BioAnalysis: Mastering Sample Preparation. Techniques to Elevate Your Bioanalytical Assays #lcms

Hi, my name is Ryan Chu and I'm the Director of Chemistry here at Emory Pharma. Welcome to our bioanalytical series. Today we're going to be talking about mastering bioanalysis method development, and we're going to focus on the key insights on optimizing sample preparation. Our goal in this video series is is to not only provide insights, but also to educate our clients, our partners, and you watching this video on what we do here at Emory Pharma and how we do it. Come take this journey with us. When we think about bioanalysis method development, there are a variety of key steps that we've emphasized here. The first is sample collection. We're talking about how we collect samples from these animals in vivo, what relevant time points, how the samples should be stored, what type of anticoagulants. If you're talking about blood collection, in fact, where should you even take the site for these bioanalysis samples? Following that, we're going to talk about sample preparation and that's the key focus of this video. In the video series, data acquisition. What types of instruments do you need to collect the data that you're interested in? Whether that's ELISA's, NMR, GCMS, or LCMS, together with sample preparation and data acquisition, method validation is important. What considerations do you need to have to appropriately validate a method? I encourage you to watch a previous video in our video series where we talk about following the ICH M10 bioanalytical guidelines for method validation. And then finally, once your method is validated, what type of data analysis is appropriate? How are you using this data? What types of parameters do you care about? What is it that we should focus on? But again, today we're going to focus on sample preparation. If there are any key steps that we've missed that you're interested in, please comment below. And we'd be glad to film a video where we explain details about that process. So the first question, what is bioanalysis? Right at its core, we are measuring drugs, metabolites, biomarkers in a biological matrix. Biological matrices could be plasma, whole blood, excreta, such as urine or feces, or even as complex as tissues. Measuring these markers or these metabolites or drugs are absolutely critical to understanding drug behavior in pk, pharmacokinetics, pharmacodynamics, pd, toxicology, and even clinical studies. Applications for bioanalysis include assisting in drug development, measuring drug levels in clinical trials for safety and efficacy, therapeutic drug monitoring so ensuring drugs stay within the therapeutic window, the balance between efficacy and safety, and then finally, diagnostics quantifying these biomarkers for diagnosing diseases or monitoring disease progression. So we now get to focus of this presentation, which is sample preparation. So before we get started, what are the challenges with sample preparation? We need to know what the challenges are before we figure out how to solve them. So one of the major challenges with sample preparation is the matrix itself. So the complexity of the matrix. As you're probably well aware, we have a variety of biological matrices available to us and can you. And each one of them comes with unique properties that add challenges to sample preparation. Those matrices can contain various proteins, lipids, salts, all of which can potentially interfere with the analyte we're trying to detect. Because of the complexity of the matrix, we have a variety of matrix effects. So matrix effects are things that cause either suppression. So we're decreasing the signal or enhancement where we're enhancing or growing the signal when we do things like mass spectrometry, because that's caused by coelution of any of those previous matrix effects or matrix complexity issues that are causing inaccurate quantitation. We now have to think about the balance though between cleanliness and recovery. Right. Sample prep in at its core is cleaning up the sample. You're trying to remove things that you are not necessarily focused on measuring. But over cleaning has the negative effect of potentially reducing the analyte recovery. So your analyte signal could be suppressed as well. However, inadequate cleaning can leave interferences which are affecting your sensitivity. So again, you want to clean samples but not clean too much where now you've lost your signal. So that balance is absolutely critical for sample preparation. So what do you do? Right, so you need to select the appropriate sample sample prep method based on your matrix and based on your analyte. Oftentimes people use internal standards such as deuterated or labeled compounds to correct for these matrix effects. So what are some common sample prep techniques that you can do? The first, most common is protein precipitation. What we're doing here is we are precipitating out all of the proteins or things that are not of interest as a way to clean up our sample. So commonly used solvents for protein precipitation can include acetonitrile, methanol, perchloric acid. The major benefit of protein precipitation is that it's fast, it's straightforward, it's simple. This is the most commonly used preparation technique for things like plasma, serum or even whole blood analysis. While it is successful at removing proteins, it does come with the potential that some residual impurities can remain. So there is that trade off and Something for you to consider when you're developing your method and choosing protein precipitation as a sample preparation technique. The second most common is liquid liquid extraction, so this is often abbreviated lle. Commonly used solvents could be ethyl acetate, hexane, dichloromethane, dcm, chloroform, or other organic solvents such as those. What this focuses on is separating between the organic and aqueous phases, so allowing the analytes to go into that organic phase. This is extremely effective for extracting non polar analytes, but may require some optimization as well. So again, a little more complex than protein precipitation, but still successful, depending again on what your analyte is. Third most common, I would say is SPE or solid phase extraction. So here we're actually using a solid phase, such as this SPE cartridge. So a solid phase extraction cartridge, which is packed with either C18 or C8. For reverse phase, you can have silica packing for normal phase or even immunofinity. What this does is it allows extracts to be cleaned by selectively binding the analyte to the solid phase resin. This is going to be the most thorough cleaning process, but also with that trade off, potentially leading to the lowest recovery. So even though your samples are highly pure, you're going to have less of those matrix effects. This does take a lot more time. It is more resource intensive. And again, depending on the sensitivity of your analytes, you may actually lose some of them, especially at the low, low levels. So we often have clients that approach us with analytes of interest that are endogenous, meaning that they are naturally occurring within us. So how do you distinguish those differences? What do you do? How do you quantitate? Right, so endogenous analytes, as I mentioned, are already present in the biological matrix. This does make quantitation challenging. Again, how do you differentiate between those endogenous levels? Well, you can do a few different strategies, one of which is spiked concentrations. So you can spike in known amounts within that matrix and take that additive effect for quantitation. Additional solutions are using baseline subtraction, where if you know the endogenous amount, you can subtract that. After quantitation, you can prepare calibration curves in matrix matched samples. So again, you can pool a variety of different biological matrices, let's say plasma, and use that as your sort of representative physiological matrix to reflect the conditions of your samples. Or finally, you can use a novel idea which is using a surrogate matrix or something that doesn't have that analyte, but is meant to mimic the matrix of your choice. I encourage you to click on this link or the link below to watch the video on method validation where we actually talk about using a surrogate matrix. This here is an example of a bioanalysis workflow. So let's pretend you have your test compound or your analyte of interest. You have this in some biological matrix. We talked about the addition of an internal standard, and in this case we focused on protein precipitation as our sample prep prior to data acquisition using LC Ms. Ms. When you think about your sample preparation, other key considerations are your analyte stability. How stable is your analyte throughout this sample preparation process? How stable is your sample prior to the sample preparation process? So things that are important are looking at storage conditions, freeze, thaw cycles. These are absolutely critical to prevent degradation, especially for sensitive analytes, and they could potentially impact the quantitation of your method sensitivity and specificity. So sensitivity here being what's the lowest amount you can detect? Specificity is how specific you are in identifying your analyte of interest within this complex matrix. These are key considerations in ensuring your method from the sample prep do not cause your analyte concentrations to be fall below your limit of detection, but also cleaning up your samples enough where they are specific to being able to detect what you're interested in. So again, the balance between cleanliness and recovery. And then finally, at the conclusion of identifying and developing a sample preparation that's suitable for you, method validation According to the ICH M10 bioanalytical guidelines will ensure that your method is robust and meets regulatory standards. So in conclusion, I hope that following along this video you've learned that sample preparation is absolutely critical to successful bioanalysis method development. The choice of your sample preparation technique is absolutely critical as it influences your analyte recovery, it influences the purity of your sample, and with the goal of reducing matrix effects, proper method development and validation ensures accurate results. And again, method validation is the final step to ensuring regulatory standards are met. Careful optimization of sample prep and validation leads to reproducible data, which is absolutely critical for successful studies. I encourage you to contact Emery Pharma for any of your bioanalytical needs as we have a lot of experience throughout the entire bioanalysis workflow, but particularly with sample preparation. Thank you for watching.