Expert Insight: Analytical workflows for anti-doping, using LC tools

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Dr. Abigail Burrows, postdoctoral scholar, University of Kentucky, and Prof. Scott D. Stanley, Director of University of Kentucky Equine Analytical Chemistry Lab

A major part of mass spectrometry-based analysis is the ability to separate analytes of interest from other analytes and complex matrices. The two most used separation techniques comprise Ultra High-Pressure Liquid Chromatography (UHPLC) and Nano Liquid Chromatography.

Since its inception, UHPLC has shown very high efficiency in separating a wide range of organic compounds. UHPLC can separate many drugs that are not amenable to Gas Chromatography (GC) based separation owing to their polarity, volatility, or high molecular weight. Research laboratories have been successfully implementing nano-liquid chromatography for separation of peptides resulting in confident analysis of protein mixtures or samples with very small concentrations of targeted peptides. The combination of UHPLC or NanoLC with the Thermo Scientific™ Orbitrap Exploris™ 480 mass spectrometer results in robust, reliable, reproducible LC-MS assays for every clinical research and forensic toxicology laboratories, regardless of the analyte or matrix complexity and user expertise.

In this webinar, Prof. Scott D. Stanley, Director of University of Kentucky Equine Analytical Chemistry Lab, and postdoctoral scholar Dr. Abigail Burrows present the latest methods for equines anti-doping analysis, the importance of liquid chromatography and the analysis of challenging samples.

Read on for highlights from the Q&A discussion at the end of the live webinar or register to watch the full webinar on demand >>

Q: In your presentation you showed that nano LC-MS is needed to reach high sensitivity and find biomarkers that can indicate the usage of doping substances. How difficult was it to start using that nano LC-MS technique in the lab? Do you think scientists with mainly UHPLC-MS experience can run a nano LC-MS experiment using the setup like the one that you used? Finally, are there any considerations that we should consider before starting nano LC-MS experiments?

AB: I can speak from experience on this, during my Ph.D. I used a high flow chromatography system. The principles of chromatography are the same, but there are certain guidelines and tricks an individual should know before beginning to run a nanosystem. A couple of key learning points that I have acquired are that you need to account for the delay time from the LC to the MS because you are using such low flow rates. In our system, there is approximately a 15-minute delay that you need to account for in MS acquisition. With the nanos you can also use a track column where you can pre-concentrate your samples. You will also need to account for that in your methodology program.

Alongside this, you should consider the probe positioning, source settings, and the cleaning procedures. These are all more particular than in high flow, so you need to set your source settings and evaluate your spray each time you reattach the column. Typically, in high flow, you can leave your voltages and gases the same for all your methods, this is distinctly different in nano. For Nano you need to evaluate those spray settings each time you set up your system to run. In my opinion, it has been a good learning experience to learn the nano and the intricacies of the system, to be able to compare and contrast between the high flow.

Q: Is it advantageous to use a trap column when analyzing complex samples? What are the pros and cons of this setup?

AB: Yes, I have worked with serum for quite a long time. You want to eliminate as much noise as you can, especially when you are trying to analyze proteins or peptides that are present in a low abundance. As I have mentioned before, this works as a pre-concentration step. Your peptides will bind to the trap column and then wash away anything that you do not want to go into your analytical column. Therefore, you can get away with not using it, but your data will probably be more complex and more challenging to analyze. I would advise that it is useful to use the trap column, especially with serum samples.

Q: I assume that the carryover in anti-doping analysis is an extremely important topic as the conclusion of the presence or absence of target substance in samples must be done unambiguously. Could you comment on how you eliminate carryover with an optical flow LC-MS analysis and if the carryover is an issue for your LC-MS setup in the lab?

AB: Carryover is a critical problem in all LC systems. In our systems we have binary pumps. Therefore, we are unable to wash our column with a solid flowing through the system. Instead we run a wash method after every 10 injections and use a solvent called ChromaCare, which you can buy from Thermo. ChromaCare is a mixture of isopropanol, acetonitrile, and acetone.

We run an up-down method where we have a high concentration of organic and then a low concentration of organic, some people call this a seesaw gradient. This has enabled us to reduce the effective carryover. If you do have a quaternary pump system, you could put this solvent onto your pumps and flush it through that way. But that is what I used in my high flow. I found that this works well and keeps the nano precision very similar between runs.

Q: What are some of the challenges you have encountered during method development and synchronizing the LC and MS methods?

SS: Challenges can be avoided upfront if you put some planning into the project and identify the targets and some of the specific gains. The best way to avoid some of those challenges with your LC configuration would be to decide ahead of time what your chromatography targets mean. Meaning selecting a column or experimenting with a couple of columns, defining the mobile phases, and then sticking with those mobile phases. Also, have an idea of your targets and your runtime needs. For example, how many samples do you need to run in a specified period? You can eliminate a lot of the early challenges of synchronizing your methodology with your LC in your mass spec. Mass spec can easily be set up by the direct infusion route using a syringe pump. Therefore, you can have all your analytical specified targets incorporated quickly, this can also be joined with your chromatography configuration simply. I think if you do a little planning before you start your development, this can ensure it is done quickly.

Q: You presented a great overview of research tools to LC-MS analysis and the identification of prohibited substances. Do you think these methods and similar hardware, or software LC-MS setup can be transferred to laboratory for routine LC-MS anti-doping control? Would the reproducibility and robustness be sufficient to analyze large sample cohorts?

SS: All the technology including the Orbitrap technology, LC technology with a vanquish, and the nanosystem are easily available to anyone that wants to utilize this. Getting the hardware up and running is not really a challenge for any laboratory nowadays. There is some software configuration for your specific targets and needs, however this can be done with the initial installation or with some assistance from one of the representatives to get you off to a good start. I think other than anti-doping control labs, in which most of them use technology that are exactly like this or very similar to this, whether it's human anti-doping or equine anti-doping programs, they are all fairly easy in getting those set up. I think it also goes universally to other applications as well, toxicology, pharmacology, environmental analysis can all use this type of approach to get their analytical problem solved.

Q: What are the benefits of having an MS instrument equipped with two vastly different LC systems? Why would this be advantageous to other high-throughput labs?

SS: The good thing is that there is a quick conversion to a completely different problem-solving. You can handle small molecules very easily with the Vanquish and then complex proteins and some peptides with the nanosystem. Switching between these is very simplistic. We can have the instrument handling one set of problems one day and another set of problems another day. We also do the same thing with our discovery proteomics and our triple quadrupole instrument because we share the Vanquish on a mobile cart so that we can move it between the instruments depending on exactly what experiments and analysis that we're trying to conduct.

AB: I agree with what you said. And I think it is really beneficial to have that flexibility in our systems. We can develop robust discovery methods and be able to select targets with high confidence. We are also able to transfer those over to a targeted system in the same lab, knowing that the targets that we selected should be detectable using another system. I really like having that capability. The MS instrument also gives us the ability to develop different types of experiments depending on the questions we are asking. Therefore, we are not limited by one system setup, which is beneficial from a research perspective.

SS: Regarding the question about reproductivity and robustness we found that even by switching back and forth between the systems, we're able to consistently get reproducibility in our methodology without having to spend a lot of time conditioning or re-establishing the equipment. We also do a tremendous number of validation experiments where precision and accuracy are required. We have high precision and accuracy with the combination of equipment that we are utilizing right now.

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