Ranitidine Stability Study

I'm Ishani Nandida, the Assistant Director for Bioanalytical Chemistry here at Emory Pharma. Today I'll be discussing the study of ranitidine stability quantification of N nitrosodimethylamine, a probable human carcinogen, in ranitidine drug products and biological matrices by UHPLC and sns. So most of you have probably heard of or used Zantac, which is a ranitidine drug product. It's typically sold as an OTC drug for prevention of heartburns or as prescription for prevention of stomach ulcers. So the mechanism of action that it follows is it basically prevents the binding of histamine to H2 receptors on parietal cells, thereby reducing the influx of stomach acid. Renitidine drug products have been mentioned in the World Health Organization's list of essential medicines and has been recognized for being highly safe and efficient. It's the 48th most prescribed medicine in the US with over 16 million prescriptions as of 2017. Ranitidine drug products have been in the market since the 1980s. However, it is important to note that the regulatory conditions in the 1980s may not have had proper analytics or controls over impurity analysis, stability and shipment of these drug products. That's why, nearly 40 years later, high levels of N nitrosoldimethylamine NDMA was found in ranitidine drug products. This was first announced by the US FDA on September 19th of 2019. The international agency for Research on Cancer described NDMA as a probable human carcinogen. The US FDA's acceptable daily intake limit of NDMA is 96 nanograms per tablet. The recommended venitidine dosage is 150 milligrams. However, it can be up to 6 grams under certain severe conditions. Initially, the reported NDMA levels were performed using Headspace GC MS, where the results were up to 3 million nanograms per tablet. We thought that these levels were highly astronomical, otherwise we would have seen quantifiable detrimental health outcomes since the 1980s when this drug product was first in the market. At this stage, we thought that the levels of NDMA detected using headspace GC Ms. Is likely an artifact of elevated temperature. This is because typically headspace temperatures are 120 degrees Celsius or higher. So we investigated NDMA analysis using LC Ms. And lchrms, which avoids the exposure to elevated temperatures. And in this case, our initial hypothesis was that NDMA is likely a processing impurity and this approach was very similar. To that of Valsartan and other angiotensin receptor blockers. So as I mentioned before, our initial hypothesis was that NDMA is likely a processing impurity. However, after thorough structural analysis of ranitidine, we found two main functional groups that may be responsible for the formation of ndma. These functional groups are the nitro and the dimethylamine functional groups, and it's very likely that under elevated temperatures that headspace provides of 120 degrees Celsius or higher that we are very well forming up to 3 million nanograms of NDMA per tablet. Now, it's important to note here that no drug product is realistically exposed to such high heat conditions. But commercial shipment encounters temperatures as high as 70 degrees Celsius over summer and also there's no clear requirement of how these drug products should be stored. So most often these drug products are left in the car over hot summer months. We developed a rich PLC Ms. Ms. Method to quantify NDMA in ranitidine drug products. So for this method we take our drug product at a concentration 150mg ML in methanol and we store, extract and filter to prepare for LCMS analysis. For quantitation we utilize an internal standard which is a deuterated version of ndma. So we also utilize mrm, for which the transitions and source and Ms. Conditions are provided in the table below. Our method allows us to achieve a linear range between 1 ng per ML to 1000 ng ML. This translates to quantification of NDMA as low as 1 ng per tablet. Our extraction recovery results were greater than 99% with RSDS lower than 10%. Additionally, we performed accuracy and repeatability experiments with three different concentrations and in this case our accuracy results were greater than 80% and relative standard deviations were lower than 5%. These results indicate that the method is highly feasible and accurate as it allows for accurate and reproducible extraction and quantification of NDMA. In ranitidine drug products. We looked at temperature and humidity parameters to assess the stability of ranitidine. So we looked at ranitidine drug products such as Zantac and Topcare, and we also looked at ranitidine drug substances such as USP Ranitidine. We placed all these drug products and drug substances through three different conditions. So we first looked at 40 degrees Celsius under ambient humidity condition, which was done to simulate the storage of these drug products under hot summer months. We also looked at 70 degrees Celsius under ambient humidity conditions which was done to simulate the shipment of these drug products on hot summer months. Lastly, we looked at 30 degrees Celsius with 100% humidity. This was done to simulate the storage and shipment of these drug products under high humidity conditions. At the end, all these drug products and drug substances were analyzed using LC Ms. Ms. Now this was particularly challenging as all of these drug products and drug substances had initial levels of NDMA which was substantially higher than the acceptable daily intake limit as set by the fda. So, upon analysis of both Zantac and Topcare under elevated temperatures such as 40 degrees Celsius and 70 degrees Celsius, we see increasing levels of NDMA. In fact, for Zantac, if you look at past day four, the levels of NDMA exceed greater than one microgram per tablet. Our results now prove that ranitidine is in fact heat unstable compound which degrades to form NDMA under elevated temperatures. To further confirm our hypothesis here, we wanted to also test ranitidine drug substances. So even when USP Ranitidine was placed under the same stability assessment at 40 degrees Celsius and 70 degrees Celsius, you can see that NDMA levels still show the same trend where the results were reported in nanograms of NDMA per milligram of ranitidine by day 45. At 40 degrees Celsius, you can see you can generate an equivalent of 930 nanograms of NDMA per tablet had this been in the form of a vernitidine drug product left in your car during the heat of summer. So you can see that without even the presence of excipients, NDMA was still being formed and still being found as a degradation product of ranitidine. So we also assessed and found out what the presence of humidity does to the levels of NDMA in USP Ranitidine. We placed our USP Ranitidine under 30 degrees Celsius at 100% humidity. And as you can see, the levels were greater than 50 nanograms per milligram of ranitidine past day 22 when compared to 40 degrees Celsius. There's a steeper climb with humidity that may imply that humidity may play a larger role in temperature in the formation of ndma. It's important to note here that humidity tests are very important in this case as shipment and storage of these drug products are largely impacted by these conditions. So you can imagine in countries such as India, where there is consistently 100% humidity, you can imagine the kind of implications this may have on the shipment and storage of these drug products as a probable human carcinogen builds over time. Besides storage conditions, we also explored additional parameters that may lead to the formation of NDMA from ranitidine drug products. So there was a study done by Professor Mitch from Stanford University which explored the oral intake of ranitidine and how it affects the urinary excretion of ndma. The study found that ranitidine undergoes nitrocation under stomach relevant ph conditions causing the formation of NDMA over time and this was tested in vitro. They found a 400 fold increase in NDMA formation. So based on this study our question was whether or not stomach relevant ph conditions caused the formation of NDMA upon ranitidine consumption. So to study that phenomenon we employed an in vitro analysis in simulated gastric fluid to study the fate of ranitidine upon consumption. So we know that ranitidine drug products are typically taken after food consumption. So in this case we were trying to mimic the properties of a fed state in our SGF medium for possible cases of nitrocation of ranitidine drug products. So our SGF medium was composed of 50 millimolar potassium fluoride and 85 mm hydrochloric acid adjusted to a pH of 1.2 and this was based on the USP and the FDA guidance for industry. Additionally, our initial studies indicated that nitrites may play a huge role in the nitrosization of ranitidine upon consumption. Nitrites are introduced into our body through the consumption of processed meats and vegetables, so therefore we needed to introduce nitrites into our SGF medium. The concentration concentration of sodium nitride was based on the World Health Organization. So to explore the levels of NDMA and simulated gastric fluid we developed a UHPLC MSMS method for quantification of ndma. So for our workflow our drug product was at a concentration of 30mg ML in simulated gastric fluid. It was incubated at 37 degrees Celsius for two hours and then it was processed process for LCMS analysis for quantification of NDMA we then investigated the in vitro analysis with stimulated gastric fluid which indicated that NDMA formation can occur upon consumption of ranitidine. So as you can see in the graph below, the levels of NDMA in water represent the initial levels of NDMA in the ranitidine drug product, which in this case is 123 nanograms per tablet. Upon incubation in simulated gastric fluid, you can see that the levels essentially remain the same. However, when incubated in simulated gastric fluid with the introduction of sodium nitrite, you can see that the levels increase by over five times. So, in conclusion, ranitidine has potential to form high levels of NDMA in vivo under stomach acid conditions with the presence of nitride. So we also investigated whether nitrocation occurs with other H2 receptor blockers in simulated gastric fluid with nitrite. We explored quite a few of the H2 receptor blockers, but in this case I'm showing examples from two of them. So here I have examples for cimetidine and famotidine. And as you can see, neither incubation in stimulated gastric fluid nor with the influence of added sodium nitrite promotes the formation of ndma. In cimetidine and famotidine only the structure of ranitidine shows susceptibility towards nitrocation under stomach acid conditions with nitrite present. Since our in vitro results indicated that branitidine has potential to form NDMA in stomach conditions in vivo, we developed a uh, Pilsen Ms. Ms. Method to quantify levels of NDMA in urine. So now we're using our method to perform a study in collaboration with Professor Mitch from Stanford University to investigate whether renatidine consumption results in high levels of NDMA in urinary excretion. So, in conclusion, ranitidine drug substances and drug products are inherently unstable under elevated temperatures. We saw that the presence of humidity also is responsible for increasing NDMA levels. Additionally, our initial in vitro studies in simulated gastric fluid using ranitidine drug products indicated ranitidine susceptibility to nitrousation under stomach acid conditions with the presence of nitrites. We also found that the structure of ranitidine is more prone to nitrezation under such relevant stomach ph conditions when compared to other H2 receptor blockers. And in this case, this was compared to cimetidine and famotidine. Additionally, our ongoing study with Professor Mitch will be examining whether ranitidine undergoes nitrocation in vivo upon consumption to form ndma. Lastly, I wanted to mention that this is a global health concern, a concern over a drug product that has spent nearly 40 years in the market and is only recently found to be unstable where it degrades to form high levels of a probable human carcinogen. Now, to date, there has been no proper epidemiological studies, however long story short, we know that prolonged use of these ranitidine drug products does have possible links to cancer. Thank you for listening.