Avoiding N-nitrosodimethylamine formation in metformin pharmaceuticals by limiting dimethylamine and nitrite.

Since late 2019, concerns regarding trace levels of the probable human carcinogen N-dimethylnitrosamine (NDMA) in Metformin-containing pharmaceuticals have been an issue if they exceeded the maximum allowable intake of 96 ng/day for a medicine with long-term intake. Here, we report results from an extensive analysis of NDMA content along the active pharmaceutical ingredient (API) manufacturing process as well as two different drug product manufacturing processes. Our findings confirm that Metformin API is not a significant source of NDMA found in Metformin pharmaceuticals and that NDMA is created at those steps of the drug product manufacturing that introduce heat and nitrite. We demonstrate that reduction of nitrite from excipients is an effective means to reduce NDMA in the drug product. Limiting residual dimethylamine in the API has proven to be another important factor for NDMA control as dimethylamine leads to formation of NDMA in the drug products. Furthermore, analysis of historical batches of drug products has shown that NDMA may increase during storage, but the levels reached were not shelf-life limiting for the products under study.

NDMA analytics in metformin products: Comparison of methods and pitfalls.

BACKGROUND: For nearly three years, the concerns regarding trace levels of N-nitrosamines in pharmaceuticals and the associated cancer risk have significantly expanded and are a major issue facing the global pharmaceutical industry. N-nitrosodimethylamine (NDMA) found in formulations of the popular anti-diabetic drug metformin is a prominent example. This has resulted in product recalls raising the profile within the media. Issues of method robustness, sample preparation and several unexpected sources of nitrosamine contamination have been highlighted as false positive risks. It has become apparent that the identification of the root causes of artefactual formation of nitrosamines must be identified to mitigate risk associated with the analysis. METHODS: A comparison study between four laboratories, across three companies was designed, employing orthogonal mass spectrometric methods for the quantification of NDMA in two metformin immediate release (IR) formulations and one extended release (XR) formulation. These were 2x LC-MS/MS, GC-MS/MS and GC-HRMS. RESULTS: Good agreement of results was obtained for the IR formulations. However, we measured higher concentrations of NDMA in the XR formulation using GC-MS/MS compared to LC-MS/MS. We could show that this was due to artefactual (in situ) formation of NDMA when samples were extracted with dichloromethane. Removal of dimethylamine (DMA) and nitrite from the extracted sample or the addition of a nitrosation scavenger are shown to be effective remedies. NDMA in situ formation was not observed in 10% MeOH or acetonitrile. CONCLUSION: Metformin pharmaceuticals contain traces of the API impurity DMA as well as inorganic nitrite from excipients. This can lead to artefactual formation of NDMA and hence false positive results if DCM is used for sample extraction. Similar artefacts are likely also in other pharmaceuticals if these contain the secondary amine precursor of the respective nitrosamine analyte.

Forensic analysis of carbohydrate-deficient transferrin (CDT) by HPLC--statistics and extreme CDT values.

BACKGROUND: Carbohydrate-deficient transferrin (CDT) is the most specific serum marker of chronic alcohol abuse so far. There is little knowledge about extreme CDT values of >20% and the more >30% CDT. METHODS: Serum CDT/transferrin ratios from 19,236 serum samples sent to our laboratory for routine CDT analysis were determined by HPLC. About 75% of these serum samples were from traffic or employment medicine investigations. A CDT value frequency histogram was computed and extreme CDT values were clinically validated. RESULTS: Fourteen thousand four hundred and sixty-one CDT results were normal (< or =1.7%) and 4775 increased (1.8-36.9% CDT). Most frequent normal and increased results were 0.9% CDT (n=1964) and 1.8% CDT (n=356). CA. 70% of the pathological results were between 1.8% and 5.0% CDT, ca. 88% between 1.8% and 10.0% CDT, and 98% between 1.8% and 20.0%. CDT values >20.0% appeared in 79 cases and results >30.0% in two cases (33.8% and 36.9%). In each case of CDT values >20%, chronic alcohol abuse was the underlying cause as confirmed by anamnestic exploration. CONCLUSIONS: CDT/transferrin ratios are usually <20%. Higher values can appear in rare cases. CDT results >30% can be due to alcohol abuse but should be considered as remarkable single observations. Visualization of the transferrin isoform patterns by HPLC allows the detection of pathological transferrin isoform patterns and of genetic transferrin variants. This is essential for a reliable interpretation of (extreme) CDT values. CDT analysis by immunoassays without physico-chemical confirmatory analysis is no longer acceptable.

Determination of serum amantadine by liquid chromatography-tandem mass spectrometry.

BACKGROUND: Amantadine (1-adamantylamine) is used for treatment of influenza, hepatitis C, parkinsonism, and multiple sclerosis. Current amantadine analysis by HPLC or gas chromatography (GC) requires a laborious sample pretreatment with extraction and/or derivatization steps. We established an LC-MS/MS method without protein precipitation, centrifugation, extraction and derivatization steps. MATERIAL AND METHODS: 50 microl sample+50 microl of 0.4 mg/l 1-(1-adamantyl)pyridinium bromide as internal standard+1000 microl water (96-well plate). Of this 25 microl+500 microl water (96-well plate; final serum dilution 1:462). LC-MS/MS: Surveyor MS pump, Autosampler, triple-quadrupole TSQ Quantum mass spectrometer (Thermo Electron). Autosampling: 2 microl of each sample. Chromatography: isocratic water/acetonitrile (60/40 v/v) with 5 g/l formic acid, flow rate 0.2 ml/min, run time 3 min, Phenomenex Luna C8(2) (100 x 2.0 mm (i.d.); 3-microm bead size) column. Mass spectrometry: electrospray atmospheric pressure ionization, positive ion and selective reaction monitoring mode, ion transitions m/z 152.0-->135.1 (at 22 eV amantadine) and 214.1-->135.1 (at 26 eV internal standard). RESULTS: Calibration curves were constructed with spiked serum samples (amantadine 50-1000 microg/l, r>0.99). No carry over (5000 microg/l). No ion suppression with retention times similar to those of amantadine (1.8 min) and the internal standard (2.1 min). Detection limit 20 mg/l, linearity 20-5000 mg/l, intra-assay/inter-assay CV<6%/<8%, recovery 99-101%. Method comparison: LC-MS/MS=1.23 x GC-45 (Passing-Bablok regression). No significant bias between GC and LC-MS/MS (Bland-Altman plot). CONCLUSION: We consider the sample pretreatment without deproteination, derivatization and centrifugation steps and the specificity of the tandem mass spectrometry as the most important points of our amantadine analysis method.