Episode 10: Insights into Toxicology Lab Practices and Enzyme Developments (featuring Professor Thomas Rosano)

Andrew Lee: [00:00:00] Welcome to the podcast series. Imagine More Create Solutions. We’re gonna talk about enzymes and drug testing, especially related to laboratory operations, clinical diagnostics of urine drug testing, and running a good toxicology services and toxicology laboratory practice. Hello, my name is Andrew Lee, co-founder and Chief Scientific Officer at IMCS.

In this episode, I host a very special guest, Professor Thomas Rosano. He’s a professor Emeritus of Pathology and Laboratory Medicine at Albany Medical College. So we’ve come to meet each other, because IMCS is a supplier, we manufacture and provide key reagent called the IMCSzyme and IMCSzyme RT. This is a critical component of the urine drug testing market, and I have in front of me is 27 page CV.

But instead of me doing all the talking, I’ll pass it on to Professor Rosano, please.

Thomas Rosano: Thank you, Andrew, for the invitation to be a guest on your podcast series. I’ll give just a synopsis of [00:01:00] background. I started my career as a clinical chemist and a toxicologist with a fellowship at the University of Washington in Seattle back in the 1970s, and I’ve been practicing toxicology ever since.

Over 30 years, I’ve served as a clinical chemistry and toxicology director at the Albany Medical Center Hospital, where I remain in the college as the Emeritus Professor in the department. In 1998, relevant to experience, I established a post-mortem toxicology service for a 22 county region and served as the director of that service for over 25 years.

In 2018, I was promoted to Emeritus Professor and became director of the National Toxicology Center, which is just across the street from the medical center. Early this year I stepped down as director at NTC to move back to a more academic setting with a focus on research and teaching and toxicology.

Andrew Lee: [00:02:00] Wow. Professor, I mean, that’s a long history and you’ve covered quite a bit of time and experience, so can you touch on how this toxicology industry has evolved over those years?

Thomas Rosano: Probably the most, uh, striking thing to me in that evolutionary process is the exponential growth in the number and the diversity of both therapeutic as well as designer drugs that have come into use and abuse over the many years.

The field of analytical toxicology has also evolved along with that in response to the new and novel psychoactive agents. And with that, there has been a significant increase in both the sensitivity, selectivity of MS technology and the analytical application methods that we are currently using today in order to adjust to that particular area.

For example, when I started in toxicology, we were using GC-NPD [00:03:00] for general unknowns, and that was quite a few years ago, but quickly moved to GC-MS technology. I took part along with other toxicologists in advancing from GC to LCMS technology for general unknown screening. Thought to be heresy at that time in terms of using LC, but we went and developed, validated nominal mass LCMS methods, and then went on to further advance with high resolution MS with an ever expanding library of exact mass fragment data that we use today on it.

There are other areas of forensic and clinical toxicology, but that have been moving away from presumptive testing. So I’ve seen that in process and we’ve been part of that movement away. We’re now using quadruple, tandem MS as well as QTOF, as needed in, in testing, not only in confirmation testing, but in initial testing.

And we’re [00:04:00] also developing the use of not only abused drugs, but psychiatric agents. Which are important in the evaluation of addiction itself, and we’re moving ahead even in terms of non-targeted methods of high resolution as playing an increasing role, especially because of the newer illicit drugs that we’re seeing in our casework.

So there’s been quite a change in the technology over that particular period of time. Along with that is the change in the number of matrices that we’re actually testing for. In post-mortem work, we’ve always done a wide range of blood and solid tissue analyses. Even now in other areas, we’re using hair testing for employment testing purposes, using oral fluid testing,

also now actually accepted for testing in the Federal Workplace Drug testing program, and also in drugs and driving and addiction monitoring. So we’ve lived through a significant challenge that had continues [00:05:00] to cycle through many agents, and I think that’s the resounding aspect of this time period. When I first started barbiturates were the drug of abuse at that particular time.

But we’ve progressed through designer amphetamines, designer benzos, bath salts, fentanyl analogs, synthetic cannabinoids, and we’re currently in a hemp-based cannabinoid, isomer and analog era. So this evolution is far from over, but it is a continual development of both societal drug use as well as toxicologic reaction to that.

In terms of developing our practice, it has been a significant era.

Andrew Lee: Sounds like you’ve experienced kind of phases of the societal uptake in, or usage in various drugs and how these kind of periods have certain drugs that pop up. And then now you mentioned these designer drugs and [00:06:00] barbiturates, and then also the synthetic cannabinoids and the hemp-based cannabinoids.

Those are popping up more frequently. Is that the case?

Thomas Rosano: Yes. And you know, it’s a surveillance that goes on and it is information that comes from many, uh, governmental agencies from other laboratories, uh, and the exchange of information between toxicologists and what’s happening in our own area, because quite honestly, we can have a significant infusion of designer drugs that are not seen in other areas.

So it is a continual surveillance process from an analytical standpoint that we’re challenged with in this practice.

Andrew Lee: And these processes, especially with the new tools you mentioned, non-targeted. So especially if you don’t know these new drugs or these designer drugs, they’re novel. You don’t have a purified standard for it.

And so how you find it is using some of these new tools, right?

Thomas Rosano: Yes, high resolution mass spec with, its really mass precision and ability to determine elemental composition and ability with fragmentation [00:07:00] to, in silico identify potential sources of that particular peak have been important. I’m not saying that it’s a simple process, but I’m saying that the tools are continually evolving and it’s the application of those tools that’s important in our field.

Andrew Lee: You mentioned that you’ve actually done two different areas of work. One is in the toxicology space, but even within toxicology there’s the clinical toxicology and then there’s the forensic toxicology, kind of the broad stream of those two streams. How would you differentiate between those two? There’s a lot of overlap, but then at the same time, what is the difference between those two fields?

Thomas Rosano: I have practiced throughout my early career in clinical toxicology and starting in the nineties in forensic toxicology, and they do have similarities and differences, but fundamentally they are based upon analytical toxicology for the determination of drugs and agents and poisons that are there. The major [00:08:00] difference is the practice setting, uh, one in patient care setting and the other in the courts.

One for purposes of healthcare and the other for purposes of law. But those boundaries really are blurred sometimes. And we do have patients who, with toxicology findings that become matters of law and testimony related to ’em. But there is a difference in the protocols that we do use in clinical versus forensic casework.

For example, chain of custody and confirmatory testing is a mandatory part of the forensic practice, and in contrast, turnaround times set priorities for what testing is going to be potentially available on the toxicology, uh, side. At the Albany Medical Center, where I was the director of laboratories and director of Clinical Chemistry and forensic toxicology, we practiced both clinical and forensic, and we did therapeutic drug monitoring,

alcohol and [00:09:00] glycol testing, lead poisoning, emergency toxicology employment testing along with a postmortem toxicology service for a 22 county region. So you can call those separate entities, but we really did them in a facility that had a clinical laboratory where we had 24 hour services. We had a forensic laboratory, a lock facility, but we really used many of the same analytical techniques in many of the same matrices in terms of samples for those.

My experience at the National Toxicology Center, which is my more recent experience where I transitioned to. The focus has been also on clinical and forensic toxicology, but, uh, includes urine, oral fluid, and hair testing. And it’s, uh, intended application is for the drug monitoring, addiction and pain management.

Also for forensic casework in drug courts, probation departments, chemical [00:10:00] dependency, social services, child custody. So my experience over the years is in both areas, but again, I would emphasize that the foundation really is our analytical methods and their development, their validation, their quality control, their casework application.

And also their research application is another aspect, especially in an academic setting.

Andrew Lee: It looks like you’ve actually done a lot of publications, like you just said over the years as you worked at the National Toxicology Center, as well as Albany Medical Center and these research procedures or these actual laboratory procedures and the analytical methods, you’ve turned them over to peer reviewed journal articles as well as book chapters.

But more recently, what sort of direction is your research taking you now? Is it still within toxicology and is it still in this postmortem toxicology service, or is it specific more towards like therapeutic drug monitoring? What sort of [00:11:00] research are you evolving into?

Thomas Rosano: You’re right, I, I’ve involved in research in both clinical and forensic toxicology over many years, and I’ve tried to communicate each year findings from those.

I have what I call holiday writing when everyone else is enjoying Christmas. I’m enjoying writing up the things that I did during the year, but it’s allowed for a consistent communications. Most recently, the focus has been on the application of definitive drug testing, and I’m really referring to MS based methods where you have specific identification of drugs in those definitive tests, not only in confirmatory testing, which we’ve been using consistently for years.

Also in the initial testing process and, uh, this focus on definitive testing really comes from my experience over the years in, in medical examiner death investigation, where definitive testing by mass spec has been routine in both the initial screening process and the confirmatory process. And two of recent studies that I’ve [00:12:00] done in clinical toxicology have involved

review of case work in, for example, 5,000 Addiction Medicine cases, which was published in 2022 in Pain Medicine, and another study of definitive drug testing in 400 emergency room patients, which was just published this year in JMSACL. And both of those studies have revealed the high prevalence of

therapeutic and illicit drugs use in the clinical population based on definitive drug testing. What they’ve also shown, however, is that if you compare the findings of definitive initial, definitive testing with the, the routine practice of immunoassay testing, you find a a high false negative rate of drug detection with presumptive testing by immunoassay. And it clearly has showed the need for expanding clinical drug [00:13:00] testing and the use of definitive methods.

Recognizing it’s a challenge, but it is essential based upon what we’re finding in the studies of prevalence of drugs and the diversity of drugs.

Andrew Lee: So for both of these clinical toxicology studies and those reports, you mentioned high false negative rates, especially by immunoassay testing. Do you mind expanding and clarifying?

So first, what is net meant by false negative or false positive? Is it as bad as it sounds? And how is it different from false positive or false negative? And why is it so concerning to the audience or just to us as a community?

Thomas Rosano: Well, I think this point is important to clarify, especially for false negative problem and presumptive testing, which is largely unrecognized.

And yet is a significant in treatment impact on it. I, I would start by emphasizing the guidelines that we use as a standards of practice in toxicology and in initial testing for simple [00:14:00] principle. And yet it’s an important principle. We want to use methods which have sufficient sensitivity to detect drugs that we are interested in identifying or may have an impact in that particular setting, whether it’s legal or medical.

The high method cutoffs and the limited scope of a drug screening by immunoassay really undermine this principle and lead to under detection of important drug use. Now, confirmatory testing has been used. In forensic practice and to some extent in clinical practice, but it will only eliminate false positive reports.

The false negatives, however, can be as just as misleading to a clinician as a false positive. They can be just as misleading clearly. In other areas of, in areas of forensic toxicology, we are routinely blind to how many drugs we miss. But in our recent studies with definitive versus immunoassay testing [00:15:00] in both the addiction medicine patients and in the emergency room, and sometimes I refer to that as ED patients, in those cases, the rate of both false positives and negatives has been found, and the rate of false negatives is significant and under recognized, and that’s why I am, that’s why I’m making this emphasis.

Both of the recent studies showed a high rate of false negatives for immunoassay testing. And let me give you a couple of examples. In the ED 22, again, the emergency department, 22 of the 135 cocaine users were missed by immunoassay screening. And in the addiction medicine casework, 33% of cocaine users were undetected.

The incidents of false negatives in benzos, benzodiazepines and amphetamines was even higher at 40 and 38% respectively based on the ED study. Now, the scope of immunoassay is another problem, [00:16:00] and you asked me to define false negatives. If the test is only intended to cover a certain area of class of drugs than if you have negatives in other classes of drugs, you would not call it a false negative, but remember the principle

of toxicology for screening, is to identify drugs that may be of significance to that particular questions in either the medical or the forensic setting. And in the case of assay, there is a problem relative to this additional missing or lack of scope of the assays them themselves. For example, in the emergency room patient study.

66 of the patients who were on fentanyl, which was all the patients we identified by definitive testing, were completely missed in the clinical practice setting because fentanyl screening was not included in the immuno acid panel. So call that a false negative in terms of [00:17:00] practice, maybe call it something else in terms of not having the methodology, but it’s needed for that.

One overall look if you look at all the drugs, because we found 1,350 drug uses, that’s an interpretation of all the metabolite and drug findings, and we had in those 1,350 cases, we had 794 drug uses that were missed by immunoassay, completely out of the scope in terms of what the testing was performed, but also missed in terms of potential impact on patient care.

In the 28,000 positive findings that we had in the 5,000 case study, 13,000 findings were outside the scope of the immunoassay testing. So clearly, when you start to look in terms of the, whether we’re detecting drugs, we are certainly have some work to do in terms of improving our analytical [00:18:00] methods in, at least in the clinical setting where definitive testing is not used initially.

Andrew Lee: So you’re able to find a lot of these missed analytes when you’re using amino assay, and you’re comparing that against the definitive testing. The definitive testing is using the high resolution mass spectrometry. Is that correct?

Thomas Rosano: Oh, definitive testing is actually done by tandem MS. A targeted methodology in our practice targeted for 105, uh, drugs and metabolites, and that 105 is a variable that that continues to get updated with newer drugs or otherwise.

But a large panel of drugs and metabolites are tested in that definitive test.

Andrew Lee: Got it. Got it. Okay. So do you think it’s a common practice that a lot of these are being missed in the emergency rooms or emergency departments as they’re coming in because they’re heavily relying on immunoassays and not by mass spectrometry.

Thomas Rosano: Yes. The study actually clearly shows that in the emergency department, that [00:19:00] is the, the finding. Now that’s a university level practice, so it may have a higher incidence. It’s, it depends upon the patient population that comes in. It’s just a sampling of a single university hospital, 400 consecutive patients that were thought to be, there was enough concern about potential drug use that they ordered a urine drug testing on them.

But other experience that we have is also that we’ve been using definitive testing as an expanded confirmation where we confirm the immunoassay test and then go on to use the entire panel to identify any additional other agents. And that kind of protocol of expanded confirmation testing also shows that assay in other settings, in clinical settings and outside of these two studies is a problem.

Andrew Lee: And in your study, you also reveal a multi-panel drug use, especially involving serotonin. Or different type of serotonergic [00:20:00] drugs. Can you comment on your finding?

Thomas Rosano: Uh, yes. That was surprising to us honestly in that study. Uh, our finding was that there’s largely an unrecognized risk of drug induced altered mental status in the, uh, emergency room patients that were in this particular study. The condition is called the serotonin syndrome.

Which involves a mental status changes along with autonomic and neuromuscular abnormalities, and the syndrome can be caused by couse of serotonergic drugs, along with antidepressants, which are also serotonergic drugs such as SSRIs or SNRIs. And antidepressant users are at risk of developing this syndrome when other serotonergic drugs are co-used.

Yet antidepressants are rarely tested in clinical setting, especially in the emergency room setting. Our experience in [00:21:00] initially in the addiction medicine patients surprised us and showed that 50 over 50% of the patients who were in the study were on psychiatric medications. So in addition to pain medications, they may be moving down the hall to psychiatry and also having psych medications.

And those psychiatric medications are not monitored routinely. We have incorporated them into our methodology. So the panel, definitive panel that we have used in these studies includes SSRIs and SNRIs, and the study in the emergency department shows that 112 of the 400 patients were on these psychiatric drugs.

We discovered in those patients a high incidence of co-use, of illicit or abused drugs that were also serotonergic agents. In 67% or 75 of the [00:22:00] 112 of these patients were using an additional abused or other drug that has effects on the serotonin system and therefore can cause and result in a high risk of serotonin syndrome.

So unless a good medication history is taken in the ED, the treating physician, and treating physicians across the country. This is a broad-based problem, are routinely unaware of this multi-serotonergic drug use when only immunoassay testing is performed. We also found that 33% of these patients, uh, were using multiple, not just one or two, but multiple.

And we had a patient who had 11 serotonergic drugs on board, which obviously could affect their mental status. In the emergency room, the evaluation for altered mental status is an important determination and being unaware of the fact that a drug source of this may be [00:23:00] at play, I think has some implications for care in it.

So it was, again, a, an interesting finding, and we did include it in the publication of our findings.

Andrew Lee: So in this finding, in these reports, the immunoassays are used because they’re quick, easy to use, and the readout is very fast, especially for emergency room or urgent care. You need that information as quickly as possible.

But what you’re providing with the definitive care approach is a more expensive process also involving complex instruments like a max spectrometer, and that’s typically not run by non-technical staff members or typically available in any of these emergency rooms. So is that kind of crossing into sort of laboratory test or laboratory developed test?

Rather than really quick turnaround time, like immunoassay tests, how would these kind of challenges be addressed?

Thomas Rosano: Yeah, so I agree with you that it is a, a challenge. And test turnaround time and cost both of these [00:24:00] constraints are the reason that we are using immuno presumptive testing by immunoassay.

However, there have been a significant number of advances in, in definitive drug screening that now will allow its use in the clinical setting, in my opinion. And it’s not just my opinion, but it’s my experience because we have been using it. Automated sample prep has made fast and simple. Rapid hydrolysis and dilution protocols allow for us to initiate the testing very rapidly and to prepare samples for testing.

Novel Methods of Matrix normalization, which we’ve also developed, allow us to do accurately perform both identification and quantification of drugs by definitive methods. Our chromatographic methods are fast. We now have less than four minutes for a chromatographic run that includes hundreds of drugs.

And as importantly, I think the real challenge in the [00:25:00] end is that MS Data export and managing that data for the amount of acquisitions that are done is, is thought to be quite challenging, and it is, but we do it with an algorithm and with a template that we just pop the data in and it pops out all of the, the findings, the quality control, all those things that we need to do to make it a clinically validated test.

I submitted this testing years ago to New York State. The head of the unit came to our laboratory to make sure that they really felt confident that we knew what we were doing, and we have been performing definitive testing, especially in pain management and addiction medicine, where you can get a specimen and then you do it later.

You’ve made a couple of other points though, as to whether you can do this rapidly or not. And we have developed protocols in which we can add onto an existing run or start a new run with a [00:26:00] calibration technique that allows us to stay within as much as one hour and sometimes up to three hours in terms of turnaround time.

So it is possible, but your point is well taken. They’re not FDA approved. We have laboratory developed test issues. In New York State, it was an acceptable laboratory developed test and could be used in a clinical setting. So I’m not suggesting that this is for every emergency department in every hospital, but I think university hospitals recognize this problem, and I think a number of university hospitals have taken approaches to address it as we have.

Andrew Lee: So in a similar vein of the toxicology research, you’ve also published on the chromatographic analysis of propofol and also the metabolites of propofol in urine. Now, propofol is a different drug class, but you published this and focused on the specialized testing of this target. So you monitored the metabolites of propofol.

What was your [00:27:00] logic behind selecting this target and the metabolites?

Thomas Rosano: Well, propofol monitoring is very limited. It has importance in the monitoring, for example, of professionals, anesthesiologists, nurse anesthetists, operating room staff OR staff, uh, that have access to drugs and a knowledge of IV therapy.

So it is not a highly pervasive drug use or abuse, but it, it does occur, and anesthesiologists to need to go through a monitoring program when this happens. And we need methodologies. Propofol is used in the OR on a regular basis. It’s used both to induce and sometimes to even maintain anesthesia during an operating procedure.

It’s a highly lipophilic drug and its use is, as I said, for induction, and it has a very rapid effect. It’s called arm to brain in terms of almost an [00:28:00] instantaneous anesthesia that it gives, but the effects go away very rapidly in order to maintain it. If you’re using it beyond induction, they need to also infuse it.

And in high quantity, up to gram doses and boluses that are used in the OR. So we’ve been working with the anesthesiologists at the Albany Medical Center and developed and validated a method for detecting drug use in individuals who are using propofol. We were able to detect the phase two metabolites in urine for an extended period of time.

That was one of the major findings out of this, is that although it’s arm to brain. Although propofol itself is so lipophilic that you’re not gonna find it, it, it ends up distributed into adipose tissue or metabolized glucuronidated, sulfated, and also goes through the cytochrome system. Very rapidly that glucuronidation takes place, [00:29:00] and with the method that we developed, we were able to detect for up to a month after the surgical procedure, the presence of it.

So it is the metabolites, inactive metabolites that are either leaching out of adipose or small amounts of propofol, leaching out of adipose that are then metabolized. But in any case, allows us from an analytical standpoint to identify over a long window of time propofol use. Now, we also during that study, looked at multiple, not only glucuronidated but sulfate metabolites and, and actually found an additional metabolite that has not been reported specifically, and it was from nominal mass, or the use of quadruple LCMS-MS,

that we were able to find it as an isomer with a different chromatographic position. We have since used a high resolution mass and also add information [00:30:00] to that identification. So we are monitoring it by monitoring propofol, by monitoring its directly. The glucuronide metabolites, initially propofol itself is glucuronidated directly ’cause it’s hydroxylated.

Therefore, that’s probably the reason why it is so rapidly eliminated in, in its effects, is that throughout this systemic system, it is being metabolized very rapidly itself. But we also monitored for the sulfate, which is also significant concentrations of the sulfate, are present. And we found phase one metabolites.

Going to the cytochrome P four 50 system that have been reported, and as I just said, an additional one that we think has not been reported, where on the isopropyl side chains, there is also a hydroxylation going on through the cytochrome P four 50 system, followed by glucuronidation. So there’s a fair amount of work that’s gone into it, but it’s resulted in [00:31:00] fundamentally in a procedure that can be used for the monitoring and really with a long detection window

for propofol use.

Andrew Lee: So you talk about sulfation and glucuronidation as part of the drug metabolism and inactivation, and in that part you actually mentioned cytochrome P four 50, which does the phase one modifications to the parent drug. And then there’s also phase two metabolism, which does the sulfation, and I think there’s also glucuronidation and then there’s a couple other modifications to that to make it more water soluble and then excrete through the urinary system.

So when you monitor the phase two metabolites. What do you do to detect the phase two metabolites? Can you comment a little bit more about that?

Thomas Rosano: We are testing for glucuronidated metabolites, but not directly in most cases, and that’s true in both clinical and forensic practice. What we’re doing is through hydrolysis, we are monitoring for the combined amount of both free and [00:32:00] glucuronidated form.

And we do that for not only drugs themselves, if they’re able to be glucuronidated, for example. We are testing free and glucuronidated forms, but also for phase one metabolites that are glucuronidated. We are testing them in both the free form and the glucuronidated form. We can and do test for phase two metabolites by LC-MS/MS and they are tested routinely.

Especially when they are very predominant or when hydrolysis results in current drugs like propofol that are highly lipophilic. Or in cases where we have drugs once hydrolyzed that are highly hydrophilic. So examples are propofol, very lipophilic drug. The glucuronide is much easier to monitor by our LCMS procedures and drugs.

I’ll call it a drug ethanol, where [00:33:00] ethyl glucuronide and ethyl sulfate. One also because of their size in terms of mass. But uh, in terms of our ability to very accurately identify the metabolite versus the parent drug itself. And another thing I would bring up regarding the glucuronidated metabolites is there stability, and we have had experience over time that using quality control material for glucuronidated metabolites, we find them that not always as stable on storage.

So one has to be very careful that in your assay for direct assay of glucuronidated metabolites, that you ensure that your methodology and your conditions of storage are validated for stability of your conjugated analyte. Therefore, it’s especially important and critical to test to afford this stability.

And in the case of the propofol metabolites, we were fortunate and found that they were fairly stable and met the [00:34:00] validation criteria for our assays. So there’s a variety of issues with. With their measurement. Not to say also that it will expand the number of analytes if you try to measure all of the glucuronide and free forms of the drug, and for all those reasons, I think there is more of a limited use of direct measurement.

But hidden behind our screening program is that hydrolysis an important aspect of the program that is responsible and allows us to get some indication of the amount of glucuronidated metabolite.

Andrew Lee: Thank you for highlighting that because I didn’t wanna mention it directly. But yes, there is that hidden step of hydrolysis or not so hidden step of hydrolysis to convert the phase two metabolites to the parent form.

You also pointed out quite interestingly, that even the phase one metabolites, some of ’em do get glucuronidated, and you’re also hydrolyzing to monitor those phase one metabolites. And I wanna add a little bit more [00:35:00] about the sulfated versus the glucuronidated metabolites. The glucuronidated metabolites are more common, especially in the forensic and clinical toxicology space.

More recently, I’m seeing a little bit more on the sulfation. These phase two metabolism, and it’s a bit more added significance for newborns or pediatric cases. There’s of course, acetylations and other events, but when they’re younger, when our body is younger, I guess the acetylation in glucoronidation detoxification pathways are not fully developed.

So a lot of these opiates are more sulfated rather than glucuronidated for the younger population. And I’m wondering if you looked into that or not, or what is your information on that space?

Thomas Rosano: We do have a newborn casework in our forensic casework, and usually it’s in the evaluation of an exposure from a household member who’s a drug user.

But as you point out, direct testing for phase two metabolites is limited, especially for sulfated [00:36:00] metabolites. So we are missing information when there is an altered metabolism compared to the adult. And the only approach that we really have, which is, has value in this is high resolution mass spec where we target because we have a library, but we’re able to include in those libraries the glucuronidated and sulfated forms of metabolites, and if they are chromatographically present and separated

and resolved, we do have some ability. It’s possible that sulfated metabolites are a significant contributor to metabolism and would help us in the detection of drugs, in urine drug testing. And I guess I would ask you whether there may be any studies that we may do in the future that would allow us to reveal

sulfated drugs, for example, through reagents that could be incorporated in a very similar fashion to now, which are very convenient approaches to sample [00:37:00] preparation. So, uh, help us if you can.

Andrew Lee: Thank you for that, especially pointing out some of those reagent needs and some of these new areas that haven’t really been explored.

I guess the bigger question then is what is your current interest? What are you currently working on, and then are you presenting this anytime soon? I guess an upcoming conference, like Society of Forensic Toxicology? Will you be attending this and presenting any of your recent and latest work?

Thomas Rosano: Uh, yes.

I’ve regularly attended SOFT. We’ll be attending this year in Portland, Oregon. SOFT is a great opportunity for toxicologists to meet and exchange information, and we do have a poster presentation this year, and it’s quite relevant I think, to this podcast. It’s the importance of glucuronidase hydrolysis in the detection and quantification of drugs in urine.

And in that report that we will be giving and hopefully publishing later in the year, we extended the study that we [00:38:00] did in the 400 emergency department patients by taking those samples and retesting them both with and without Glucuronidase hydrolysis. And this allowed us to determine the free analyte contribution to our test response.

We also wanted to understand the contribution of the phase two, uh, conjugated metabolite to urine drug testing for each of the drugs and phase one metabolites that are in our panel.

Andrew Lee: So, professor, do you mind highlighting some of those points and conclusions of the poster that you’ll be presenting at SOFT?

Thomas Rosano: It was quite revealing. We found that glucuronidated metabolites contribute to greater than 50% of the test response. For more than half of the 76 analytes that we had enough clinical data on with and without hydrolysis. And we also found that that there was a predominance of glucuronidated metabolites, so they were [00:39:00] the major contributors to the drug finding in urine in a third of the analytes that went across the classes of cannabinoids, benzodiazepines, opioids, and the antipsychotics, which we have in our panel.

The detection sensitivity without hydrolysis decreased significantly for 32 of the analytes with greater than 50% false negative rates without hydrolysis. So we went from positive drugs to negative drugs in more than 50% of the patient samples when we did not use hydrolysis. The strikingly, cannabidiol, α-hydroxy alprazolam.

11 hydroxy THC and Buprenorphine had false negative rates of a hundred percent without hydrolysis, so there was no ability to detect those drugs in urine without the hydrolysis step. We also found that Glucuronidation correlated directly with quantitative [00:40:00] performance, and I know we don’t rely heavily on quantitation, but we do report quantitative results on it.

But it was interesting to see how much of an impact they had. We found that greater than 99% decrease or decrement in the concentration of 16 of the analytes when hydrolysis was not performed. So it very severely affected our ability to even be able to quantitate. We may have detected them, but we are not at the level of quantitation.

The levels of the free drug are so low. This study shows that glucuronide hydrolysis is a major contributor to analytical detection and connotation for many of the drugs and metabolites that we are testing in our programs.

Andrew Lee: So you really highlight the importance of hydrolysis in this upcoming poster presentation at SOFT.

I’m just curious, there’s been prior work with the importance of the need for adding glucuronidase, but your work actually takes a broader and more expansive approach, right? So do you mind [00:41:00] highlighting some of that difference that you’ve identified in your recent work?

Thomas Rosano: Yes, you’re right. Hydrolysis is a common practice, so one would say we’re using it, so let’s not worry about it.

But our knowledge of free versus glucuronidated analytes has not determined in our routine urine drug testing practice. In our study, we wanted to know the relative contribution and what contribution it was making, both to the detection of the drug and the quantification for a large number of drugs and phase one metabolites that we’re routinely testing for.

Many of the analytes that we had in the study, we do not even have commercial reference material for the glucuronidated form of that particular analyte. And direct testing or results of even optimizing your method for those analytes is impeded by the lack of reference material. We have a figure that’s in the paper that we think will be very useful to others in toxicology [00:42:00] to not only see what the impact is

of the glucuronidation on the assay itself, but to give a sense for how much glucuronidated metabolites are really being excreted. The salt is great at telling us that they are glucuronidated and which ones are heavily glucuronidated, but this is an actual kind of quantitative look. Relying upon the current use of reagents that are very rapid and known to be as optimized as possible for the hydrolysis, it gives us a look at, at actually,

quantitatively how much glucuronide we find by this hydrolysis procedure. So I think that the data is valuable and we do need to continue to use hydrolysis. We do need to continue to validate our methods for the hydrolysis step itself, and we need to be cognizant of the importance of quality controlling that so that we do not have the [00:43:00] detection problems that would result

if hydrolysis is insufficient or incomplete. I’ve been following your work on beta glucuronidase variants produced by site directed mutagenesis, and we have applied rapid room temperature hydrolysis in this study to have as efficient as possible hydrolysis step. I hear that your team has an abstract at SOFT on Glucuronidase performance, and we’ll be interested in seeing that paper presented.

Andrew Lee: Yeah, thanks for following our work and really highlighting that. It’s a really compilation of years of work on several different enzymes. We’ve actually got probably over a hundred different beta glucuronidase that we’ve characterized. I’ve also presented on a workshop where the industry standard report, Phenolphthalein glucuronide , hydrolysis.

That correlation is very poor. So everybody asks, what is your enzymatic activity? But the activity is measured using an arbitrary substrate, [00:44:00] especially like colormetric substrate phenolphthalein glucoronide. So we have an enzyme that’s like a million units on that particular target, but it has no activity on any of the drugs of abuse on opiates.

It won’t hydrolyze it. And then we’ve also classified enzymes based on their ability to hydrolyze O-link versus N-link glucuronides. There are several analytes with N-link glucuronides, common in the nicotine metabolite, and we see a pH preference for these N-linked glucuronides. So a little bit more basic, and it will hydrolyze better. If you go acidic,

it doesn’t hydrolyze as well, or sometimes it doesn’t hydrolyze. And then the O-link, especially for the opiates, there’s actually slightly different linkages on the opiates, like the three hydroxyl groups versus the six hydroxyl group. A lot of these metabolites being hydrolyzed, we’ve spent probably a good decade or more on really exploring the reagents on it, and we’re glad that you’re using it for room temperature, hydrolysis and doing some of those detections.

So I’ll move on to the other work that you’ve highlighted are the [00:45:00] cannabinoids and the isomer selective LC-MS-MS analysis. So this, uh, CBD, right? Everything’s kind of hot with hemp and Cannabinoid or marijuana metabolites or derivatives thereof. And you actually published a work on detecting structural variants of these drugs.

Do you mind providing some quick highlights of that work and your work in forensic toxicology casework?

Thomas Rosano: This is an interesting etiology of a new class of designer cannabinoids initiated inadvertently here in the US by congressional legislation called the 2018 Farm Bill. The legislation defined hemp as C sativa with concentrations less than 0.3% Δ9-THC. And ambiguously deemed that hemp derivative products were no longer considered

scheduled substances. Following this legislation in the forensic laboratories [00:46:00] primarily, but I think it was happening really across the measurement of anyone doing mass spec for cannabinoid confirmations we’re finding interferences in their THC confirmations, both in urine and in blood. And we showed in our reported in our JAT publication that the interference was Delta eight isomer of THC.

In our case, in urine, it was the CARBOXYLATED Delta eight THC isomer that was overlapping in the illusion. We reported the chromatographic conditions. It’s challenging to separate the isomers baseline, separate the isomers. So we reported our chromatographic conditions and showed that 14% of our cases had the presence of the Delta eight isomer.

So it was a prevalence that we see in our regions. We’ve since repeated that in a number of studies that ED study had some Delta eight and it also, and others have [00:47:00] reported in medical examiner cases in blood, and Delta eight is easy to produce illicitly in terms of acidification. And it just depends on how long you incubate it for

with heat that you can produce varying amounts of Delta eight, delta nine, and we showed that variation that we saw across our casework in terms of the metabolite of Delta eight and delta nine. But there are other isomers and analogs continue to be identified. And CB one receptor potency is quite significant.

Compared to THC itself or some of these newer analogs. So again, we’re one of a number of groups that reported on how we address this problem. And the general knowledge of it has led to improvement of methods across many laboratories. So it’s an evolving area of drug use, and I consider it another era in this case of cannabinoid, [00:48:00] isomers and analogs.

Andrew Lee: Thank you so much. It’s just tying back to our earlier discussions Throughout the history, you’ve looked at these other analogs or other casework, and at this point you’re seeing quite a bit of cannabinoid and these analogs and quite interesting between the Delta nine and Delta eight, it’s a very subtle chemical difference, but it also has a very similar potency, if I’m not mistaken, but also legal implications.

And these are some of the cases that you’re highlighting, and it sounds like you’ll forever be busy with a lot of these legal ramifications as well as these chemical changes. But I also wanna talk about this and comment on this matrix normalization technique and then your accurate LC-MS-MS detection.

So this matrix normalization matrix referring to whether you get it from blood samples, urine samples, saliva samples, and then you have to normalize it because there are gonna be variations across the board. And how do you get it to be consistent or just normalized? So this technique, do you mind talking a little bit about it?

Thomas Rosano: Uh, yes. The technique [00:49:00] is called Threshold Accurate Calibration. That was what I named it in the first publication, and we’ve reported on it in its validation in, I think now five studies that we have done using it. The abbreviation is TAC. We developed a technique as an alternative to stable isotope internal standardization.

It’s especially useful for high volume multianalyte testing where analyte matched internal standards are clearly needed analytically, but are very costly to have, and we’ve shown many times that you need to have that internal standard in the source. Along with the analyte in order to have it represent the impact of either suppression or enhancement that goes on.

So using a shared internal standard somewhere else in the chromatogram really leads to some significant problems, as you would see in some of our publications and reports on this. It’s [00:50:00] a technique that’s similar to standard addition and that uses the analyte addition to correct for any matrix effect on it.

A sample aliquot is fortified with a controlled amount of reference analyte and then reanalyzed. The calibration procedure is like internal standard, and it uses an iron response ratio of unfortified to fortified analyte. Unlike standard additions, where each case sample is individually calibrated

in making it a very cumbersome process. The use of the ratio allows an accurate quanification across all of the samples that are in the analytical run. So it’s just a standardization as you think of standardization, but it’s using the ratio of the analyte to the added analyte rather than the analyte to a, hopefully an analyte matched internal standards.

We’ve been using TAC for years in our clinical and forensic casework and to have many LDT laboratory [00:51:00] determined methods, techniques that use this particular for both screening and quantification. We think accuracy at the threshold for calibration is as important as quantitative accuracy across the calibration curve, and we use it in both cases and it allows us to have good accuracy with it.

Andrew Lee: So this is a technique that you rely on, especially if you don’t have that reference standard, like a Derated reference standard. This would be very useful because you have that reference material so you can actually fortify a sample and say, okay, you’ve got. This ion response ratio difference between the un fortified versus the fortified?

Thomas Rosano: Yes. We have in current protocols, a year and a half or two years ago, reported the details of how to perform this, and it really put it for use, not only in the clinical and forensic toxicology setting, but in research settings and in drug discovery, because many times you’ve got a new drug and you could accurately identify it by just having the pure drug itself.

Then using [00:52:00] that as a reagent for supplementing. So it has many applications and hopefully it will be of value to others. It certainly has been of value to us.

Andrew Lee: Well, Professor Rosano, thank you so much for all of that information and it was just very intriguing to hear about some of your latest work. It was definitely a blast having you here at IMCS, the podcast, Imagine More Create Solution podcast series, and please check out his poster at SOFT in Portland, Oregon and say hello, and please ask him additional questions.

Again. Thank you so much Professor Rosano for this opportunity.

Thomas Rosano: Thank you, Andrew. You know, it’s very seldom that any conversation with me is called a blast, but it’s really a pleasure to participate in the podcast and I think education is a career long endeavor for all of us and a mutual exchange process, and I’ve learned much from you over time regarding the advances that are made in enzymatic hydrolysis.

I appreciate the opportunity to participate in the podcast. I hope [00:53:00] that you and the listeners gain some information today that may be useful in practice.Andrew Lee: Well, let’s wrap it up, folks. I hope you enjoyed this episode. If you want to stay connected, follow us on LinkedIn and for more episodes, find us on Spotify or visit our website www.imcstips.com. Catch you on the next one. Take care.