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 The method for determination of triazolopyrazine was previously unpublished, the method for determination of NTTP was published only for analysis of active pharmaceutical ingredient Sitagliptin and not the drug forms.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.
An effective analytical method for the quantification of N-nitrosodimethylamine (NDMA) using a liquid chromatography coupled with tandem mass spectrometry was developed and applied to a process optimization study of the production of metformin film coated tablets in order to identify the key factors behind the NDMA formation in metformin products. The method uses a linear gradient elution with mobile phases 0.1 % formic acid in water for chromatography and methanol for chromatography and a column Acquity UPLC HSS T3 1.8 μm. The use of the tandem mass spectrometry in a positive ion mode with an atmospheric pressure chemical ionization allows for the use of an isotopically labelled internal standard and an external calibration standard. The method was validated according to the guidelines of International Council for Harmonization in terms of limit of detection and quantification, linearity, precision, accuracy and method selectivity. To further justify the effectiveness of the method, a comparison between two laboratories was performed using a linear regression testing. Both methods give comparable results. 469 samples of both metformin active pharmaceutical ingredient and film coated tablets were analysed and the key factors behind NDMA formation were identified. Hypotheses explaining the mechanism were formulated and confronted with measurements and scientific literature. Protective measures to prevent NDMA contamination in metformin products were drawn.
4'-(azidomethyl)-[1,1'-biphenyl]-2-carbonitrile (GTI-azide-1) and 5-(4'-(azidomethyl)-[1,1'-biphenyl]-2-yl)-1H-tetrazole (GTI-azide-2) are potentially genotoxic impurities that can be present at trace levels in the active pharmaceutical ingredients and drug products of sartans containing a tetrazole group. A method of high-performance liquid chromatography coupled with mass spectrometry, that allows the determination of those genotoxic impurities at sub-ppm level relative to the active pharmaceutical ingredient, was developed. The method utilises a very efficient liquid chromatograph Waters Acquity I-Class coupled with a highly sensitive tandem mass spectrometer Xevo TQ-XS. The separation was achieved on a column Acquity UPLC BEH Shield RP18 1.7 μm employing a linear elution gradient. The mass spectrometer was used with a heated electrospray ionization. The method was found to be sufficient in terms of sensitivity, linearity, precision, accuracy, selectivity and robustness and is easily applicable in the pharmaceutical quality control environment. The method allows for accurate quantification of both impurities GTI-azide-1 and GTI-azide-2 at levels below 1/10th of the specification limit, which is crucial in the context of pharmaceutical analysis. The limit of quantification was determined to be 0.033 ppm and 0.025 ppm for GTI-azide-1 and GTI-azide-2, respectively.
New unknown impurity at m/z 421.15 was observed during the accelerated stability analysis (40 °C/75% relative humidity) in the multi-component tablets of amlodipine besylate by reversed-phase ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS). UHPLC-MS and nuclear magnetic resonance (NMR) techniques were employed to identify and fully characterize the degradation compound. The degradation product was unambiguously identified as 3-ethyl 5-methyl 4-(2-chlorophenyl)-6-methyl-2-(morpholin-2-yl)-1,4-dihydropyridine-3,5-dicarboxylate and mechanism of its formation was proposed. It was confirmed that the degradation product was formed by the reaction of amlodipine with formaldehyde originating from the excipients present in the dosage form.
- MeSH
- amlodipin chemie MeSH
- časové faktory MeSH
- chromatografie s reverzní fází MeSH
- farmaceutická chemie metody MeSH
- fixní kombinace léků MeSH
- formaldehyd chemie MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
- hydrochlorthiazid chemie MeSH
- kontaminace léku * MeSH
- magnetická rezonanční spektroskopie MeSH
- molekulární struktura MeSH
- pomocné látky chemie MeSH
- stabilita léku MeSH
- valsartan chemie MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- časopisecké články MeSH
