Cannabis sativa L. is a plant belonging to the Cannabaceae family known primarily for its recreational use due to the psychoactive properties of Δ9-tetrahydrocannabinol (THC). Despite this, several compounds belonging to the category of phytocannabinoids have shown in recent years a number of potentially promising therapeutic effects that have increased the interest in the pharmaceutical field towards this plant. However, the content of these compounds is very variable and influenced by different factors, such as growing conditions and time of the year. An indication of the status and age of Cannabis samples is provided by the content of CBN, a minor phytocannabinoid and degradation product of other phytocannabinoids, including THC. In this research work an innovative, solid state analytical approach has been developed to observe and evaluate the variations in the content of two phytocannabinoids (CBN and CBD) in Cannabis-derived products over time. In order to simulate the ageing of the Cannabis samples, an artificially accelerated ageing procedure has been developed and optimised by using high temperatures. The analyses were carried out using an innovative ATR-FTIR method for solid state analysis, enabling direct analysis of a solid sample without any pretreatment phase. This study has allowed the development of an innovative analytical approach for the evaluation of the age and state of conservation of Cannabis samples and may be a useful tool both in the industrial, pharmaceutical and forensic fields.
- Klíčová slova
- ATR-FTIR, Accelerated ageing, Cannabis sativa L., Phytocannabinoids, Solid-state analysis,
- MeSH
- Cannabis * chemie MeSH
- časové faktory MeSH
- kanabidiol analýza chemie MeSH
- kanabinoidy * analýza chemie MeSH
- rostlinné extrakty chemie analýza MeSH
- spektroskopie infračervená s Fourierovou transformací metody MeSH
- stabilita léku MeSH
- tetrahydrokanabinol analýza chemie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- kanabidiol MeSH
- kanabinoidy * MeSH
- rostlinné extrakty MeSH
- tetrahydrokanabinol MeSH
Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths in the world. HCC is often diagnosed late because patients with early-stage cancer have no apparent symptoms. Therefore, it is desirable to find a reliable method for an early diagnosis based on the detection of metabolites - biomarkers, that can be detected in the early stages of the disease. Untargeted metabolomics is often used as a tool to find a suitable biomarker for several diseases. In this work, untargeted metabolomics was performed on blood plasma samples of HCC patients and compared with healthy individuals and patients with liver cirrhosis. A combination of liquid chromatography and high-resolution mass spectrometry was used as an analytical method. More than a thousand peaks were detected in the blood plasma samples, from which mainly amino acids, carboxylic acids, lipids, and their derivatives were evaluated as potential biomarkers. The data obtained were statistically processed using the analysis of variance, correlation analysis, and principal component analysis.
- Klíčová slova
- Biomarkers, Cirrhosis, Hepatocellular carcinoma, LC-MS, Metabolomics,
- MeSH
- analýza hlavních komponent MeSH
- chromatografie kapalinová metody MeSH
- dospělí MeSH
- hepatocelulární karcinom * krev MeSH
- hmotnostní spektrometrie metody MeSH
- jaterní cirhóza krev diagnóza MeSH
- lidé středního věku MeSH
- lidé MeSH
- metabolomika * metody MeSH
- nádorové biomarkery * krev MeSH
- nádory jater * krev MeSH
- senioři MeSH
- studie případů a kontrol MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- nádorové biomarkery * MeSH
A case study on Sitagliptin drug products and Sitagliptin/Metformin drug products concerning contamination with N-nitrosamines was performed using two newly developed analytical methods for determination of N-nitroso-triazolopyrazine (NTTP; 7-nitroso-3-(trifluoromethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine) and its precursor triazolopyrazine (3-(trifluoromethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine). The method for determination of triazolopyrazine was previously unpublished, the method for determination of NTTP was published only for analysis of active pharmaceutical ingredient Sitagliptin and not the drug forms. Solving the N-nitrosamine contamination is requested by regulatory authorities all over the world and thus is vital for all pharmaceutical companies. The solution always requires a sensitive analytical method. Both newly developed methods use liquid chromatography coupled with mass spectrometry (single quadrupole analyzer in case of triazolopyrazine and triple quadrupole analyzer in case of NTTP). Separation of triazolopyrazine was achieved on a column Acquity CSH C18 using a mobile phase consisting of aqueous ammonium formate buffered at pH 4.2 and acetonitrile. Detection was performed using positive electrospray and selected ion monitoring at m/z 193. Separation of NTTP was achieved on a column Acquity HSS T3 using a mobile phase consisting of 0.1 % formic acid in water and methanol. Detection was performed using positive electrospray and multiple reaction monitoring at transitions m/z 222.15→42.05 (collision energy 17 eV) and m/z 222.15→192.15 (collision energy 11 eV). Two issues specific to NTTP and triazolopyrazine previously not described in scientific literature were successfully troubleshooted. Spontaneous degradation of Sitagliptin to triazolopyrazine and methyl (R)-3-amino-4-(2,4,5-trifluorophenyl)butanoate was solved by using N,N-dimethylformamide as sample solvent during development of the method for quantitation of triazolopyrazine. A bad peak shape of NTTP due to the presence of rotamers of NTTP was successfully troubleshooted by increasing column temperature. Both methods were used during an optimization study of manufacturing of Sitagliptin and Sitagliptin/Metformin drug products. The goal of the study was to decrease NTTP content in the final drug product under the strict legislative limit set by Federal Drug Agency. The efficacy of several solutions was proven, but could not be fully disclosed due to Intellectual Property Protection policy of Zentiva. Instead, a brief review of recently published strategies to cope with N-nitrosamine contamination is presented.
- Klíčová slova
- NTTP, Nitrites, Nitrosation, Scavengers, Sitagliptin, Triazolopyrazine,
- MeSH
- léčivé přípravky MeSH
- metformin * analýza MeSH
- nitrosaminy * MeSH
- příprava léků MeSH
- pyraziny MeSH
- sitagliptin fosfát MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- léčivé přípravky MeSH
- metformin * MeSH
- nitrosaminy * MeSH
- pyraziny MeSH
- sitagliptin fosfát MeSH
Unusual glucose-substituted cardiolipins (Glcx-CLs) in three genera of thermophilic bacteria, having more than one glycosidically linked glucose to the hydroxyl of the central glycerol of Glcx-CLs were identified for the first time in thermophilic bacteria of the genera Geobacillus, Meiothermus, and Thermus. The number of glucoses reached up to five units. The structure of glycosidically linked oligosaccharides was determined based on shotgun analysis MS (electrospray high-resolution tandem mass spectrometry), partially methylated alditol acetates were identified by GC-MS, both electron ionization (EI) and positive chemical ionization (PCI), hydrophilic interaction liquid chromatography (HILIC) separation and identification of CLs glycosides by high resolution MS-ESI, and digestion by specific glycosidases.
- Klíčová slova
- Electrospray mass spectrometry, Glucosyl-cardiolipins, Hydrophilic interaction liquid chromatography, Partially methylated alditol acetates, Thermophilic bacteria,
- MeSH
- Bacteria MeSH
- chromatografie kapalinová metody MeSH
- glukosa MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací metody MeSH
- kardiolipiny * analýza MeSH
- plynová chromatografie s hmotnostně spektrometrickou detekcí MeSH
- tandemová hmotnostní spektrometrie * metody MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- glukosa MeSH
- kardiolipiny * MeSH
Radioimmunoconjugates represent a promising class of therapeutics and diagnostics. The characterization of intermediate chelator-antibody products, i.e., without the radionuclide, is frequently omitted, bringing significant uncertainty in the radioimmunoconjugate preparation. In the present study, we explored the utility of reversed-phase (RPLC) and hydrophilic interaction (HILIC) liquid chromatography with UV detection to characterize ramucirumab stochastically conjugated with p-SCN-Bn-CHX-A"-DTPA chelator (shortly DTPA). The conjugation was well reflected in RPLC chromatograms, while chromatograms from HILIC were significantly less informative. RPLC analyses at the intact level confirmed that the conjugation resulted in a heterogeneous mixture of modified ramucirumab. Moreover, the RPLC of DTPA-ramucirumab confirmed heterogeneous conjugation of all subunits. The peptide mapping did not reveal substantial changes after the conjugation, indicating that most parts of ramucirumab molecules remained unmodified and that the DTPA chelator was bound to various sites. Eventually, the RPLC method for analysis of intact ramucirumab was successfully applied to online monitoring of conjugation reaction in 1 h intervals for a total of 24 h synthesis, which readily reflected the structural changes of ramucirumab in the form of retention time shift by 0.21 min and increase in peak width by 0.22 min. The results were obtained in real-time, practically under 10 min per monitoring cycle. To the best of our knowledge, our study represents the first evaluation of RPLC and HILIC to assess the quality of intermediates during the on-site preparation of radioimmunoconjugates prior to radiolabeling.
- Klíčová slova
- Characterization, Chelators, DTPA, Hydrophilic interaction liquid chromatography, Radioimmunoconjugates, Reversed-phase liquid chromatography, p-SCN-Bn-CHX-A"-DTPA,
- MeSH
- chelátory MeSH
- chromatografie kapalinová metody MeSH
- chromatografie s reverzní fází * metody MeSH
- hydrofobní a hydrofilní interakce MeSH
- imunokonjugáty * MeSH
- kyselina pentetová MeSH
- ramucirumab MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- chelátory MeSH
- imunokonjugáty * MeSH
- kyselina pentetová MeSH
Monoglycerides (MGs) such as glycerol monolaurate (GML) and glycerol monostearate (GMS) have been used as excipients in oral formulations because of their emulsifying effect as well as their ability to inhibit the precipitation and intestinal efflux of drugs. Excipient-drug compatibility studies, however, have been underexplored. In this study, benznidazole (BNZ) was selected as a drug model due to the difficulty in improving its solubility and because of the potential impact on public health (it is the only drug currently used to treat Chagas disease). The effect of different processing conditions (maceration, ball milling, and melting) on the physical-chemistry properties of BNZ/MGs mixtures was investigated to guide the rational development of new solid formulations. GML was more effective in improving the solubility of BNZ, which could be due to its more malleable structure, less hydrophobic nature, and greater interaction with BNZ. The formation of hydrogen bonds between the imidazole group of BNZ and the polar region of GML was confirmed by spectroscopy analyses (IR, 1H NMR). The higher the monoglyceride content in the mixture, the higher the BNZ solubility. Regardless of the method of processing the mixture, the drug was found to be crystalline. Polarized light microscopy analysis showed the presence of spherulites. Overall, these findings suggest that preparation methods of BNZ:MGs formulations that involve thermal or/and mechanical treatment have a low impact on the solid properties of the material, and this allows for the production of formulations with reproducible performance.
- Klíčová slova
- Benznidazole, Compatibility study, Monoglycerides, Solubility,
- MeSH
- glyceridy MeSH
- monoglyceridy * MeSH
- nitroimidazoly * MeSH
- pomocné látky MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- benzonidazole MeSH Prohlížeč
- glyceridy MeSH
- monoglyceridy * MeSH
- nitroimidazoly * MeSH
- pomocné látky MeSH
Toxicity of β-blockers is one of the most common causes of poison-induced cardiogenic shock throughout the world. Therefore, methodologies for in vivo removal of the drugs from the body have been under investigation. Intralipid emulsion (ILE) is a common commercial lipid emulsion used for parenteral nutrition, but it has also been administered to patients suffering from drug toxicities. In this work, a set of β-blockers of different hydrophobicity's (log KD values ranging from 0.16 to 3.8) were investigated. The relative strength of the interactions between these compounds and the ILE was quantitatively assessed by means of binding constants and adsorption constants of the formed β-blocker-ILE complexes. The binding constants were determined by capillary electrokinetic chromatography and the adsorption constants were calculated based on different adsorption isotherms. Expectedly, the binding constants were strongly related to the log KD values of the β-blockers. The binding and adsorption constants also show that less hydrophobic β-blockers interact with ILE, suggesting that this emulsion could be useful for capturing such compounds in cases of their overdoses. Thus, the use of ILE for treatment of toxicities caused by a larger range of β-blockers is worth further investigation.
- Klíčová slova
- Adsorption isotherm, Capillary electrokinetic chromatography, Distribution constant, Drug toxicities, Intralipid emulsion, β-blockers,
- MeSH
- beta blokátory MeSH
- chromatografie MeSH
- fosfolipidy * MeSH
- lidé MeSH
- sójový olej MeSH
- tukové emulze intravenózní * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- beta blokátory MeSH
- fosfolipidy * MeSH
- sójový olej MeSH
- soybean oil, phospholipid emulsion MeSH Prohlížeč
- tukové emulze intravenózní * MeSH
Natalizumab is a humanized recombinant monoclonal IgG4 antibody used in the treatment of multiple sclerosis. Commonly used methods for natalizumab and anti-natalizumab antibodies quantification are enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay, respectively. Measurement of therapeutic monoclonal antibodies can be challenging due to the resemblance to human plasma immunoglobulins. Recent developments in mass spectrometry enables to analyze vast variety of large protein molecules. The aim of this study was to develop a LC-MS/MS method for determining natalizumab in human serum and cerebrospinal fluid (CSF) and apply it to clinical settings. For successful quantification, it was necessary to find specific sequences of peptides in natalizumab. This immunoglobulin was treated with dithiothreitol and iodoacetamide, cleaved with trypsin into short specific peptides and determined on a UPLC-MS/MS system. An Acquity UPLC BEH C18 column at 55 °C and gradient elution was used for analysis. Intra- and interassay accuracies and precisions were tested at four concentration levels. Precision was determined by coefficients of variation and was in the range of 0.8-10.2 %, with accuracy in the range of 89.8-106.4 %. The concentration of natalizumab in patient samples ranged from 1.8 to 193.3 μg/mL. The method was validated according to the European Medicines Agency (EMA) guideline, met all acceptance criteria for accuracy and precision, and is suitable for clinical applications. In comparison to immunoassay, which can be elevated by cross-reaction with endogenous immunoglobulins, the results of developed LC-MS/MS method are more accurate and specific.
- Klíčová slova
- Antibodies, Liquid chromatography, Mass spectrometry, Multiple sclerosis, Natalizumab, Serum,
- MeSH
- chromatografie kapalinová metody MeSH
- humanizované monoklonální protilátky terapeutické užití MeSH
- lidé MeSH
- natalizumab terapeutické užití MeSH
- peptidy terapeutické užití MeSH
- roztroušená skleróza * farmakoterapie MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- humanizované monoklonální protilátky MeSH
- natalizumab MeSH
- peptidy MeSH
A new approach for testing drug sensitivity to autooxidative degradation in the solid state is demonstrated in this work. A novel solid-state form of stressing agent for autooxidation has been proposed, based on azobisisobutyronitrile loaded into mesoporous silica carrier particles. The new solid-state form of the stressing agent was applied in degradation studies of two active pharmaceutical ingredients: bisoprolol and abiraterone acetate. The effectiveness and predictivity of the method were evaluated by comparing impurity profiles with those obtained by traditional stability testing of commercial tablets containing the investigated APIs. The results obtained by the new solid-state stressor were also compared with those obtained by an existing method for testing peroxide oxidative degradation in the solid state using a complex of polyvinylpyrrolidone with hydrogen peroxide. It was found that the new silica particle-based stressor was able to effectively predict which impurities could be formed by autooxidation in tablets and that this new approach is complementary to methods for testing peroxide oxidative degradation known from the literature.
- Klíčová slova
- Abiraterone acetate, Bisoprolol fumarate, Impurity profile, Mesoporous silica, Oxidative stress testing, Solid-state degradation,
- MeSH
- oxid křemičitý * MeSH
- oxidační stres MeSH
- peroxidy * MeSH
- tablety MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- azobis(isobutyronitrile) MeSH Prohlížeč
- oxid křemičitý * MeSH
- peroxidy * MeSH
- tablety MeSH
Ixazomib is the only orally active proteasome inhibitor used in clinical practice as an anticancer drug. The novel, rapid UHPLC-UV assay for ixazomib was developed and applied to the forced degradation study followed by HRMS identification of the main degradation products. Oxidative deboronation and hydrolysis of the amid bond were found to be the principal degradation pathways. The chemical standards of the main degradation products were prepared. The method was validated for the simultaneous assay of ixazomib and its degradation products within the concentration ranges of 2.50-100.00 µg/mL (ixazomib); 0.75-60.00 μg/mL (Impurity A and B) and 1.25-60.00 μg/mL (Impurity C). The stability study revealed that ixazomib in solution is: 1) relatively stable in neutral and acidic environments, 2) its decomposition is accelerated at higher pH, 3) it is sensitive to the effects of oxidants and light, and 4) the degradation of ixazomib follows the first-order kinetics under neutral, acidic, alkaline, and UV stress. Contrary, the solid substance of ixazomib citrate was relatively resistant to heat (70 °C), heat/humidity (70 °C/75 % RH), and UV irradiation for 24 h. This study presents the first MS-compatible UHPLC method for the quantification of ixazomib and its degradation products. Furthermore, it provides data about the inherent stability and kinetics of degradation of ixazomib in a solution that may be useful in further investigation of this drug, or the development of novel proteasome inhibitors based on the ixazomib structure.
- Klíčová slova
- Forced degradation, High resolution mass spectrometry, Impurity, Ixazomib, MLN2238, MLN9708,
- MeSH
- antitumorózní látky * MeSH
- glycin * MeSH
- hydrolýza MeSH
- inhibitory proteasomu MeSH
- oxidace-redukce MeSH
- sloučeniny boru MeSH
- stabilita léku MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- antitumorózní látky * MeSH
- glycin * MeSH
- inhibitory proteasomu MeSH
- ixazomib MeSH Prohlížeč
- sloučeniny boru MeSH
