Hello, from the National Archives Public Programs and Education Team. My name is Missy McNat and I'm an education specialist in Washington, D.C. And welcome to the July National Archives Comes Alive Young Learners Program. You can find information about future programs on the National Archives website, archives.gov under events, and on the National Archives Facebook page. Today, we will meet President Theodore Roosevelt portrayed by Joe Wegan, historian and Theodore Roosevelt impersonator since 2002. And over the years, Mr. Wegan has portrayed Theodore Roosevelt in all 50 states. President Theodore Roosevelt was a soldier, a scholar, an explorer, scientist, conservationist, historian, and author. He was often known as Teddy and TR, and he remains our nation's youngest president. We will hear about many of President Roosevelt's accomplishments today, including the fact that he was able to double the number of national parks, despite his enemy's foes in Congress who declared not one red cent for scenery. The National Archives has in its holdings numerous documents related to Theodore Roosevelt. And here are two of the documents. On the one is a portrait of Theodore Roosevelt with his iconic pince-nez glasses. And the other is a Clifford Berryman political cartoon showing Theodore Roosevelt heading down the Mississippi River on a steamboat. And in front of the steamboat are partially submerged logs and branches that show some of the issues that Roosevelt faces during his administration. But look closely and you will see a teddy bear or cub bear sitting on the bow of the steamboat. Clifford Berryman used that cub bear to symbolize Theodore Roosevelt throughout Roosevelt's administration in his political cartoons. And eventually a toy store picked it up and it became the stuffed teddy bear. In this slide we see the Docs Teach activity for today's program about Theodore Roosevelt, our conservationist president. And we will share this slide again at the end of the program. At the end of President Roosevelt's presentation we will have a question and answer session with him. So please write your questions in the YouTube chat box and we have a National Archives staff member who is monitoring it and let us know where you're watching from today. This program is brought to you by the National Archives Public Programs and Education Team, the National Archives and the National Archives Foundation. And now it is my great pleasure to introduce to you the 26th President of the United States of America, President Theodore Roosevelt, a truly remarkable man whose unparalleled energy, dynamic personality, quick wit and intelligence enabled him to accomplish a great deal during his administration and he was the most popular president up to his time. Thank you President Roosevelt. Thank you Ms. McNat and thank you to each and every one of you who are viewing today or who may view this program in the future. I am indeed Theodore Roosevelt. I come to you today from Madora, North Dakota. It's the gateway to Theodore Roosevelt National Park and it is the future home of the Theodore Roosevelt Presidential Library and Museum which will be working very closely with the National Archives to meet the mission of a presidential library here in the beautiful Badlands. We can come back to it but your viewers, your listeners might know that I said after my presidency I would have never been president but for my experiences in North Dakota and that I wrote of my time as a rancher, a writer and a hunter here in the Badlands that this is where the romance of my life began. The theme today is conservation and I am the conservation president. When we say conservation we are speaking of our natural resources, the water, the lumber, the minerals, the wildlife. All of that is something with which I was greatly concerned and my concern and interest in nature began as a little boy. In New York City, New York, then in my childhood, New York City was a city of a million people and half a million horses. The city was covered in sewage and we burned coal fires in the fireplaces such that the city was covered with a heavy black soot but even under those conditions a small child like me could find nature in nearby Gramercy Park or in the great Central Park designed by Mr. Olmsted, a wonderful playground still, or along the rivers, the East River and the Hudson River that define Lower Manhattan Island and as a little boy I collected snakes and bugs and turtles and birds, squirrels and rats and mice, alive or dead it did not matter. I brought these creatures home and had on the fourth floor of my home something that we called Roosevelt's Museum of Natural History. A very wide ranging, there was any mother might suspect a truly malodorous, a very smelly collection. You had to be careful coming to my house. If you poured yourself a glass of water from the pitcher, a snake might fall out of the pitcher into your glass for that's where I was keeping my snake cool. If you reached into the icebox, the refrigerator, you might pull out a collection of mice awaiting my experiment. One afternoon as a little boy I was walking up Broadway Avenue. Mrs. Hamilton Fish, the wife of the United States Secretary of State was walking down. I thought I should have some display of gentlemanly conduct that report of my gentlemanly conduct might go home to my mother much to my own benefit. I cried before the fall as I passed Mrs. Fish in the street. I tipped my hat and wished her a good day and when I did so, two frogs fell out from beneath my hat and fell upon her shoes. I'm afraid that the only report that went home was of my un-gentlemanly conduct that day. Amongst my prized possessions in our Roosevelt Museum of Natural History was the skull of a harbor seal. I was but a boy five, six years old when sent down to the fish market to purchase something for the family for the supper table. I saw a dead harbor seal. I was so struck with awe and wonder at the sight of that creature that I ran back home, my shopping errand forgotten, grabbed my sketch pad, my pencil, and my folding ruler and I made measurements of drawing of that seal and had wonderful imaginations about the places that that seal had swum. And then I begged the fishmonger for that seal, but he used that seal in its parts until such time as the only thing left was the skull, which he gifted to me. As a little boy, you might know that enjoying nature under those conditions in the city was quite a challenge for me because I was quite a sickly child.  Today, thankfully, the asthmatic child has the inhaler to restore breath hopefully quickly. In my day, we had no such medicine. In my day, the treatment for asthma was worse than the disease. Can you imagine giving a small asthmatic boy a cigar to smoke? The idea was that coughing would stimulate my breathing. I was given bitter black coffee to drink again as a stimulant and I was given Ipacac syrup to drink. Most of the children watching won't know Ipacac syrup. Ipacac syrup is something that you drink in order to see your lunch twice. Unpleasant memories for me to this very day. When I was a small boy, three, four years old, I can recall hearing my mother and father out in the hallway outside my bedroom speaking as if I might not survive the night. When my asthma was at its worst, my father in the middle of the night would have the horses brought round to the front of the house and tied to the carriage with me bundled in blankets in the front seat of that carriage seated beside my father. My father would speed that carriage through the streets of New York in the hopes of rushing air to my young and sickly lungs. Today, physicians tell me that rather than the speed of the conveyance, it was likely the excitement of the ride that naturally produced adrenaline in my bloodstream and assisted me to breathe. It was nature that helped restore my health as a child. Very often, the family would go on long hikes in the hills of northern New Jersey along the Hudson River and out in our beloved Oyster Bay Long Island. We rented a summer cottage there and eventually I would build my home. Today, a national historic site ably administered by the National Park Service, the National Park Service with its first park in Yellowstone in 1872 and then on through our modern era has helped preserve wild places. And this is from the legislation creating the National Park Service, wild places for the use and benefit and enjoyment of the people. That's part of conservation saving the wild places. I'll come back to the point that part of conservation is using what's been given us in those wild places, the lumber and the minerals and the opportunity to harvest electricity from the rivers and dams and that sort of a thing. But it was in my youth, what I wish to convey to the audience, is that as a little boy growing up in New York City, that is where the romance of the relationship between me and nature, wild creatures began. And also there's a great deal of preservation being done of our natural history. That's what we studied. That's what we call the study of nature in my day, natural science. And very often it was the hunter who gave us a window into the natural world. For example, John James Audubon. I would suggest that the children watching today find some of his pictures that he drew the birds of North America being a very important work. But in order to draw many of those birds, in his day, the only way to do so was to shoot the bird perhaps from a tree or in flight. And then having the dead bird taxidermy that bird, study it, mount it in such a way that the feathers and the coloration could be studied and drawn. And that was in its day as close as one might get to making a record of what a particular species of bird looked like. And I took to this very same science when I was a teenager, a boy of 13. I was given my first shotgun and I used that gun to collect birds, including when my family traveled to Egypt and we took a bark up the Nile. I shot and mounted using taxidermy in the Egyptian plover. And if you visit the American Museum of Natural History in New York City, you can see that Egyptian plover that I harvested and mounted using taxidermy. They are in the American Museum of Natural History. By the way, the charter for that museum signed in 1869. But that charter was signed in the Roosevelt family living room, where I had been a little boy. The museum includes typical specimens of animals that I assisted to harvest, most especially the African collection on a hunt and the safari that I made with my son Kermit after my presidency. I wish to address before the time runs out of my formal remarks, of how I also came to respect nature a great deal after coming here to the badlands of what was then Dakota territory. Not yet a state, North Dakota and South Dakota joining the Union of States with statehood in 1889. My active ranching was done here between 1883 and 1897, a period when I was roughly 23 to 28 years old. When I first came here to the badlands, I came to hunt, and to hunt a bison, a buffalo known as a bison-bison. I wanted a trophy of that great North American mammal hanging in my trophy room in my home being built in Oyster Bay, Long Island. And I'm sorry to say that I wanted to hunt a bison before there were no bison to hunt. I knew that the bison was on the verge of extinction, of no longer being alive on the planet. I wanted to hunt a bison so badly, and I hunted that bison and hosted that bison, have that bison mounted in my home, now a museum. But I paid pennants. I made up for the fact that I had not yet developed my thoroughgoing ethic as a conservationist. I helped to found Boone and Crockett, named for my heroes, Daniel Boone and Davy Crockett. Indeed, that organization, the nation's first fair hunting and conservation organization, has done more than any organization to help preserve the big game. That means the wild animals, some of whom are managed through a process in part that includes hunting. That perhaps provides food for the table for many tens of thousands of families throughout this country. We saved the bison from extinction when I was your president. I was able to order an amount of breeding stock. That's both mother bison and father bison, cows and bulls, that they be shipped to the Bronx Zoo, headquartered in New York City at the Bronx Zoo, my friend Professor William Hornaday, and head of the New York Zoological Society. We acquired breeding stock, more fathers and mothers, cows and bulls from private herds in Texas and Oklahoma. We shipped them to the Bronx Zoo. Their Professor Hornaday experimented with the species, bred the species. Many more baby bison were born. We call them calves. And they were sent back to repopulate Yellowstone, to repopulate Wind Cave, one of my five national parks. Also, Sully's Hill. We had bison game preserves in Montana and Oklahoma, where the species was propagated. Today, if you visit Theodore Roosevelt National Park, you can hear these bison, they still speak with a distinct Bronx accent. Followed me through the bull reeds on that one, didn't you? When I traveled as a young man between the badlands of the Dakota Territory and my native New York City to see to family affairs and to avoid some of the bitterest of the Dakota winter, that's when I became greatly concerned. I saw that much of the countryside between had been hunted out. The animals made scarce. The forests clear cut. The rivers polluted by a runoff and industrial waste. I knew that future generations, meaning you, that you would be ashamed of us, not for what we had used, but for what we had wasted. So as your conservation president, when I was able, I was able to protect 150 million acres of national forests, declare 18 national monuments, including the Grand Canyon. I was able to set aside 51 bird sanctuaries, now known as our first 51 national wildlife refuges. And I think I would conclude my prepared remarks by reviewing how the first came into being. I love birds. As a little boy, you heard of my glasses, my pince-nez glasses, that means pinch nose, and they do. But as a little boy, I didn't know that I could not see well until I was 13 years old. I went about learning my birds, not by sighting them, but by listening to them. And I became quite a bird watcher and bird listener throughout my lifetime. And the loss of one songbird for me would be like the loss of some great body of works by some great author. And so I wanted to help save the birds. In 1903, when I was your president, I was day-losed at the White House by letters and telegrams from members of the Florida Ornithological Union, members of the Florida Audubon Society. These are both bird watching and bird science organizations. My friend, Dr. Frank Chapman, was the head of the American Museum of Natural History's Ornithological Department, Bird Department. He did his fieldwork. He studied the birds on the coasts of Florida, from where he and the others complained and reported that for another season, millions of nesting birds would be slaughtered and their nestlings left to starve. All that mostly women might wear the feathers of those dead birds on their hats and on their dresses, sometimes wearing entire dead birds as adornment. There's no explaining fashion. But greed is understandable. An ounce of white egret feathers meant for adornment, they were worth more than an ounce of gold. Plume hunters, poachers, people who hunted against the regulations, they had wars with the game wardens. Two Florida game wardens and one South Carolinian were killed during my presidential administration by these poachers. So I asked my attorney general, was there anything in the laws of the constitution of the United States which would prohibit me from naming a small mangrove island, the last nesting place on the Atlantic of the brown pelican as a federal bird sanctuary. This little island south of Melbourne, north of Euro Beach off the coast from Sebastian. I, when informed there was no express prohibition against naming it so, I said I thus declare it. The first of 51 bird sanctuaries declared during my presidential administration. Nature is a wonder. And our parks and a fish and wildlife service, our forest personnel, they all assist us to make sure that our children and grandchildren can enjoy the wild places. And also in the spirit of conservation, some of you youngsters might study the life of my national forester, Gifford Pinshow. But we understood that conservation meant the wise use and utilization of our natural resources. That indeed was the responsibility of each and every generation to use those natural resources, especially of the public lands. But to do so in a way that left those natural resources, the water, the air, the timber, the land, in better condition than we found it and not in worse. And in summary, that's what we mean by conservation and why I am still remembered as your conservation president. But as Ms. McNat said in the introduction, perhaps I'm actually best remembered for the legacy of the teddy bear. And that seems like a square deal to me. With that, I think seeing that I've done what an old politician does, I've gone and run on now, haven't I? But I'd be delighted at this point to take any questions from our viewers, our listeners, any from our guests who are online now. Well, thank you. That was wonderful. And I learned so much about your early life and that was great and all the conservation.  So anybody else wanna jump in? We'd love to know where you're from, so thank you. So we do have some questions and I had mentioned that you are still the youngest president and how old were you when you became president? That's one of our first questions. 42 years and 11 months. You know I sadly came to the White House through the graveyard. I was the vice president when President McKinley was assassinated in September of 1901. So for those that keep track, I'm not the youngest president elected, that would go to President John F. Kennedy, the youngest president ever elected. I ascended to the presidency and can claim correctly to be the youngest president in our history to date. But maybe we have some young viewers who themselves may someday serve as president at the age of 40 or 41, which is the constitutional qualification. Or is it actually, I think it may be as young as 35. I must check my constitution. I believe it is 35. 35? Yeah, so those young folks out there, you can start working towards that. So here's another question. You mentioned the national parks that were created when you were president. Were you able to visit all of them at some point during your life? It's interesting that you mentioned that for of the five national parks that I created, I would say that I visited none of them. They were all reported to me. They were all rather remote in their day. So this includes Crater Lake National Park in Oregon, Mesa Verde National Park, the Pueblo Ruins in Colorado, and Wind Cave in South Dakota, which was also in a remote part of the Dakota territory, Platte National Park, the Hot Springs in Sofa, Oklahoma, and Sully's Hill, which is now a game preserve renamed White Horse Hill. So oddly, I never had a visit to any of my, I was only able to name five national parks. There were five in existence. Of course, I was able to visit Yellowstone and camp there for two weeks. I was able to visit Breton National Wildlife Refuge, the bird sanctuary off the coast of Louisiana. But an interesting question that I've never been asked before. I did not visit any of the national parks that I created. Ah, okay, well, thank you. So we have a question now from Reagan and she, Reagan, I don't know if it's Reagan's he or she, wants to know what it was like to be the 26th president and then going along with it, did your kids really drop water balloons on the guard's heads and misbehave? I never heard that one before about dropping the water balloons, but. The children at the White House and this to Reagan, I was the 26th president. I served in the White House from late September of 1901. We allowed Mrs. McKinley to take all the time that she needed as a bereaved widow to move out of the White House. But when we moved in and for the following seven and one-half years, Mrs. Roosevelt and I reared six children at the White House ranging in ages from four to 17. The head White House usher, Mr. Ike Hoover, he was the head usher for decades. He wrote to the Roosevelt years. It was no time for a person with a nervous constitution to be working at the White House.  Well, came careening down the grand staircase, riding on cookie trays, stolen from the kitchen as if it was a great toboggan. And it was the little boys, the youngest Archie and Quentin, seven and four years old when we entered the White House as those boys grew, so did their mischief. They and their friends known as members of the White House gang and my recollection, not of water balloons, but of a giant 30-pound snowball, mostly ice, that when I left the White House after a heavy midwinter snow and ascended an open-air carriage for an appointment, one Washington DC police officer gave me a crisp salute. Just as the tips of his fingers hit the brim of his helmet, so did this giant 30-pound snowball drop from the White House roof. The snowball smashed through the officer's helmet and left it knocked out cold on the driveway. I did not need to investigate. I turned over my shoulder, I said, boys, get down here immediately. Down the little boys came, they apologized profusely and sincerely. And they meant to frighten us for fun, not to harm anyone. I looked into the issue and I discovered Reagan that that officer was long overdue for a promotion. It's good to be president. And as far as being the 26th president, I don't think any president before or since ever enjoyed themselves as much as your president as I did. I said, when you work, work hard, when you play, play hard, when you work, don't play at all. But when the work day was over, I played hard as your president. In the White House, I boxed with army and Navy officers. I wrestled jujitsu and sumo with great bare-chested men brought over by the Japanese ambassador. And I took my point-to-point hikes out across places like Rock Creek Park, scrambling across the rocks and through the river. And when we played point-to-point, the rule was that whenever you came to an obstacle, a downed tree, a cliff, a barn, a haystack, you never went around the obstacle, but always and only over it, under it, or through it. It makes for great dirty fun. I recommend the viewers play that at home. And I will say in all earnestness, Reagan, there was very important work to be done when I was your president. Those years that I served from late 1901 until early 1909, well, that's nearly a decade. And the century is known as the Great American Century. And I think perhaps the work that we did in the Roosevelt administration during those seven and one-half years helped to launch the efforts that would become known as the Great American Century by building up our Navy, by building the Panama Canal, by filling in the country and caring for this issue of conservation and indeed taking the world stage to, for example, negotiate peace between Russia and Japan with the Treaty of Portsmouth, New Hampshire, which earned me the Nobel Peace Prize, but more importantly, saved hundreds of thousands of lives amongst the combatant nations. There was important work to be done. So while I had fun being your president, I was also glad to hopefully make some contribution to the accomplishment of important work being done. Yeah, it's amazing the numerous accomplishments. So somebody from Connecticut has joined us and this sounds, this next question sounds like someone who has really knows a lot about President Roosevelt and it says, Mr. President, I think I remember seeing a picture of you and your brother as children viewing the funeral procession of President Lincoln from your home in New York City. Can you speak to that? It is quite a famous photo taken during the April funeral parade, bringing the martyr presidents remains back along the route to Springfield, Illinois, the route that he had come in 1861 to take his inauguration. It was from the second floor of my grandfather's house at Union Square that when you see the photograph, you can see the silhouettes of two small boys watching the parade below. That's me and my brother, Elliot, sometimes called Nell or Nelly in the family. What you do not see is that my future bride, Edith Carroll, she had been watching from the windows, the parade below, but she was younger than Nelly and I and she was distraught by the sadness of the occasion and the sight of the wounded Union soldiers in the parade, the sad funeral music being played. She was crying and shrieking. It was annoying as greatly. So Nelly and I picked her up and locked her in the closet, a sin for which she apparently forgave me in the intervening years, but an event of which she would still say in later years that horrid little boy, meaning me. That's a great story. I've never heard that one before. That's terrific. So you talked about all the incredible accomplishments during your administration. You had mentioned many of them from, you know, earning the Nobel Peace Prize and the parks, but is there one that you think was more important than any of the others? Most definitely so. And that is the building of the Panama Canal. As you know, it had been a vision of mankind, at least of that portion of mankind that was sailing the oceans to somehow find a pathway through the isthmus of Central America, since the explorations of the Spaniard, Balboa, whose name is remembered now in Panama. And so the United States had even since the time of President Grant been exploring routes, perhaps through Nicaragua, taking advantage of the giant in Linda Lake, Nicaragua, but the French had attempted to build the canal in the 1890s. And this was under the engineer, De La Cepes. He and his crews had built the Suez Canal, opening up that portion of the world to commerce and transportation. And the French thought that they would build a similar sea level canal, but we thought better after the French failed and after more than 10,000, mostly Frenchmen, but also nurses, they died of yellow fever. They did not understand the disease. So when we began the work of the canal again in 1903, completed under President Wilson in 1914, it was not only a great work of engineering and excavation, but also a work of medicine. Dr. William Crawford Gorgas, later Surgeon General of the United States by my appointment, Health Commissioner to the Panama Canal Commission. It was he having worked in Havana with the Cuban doctor Carlos Finlay and with the famous United States surgeon in the army, Walter Reed, for whom the famous veterans hospital in Washington is named. These men together had the right idea that yellow fever was transmitted by the bite of the ages, Egypt time mosquito. And so it was Dr. Gorgas that saw all the rain barrels poured out, all of the ditches sprayed with oil and kerosene to keep the mosquito from breeding, while even putting screams on the doors and windows for the first time so that by 1907, there were zero cases of yellow fever amongst the American workers that helped to lead the effort to dig the canal. And on this point I must acknowledge that workers from Southern Europe and from the Caribbean islands, it really was a work done internationally. And many of those men working with shovels and dynamite and many sacrifice to accomplish what I thought the greatest work of my administration and that completed under Taft and Wilson, the building of the Panama Canal primarily because it cut to one third the amount of time necessary to move our Navy, our naval assets, our battleships from one ocean to the other because the protection of the sovereignty of the United States, our ability to rise to the challenge, if given, that can only be met by the United States Navy. Moving that Navy from one ocean to the other during the Spanish American War, it became highly evident that that was a primary responsibility and achievement that was most necessary for the defense in the future of the United States of America. Oh, that's great, yes. I mean, that was a huge, huge accomplishment. That's for sure. So, Reagan has jumped in again and she tells me she's an eight-year-old girl. So, but she has asked another bit of a question and that is, do you have any advice for someone who wants to become president? Most definitely so. And that is to listen to your mother and your father to find joy in reading books. I really have no care for the subject matter about which you like to read. The varieties of taste in books is just about like the variety of taste in art or food. But if you like to read and if you listen to your mother and your father, very likely you could be a citizen who would serve as a good and competent president. But most especially, I would say, Reagan, go forth forgetting any idea about being president. Start out the way I did. I wanted to be a good son and a good brother. Eventually, in my life, I had the opportunity to attempt to be the best I could be at being a good husband and a good father. When Ms. McNat asked me about the most important accomplishment, as president, it was the Panama Canal. As a human being, as an American, my greatest accomplishments were in these familial relations to have had a wonderful marriage with my wife, to have known at the time of the passing of my father and my mother that each one of them considered me to be a good son. Oh, as pedestrian as it might see in this hurly burly of a world in which we live, the love that we have in our homes for one another, the encouragement that we receive from one another and then the duty that we have because Reagan, to whom much is given, much is expected. There's not a lot of responsibility for an eight-year-old except to listen to mother and father and do those chores which you're assigned. In my family, though, we often wanted to express the love and care we had for one another and that meant more than just saying I love you. It often meant that in the evening after my father had labored hard all day that we children might do a recitation of poetry or share with the mother and father some of the Bible verses that we had committed to memory earlier that morning in our studies.  And as you continue on in your education, if you're able to take care of yourself and your family's able to take care of things about them, well, then at your earliest opportunity, stop thinking about how you might help others, your classmates, your neighbors, maybe some older lady or old gentleman who lives down the street who has no children or grandchildren of her or his own. Maybe there's a little chore you could do for your neighbor. Maybe there's something you could do for your teacher at school. You do those things well and then maybe you'll be ready to do the things well that are required to be done well by the president of the United States. Well, that's fantastic advice. So we have time for just maybe two more questions and one is somewhat similar to the one that we were talking about. But before we get to that one, can you recommend any books for both younger and adult readers who would like to learn more about and study your life? I shall. First, of course, my autobiography. Not that I'm receiving royalties on it anymore, but written in 1913 after my 1912 presidential campaign, while it does not include the last seven years of my life. It does include one of those stories about my youth in New York City, about my time, cattle ranching. With regards to a book that I think is accessible for the families, there's one called, Mornings on Horseback by David McCullough. Now that might be a book read aloud by a parent to a child, as is another book called Hero Tales. I was at dinner with Mrs. Roosevelt and with Mr. and Mrs. Henry Cabot Lodge, Fanny Lodge. And Henry and I, I called him Cabot. Cabot and I, we said that we should write a little book of biographies of our heroes. Well, Mrs. Roosevelt and Mrs. Lodge said, well, stop talking about it and get about the work. And we did when I was a United States Civil Service commissioner in the early 1890s. Cabot and I published Hero Tales. Most of our heroes are from the Revolutionary War and the Civil War, but you'll also find one of my heroes, Francis Perkins, who was a historian and who wrote The Oregon Trail. Parkman, I'm sorry, Francis Parkman. And you'll find that he's one of my heroes because Parkman wrote his history at a time when he was tremendously ill, nearly blind, neurological issues. He had enough strength to get out of bed and dictate a line or two of history before crumbling from the effort. Well, he's a hero too, so it's not just the generals and the heroic soldiers and sailors of the wars that are our heroes, but that's a wonderful book. Those are the two that I'll recommend. And for those with a sense of adventure, by Candice Millard, she wrote the wonderful book, The River of Doubt. It tells the story of my explorations of a river in the Amazon never previously explored, Colonel Candido Hondon, the Brazilian army, he for whom the Brazilian province of Hondonia is named. He and I were joined by my son, Kermit, and we successfully explored the Rio da Duvida. And today you can look it up on the map as Rio Roosevelt. We put the family name on the map. Candice Millard's The River of Doubt, a wonderful book. Well, thank you. I hope everybody's been writing those down. And then our last question, but actually kind of connects with what the advice you were giving to Reagan earlier about what to do as a president, but is there any additional advice that you would give to young people today in 2021? Find where you can be of assistance to this country. We live in a land of freedom and opportunity that has come to us, not free, but at great sacrifice by previous generations. And so to reiterate to whom much is given, much is expected. It is very likely that Reagan and some of our other young viewers and listeners, they perhaps are receiving some of the greatest educations available in the world. And I hope that they understand that when it comes to graduating their high school or graduating their college, and then going about the work that they've chosen in their lives, that if they want to bring honor to their alma mater, to the schools that they attended, then they must find some way to make the world a better place for their having been here. And that means service, whether it's service through your school, your own initiative, through your religious community, your temple, your mosque, your church, your synagogue, get involved in some way in the activities that make this country a better place. And then good things will happen from it, including opportunities to serve perhaps in some capacity that makes a big difference. But I'll tell you, Ms. McNat, I think a member of a school board can make just as big a difference in this country as the president of the United States. The challenges are many, the workers are too few. That, yes, that's true. We all do need to work in many different ways, in whatever way we can to make our world a better place. And you're right there, whether it's through conservation efforts or whether it is through something that you do in service to others, absolutely. So wonderful advice, wonderful, wonderful. And thank you. I cannot thank you enough for joining us today. President Roosevelt, you have shared so much with us. And I know that our viewers have had a delightful time during this past 40, 45 minutes or so. So thank you again. And we appreciate you joining us. And goodbye to everybody out there and thank you to everybody who joined us. And I hope you'll join us for our next program on August 26th, where we meet somebody very different, Ida B. Wells. But yes, yes. Fantastic. Thank you very much. And I just recognize if I may, by presidential prerogative request, a last word. Sure. A bit of inspiration, if I may, to your viewers. Keep your eyes on the stars and your feet planted firmly on the ground. Believe you can, you're halfway there. Do what you can with what you have, where you are. And thank you so very much. Well, that's wonderful advice. Thank you, thank you. A good rest of your day and take care. Goodbye, everyone.


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.