Metformin drugs under simulated gastric conditions can generate high nitrite-dependent levels of N-nitrosodimethylamine

N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA), group 2A carcinogens, were detected in finished drug products, including metformin, ranitidine, sartans and other drugs which caused multiple recalls in the USA and Europe. Important studies also reported the formation of NDMA when ranitidine and nitrite were added to simulated gastric fluid. Our objective was to screen finished drug products from Europe and USA for nitrosamine impurities and investigate the formation of NDMA in metformin finished drug products when added to simulated gastric fluid. One dosage unit of 30 different commercially available drugs, including metformin, sartans, and ranitidine were tested for NDMA, NDEA, and dimethylformamide (DMF) impurities, using a liquid chromatography-mass spectrometry (LC–MS) method. Then, 6 metformin finished drug products were tested in stomach conditions for 2 h at 37 °C in a 100 mL solution with a pH of 2.5 and different nitrite concentrations (40, 10, 1, 0.1 mM) and tested for NDMA, and DMF using LC–MS. We measured NDMA, NDEA, and DMF in 30 finished drug products. NDMA and DMF were quantified for metformin drug products in simulated gastric fluid with different nitrite concentrations. None of the 30 drugs showed concerning levels of NDMA, NDEA, or DMF when tested as single tablets. However, when metformin tablets are added to simulated gastric fluid solutions with high nitrite concentrations (40 mM and 10 mM), NDMA can reach amounts of thousands of nanograms per tablet. At the closest concentration to physiologic conditions we used, 1 mM, NDMA is still present in the hundreds of nanograms in some metformin products. In this in vitro study, nitrite concentration had a very important effect on NDMA quantification in metformin tablets added to simulated gastric fluid. 1 mM nitrite caused an increase above the acceptable daily intake set by the U.S. Food and Drug Administration (FDA) for some of the metformin drugs. 10 mM, 40 mM nitrite solutions generated NDMA amounts exceeding by more than a hundred times the acceptable daily intake set by the FDA of 96 nanograms. These findings suggest that metformin can react with nitrite in gastric-like conditions and generate NDMA. Thus, patients taking metformin could be exposed to NDMA when high nitrite levels are present in their stomach, and we recommend including a statement within the Patient Package Inserts/Instructions for use.

In 2018, angiotensin receptor blockers (ARB) joined the list of nitrosamine impurities sources when some valsartan 6 and losartan 7 drug products were found contaminated with NDMA or NDEA, and recalled.Approximately 15 million patients 8 had a written prescription for losartan or valsartan finished drug products at the time of the recall.
After ARB recalls, in 2019 and 2020, reports showed unacceptable nitrosamine levels in over-the-counter preparations of ranitidine 9 and metformin 10 .While metformin was voluntarily recalled by some drug manufacturers 11 , the U.S. Food and Drug Administration (FDA) ordered the removal of all ranitidine preparations 12 .More than 20 million patients 8 were taking metformin and over 1 million patients 8 had a prescription for ranitidine in 2020 in the United States.
Similar measures were taken by the European Medicines Agency (EMA), i.e., suspending all ranitidine products in the European Union 13 and asking manufacturers to test sartan and metformin drug products for nitrosamine impurities 14 before releasing them on the market.
To assess the effectiveness of these measures within the European and American drug markets, we tested different commercially available, and post-market drug products.Investigated drugs included 13 metformin products, 17 sartan products, 1 injectable ranitidine product, 1 calcium-channel blocker, 1 anti-Parkinson agent, 1 antimicrobial agent, and 1 diuretic.The impurities we searched for were NDMA, NDEA, and DMF.
Structural similarities to ranitidine 15 e. g. the dimethylamine group, and the proven reaction between DMA and nitrosating agents 16 led us to test the potential of metformin finished drug products to generate NDMA in simulated gastric fluid.

Methods
Commercially available finished drug products from Europe/Romania (30) were tested for NDMA, NDEA, and DMF as single tablets.Metformin finished drug products were evaluated for NDMA production and DMF in simulated gastric fluid.We tested various available metformin drugs with several concentrations of sodium nitrite, such as 40 mM, 10 mM, 1 mM, and 0.1 mM, in stomach conditions, including a few metformin drugs from the USA market, as specified in the Results section.

Chemicals and reagents
Two nitrosamines (NDMA, NDEA), and DMF were selected as target analytes in this study.Reference standards of NDMA, NDEA, and isotope-labeled DMF standard D 7 -DMF were purchased from Sigma-Aldrich (St. Louis, MO).Isotopically labeled NDMA standard 13 C 2 -D 6 -NDMA was purchased from Cambridge Isotope Laboratories (Tewksbury, MA).Formic acid (LC-MS grade) was purchased from Millipore Sigma (Burlington, MA).Methanol (LC-MS grade) was purchased from Honeywell (Charlotte, NC).Milli-Q water (18.2MΩ) was generated from a Milli-Q IQ Element system from Millipore Sigma.

Sample preparation
The single drug tablet was accurately weighed.After the methanol dilution to approximately 100 mg active pharmaceutical ingredient (API) per mL of methanol, samples were homogenized.The last tube of rinsing methanol was used as a laboratory reagent blank (LRB) tested with the drug samples.A quality control positive (QCP) sample is also prepared from a recalled lot of Valsartan.Further dilution to 25 mg API/mL is achieved with Milli-Q water, followed by vortexing, centrifugation, and filtration through a 0.2 µm nylon filter to obtain a sample extract that was spiked with an isotopic internal standard mixture containing 666 ng of 13 C 2 -D 6 -NDMA and 333 ng of D 7 -DMF.The vial subjected to the LC-HRMS contains a sample of 500 µL with 26 ng/mL 13 C 2 -D 6 -NDMA and 13 ng/mL D 7 -DMF.The final internal standard concentration in each sample is 40 ng/mL.
For stomach conditions testing, metformin tablets were kept for 2 h at 37 °C and pH 2.5 in 100 mL solutions with 4 different sodium nitrite concentrations: 0.1 mM, 1 mM, 10 mM, and 40 mM.
Determination of target analytes is performed by SCIEX X500R QToF HRMS operated in positive atmospheric pressure ionization (APCI+) mode for the ionization of NDMA, NDEA, DMF and their isotopic labeled internal standards.
For a comprehensive description of the LC and HRMS parameters consult the supplemental data and the methods described within the reference 17 .

Quality assurance and quality control (QA and QC)
All calibration and QC samples are prepared in 25/75 (v/v) methanol/water, following a protocol previously described in detail here 15 .

Statistical analysis
Data are presented as mean and standard deviation (SD) of 3 independent experiments, unless specified otherwise.Data were collected and analyzed using Excel (Microsoft Corp).In Fig. 2, we present the data from the stomach conditions testing with 3 different nitrite concentrations.At 0.1 mM sodium nitrite, no NDMA was detected in any of the 6 drugs. 1 mM nitrite caused an increase above the acceptable daily intake set by the U.S. Food and Drug Administration (FDA) for some of the metformin drugs, while the 10 mM nitrite and higher concentrations led to NDMA amounts way above the acceptable daily intake set by FDA of 96 nanograms. .

Discussion
Various reasonable and common situations exist in which concentrations of nitrite could be significantly elevated.
As an example, consuming one pound of dry-cured meat that contains the regulatory limit of nitrite (625 ppm) 20 would bring 800 mL of stomach volume to a nitrite concentration of over 5 mM.All drugs tested in the presence of 40 mM of sodium nitrite in stomach conditions show NDMA levels in the thousands of nanograms per tablet, amounts which significantly exceed the acceptable daily intake established by the FDA of 96 nanograms.For example, Metformin 6 and Metformin 1 have 4255 ng/tablet and 3470 ng/tablet of NDMA, respectively.However, a concentration of 40 mM is also hard to reach in the stomach, in vivo.Thus, we repeated the experiment with 2 selected metformin drugs and 4 metformin drugs from the USA market at Figure 2. N-Nitrosodimethylamine formation from metformin at pH 2.5 in simulated gastric fluid and in increasing nitrite concentrations 0.1, 1, 10 mM.A plot of the variable NDMA amount at 2.5 pH in 100 mL simulated gastric fluid with different metformin tablets and 10 mM, 1 mM, and 0.1 mM sodium nitrite after 2 h incubation at 37 °C.The data is presented as the mean of 3 independent samples.For the data with mean and SD of 3 independent samples see Table 2

Limitations
The in vitro model used in this study has inherent limitations and does not reflect all aspects of human physiology.However, it does provide a method for assessing the potential for physiologic nitrite reactions with drugs in the gastric fluid that lead to NDMA formation.

Conclusion
In this in vitro study, nitrite concentration had an especially important effect on NDMA quantification in metformin tablets added to simulated gastric fluid. 1 mM nitrite caused an increase above the acceptable daily intake set by the U.S. Food and Drug Administration (FDA) for some of the metformin drugs, while the 10 mM nitrite and higher concentrations led to NDMA amounts exceedingly elevated compared to the acceptable daily intake set by the FDA of 96 nanograms.These findings that metformin can react with nitrite in gastriclike conditions and generate NDMA.Thus, patients taking metformin could be exposed to NDMA, when high nitrite levels are present in their stomach, therefore leading to exposure to higher-than-normal concentrations of NDMA.This metformin-nitrite interaction in the gastric medium is even more relevant when considering how the biguanide is prescribed i.e. it should be taken with food 21 .Further studies are needed to evaluate the interaction between nitrite content of the meal and its interaction in the gastrointestinal tract with metformin or its degradation product, DMA.

Figure 1 .
Figure 1.formation from metformin at pH 2.5 in simulated gastric fluid and 40 mM sodium nitrite concentration.A plot of the amount of NDMA at 2.5 in 100 mL simulated gastric fluid with 8 different metformin tablets and 40 mM sodium nitrite after 2 h incubation at 37 °C.The data represent the mean and SD of 3 independent samples, and the error bars show the SD of the measurement.The drugs are presented the x-axis.

Table 1 .
Chemical impurity analysis of the first 30 finished drug products from the European Market.Results for one sample.ND Not Detected, LOQ Limit of Quantification, value given when the analyte is detected but not in amounts high enough to quantify. .

Table 2 .
The data represents the mean and standard deviation of 3 independent samples.0.1 mM column is not shown since the values were ND.ND Not Detected.