 We'll see my screen. Can you confirm it that young place? Yes, I see it. Yeah, we see it. Okay, great. Well, thank you, John and Christian for inviting me to speak on testing methods for natural. So many period is analysis in from us. The articles today, I'll be giving a special focus on the 4 testing methods published in the chapter 1469. And this chapter, it became online today this morning, and it's already available in us, enough online for you to to check the final chapter. And I will also be discussing about analytical challenges involved in testing methods for that shows them in analysis from a city cause. And I'm going to start by providing you a brief overview of the USP natural and in activities. Then you're going to discuss about method performance characteristics for those for those methods. And here, I'm going to highlight some analytical factors, which can impact sensitivity and selectivity, accuracy and precision as well. And then we are going to deep dive into some details related to the testing methods published in the chapter 1469. You're going to discuss about some analytical challenges. And here, I would like to share with you some considerations and recommendations for those testing methods. Well, the 1st, it was this response to address the nature of the many purity safety concern was the creation of the joint experts to be comedy on that shows the many periods, which is consisted of different experts from industry with a very strong expertise in and drug manufacturing tracing. Analysis and also is consisted of representatives from FDA and also and the primary goal of this to be comedy was to develop a roadmap for us to guide us in creating public standards regarding nitrosamines and also to assist us in in some efforts regarding some activities related to nitrosamines. And the 1st step was to draft the general chapter 1469 on a choice of any priorities, which will bring a current industry and for thinking on nitrosamines to discuss about different sources of nitrosamines risk assessment. And you're going to see that we published here for testing methods and in those testing methods, we are describing the use of some different standards for nitrosamine, impurities and a USP developed 8 USP have from standards for nitrosamine impurities. As you can see here in this list and the most recent released standard is the NMPA have front standard and also the isotopic labeled in DMA, which is very often used as internal standard to compensate for for matrix effects and any kind of is stretching efficiency that we may face and we are going to discuss about that too. And I also do like to anticipate today that USP has a new impurities program called from a pseudoconalytic or impurities or for short, where we are also developing some nitrosamine impurities, which will be soon available. Well, this year is a timeline for the general chapter publication. So, as you know, last year, USP published the proposal chapter in the pharmacopoeia forum on 1st September and after 90 days, after the public consultation, all the comments were gathered and addressed by the choice of the comedy on nitrosamine impurities and after reviewing and addressing all comments, the chapter was moved to be balloted by the chemical analysis general chapter expert comedy. And recently this, this expert comedy approved the chapter and as already shared with you today, this chapter became online and you can find it in the USP and after 2021 issue 3. And it will become official on 1st December of this year. Well, beyond the chapter 1469 and in the 8 different standards that you must be developed, there are many efforts currently underway to provide supports, tools, best practices to provide support manufacturers and regulators to analyze the risk of the potential presence of nitrosamines and pharmaceuticals and also to test as needed to help them to control the nitrosamine levels below a safe level, right? And USP has been delivering several scientific discussions in different formats in workshop format webinars as today. And we are also working on several publications and 1 that I would like to highlight today, it is the nitrosamine tutorial videos that you're going to find on YouTube. Here you have the link. So, you're going to share the slide deck afterwards and then you can have access directly to the links of this presentation. And this will direct you to the YouTube page where you're going to find some several videos discussing about the different different sections of the chapter 1469. And I also do like to anticipate here that USP is also working on the review article, which we are discussing about the different published methods in the literature and providing a very robust discussion. Around the analytical challenges and factors, which may impact on sensitivity and selectivity. Well, in the USP education environment, USP has developed a course, which is now available on demand, which we will discuss about the ice age guideline Q3 and and seven. And we have a pretty new course on the chapter 1469, which will be delivered in 2 weeks on 15th and 16th June. And here you're going to have the link, the access to the link, and then you can learn more in the web page for this link here for this course and learn about the agenda and structures and so on. And USP is also continuously developing and validating methods for specific products for running to team, manufacturing, and also exploring the application of methods to detect nitrogen and impurities insolvent in water, which are commonly used in the manufacturing process. And, and might also be source of nitrosamines or their precursors, right? And I also do like to highlight the stimuli article published in 2019, discussing about a method to evaluate nitrosamines and also poly aromatic hydrocarbon, which is another class of genotoxic impurity. In the list of merit components for inhalation, packaging and delivery systems, and I'm highlighting this today because this might trigger in the future, the reveal of the chapter 381 on elastomeric closures for injection. And the other activity that I would like to share with you is the nitrosamine exchange knowledge community, which is a great place for us to share knowledge around risk assessment. It's also a great place for us to discuss with other experts around this topic and also learn a lot, right? So, here you find the link where you can register yourself and become a member and participate in all of those discussions. And today we have our colleague, who is the exchange community member and he will be presenting some additional details after my presentation around this community. Well, this is the content that you're going to find in the chapter 1469. So we have nine sections, right? And the first section would be a brief introduction, basically bringing the scope of the chapter, which is to provide a guidance to assess the materials and to ensure that we can identify the potential nitrosamines that might be present in the products. And also provide some guidance on analytical procedures and how to establish controls. Section 2, you're going to find a table with some nitrosamines. The most commonly found nitrosamines will not be an extensive table with all nitrosamines, but we are going to find the urchinical information as well. And section 3, you're going to discuss about sources of nitrosamines. And here, you're going to find a very comprehensive table with the different types of sources of nitrosamines along with their risks. Section 4 are discussing about risk assessment and section 5 approaches for calculation of limits of nitrosamines. And then section 6, 7, and 8, they are focused on an analytical procedure, which is the focus of this webinar today. And we know that basically, based on the FDA and EMA recommendations, they are proposing that 3 steps approach for us to potentially identify nitrosamines and pharmaceuticals. And till we can develop a strategy to mitigate the deformation of nitrosamines. And the 1st step would be the identification risk assessment and identifying the potential formation of nitrosamines. And in case we see that we have the possibility to find nitrosamines in the products, we need to conduct confirmatory testing, right? For this reason, it's so important that we learn more about key aspects of those testing methods. And this will be the focus of the next slides. And before we just discuss more about the testing methods in the procedure, in the general chapter 1469, the 1st question we should try to answer here is, what should be the required sensitivity for analytical methods, right? So, we know that we need to control the nitrosamine levels at or below the acceptable intake, which will be at PPM level, right? It means that we are working with trace superiority analysis. And then the question is, which kind of analytical parameter should be used to define the required analytical sensitivity for those methods? Should be the limit of configuration or detection. And if you have a look at the ICH Q2 for validation, we're going to find the concept of LOQ, which is the minimum level at which an analyte can be quantified with a sample accuracy. And for this reason, it's been recommended to use the LOQ to define the required analytical sensitivity for these testing methods. And we also know today that for some APIs and products, it might be not totally avoidable the formation of nitrosamines. And for this reason, it's also not recommended to use the limit of detection for setting limits and technical limits. And that's been said, we see here that as a minimal requirement, we need to have methods, which will have an LOQ at or below the acceptable intake, right? But we also need to meet regulatory recommendations. And we know that depending on the country that you are, you need to follow the local regulatory recommendations, right? And they might be slightly different. And just to illustrate that, so here we have the discussion that we find in the guidance published by the FDA on nitrosamines regarding required sensitivity for testing methods. So there you're going to find a discussion saying that for products with maximum daily goals or MDT for short below 880 milligrams per day, the method should have an LOQ at or below 0.03 ppm. And for products with maximum daily goals above 880 milligrams per day, we should have methods with LOQ as low as reasonably practical. And there, the strategy to control the nitrosamines level would be to identify the limit of quantification as a sort of reporting threshold. And in case we detect the nitrosamine level above this threshold, we need to have a mitigating strategy to control the nitrosamine level well below the acceptable intake. Well, the EMA also published several recommendations in the assessment report last year. And here they are discussing that we should have methods for our products with LOQ at or below the acceptable limit, right? But depending on the purpose of testing, we might need to work with different methods with different sensitivities. The first option is when we use a testing methods in the routing control, then we need to work with methods with LOQ at or below the acceptable limit. And we can also use methods to justify skip testing. And here the testing methods you have an LOQ at or below 30% of the limit. And the third option is to use methods to justify omission from the specification. And here we have a greater challenge, right? Because we need to work with methods with higher sensitivity and LOQ at or below 10% of the limit. And in this assessment report, we are going to see that we can discuss about some exceptions with the local regulatory agency, especially when we are working with some maximum products with high maximum daily tolls. Well, over the last two years, we saw that many different testing methods were published by different regulatory agencies, quality control labs. And basically, as you can see here, some examples, basically, the most of them are exploring the application of chromatography coupled to mass spectrometry. Basically, this is due to the challenge that we have because the nitrosamines will be present at very low level at PPM. And one of the main challenges here will be through it, the suitable sensitivity that we need to control them, right, at those low levels. And here we also have the chapter, USP chapter 1469, with four testing methods, also exploring chromatography coupled to mass spectrometry. And we are also publishing here methods using different mass pletiforms, a mass analyzer that we are going to discuss about that later. Well, and here are some performance characteristics that we need to take into consideration. This is an example for a quantitative method, and we saw that the main challenge will be sensitivity, right? We need to have a methods with good sensitivity to detect metrosamines at PPM level and also meet the proposed regulatory recommendations. And something that I would like to point out here is that neither the limit of detection nor the limit of quantification will be constant values, right? So they can vary over time, depending on a plenty of factors. And it's important that we know which kind of factors those are, right? Depending on the use of different systems from different manufacturers, we can impact significantly on a low kill sample preparation protocol can greatly impact on sensitivity. And the second characteristic is selectivity. We need to assure that our method has the ability to assess on a publicly the analyte in the presence of components, which might, which might be expected to be present. And here we need to have methods which are selecting for the target nitrosamines and which would be those target nitrosamines. Those would be the ones that we identify during risk assessment, right? And the third point here is that we need to have methods which are accurate and precise, right? And because we are working with LCMS and GCMS, we know that matrix effect may take place, right? And may impact on sensitivity, accuracy and precision. And one of the biggest challenge that we have here is regarding the sample preparation protocol, for example, where we need to develop a method trying to enhance distraction efficiency. While maintaining a good reproducibility and recovery of the trace impurities. Regarding linearity, we need to conduct the linearity studies. I just would like to mention that for a quantitative method, it's important that we include the acceptance intake within the concentration frame, right? And at the end, we also need to have a robust method, right? We need to ensure consistent results and a lack of robustness can really impair all of those of performance characteristics that I mentioned before. And here are some key aspects of developing a method. After establishing the objective of the method, which can also be called as analytical target profile, we need to conduct risk assessment as well. Because we need to start to get on prior knowledge. We need to understand which kind of naturalism and impurities might be present in the product. Also, recipients, the relation products in any other kind of processing purity, right? And then you're going to evaluate their physical chemical properties, including the API. Because this will guide us to select suitable analytical techniques. And we know that one of the main challenges will be sensitivity, right? And again, for this reason, we are exploring the application of chromatography coupled to mass spectrometry. For example, for the majority of nitrosamines, as we can see here, one example, the majority of nitrosamines will have only the end nitrosal group as the chromophore group, right? And this has a very low molar absorptivity. In other words, we know that this will impact in a poor detectability using a CUV. So, that is why we are applying mass spectrometry to increase sensitivity and selectivity as well. And other main challenge would be to develop methods with broad coverage of nitrosamines and with a great applicability to different APIs and products if necessary. And still during the method design, something super important is that we can select the critical attributes. We already discussed it about them, selectivity, sensitivity. Now that we are working with chromatography, there are some aspects related to selectivity. Basically, we are going to try to develop a method in which we are going to have a very good resolution between the target compounds, the nitrosamines and other adjacent spikes, right? But sometimes in case there is some collusion between a nitrosamine and an interfering compound and they are isobaric compounds, then we need to rely on good mass resolution, right? And with that, we can just select the critical parameters, the analytical factors, which can impact on selectivity, sensitivity, accuracy and precision. And here you have a comprehensive list, because we have factors regarding sample preparation, chromatography, mass spectrometry and even data processing can impact on selectivity, for example. And here, under mass spectrometry, we see that we can work with different ionization sources, right? This can impact greatly on sensitivity, selectivity. We can work with different MS analyzers and different MS resolving power, this can break impact on selectivity. Acquisition data strategies would be also a strategy to enhance sensitivity and selectivity and we need to try to reduce and minimize the matrix effect by looking to all those conditions here during method design. Well, here we have a brief overview of the mass spectrometer stages. We know that the first step here is to introduce the sample into the MS system and we can use different techniques, right? We can use a separation technique, such as chromatography, and we'll be exploring this for this application for nitrosamine analysis because it is will be one of the strategies that we can use to reduce matrix effect. And increase the ionization efficiency of compounds and in the ionization source in the mass system, the sample molecules will be ionized. And here is a short list of some ionization sources that that you can find available and the ions will be separated and analyzed in the MS analyzer, right? According to their mass to charge ratio, and we can also use several different types of MS analyzers. They will be operated under different physical principles, but this is not the focus of today's talk and we are going to discuss about some characteristics of some of them and understand how we can increase sensitivity and selectivity using them. And then you're going to detect the ions. You're going to process the data. We can work here with chromatograms, mass spectrum, and something important to mention data acquisition strategies because you're going to reveal some targeted data acquisition strategies because we can rely on that to increase sensitivity and selectivity, which is pretty important for this application, right? Well, just some key aspects related to the differences between 2 ionization sources commonly used in a CMS based methods apply to analyze nitrosamines. So, a lateral spray and atmospheric pressure, chemical ionization. Well, the biggest difference between them is that in ESI process, the ionization we will call in solution, right? And because of this, we can say that in ESI process, there is a crazy competition for charge, right? And in the APCI process, the analyte and solvent will be first vaporized into the gas phase, right? And the solvent will be ionized and consequently will ionize the analytes. And because the electron spray process, the ionization occurring solution, we can say that is less tolerant to matrix effect and we may use APCI as a strategy to increase sensitivity of some nitrosamines, those which are not acidic or basic compounds. Well, regarding MS analyzers, we know that we can classify them into low. In high resolution MS systems, right? And what is resolving power? Resolving power is the ability of a system to distinguish between 2 peaks at MOV values differing by small amount. And to illustrate that, let's assume we are analyzing 2 compounds with an MOV value very close, right? For example, 1 compound will have an MOV value at 75.05. The other one 75.06. If we analyze this using a low resolution MS system, because it has a lower ability to distinguish between those 2 signals, probably you're going to see a unique peak, which will not be able to distinguish between those 2 compounds. In other words, we can say that we have lack of selectivity. And when we analyze using a high resolution MS system, we have a higher ability to distinguish between those 2 peaks and we can resolve them and we have greater selectivity, what is pretty important to quantify the nitrosamines, right? In case we have the collusion of interfering compounds, however, we can also work with funding MS systems. Basically here we can conduct fragmentation experiments. We can use, for example, a triple pod, ion trap. We also have the hybrid systems, which is consistent of different systems from different types, for example, Q12, a Q orbit trap, and we're going to reveal some strategies to use low resolution MS system. And increase selectivity, and then this will be able to allow the quantification of nitrosamine as well. For some aspects related to the use of a high resolving power system, let's assume this is a hypothetical case that we are analyzing NDMA. And using here a low resolution MS system, with a low resolution MS system, we also have a lower accuracy to estimate a mass, right? And with this, we will be working with a low mass effort. And when we analyze this using a high resolution MS system, you're able to have a higher accuracy in the estimation of the MOV value, right? The experimental MS to charge ratio, which will be closer to the theoretical MOV value of the compound, right? Which is the ZACMAS. And here we are working with a lower mass effort, and this will increase the confidence to propose the identity of compounds. So in other words, we have higher selectivity, right? So why using high resolving power? We can improve accuracy mass measurements, which is translated into selectivity, good selectivity. We can resolve isotopic labeled species and improve accuracy of quantification. And let's say that we are developing a method to quantify NDMA now. And based on the LC conditions that we developed, there is a collusion between our target nitrosamine NDMA with an interferic compound. In this case here, dimethylformamide, right? By using a low resolution MS system, it can be that the monoisotopic peak of NDMA will not be distinguished from the isotopic peak of DMF. Here we have lack of selectivity and DMF will be quantified as NDMA. We are overestimating the NDMA content, right? And by working with higher resolving power, we are having here greater selectivity. And this is exactly what the FEDA published in this paper here, a private lab published and reported actually high content of NDMA in some metformin drug products. And the FEDA just investigated by running the analysis again, but now using higher resolving power, 45,000. And what they saw is exactly that NDMA was co-alutting with DMF and we needed then a minimum resolution of 45,000 to be able to distinguish and resolve well those two signals, right? And other reason for seeing lack of selectivity, it is the use of inappropriate mass tolerance when doing the data processing. Even when we use high resolution MS systems and we can separate the interfering compounds with an M-overse value very close to the target compounds, during the processing, the data processing, when we are extracting the chromatograms to quantify the target impurity, for example, if we use a very broad mass tolerance window, right, we are going to filter all interfering compounds. And because the mass difference between those two compounds will be 21 ppm, if we use a mass tolerance window above 21 ppm, we are filtering this signal here and quantifying as NDMA. So this is one of the reasons for us to face lack of selectivity, even using high resolution MS systems. So we need to take care of that. Well, let's go back to the chapter and now deep dive into the testing methods, section 7, you're going to find, basically, a discussion about importance of validating the alternative methods by using suitable guidances. And you also find some method performance characteristics to validate quantitative methods and also limit tests. And under the section 7.1, you're going to find some considerations for sample preparation. And we know that when we are working with trace impurity analysis, this is really a critical step, right? We need to avoid to lose the nitrosamines or the impurities, right? During sample handling, sample preparation, we also need to avoid the potential formation of nitrosamines as artifact during sample preparation. And in this section, you're going to find some considerations regarding the characteristic of some APIs. Here we have, for example, a runited in molecule, and we know that runited in molecule has a dioclamine group embedded in its molecule, right? And this API is thermally biome. It means that it may be great at high temperature. And by analyzing this using GCMS, we are applying high temperature in different parts of the system, such as injection port, headspace, and so on, it may be great forming here a secondary amine. And we know very well that this might be one of the precursors that in presence of a nitrosate agent under certain conditions may lead to the formation of NDMA or nitrosamines as artifact, right? And we need to bear in mind that other APIs might have the same characteristic. Well, for runited in, it's recommended then to analyze them using LCMS instead of GCMS to overcome this limitation. And for other APIs, they might also have dioclamine groups in the molecule. And here it's been recommended to avoid the use of total dissolution sample preparation protocols where the API would be totally dissolved in solution, right? We need to try to work with selective extraction protocols and processing priorities might also be precursors of nitrosamines or even counter ion of the soft form of the API. Well, here we have finally the four testing methods that we have in the chapter. Procedure one and procedure three, we are exploring the use of liquid chromatography coupled with mass spectrometry. Procedure one is using electron spray and a two orbit trap. Procedure three, it is using ionization source called APCI and a triple quad, which is a low resolution of mass system. And you're going to see which are the strategies for the acquisition we are using to increase selectivity and sensitivity. And procedure two and procedure four, we are exploring gas chromatography coupled with mass spectrometry using a triple quad as well. And procedure two, we are exploring headspace and for direct injection. Well, let's deep dive into the procedure details. Here we have the procedure one, which is using liquid chromatography coupled with high resolution of mass system. And the first point I would like to highlight here is the diluent used, right, which is methanol. And we know that sartans will be totally soluble in methanol. So we are, we have in solution the sartans, the API, which is very concentrated, right? And also the nitrosamines. We don't have so much headaches with stretching efficiency issues, right? But we need to avoid to inject the API, which is pretty concentrated into the mass system. Here we can use divert faults, for example, to divert the LCF with the API to waste. And avoid this from entering into the mass system because we know that over time the iron source can get dirty and this will impact greatly on sensitivity. Now, here, we are using reverse at phase from autography. We are using a pentafluorophane new column and detector high resolution of mass system. We use a Q orbit trap and this is the same procedure that was published by the FDA. It was actually adapted from from the FDA, as we can see here. And we are using electron spray ionization and we are using here as data acquisition strategy to modes of data acquisition, selected I am monitoring mode and parallel reaction monitoring mode in order to increase especially sensitivity and also selectivity. And let's give a closer look into the system to understand how we can increase sensitivity and selectivity. Well, first thing we know that a Q orbit trap, it is a high resolution of mass system, right? So we have great selectivity here. However, we know that high resolution of mass system might have also higher costs, right? It might not be widely available in the industry. And here, a strategy to increase sensitivity would be to analyze the data by using selected iron monitoring mode where in the first quadrupole, you're going to select the target items to be quantified, right? By setting here a narrow scan range, we are here filtering the interfering compounds, increasing sensitivity, right? And the ions will be then scanned at high resolution using an orbit trap. So we can say that in this mode, the MS is not spending time scanning the entire mass range and we are increasing a lot in sensitivity. Using a Q orbit trap, we can also have our data using parallel reaction monitoring mode. And here, in the first quadrupole, we will also be selecting the target ions for quantification. And in the collision cell, applying a certain collision energy, the precursor ions might fragment and we can scan the fragment ions at high resolution using orbit trap. And with this, we are increasing a lot sensitivity and sensitivity and selectivity. Well, let's go back to the procedure one. I would like to show you this note here, data processing. As I discussed, the use of inappropriate mass tolerance for data processing might be one of the reasons for lack of selectivity. And for this reason, you're going to find this note here saying that you should extract the ion chromatograms by setting a suitable mass tolerance of maximum of 15 ppm. Well, let's have a look at the procedure three, which is using liquid chromatography coupled with a triple quad, which is a low resolving MS system. And here we see that we are using a triple quad atmospheric pressure chemical ionization. And the strategy we are using here to increase selectivity and also sensitivity. It is a use of or acquisition of data using multiple reaction monitoring mode, as you can see here. So let's give a closer look into the triple quad to understand that. Well, we discussed that the triple quad, it is a low resolution MS system. It has a lower ability to distinguish between two peaks at MOVC values referred by small amounts. In other words, we can say that it has a lower selectivity when compared to a higher resolution MS system. However, it has also a lower cost, and it's more widely available in this trade, right? And here we can acquire data by using the selected reaction monitoring mode, where in the first quadruple, you're going to select again, the precursor ion that we would like to quantify, right? And under the collision cell, we will apply a certain collision energy and the precursor ion may fragment. And the fragmentation reaction will be totally dependent on the structure of the precursor ion and nature, right? And this will bring somehow a specificity to the analysis exactly what we need here to quantify right metrothermines. Because the fragment ion can be then monitored, this transition can be monitored in the last quadruple. And with this, we are increasing a lot in selectivity because we have now less interference of colluting compounds. This can work like a double mass filter, right? And increase selectivity. And for the same reasons, it will also increase sensitivity because now we have better signal to noise ratio, allowing us to quantify with lower limits of quantification and also using or experience wider linear range of quantification. Well, you're going to see that in procedure three, two and four, we are using this data acquisition strategy, but called a multiple reaction monitoring mode, which is basically multiple selected reaction monitoring transitions. For example, we can monitor the first transition between the precursor ion 103, which is related to NDEA, to the first fragment ion, which is 75. But we can also monitor other transitions, right? For example, the transition between 103 and 47, fragment 37. And with this, again, we are increasing even more the selectivity. Well, here, we are still in the procedure three. I would like to show you that we are using the diluent, final diluent will be 1% of formic acid in water. And here, we are using a selective extraction protocol because these are times are not soluble in this final diluents. And because it's not soluble, we should expect that we have these are times full of dust, dust, like person to have actually a high extraction efficiency, right? And because these are times are not soluble in this diluent, it's common deformation of clumps or even dry spots, right? Which will be actually a physical barrier avoiding the interaction between the solvent and the nitrosinates, right? This can lower the quantification results artificially. And another recommendation here is to use smaller vessels because using larger vessels, these are times real deposit on the walls of these larger vessels, which will have larger surface area, right? And again, this might complicate the extraction of nitrosinates. Well, here's some recommendations regarding sample prep. So we know now that based on the procedure one, for example, that we can work with total dissolution extraction protocols, where the drug substance is soluble in the extraction solvent, right? Again, so here we don't have so much issues related to extraction efficiency, but however we need to prevent that the drug substance will enter into the MS system, right? And we can also use selective extraction protocols. We can use a solvent, a final solvent, where the drug substance is somewhat soluble in the solvent or the drug substance is totally soluble in the solvent. And here there are two critical factors which can really impact on the extraction efficiency issue, which is the extraction time and mixing, right? So we need to allow to the correct and suitable extraction time. It must reach the equilibrium, right? Before we sample the sample to inject in the MS system. And here we also observe that during a curious spike experiment, right? Some nitrosamines might absorb onto the drug substance. And it's pretty important here to compensate for any kind of matrix effect and extraction efficiency issues that we consider the use of internal standards, right? And then we can provide more accurate and precise quantifications here. Well, regarding those issues, we can also use different types of calibration procedures in case we identify the presence of matrix effect, right? We can use as already described internal standards. We can also use matrix method calibration. And I would like to share with you the matrix precipitation strategy, which is also a selective extraction protocol. But first you're going to dissolve the drug substance using a solvent in which the drug substance is soluble. And then we can add an anti-solvent or an additive, which will lead to the precipitation of the drug substance. And here for sure we have some advantages because you're going to have the full dissolution at the beginning of the drug substance. And this will assure that the nitrosamines are transferred into solution. And then you're going to cause the precipitation of the drug substance, right? This will reduce potential matrix effect that we may face impact and significant impact on sensitivity of nitrosamine analysis. And this can be more reproducible and less time consuming. However, we also need to validate this before use. Here are some aspects related to the use of internal standards. We saw that we might use internal standards. And it's pretty important, especially when we are working with our CMS and to CMS. And we see that matrix effect is taking place, right? We can use internal standards to compensate for those effects and your suppression. And then we can provide for a more accurate and precise method, right? As I said, we can use internal standards during sample prep because this can account for extraction and ionization efficiency issues. We can use prior to sample injection and account for ionization efficiency issues due to matrix effect. And we can use different types of compounds, which might be used as internal standard, as you can see here. And the most recommended ones would be the isotopic labeled compounds. And why? Because they may experience the same degree of ion suppression or enhancement when compared to the analyte and also have a similar chromatographic separation. And then this can compensate for the matrix effect and ion suppression, right? And here just to highlight some challenges when using isotopic labeled compounds, we know that sometimes they are unavailable in the market. They have high costs, right? Especially because they are pretty difficult to obtain through synthesis. And we may also face lack of confidence in the isotope purity and integrity. For this reason, it's so important that we work with quality different standards for that. Well, procedure two, we are exploring the use of gas chromatography using HAT space. Here, we are using helium as carrier gas and using a triple quad. We are also acquiring data using multiple reaction monitoring mode exactly to increase sensitivity and selectivity. And procedure four, it's also using gas chromatography using a split-less injector in a split-less mode to increase the concentration of impurity being analyzed. We are using a triple quad. And here's the same thing. We are also acquiring data using multiple reaction monitoring mode and monitoring multiple transitions to increase sensitivity and selectivity. Well, and this is my last slide. Actually, I think I'm already over time. I'd like to give a knife or some time to discuss about the community. But again, Christian and Jan will be able to share the slide deck. Then this will be only some summary of factors that we already discussed, which can impact on selectivity sensitivity, right? And again, if you would like to have some further discussions or you have additional questions that you will not be able to answer today, feel free to read it out to me at this email address here, right? Thank you very much for your attention. And I'll be more than happy to answer any follow-up question that you may have during the Q&A session. And now I'd like to hand over the floor to Nifer. Thank you, Amanda. Thank you for that really thrilled presentation. I'm looking at the questions here. So we have some really good questions coming up. So I'm not going to take too much time with this, but I didn't want to let the opportunity pass to introduce to everybody something that, an initiative that Amanda presented at the beginning of her presentation. And it's around the nitrosamine community. So as you are probably familiarized, one of the first steps required by the agencies on the different guidelines was to conduct race assessment. And this is a very important step. This will really tell what we need to test, when we need to test, and where we need to test. And in some cases, whether that's sustainable throughout the lifetime of my product or not, or that's something that I need just to check on a particular frequency. So race assessment, it's a very important step. We're starting to see some folks in industry just bypassing the race assessment and going to test samples and test their product. And while that can be an easy solution, it's really not sustainable, right, based on the amount of analysis that we probably have to do at the end of the day. So with that in mind, race assessment again, a very important step on all these addressing the challenges of nitrosamine. So the big question was, okay, I have to conduct race assessment. How do I do this? And yes, we have ICHM7 guidelines and we have different guidelines published around best practice or suggested procedures to conduct this. But there's really no place for us to come together and have a discussion on these topics. How do I address some of the questions that I might have when I'm running my race assessment? How do I, you know, every time I face a challenge, how do I overcome these challenges? And not necessarily looking at the FDA or looking at USP or looking at an official body to provide me an answer, but really my colleagues. So with that in mind, we decided to create this space, which is the nitrosamine community focusing on race assessment. And it's really a forum for scientists. So Amanda, can you go to the next slide? Wonderful. So what is the nitrosamine exchange? It's really a place, a forum where anybody that it's really working on nitrosamine can come and participate. It's a place to have discussions around different ideas, but practices, chair concerns, chairs, opinions about what's the probably the best approach to address all this. And really the idea of all this is for me, right as a user or participant of the community to be able to connect with a large audience and a large amount of expertise. USP is in the quest of gathering all the really experts out there on the topic of nitrosamine and race assessment to come together into this community so we can have that dialogue. The common team in this community, it's science. So we're not here to say, hey, I do this is this right or this is wrong. It's really a place for us to chair science, to chair knowledge around the topic of race assessment and nitrosamine. So it's a really good place to have that scientific discussion. What is it for USP? As you probably know, USP does not have a standard, a chapter on how to conduct race assessment. Neither it's on the plans to conduct or create that chapter. But it's really, we found that this was definitely a need for industry and our stakeholders. And we decided to be the host of this space for encourage and being able to have that dialogue. The next slide, please, Amanda. So it's a common question. What is it and what it's not this place? So what it is, it's a place to support USP efforts to increase the connectivity with stakeholders and help stakeholders connect with each other. It's a place that it's monitored by USP, meaning that we will monitor some of those discussions. We will try to provide the rail guards to maintain that scientific discussion. It's not a place to, once again, it's not a place to influence policy. It's not that I'm going to raise my voice and that voice is going to be raising from our regulator. This is really for a scientific discussion. It's not focused on the chapter. So again, this is not the place for us to come and ask questions about the chapter. It's not focused for that. It's really about sharing knowledge and sharing best practices among the industry and among the affected parties. If I'm a finished dosage for manufacturers, can I have those dialogues with API manufacturers, with excipient manufacturers, with packaging manufacturers? I see some questions being raised around packaging. So in fact, we are having some really interesting discussions in the forum about the packaging, the role of the packaging, and the whole strategy of nitrosamines. Is it a high risk? Is it a low risk? How do we go about identifying those kind of stuff? So again, really good discussions happening right now. Once again, this forum or this community does not by any means substitute the official processes of USP. Once again, it's not a venue to influence policy or introduce changes to the chapter. There are mechanisms for that. So with that, I will basically encourage everybody to go visit us. If you want to go before our first slide, Amanda, two slides before, so people can have the address. So there's the address. I'll encourage everybody to go visit, join the community and have a look and participate. We really want to hear your voice. We really want to hear the issues that you're having, the concerns that you're having, the feedback that you're receiving from your regulatory body about these approaches so we can all help build this knowledge together. So with that, I'll pass it to Amanda and Christian. Thank you. Thank you very much, Neifer. Thank you very, very much, Amanda, for this very great presentation, really great presentation. As Neifer mentioned, we have a couple of questions, and let's start maybe with the one referring to the stimuli article that you were referring to in the early slides, Amanda. Considering the stimuli article for elastomers in inhaled drug, is it in the plan to also include nitrosamines in the USP chapter, general chapter 381, elastomers in injection? Do we have any information? So we still don't have any formal decision around that. The comments received after this stimuli article publication, they are still being addressed and we still don't have this information. But again, as I shared earlier, this might trigger the refill of the chapter 381 where we could potentially include those methods using a thermally energy analyzer coupled with gas chromatography. Thank you. Another question that we have here, how does high resolution help with separating the isobaric components? So as I discussed, when we work with high resolution mass system, we have a higher ability to distinguish between two compounds, which will have a very high or close M over Z value. And then we are able to resolve those compounds. Okay, thank you. There's another question. Has USP considered Santé and FDA-like confidence stores that are applied to residue testing in food, for example, and in veterinary medicines? Can you please repeat the question? Yeah, it's probably to be answered with a no, I would guess. But if we have considered Santé and FDA-like confidence scores, it seems from the question that it is applied, that it is coming from the residue testing in food and in veterinary medicines. Confidence scores in Santé, that's the regulatory agency in one of the, oh, sorry, that could be the French regulatory authority. But FDA, for example, they have confidence. Let's go to the next question. I think we need to get into another question here. Maybe here's one, maybe also for Nifer. When in vivo, mutagenicity assay is negative for the nitrosamines impurity, can it then be a limit for the increase and for the, can it be limited there as per the Q3A and Q3B guidelines? I would like to. Yeah, sure. Thank you, Amanda. That's a really good question. Technically, right, if we just follow ICHM7, I think the case that the person raised, it's what it will probably be classified either class 3 or class 5 within the ICHM7. Those are really impurities that raise a flag during the destructor assessment, but prove not to be genotoxic when we go to the in vivo. However, we have seen a special attention from regulators to this kind of impurities, and there are, you know, some people might argue that some of the regulators approach cannot deviate from the ICHM7 because they are known of some of the data, right, the tox data of these impurities. So I would answer to that question technically, yes, we can classify it as a class 3 or class 5, but I think, you know, additional conversation needs to happen with regulators, given the special treatment that nitrosamines are receiving from regulatory bodies around the world. So, you know, that that approach should probably should be discussed with the regulator on the case specific. Thanks, Nifa. Maybe another one for Amanda. Which instrument LC coupled with MSMS or GC coupled with MSMS will be the first preference for analyzing nitrosamines in API and drug product? Well, this will totally depend on the nature of nitrosamines that you might see for me in your product, right? So some considerations regarding LC or GCMS. So we know that when we work with GCMS, we might have some limitations regarding volatility. So we require that the nitrosamines should be volatile or semi-volatile, right, to be eluted through the GC column and be detected. And in case we have some nitrosamine impurities that might have a higher boiling point, then probably we would have some limitations regarding using GCMS. Then we need to use LCMS. I think another point is some thoroughly bio or some nitrosamines with candy grade at high rate in high temperature. And then maybe it would be interesting to try to work with LCMS where we are working with reduced the temperature in the ionization source. And some observations that we see it is that by using comparing ESI and APCI, because APCI when we use LCMS, right, we can use both ionization sources, for example. And in the APCI, it's interesting that we try to work, for example, with a lower temperature in the ionization source in order to decrease the potential degradation of nitrosamines because normally we apply high temperature because we need to vaporize analyte and solvent into the gas phase as the first step to ionize later the solvent and then the analyte, right. So we would have those limitations. So we can say that using LCMS, we have a broader coverage of nitrosamines, right, but we need to know if we have a good selectivity, right. And something important to mention about the GCMS analysis and you can find in the section 7.1 of the chapter would be considerations for analyzing samples, right, as I described and I discussed when we have some APIs which might have a diopteramine group embedded in the molecule or the presence of a processing purity, which might be precursor of nitrosamines and they might degrade, for example, the API might degrade at high temperature. It would be interesting to use LCMS or we try to work with selective extraction protocols to avoid the dissolution of the API in order to have this present in dissolution, right. Those are some key aspects that we need to take into consideration. But again, the first step would be risk assessment regarding gathering prior knowledge about the physical chemical properties of the compounds present in the product. And then this will guide us to select the most suitable analytical technique. Thanks, Amanda. There's another question coming up here, maybe to both of you. Let's see. When it comes to determination and control of specific API related nitroso impurities, other than the seven common ones that are listed in the FDA guidance, is the use of a surrogate compound in method development, validation and quantification recommended? And if yes, what aspects would you recommend that should be taken into account regarding the selection of that appropriate surrogate in terms of uncertainty factors, method parameter setting and so on? This is a good question. When we use surrogate compounds, basically, we would expect that those surrogate compounds would behave very similar to the analyte, right. And in case, for example, you're using a surrogate, which will not have a similar chromatographic separation and will load a different time window different to the analyte that you would like to quantify and compensate, for example, for matrix effects and so on. One, it can be that if this surrogate compound is subjected to a different MS variation or ion suppression, it will not correctly compensate for a matrix effect and then we can have inaccurate or unprocessed method. So the first thing to select the suitable surrogate would be to see if this will have a similar ionization efficiency to the target ion, to the target nitrosamine, for example. And if this will be or would have the same degree, would subject it to the same degree of ion suppression in case this is happening when compared to the analyte. Thank you. One other question and maybe we can do one or two more. One other question here. We are often requested to test unconventional or custom synthesized nitrosamines. In your opinion, how should be reference standards or deuterated internal standard supply performed in those cases? If I got it right, the question is regarding the need of identifying or monitoring nitrosamines, different nitrosamines, which are uncommon nitrosamines in different products. Am I right? I would think so. This is the case. I think the first step again is risk analysis. And after risk analysis, in case those uncommon nitrosamines might be formed in the product, for sure then it's important that we have methods to be able to monitor. So methods which can control those uncommon nitrosamines under the safe level and then would be interesting and important, actually, to have a testing method which is selective, sensitive for those uncommon nitrosamines. But in case, during the risk analysis, it's not identified this uncommon impurity to be potentially formed, then actually you wouldn't need to use or confirmatory testings to prove that this impurity is not there. Okay, thank you, Amanda. One more question here. I think we have not talked about that too much here in the presentation. But how do you set limits of nitrosamine impurities and total nitrosamine impurities here? Yeah, go ahead. Yeah, no, I think we can we can there refer to the nitrosamine presentation that we had last year in autumn that NYFA gave and which was which was more an introduction into the topic where it was also explained how the nitrosamine impurity limits are coming up, especially for the valzatans. And that that is, of course, also the principle how you do it normally for for others. So we I can imagine or I can offer to refer to that presentation. Once more, it's recorded. It's an OIMIA Burbina playlist. And I can when you when I sent round the PDF, I can send a link to that presentation as well so that then the audience or the question I hear from the audience can look into that in more in more detail here. I think that's probably the best thing to do here. We cannot go to to the nitrosamine impurity limit setting, of course, now here in the question and and answers. But one other question, sorry, I just would like to add a follow up comment on that, because when we identify potential formation of more than one nitrosamine, then just would like to mention here that based on the FDA and also EMA recommendations, we should have a total this the sum of the total nitrosamine shouldn't exceed the acceptance intake of the most potent one, right, which is 26.5 nanograms per day. I just would like to to to add that. Okay, thanks, Amanda. We have maybe now I mean we need probably to close up. It's it's 12 minutes past the time. I think maybe one quick question here. Do we need to study nitrosamine impurities for topical creams and ointments? So not for for oral dosage forms. Well, I think the first thing again, it is to conduct risk assessment. And in case for this kind of product, it might also be there is a potential formation of nitrosamine. Then I would say that, yes, Nifer, would you like to to add some comments regarding risk assessment? No, I mean, I would say you you you have to have risk assessment based on these impurities. I think the expectations from the agency, it's that it doesn't really make distinction on on the type of formulation. But actually, you know, if it's chemically synthesized or biologic, so we saw that in the case of you of FDA, it's really chemically synthesized. APIs need fall under, you know, the whole umbrella of nitrosamine. In the case of Europe, it actually includes biologics. So, yeah, I would say it has to, right? It's a different kind of administration. But again, at the end of the day, we're being exposed to those nitrosamines through a different way through a topical steel. So, yeah, risk assessment, it's probably your first step. Excellent. Thank you very much, Nifer. Thank you very, very much, Amanda, once again. And also, thank you to the audience from from my side. I give now the floor once again for the closing remarks to Jan. Thank you, Christian. Yes. And thank you, Amanda. Thank you, Nifer, for a wonderful presentation. We hope everyone has enjoyed it. We just have a quick further announcement to make. We will have our next webinar on June 15th, the Valley Pharmacopoeial Reference Standards presented by yours truly Christian. Would you just like to mention a sentence or two about what it is you're going to be talking about? Yeah, thanks, Jan. I can do that. The presentation is about the value of our pharmacopoeial or pharmacopoeial reference reference standards overall. And we will talk a little bit about the origination of USP. Then we go directly into the media series and talk about conclusive results and about uncertainty aspects there and we'll draw a few conclusions from that. Excellent. And we'll also, sorry, we'll also refer a little bit to secondary standards. I forgot to mention that. Yeah, secondary, exactly. Yeah. Excellent. Participants, if you'd like to have more information, please visit our homepage at www.usp.org. And on the upper right hand, the second tab, it says events and training. If you click on that, you will get a full calendar overview of all the webinars and pharmacopoeial education courses and workshops that we are offering for you. So please try to check that out. If you have any further questions that we weren't able to feel today, please feel free to write to us at emea.usp.org and we'll make sure to get you those answers. Christian, the presentation of today will also be available later on for those registered. Yeah, we will send the PDF version, not today, but in the next days. And we will also, we have recorded the session. We will also upload it to our EMEA webinar playlist and I will give information to all registrants of the webinar when this is available. Wonderful. We will have our webinar as mentioned with Christian, and then we're going to take a break and probably resume again in fall, autumn of this year. Let's see how it goes, we hope. But that is in the pipes. We'll be updating you and hope to see you then again soon. All right. Well, with that, we conclude. Thank you so much again, everyone. Please try to fill out the survey once you exit. It's brief. It's very helpful for us. And we hope to see you soon again. Please stay safe. Please stay healthy and take good care of yourself and hope to see you soon again. Thank you. Thank you, everyone. Bye-bye.