofurfuryl macrozone reaction mixture components. Macrozones, novel conjugates of azithromycin, and thiosemicarbazones have shown very good in vitro antibacterial activities against susceptible and some resistant bacterial strains and are promising agents for further development. The post-column multiple trapping of the chromatographically separated reaction mixture components on the SPE cartridges increased the sensitivity and together with cryogenically cooled NMR probe made it possible to identify and structurally characterize main 4″- tetrahydrofurfuryl macrozone reaction mixture compounds including those present at very low concentration level. This approach has several advantages over a classical off-line procedure, efficiency and low solvent consumption being the two most important ones. All identified components were process-related. It has been demonstrated that two different kinds of compounds with respect to structure were identified, i.e., macrolide- related and thiosemicarbazone-related ones. This methodology can serve as a platform for reliable and effective macrolides reaction components structure profiling, serving as both isolation and identification tools

Rapid Structure Determination of Bioactive 4″-Tetrahydrofurfuryl Macrozone Reaction Mixture Components by LC-SPE/Cryo NMR and MS

LC-SPE/cryo NMR and MS methodologies have been developed and employed for a rapid structure determination of 4″-tetrahydrofurfuryl macrozone reaction mixture components. Macrozones, novel conjugates of azithromycin, and thiosemicarbazones have shown very good in vitro antibacterial activities against susceptible and some resistant bacterial strains and are promising agents for further development. The post-column multiple trapping of the chromatographically separated reaction mixture components on the SPE cartridges increased the sensitivity and together with cryogenically cooled NMR probe made it possible to identify and structurally characterize main 4″-tetrahydrofurfuryl macrozone reaction mixture compounds including those present at very low concentration level. This approach has several advantages over a classical off-line procedure, efficiency and low solvent consumption being the two most important ones. All identified components were process-related. It has been demonstrated that two different kinds of compounds with respect to structure were identified, i.e., macrolide-related and thiosemicarbazone-related ones. This methodology can serve as a platform for reliable and effective macrolides reaction components structure profiling, serving as both isolation and identification tools

Development of a LC-SPE/cryo NMR methodology for analysis of bioactive molecules and drugs

U ovom su radu istražene mogućnosti primjene metode LC-SPE/krio NMR za praćenje kemijskih reakcija i određivanje strukture sporednih produkata i/ili razgradnih produkata on-line koji nastaju u reakciji, s naglaskom na bioaktivne molekule i lijekove. Za razvoj metodologije odabrana su tri modelna sustava. Za svaki modelni sustav istraženi su uvjeti kromatografskog odjeljivanja komponenata reakcijskih smjesâ, zatim parametri za učinkovitu ekstrakciju odabranih analita na SPE-sorbense te utjecaj parametara ekstrakcije na osjetljivost krio-probe NMR. Za identifikaciju i strukturnu karakterizaciju izoliranih spojeva korištene su spektroskopija NMR i tehnika LC-MS. Prvi modelni sustav bili su karbohidrazidi i njihovi derivati koji imaju kelirajuća svojstva te antioksidativno i antiepileptičko djelovanje. Tijekom analize karbohidrazida istražena je osjetljivost sustava LC-SPE/krio NMR. Drugi modelni sustav bio je etodolak koji pripada skupini nesteroidnih protuupalnih lijekova. Analizom 7-etiltriptofola, polazne tvari u sintezi etodolaka, identificirano je i strukturno okarakterizirano 17 onečišćenja. Treći modelni sustav bili su makrozoni, novi amidni konjugati azitromicina i derivata tiosemikarbazida, koji imaju zadovoljavajuće antibakterijsko djelovanje na odabrane susceptibilne i rezistentne bakterijske sojeve. Analizirano je ukupno 6 reakcijskih smjesâ iz triju različitih klasa novosintetiziranih makrozona. Identificirana onečišćenja bila su sporedni produkti i razgradni produkti makrolidne i tiosemikarbazonske strukture, te dijastereomeri makrozona. Konfiguracije dijastereomerâ predložene su na temelju analize spektara NOESY i računskih simulacija molekulske dinamike. Provedeno istraživanje rezultiralo je razvojem pouzdane, brže i učinkovitije metode za analizu složenih smjesa spojeva koristeći pritom znatno manje količine uzorka i organskih otapala u usporedbi s postojećim metodama off-line. Metoda LC-SPE/krio NMR može imati širu primjenu u ekstrakciji, pročišćavanju i karakterizaciji novosintetiziranih spojeva i/ili onečišćenja iz reakcijskih smjesâ.In this doctoral thesis, the possibilities of applying the LC-SPE/cryo NMR method for monitoring chemical reactions and determining the structure of by-products and/or degradation products on-line, with an emphasis on bioactive molecules and drugs, were explored. Three different model systems were explored for methodology development. For each model system, the parameters for chromatographic separation of the reaction mixtures components, as well as parameters for the efficient extraction of the selected analytes on SPE-sorbents, were optimized. Hence, the influence of the SPE-parameters on the NMR cryoprobe sensitivity was also evaluated. NMR spectroscopy and LC-MS were used for identification and structural characterization of the extracted mixture components. The first model system were carbohydrazides and their derivatives, which are known as chelating reagents and also show antioxidative and antiepileptic effects. Sensitivity of LC-SPE/cryo NMR system was determined by analyzing carbohydrazides. Etodolac, a nonsteroidal anti-inflammatory drug, was chosen as the second model system. During the analysis of 7-ethyltryptophol, a starting material in the synthesis of etodolac, 17 impurities were identified and structurally characterized. The third model system were macrozones, new amide conjugates of azitromycin and thiosemicarbazide derivatives which exhibit good antibacterial activity against susceptible and resistant bacterial strains. Six reaction mixtures from three different classes of newly synthesized macrozones were analysed. Identified impurities were classified as macrolide and non-macrolide related by-products and degradation products, as well as macrozone diastereomers. Configurations of diastereomers were proposed on the basis of NOESY NMR spectroscopy data and molecular dynamic simulations. This research resulted in development of a reliable, faster and more efficient methodology for complex mixture analysis using significantly smaller amount of sample and organic solvents in comparison with standard off-line methods. LC-SPE/cryo NMR method can be applied for the extraction, purification and characterization of newly synthesized compounds and/or impurities from reaction mixtures

Development of a LC-SPE/cryo NMR methodology for analysis of bioactive molecules and drugs

U ovom su radu istražene mogućnosti primjene metode LC-SPE/krio NMR za praćenje kemijskih reakcija i određivanje strukture sporednih produkata i/ili razgradnih produkata on-line koji nastaju u reakciji, s naglaskom na bioaktivne molekule i lijekove. Za razvoj metodologije odabrana su tri modelna sustava. Za svaki modelni sustav istraženi su uvjeti kromatografskog odjeljivanja komponenata reakcijskih smjesâ, zatim parametri za učinkovitu ekstrakciju odabranih analita na SPE-sorbense te utjecaj parametara ekstrakcije na osjetljivost krio-probe NMR. Za identifikaciju i strukturnu karakterizaciju izoliranih spojeva korištene su spektroskopija NMR i tehnika LC-MS. Prvi modelni sustav bili su karbohidrazidi i njihovi derivati koji imaju kelirajuća svojstva te antioksidativno i antiepileptičko djelovanje. Tijekom analize karbohidrazida istražena je osjetljivost sustava LC-SPE/krio NMR. Drugi modelni sustav bio je etodolak koji pripada skupini nesteroidnih protuupalnih lijekova. Analizom 7-etiltriptofola, polazne tvari u sintezi etodolaka, identificirano je i strukturno okarakterizirano 17 onečišćenja. Treći modelni sustav bili su makrozoni, novi amidni konjugati azitromicina i derivata tiosemikarbazida, koji imaju zadovoljavajuće antibakterijsko djelovanje na odabrane susceptibilne i rezistentne bakterijske sojeve. Analizirano je ukupno 6 reakcijskih smjesâ iz triju različitih klasa novosintetiziranih makrozona. Identificirana onečišćenja bila su sporedni produkti i razgradni produkti makrolidne i tiosemikarbazonske strukture, te dijastereomeri makrozona. Konfiguracije dijastereomerâ predložene su na temelju analize spektara NOESY i računskih simulacija molekulske dinamike. Provedeno istraživanje rezultiralo je razvojem pouzdane, brže i učinkovitije metode za analizu složenih smjesa spojeva koristeći pritom znatno manje količine uzorka i organskih otapala u usporedbi s postojećim metodama off-line. Metoda LC-SPE/krio NMR može imati širu primjenu u ekstrakciji, pročišćavanju i karakterizaciji novosintetiziranih spojeva i/ili onečišćenja iz reakcijskih smjesâ.In this doctoral thesis, the possibilities of applying the LC-SPE/cryo NMR method for monitoring chemical reactions and determining the structure of by-products and/or degradation products on-line, with an emphasis on bioactive molecules and drugs, were explored. Three different model systems were explored for methodology development. For each model system, the parameters for chromatographic separation of the reaction mixtures components, as well as parameters for the efficient extraction of the selected analytes on SPE-sorbents, were optimized. Hence, the influence of the SPE-parameters on the NMR cryoprobe sensitivity was also evaluated. NMR spectroscopy and LC-MS were used for identification and structural characterization of the extracted mixture components. The first model system were carbohydrazides and their derivatives, which are known as chelating reagents and also show antioxidative and antiepileptic effects. Sensitivity of LC-SPE/cryo NMR system was determined by analyzing carbohydrazides. Etodolac, a nonsteroidal anti-inflammatory drug, was chosen as the second model system. During the analysis of 7-ethyltryptophol, a starting material in the synthesis of etodolac, 17 impurities were identified and structurally characterized. The third model system were macrozones, new amide conjugates of azitromycin and thiosemicarbazide derivatives which exhibit good antibacterial activity against susceptible and resistant bacterial strains. Six reaction mixtures from three different classes of newly synthesized macrozones were analysed. Identified impurities were classified as macrolide and non-macrolide related by-products and degradation products, as well as macrozone diastereomers. Configurations of diastereomers were proposed on the basis of NOESY NMR spectroscopy data and molecular dynamic simulations. This research resulted in development of a reliable, faster and more efficient methodology for complex mixture analysis using significantly smaller amount of sample and organic solvents in comparison with standard off-line methods. LC-SPE/cryo NMR method can be applied for the extraction, purification and characterization of newly synthesized compounds and/or impurities from reaction mixtures

Development of a LC-SPE/cryo NMR methodology for analysis of bioactive molecules and drugs

U ovom su radu istražene mogućnosti primjene metode LC-SPE/krio NMR za praćenje kemijskih reakcija i određivanje strukture sporednih produkata i/ili razgradnih produkata on-line koji nastaju u reakciji, s naglaskom na bioaktivne molekule i lijekove. Za razvoj metodologije odabrana su tri modelna sustava. Za svaki modelni sustav istraženi su uvjeti kromatografskog odjeljivanja komponenata reakcijskih smjesâ, zatim parametri za učinkovitu ekstrakciju odabranih analita na SPE-sorbense te utjecaj parametara ekstrakcije na osjetljivost krio-probe NMR. Za identifikaciju i strukturnu karakterizaciju izoliranih spojeva korištene su spektroskopija NMR i tehnika LC-MS. Prvi modelni sustav bili su karbohidrazidi i njihovi derivati koji imaju kelirajuća svojstva te antioksidativno i antiepileptičko djelovanje. Tijekom analize karbohidrazida istražena je osjetljivost sustava LC-SPE/krio NMR. Drugi modelni sustav bio je etodolak koji pripada skupini nesteroidnih protuupalnih lijekova. Analizom 7-etiltriptofola, polazne tvari u sintezi etodolaka, identificirano je i strukturno okarakterizirano 17 onečišćenja. Treći modelni sustav bili su makrozoni, novi amidni konjugati azitromicina i derivata tiosemikarbazida, koji imaju zadovoljavajuće antibakterijsko djelovanje na odabrane susceptibilne i rezistentne bakterijske sojeve. Analizirano je ukupno 6 reakcijskih smjesâ iz triju različitih klasa novosintetiziranih makrozona. Identificirana onečišćenja bila su sporedni produkti i razgradni produkti makrolidne i tiosemikarbazonske strukture, te dijastereomeri makrozona. Konfiguracije dijastereomerâ predložene su na temelju analize spektara NOESY i računskih simulacija molekulske dinamike. Provedeno istraživanje rezultiralo je razvojem pouzdane, brže i učinkovitije metode za analizu složenih smjesa spojeva koristeći pritom znatno manje količine uzorka i organskih otapala u usporedbi s postojećim metodama off-line. Metoda LC-SPE/krio NMR može imati širu primjenu u ekstrakciji, pročišćavanju i karakterizaciji novosintetiziranih spojeva i/ili onečišćenja iz reakcijskih smjesâ.In this doctoral thesis, the possibilities of applying the LC-SPE/cryo NMR method for monitoring chemical reactions and determining the structure of by-products and/or degradation products on-line, with an emphasis on bioactive molecules and drugs, were explored. Three different model systems were explored for methodology development. For each model system, the parameters for chromatographic separation of the reaction mixtures components, as well as parameters for the efficient extraction of the selected analytes on SPE-sorbents, were optimized. Hence, the influence of the SPE-parameters on the NMR cryoprobe sensitivity was also evaluated. NMR spectroscopy and LC-MS were used for identification and structural characterization of the extracted mixture components. The first model system were carbohydrazides and their derivatives, which are known as chelating reagents and also show antioxidative and antiepileptic effects. Sensitivity of LC-SPE/cryo NMR system was determined by analyzing carbohydrazides. Etodolac, a nonsteroidal anti-inflammatory drug, was chosen as the second model system. During the analysis of 7-ethyltryptophol, a starting material in the synthesis of etodolac, 17 impurities were identified and structurally characterized. The third model system were macrozones, new amide conjugates of azitromycin and thiosemicarbazide derivatives which exhibit good antibacterial activity against susceptible and resistant bacterial strains. Six reaction mixtures from three different classes of newly synthesized macrozones were analysed. Identified impurities were classified as macrolide and non-macrolide related by-products and degradation products, as well as macrozone diastereomers. Configurations of diastereomers were proposed on the basis of NOESY NMR spectroscopy data and molecular dynamic simulations. This research resulted in development of a reliable, faster and more efficient methodology for complex mixture analysis using significantly smaller amount of sample and organic solvents in comparison with standard off-line methods. LC-SPE/cryo NMR method can be applied for the extraction, purification and characterization of newly synthesized compounds and/or impurities from reaction mixtures

Maceration of Extra Virgin Olive Oil with Common Aromatic Plants Using Ultrasound-Assisted Extraction: An UV-Vis Spectroscopic Investigation

Rosemary (Rosmarinus officinalis), garden sage (Salvia officinalis), summer savory (Satureja hortensis), laurel (Laurus nobilis), and other aromatic plants were put in olive oil and exposed to ultrasounds for different duration. Filtrated oils were dissolved in cyclohexane, and UV-Vis measurements were carried out. Absorbance values corresponding to chlorophylls, carotenoids, flavonoids (370 nm), and polyphenols (around 300 nm) were measured. In addition, for some samples, total phenols were determined using Folin-Denis reagent and compared with the similar maceration procedure in water solvent (instead of olive oil). Maceration without ultrasound in olive oil for each plant was also compared with ultrasound-assisted extraction. The results show that significant amount of aromatic content can be extracted in olive oil by applying ultrasounds for only few minutes, especially for Salvia officinalis powder. The use of UV-Vis measurements is simple but enough to examine the extent of phenol content extraction through such maceration procedure

Screening of Novel Antimicrobial Diastereomers of Azithromycin–Thiosemicarbazone Conjugates: A Combined LC-SPE/Cryo NMR, MS/MS and Molecular Modeling Approach

A well-known class of antibacterials, 14- and 15-membered macrolides are widely prescribed to treat upper and lower respiratory tract infections. Azithromycin is a 15-membered macrolide antibiotic possessing a broad spectrum of antibacterial potency and favorable pharmacokinetics. Bacterial resistance to marketed antibiotics is growing rapidly and represents one of the major global hazards to human health. Today, there is a high need for discovery of new anti-infective agents to combat resistance. Recently discovered conjugates of azithromycin and thiosemicarbazones, the macrozones, represent one such class that exhibits promising activities against resistant pathogens. In this paper, we employed an approach which combined LC-SPE/cryo NMR, MS/MS and molecular modeling for rapid separation, identification and characterization of bioactive macrozones and their diastereomers. Multitrapping of the chromatographic peaks on SPE cartridges enabled sufficient sample quantities for structure elucidation and biological testing. Furthermore, two-dimensional NOESY NMR data and molecular dynamics simulations revealed stereogenic centers with inversion of chirality. Differences in biological activities among diastereomers were detected. These results should be considered in the process of designing new macrolide compounds with bioactivity. We have shown that this methodology can be used for a fast screening and identification of the macrolide reaction components, including stereoisomers, which can serve as a source of new antibacterials