The possibility of N-Nitrosation in the absence of nitrosating agents was studied on model solutions and film coated tablets containing metformin. N-nitrosodimethylamine (NDMA) and N-nitrosation precursors (dimethylamine and nitrites) were determined using previously published fully validated analytical methods. Alternative routes to N-nitrosation were found. Dimethylamine can undergo an oxidation to nitrite in the presence of strong oxidants (e.g., H2O2), as was observed during wastewater treatment in several published works. The resulting nitrite can consecutively act as a nitrosating agent. We proved that the described reaction indeed leads to N-nitrosation (NDMA formation in case of dimethylamine precursor) in model solutions made of dimethylamine and H2O2. An experiment was designed in order to prove those reactions take place in dosage forms. Film coated tablets present a highly heterogenous system with several solid phases and low water activity, which is in stark contrast to the liquid wastewater, where this reaction was originally studied. Despite that, the described reaction took place even in the tablets, but only to a small degree. The amount formed via this alternative route corresponds to less than 10 % of the total formed NDMA. The pH optimum of this alternative route lies in the alkaline range which was confirmed by the determined NDMA concentration in model solutions. The solid phase system (i.e., tablets) was found to behave differently. The addition of Na2CO3 into the tablets during manufacture resulted in tablets without NDMA (cNDMA < LOQ) even in batches spiked with both dimethylamine and H2O2. Thus, adjusting the pH of the solid dosage forms remains a sufficient measure of controlling N-nitrosamines in the product, even in product with limit amounts of oxidating agent (H2O2) and N-nitrosation precursor (dimethylamine).
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.
After a presence of highly hepatotoxic and potentially carcinogenic N-nitrosodimethylamine was detected in certain lots of sartan, ranitidine, metformin, and other pharmaceuticals, local regulatory authorities issued recalls of suspected products, and concerns of the pharmacotherapy safety were widely discussed. Since then, testing of a representative sample of each produced lot of these pharmaceuticals is required as a part of quality control processes. Hence, an interface-free CE-nanoESI system coupled with MS detection was employed for the development of a simple and economical method for quantitative detection of this contaminant in the valsartan drug substances and finished formulations used as model matrices. In this arrangement, a fused-silica capillary was used as both a separation column and a nanoESI emitter providing high ionization efficiency and sensitivity. The optimized procedure was found to have sufficient selectivity, linearity, accuracy, and precision. The established LOD and LOQ values were 0.3 and 1.0 ng/mL, respectively. The practical applicability of the method was tested by analyses of commercially available Valsacor® tablets. The results obtained prove that the developed procedure represents a promising alternative to currently available GC- and LC-based methods. Furthermore, after an adjustment of the separation conditions, the CE-nanoESI/MS system can be conceptually used for the determination of NDMA in other suspected pharmaceuticals.
- MeSH
- Dimethylnitrosamine analysis MeSH
- Electrophoresis, Capillary methods MeSH
- Spectrometry, Mass, Electrospray Ionization methods MeSH
- Drug Contamination * MeSH
- Linear Models MeSH
- Nanotechnology MeSH
- Reproducibility of Results MeSH
- Sensitivity and Specificity MeSH
- Tablets MeSH
- Valsartan chemistry MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The interaction of carcinogenic N-nitrosodimethylamine (NDMA) and N-nitrosomethylaniline (NMA) with cytochromes P450 (CYP), CYP2B4, CYP2E1 and CYP3A6, and their metabolism by these enzymes reconstituted with NADPH-CYP reductase in liposomes were studied. Using the difference spectroscopy, the lowest values of spectral dissociation constants (KS) of the binary complex of NDMA and NMA with the enzyme were found for CYP2E1. Both N-nitrosamines bind to the heme iron atom as ligands. On the contrary, the binding of NDMA and NMA to CYP2B4 gives the type I spectra. NDMA is bound to CYP3A6 analogously as to CYP2B4, whereas no difference spectra were acquired with NMA and CYP3A6. All tested CYPs oxidize NDMA and NMA. CYP2E1 exhibits the lowest Km values, 7.5 and 5.0 micromol/l for NDMA and NMA, respectively, and for CYP3A6 we found 30.0 and 10.0 micromol/l for NDMA and NMA, respectively, while CYP2B4 exhibits the lowest affinity for both carcinogens. In spite of different binding affinities of NDMA and NMA, the values of Vmax for their oxidation were, however, similar for all tested CYPs. The results demonstrate that investigations utilizing several enzymatic approaches are necessary to properly evaluate the mechanism and efficiency of NDMA and NMA oxidation by CYPs in vitro.
- MeSH
- Aryl Hydrocarbon Hydroxylases metabolism MeSH
- Cytochrome P-450 CYP2E1 metabolism MeSH
- Dimethylnitrosamine metabolism MeSH
- Microsomes, Liver metabolism MeSH
- Carcinogens metabolism MeSH
- Kinetics MeSH
- Rabbits MeSH
- Liposomes MeSH
- NADPH-Ferrihemoprotein Reductase metabolism MeSH
- Nitrosamines metabolism MeSH
- Oxidation-Reduction MeSH
- Cytochrome P-450 Enzyme System metabolism MeSH
- In Vitro Techniques MeSH
- Animals MeSH
- Check Tag
- Rabbits MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Concise International chemical assessment, ISSN 1020-6167 38
iv, 45 s. : il. ; 30 cm
- MeSH
- Chemical Safety MeSH
- Dimethylnitrosamine toxicity MeSH
- Risk Assessment MeSH
- International Cooperation MeSH
- Occupational Exposure MeSH
- Environmental Exposure MeSH
- Conspectus
- Chemie. Mineralogické vědy
- NML Fields
- chemie, klinická chemie
- environmentální vědy
- pracovní lékařství
- NML Publication type
- publikace WHO
[Embryotoxicity of N-nitrosodimethylamine in mice]
