Quest Webinar: Innovative Solutions LCMS-MS Webinar
Timothy S. Collier
0:07
Thank you so much for joining us today.
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My name is Sarah Walsh, clinical specialist within the cardiovascular, metabolic, endocrine and Wellness clinical segment at Quest Diagnostics Laboratory.
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Science itself is a field of medicine.
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It can be difficult to remember specifics on different methodologies and when to use what methodology.
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Today we meet with Doctor Tim Collier.
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He'll break down the need to know regarding the common methodology utilized in the lab, liquid Chromatography Mass Spectrometry, and highlights specific scenarios that this methodology may be recommended over.
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Another Doctor Collier is the Scientific Director of Research and Development for the Quest Cardio Metabolic Center of Excellence at Cleveland Heart Lab.
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His responsibilities include overseeing the identification and development of assays for biomarkers related to cardio, metabolic and endocrine health.
1:00
Thank you so much for joining us, Doctor Collier.
1:02
Welcome.
1:03
Thank you, Sarah, for that wonderful introduction.
1:06
I'm really excited today to speak about the power of liquid chromatography mass spectrometry in the clinical laboratory and give everyone an overview of how the technology works, how we leverage it for clinical problems in the lab that otherwise would not be able to be solved.
1:24
And you know, hopefully by the end of this talk, you know the viewers will leave with a a general understanding of how the technology is used, why we use it, what are its strengths and what are its weaknesses even in terms of our ability to deliver quality solutions to our patients and our clients.
1:46
So I'm going to go ahead and get started and give you an overview of what I'll be speaking about today.
1:53
Why do we even need LCMS and LCMS?
1:56
Is is how I'm going to refer to this instead of just saying liquid chromatography mass spectrometry all day long.
2:02
We'll also use the acronym LCMSMS, and I'll explain a little later about why we add that extra Ms.
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on the end of the of the lettering there.
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But there's really 4 main advantages to LCMS in the clinical laboratory that give us an advantage in in this field.
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One is the technology is incredibly versatile.
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It's deployable across a range of metabolites, analytes, molecules that are small molecules, lipids, small peptides all the way up to large proteins.
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It's very agile.
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So if the scenario ever arises where we want to quickly develop a new method or a new assay to measure something that to date may have never been an an analyte of interest in the medical or clinical community, This platform is modular, modifiable.
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The instrumentation is customizable and gives us the ability to relatively rapidly develop new assays to meet immediate clinical needs that otherwise may take years or longer to develop and deploy and address those unmet clinical needs.
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The instrumentation is also very high performing.
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It's multiplexible and by that I mean we can actually put multiple analytes into a single detection run or method.
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So within a few minutes, we could detect not just one single thing that we want to measure.
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We can detect dozens of things in a patient sample that we would want to measure.
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That gives us really high throughput.
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It gives us extra efficiency in the lab.
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The other thing is that mass spectrometry in general is known for its cell activity in terms of being able to make sure that we know whatever we're measuring is truly what we want to measure.
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And there's nothing else present that may be contributing to the result that we otherwise don't know about.
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And it's relatively sensitive as well relative to other common diagnostic platforms too.
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The cost of tests to quest and to our patients and clients become improved because the the cost of these things are are are generally lower.
4:21
So here's an overview of the LCMS system platform, and one of the things that you'll notice in this cartoon diagram is that there's a lot of different parts here.
4:31
On the left of the screen is the the very beginning of where a sample starts in an analytical run.
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On the top here is what we call an auto sampler.
4:41
We would put plates in the individual drawers of the auto sampler containing patient samples, calibration material, quality control standards and other samples.
4:53
That may help us determine correct positioning and sequence of the samples in the plate so that we know that when sample 8 comes through that the patient that's supposed to belong to is correctly identified.
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And then we can inject those samples into, you know, different injection ports here on the side.
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The great thing about this technology is that we can actually throw multiple liquid chromatography systems in front of a single mass spectrometric detection system and offset the timing of the injections onto the LC system so that we can utilize the detection capability way more efficiently, improving sample throughput and therefore reducing turn around time of results to our patients.
5:38
So in this particular example, we've got three separate liquid chromatography systems that are being utilized to produce results with the timing offset so that the areas of the LCMS run where our analytes of interest would be eluding from the chromatography column.
5:57
That timing is when the mass spectrometer is actually monitoring any given channel and then we can quickly switch to the next channel and align it so that those same eluding analytes are detected by the mass spectrometer and so on and so forth.
6:12
So there you know a chromatographic run is not necessarily a a method that is only the the peaks eluding.
6:22
There's always some conditioning time on the front end and some cleaning and re equilibration time on the back.
6:28
And So what we can do is make the mass spectrometer essentially blind to those points.
6:33
We don't need to see anything in those ranges.
6:36
And then we can just switch the mass spectrometers attention to the next sample where the important stuff is coming off.
6:43
And so this arrangement again it's, it's for high efficiency, it allows us to, you know, put multiple systems in front of a mass spectrometer.
6:52
It's not uncommon in a clinical lab to see four or even more streams of liquid chromatography feeding into a single detection.
7:01
So on the right here is the mass spectrometer.
7:03
In the middle here are the columns and there's a single valve on the back end that will helps us switch between each stream when the timing is correct to get the signal to the detector.
7:16
So we'll dive into this a little bit more.
7:17
Let's talk about the front end about the liquid chromatography.
7:21
So liquid chromatography is a means by which we can separate multiple components in a mixture.
7:28
Most of the time, what we receive in the clinical lab is going to be a serum sample, a plasma sample, a urine sample.
7:35
All of those biological fluids contain hundreds, thousands, 10s of thousands of individual analytes, whether they're small molecules and metabolites, lipids, proteins, peptides, you name it.
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That's all contributing to that mixture that's circulating in your body.
7:56
So one of the things that we utilize the chromatography portion of the LCMS system for is to help reduce the complexity of that mixture.
8:06
The less complex we can make the mixture, the more specific and selective we can make the detection on the back end, which means we're delivering an answer on a compound where we know exactly what that identity is, and then we eliminate any possible interference from other things that might be present in the patient.
8:26
Sample O.
8:28
This diagram here shows again a single of the multiple streams originating from an auto sampler and and the pumps that drive the liquid through this column and in the middle you it's literally a column, it's a pipe or or a piece of tubing that's you know wide in diameter anywhere from you know a couple of millimeters on up to you know 5mm or so.
8:53
And what we do is we can inject that complex sample represented here by the spectrum of closely packed colors.
9:01
And as we flow the liquid from the pumps which are various organic or aqueous mobile phases, it's the liquid that's going to carry the sample through the system that's going to allow these components in the mixture to interact in a differential way with a packing material that is crammed into this into this column.
9:26
And we can vary this packing material to be selected for different types of molecules.
9:32
And so that will allow us again to customize the analytical platform so that it is most efficient and and has its highest performance on whatever analyte we're most interested in detected, be it a hormone or a peptide or a protein or a particular, a particular lipid species.
9:51
And so as it flows through this column, these individual components of the mixture will interact differently which affects the speed at which they move through this column.
10:02
And so you can see here we've got the red material coming out a little faster followed by the yellow, the green and the blue.
10:11
And what this looks like by the time it makes it to the mass spectrometer, we detect these as essentially peaks.
10:18
And you can see the stuff that makes it to the end of the column first is the first thing that we detect.
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So if we look at this on a time scale, the red is going to elude earlier, the yellow will elude a little later, the green and then followed by the blue.
10:34
And So what this does is this gives us a rough separation based off of the separation technology that's used here, based on the the composition of the liquid mobile phase, the special chemistry of the packing material in the column and dictated by the properties of whatever it is in the sample that we want to detect.
10:58
So that's the LC column, that's the first dimension here of separation, if you will in reducing the complexity of the mixture.
11:07
From there we're going to flow into the mass spectrometer.
11:09
And what I'm going to do now is present to you a little bit more of a comprehensive view of what the mass spectrometer is actually doing.
11:20
So this looks like a really busy slide, but don't be intimidated by it.
11:23
We're going to walk through it step by step from the liquid chromatography system.
11:28
Our liquid containing our analyte goes through a high magnetic field ionization source because we can only measure items in a mass spectrometer to which we can ionize and give it a charge.
11:41
Because when we charge a particle, we then can control its path to a detector using radio fields and magnetic and electric fields and that will help us guide the the molecule all the way through to the detector.
11:55
Now there's multiple ways to ionize a sample.
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The most commonly used in the clinical laboratory is a method called Electro spray.
12:03
There are also types of ionization such as MALDI ionization, which is a more of a solid phase type of ionization, but we're not going to go into detail on that here.
12:15
But in any case, the electric field in the front of the instrument both charges the molecules and then directs them towards the opening of the instrument, where we essentially dry these droplets down to the point that the only thing that's left are individual molecules with some level of charge assigned to them.
12:32
In this case, we're going to have positively charged molecules now inside the mass spectrometer ourselves, the inside the mass spectrometer itself, at least in the the type of instrument that we use most commonly in the clinical laboratory, which is called a triple quadrupole mass spectrometer.
12:50
We actually go through what we consider a number of filters, and again we're trying to reduce complexity and separate samples out and get only the thing that we're most interested into the detector.
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So we have in a triple quadrupole.
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Of course, that implies we have three different quadrupoles, and quadrupole is simply 4 rods arrayed in such a way that we can control the electric field on any one of these quadrupoles to guide ions through the instrument to a detector that would be on the right side.
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So let's take a hypothetical here.
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We're going to spray in material that's coming off of our chromatography column.
13:34
In this case, we've got three things that are eluding at the same time.
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Now this is already an advantage for us because typically there may be dozens or hundreds of things that would elute at the exact same time.
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Or if you didn't perform a high degree of separation on the front end, you're just throwing everything in the mass spectrometer at once.
13:56
And that makes the the ability to filter and detect things much more difficult at the instrument level.
14:02
So in this case, we've got 3 molecules that came off and they all have different intact masses.
14:08
So we're talking about the essentially how much each of these things weighs as they come off of the the column.
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So we can actually set for our molecule of interest, which is actually going to be this one at the top.
14:21
It has a mass of 404.1 M over Z.
14:26
We can actually finely tune this first filter to only let things within a very small window pass through this first part of the instrument.
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So in this case, we're going to set a window from 403.8 to 404.4 M over Z units, and that's the measurement units we use in a mass spectrometer that stands for mass divided by charge.
14:49
So applying this filter allows the molecule of interest to pass forward to the next checkpoint in the in the instrument, but it also let another one that's pretty close in mass.
15:02
It's only a point to mass unit difference as the mass spectrometer sees it, but it's going to pass through to that next filter too.
15:10
So this other molecule that's in the middle here, that's got a massive 770.
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Because this filter is set so narrow, this molecule can't find a stable path through to the next stage.
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So it's actually just going to get ejected and it won't make it to the detector.
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So we've essentially blocked it from moving forward.
15:30
So now we've taken a complex mixture from the LC, reduced its complexity and just using the very front end of the mass spectrometer, we've now reduced that complexity by a third.
15:41
We now have two molecules that make it through.
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Now in the second quadrupole, we don't set it as a mass filter, but what we tend to have in this second quadrupole area is a small amount of inert gas is typically nitrogen or argon.
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And what that actually does is it serves as a material that we can collide with individual molecules and break those molecules up into their smaller pieces.
16:12
Now that is what's going to give us another degree of separation capability because each molecule when we fragment it, will generally produce fragments that become specific to that intact molecule.
16:27
And if you take a look at the pairing of the intact mass of the molecule and a fragment mass that's unique to that molecule, now you've got a very highly selective combination of masses that you can apply to that compound that only that compound should be able to survive.
16:47
And that's the case in this particular example.
16:51
We fragment both molecules into smaller fragments and we're going to set the filter in the third quadrupole.
16:58
That's only going to let a fragment that's specific to our molecule of interest pass through.
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We call these ion transitions, so we typically pair a 404.1 MO over Z.
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That's the intact molecule mass with a fragment mass, And by looking at those combinations we get a unique signal that's specific to that compound.
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Everything else, because it's outside of that filter window, can't form a stable path to make it past into the detector and therefore it's separated out.
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And so that's how we go from reducing complexity of a mixture from the chromatography system on the front end, further reducing complexity using one mass filter.
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So this is actually mass spec MS1 and then we're going to do Ms.
17:46
again on the back end on the fragments and that's why we have two MSS in the acronym LCMSMS, also called liquid chromatography Tandem mass spectrometry.
18:01
So how do we actually utilize this?
18:03
When we put it all together, this is a general overview of all of those different dimensions of separation overlaid upon one another.
18:15
So again our blood samples or urine samples, they're complex, just your blood has nearly 4000 proteins and they span A6 order of magnitude fold range and concentration from albumin to your very smallest transcription factors.
18:33
And that's just the proteins, that's not counting small molecules or lipids or or any of the other thousands of metabolites that may be circulating in your blood.
18:42
So we we're going to do this as a separation in three dimensions, starting with liquid chromatography.
18:49
If we look on this left axis here and we look at the peaks coming off, what it looks like here is that you've got four peaks.
19:00
Well, in reality if you look at a slice of this by taking a mass spectrum, the first level of mass spectrometry analysis, you can see that even within this peak there's actually lots of different intact molecules that are present.
19:16
Again, blood samples are very complex.
19:20
The second peak even more, 3rd peak, this dark green trace.
19:24
You can see even more individual intact ions there.
19:29
That are going to be potential compounds that would otherwise interfere with the direct measurement of the sample.
19:37
Even further given this complexity we want to get even more specific.
19:43
So this we're this is where tandem as spectrometry comes into play.
19:47
We can actually set that mass filter on that first quadrupole to only let our molecule of interest into the towards the detector towards the second mass quadrupole.
20:00
And then we're going to fragment that mass range.
20:04
And again, you may end up with some things that make it.
20:09
Through the fragmentation step that are specific to your compound of interest.
20:12
So that would be this item here with a star above it and this ion here with a star above it.
20:18
But these other things with XS, those are things that were Co isolated with that intact mass but aren't specific to the molecule we're interested in.
20:26
So we're not going to monitor that and let that through to the detector.
20:31
So by separating in the liquid phase and then measuring intact mass, fragmenting and measuring fragment mass, and pairing those specific fragments with the specific intact mass that we're interested in, we get a very specific measurement in a very complex mixture.
20:49
So what do we actually use this for?
20:51
What are the cases where we would actually utilize this over other technologies?
20:55
Let's look at a couple of examples.
20:59
The first example is the detection of a protein called thyroglobulin.
21:05
The reason we would like to monitor this molecule is because in thyroid cancer, which is the most common endocrine cancer in the United States, the thyroid tissue is generally resected using surgery, radiation ablation therapy applied to the patient afterwards, and the goal of this is to remove essentially all of the tissue that would have been cancerous.
21:29
So the thyroid generally gets entirely removed.
21:32
Well, we would then monitor for this protein called thyroglobulin that's only synthesized and utilized in the thyroid.
21:41
So if we've resected and removed a patient's thyroid, we should never see this molecule circulating in their system.
21:48
Again unless that tissue is growing back or in medical terms we're we're looking for recurrence of the cancer.
21:58
Now typically we would detect this by an immunoassay in which you can have an antibody with some type of indicator for, you know, generating a color in a well that antibody would attach to your thyroglobulin and you'd get a signal and you would know whether you had any thyroglobulin present or not.
22:17
Well, one of the challenges of biology is that it's complex and it's messy.
22:21
And sometimes you can end up with cases where a patient may generate thyroglobulin but their body will automatically generate antibodies to the thyroglobulin that will bind all over the protein and therefore prevent a detection antibody from an immunoassay, from even recognizing and attaching to the thyroglobulin protein, which is what we want to know is present so that we know that there's a recurrence of the patient's cancer.
22:50
So what this would result in if we were to run an immunoassay and detect and and we we had all the antibodies present, which we wouldn't know were present, that detection antibody isn't going to find that protein and you're going to end up with a result from the immunoassay that underestimates the amount of thyroglobulin present in the patient.
23:14
So what we would do is we will subject this to a mass spectrometry workflow.
23:19
So we would actually do a little bit of free analytical processing of the sample by digesting with an endoproteinase, which is an enzyme that will cleave up a protein in very predictable spots.
23:34
And the great thing is that it, it doesn't discriminate of between what proteins it goes after.
23:39
It's just interested in cleaving very specific amino acids which are present in most if not all proteins.
23:46
And what that's going to do is that's going to chew up essentially the molecule that we're interested in measuring.
23:52
So it results in these little blue peptides and it's also going to chew up the antibodies.
23:57
Antibodies are just very large proteins that recognize other molecules based on their molecular shape.
24:03
And then what we can do is we can say, OK, we want to detect the the things that are specific to the thyroglobulin using LCMS.
24:11
So then we would detect the intact mass of one of these peptides and fragment it and find specific fragments to that peptide and design an assay that would be able to measure the presence of thyroglobulin in a sample regardless of the presence of patient auto antibodies.
24:30
And that's where we can leverage the selectivity and the agility of the workflow that goes into measuring target molecules by LCMS.
24:41
We can leverage that to bypass a challenge that would be more difficult to overcome in an immunoassay environment.
24:50
So one more example that I'll talk about before we wrap up this discussion is where we can apply liquid chromatography tandem spectrometry to the measurement of steroids.
25:07
So steroid measurements such as molecules that span from progesterone 17, hydroxyprogesterone, estradiol, cortisone, testosterone.
25:19
All of these can be measured traditionally by an immunoassay.
25:25
But remember I said in the last slide, immunoassays use antibodies to detect molecules essentially based on their molecular shapes.
25:35
Well, what happens when you have molecules of a class where their shapes tend to be very similar?
25:43
Sometimes it's more difficult to find antibodies that can very specifically bind one of these structures and not at least some amount of these other chemical structures that you see here.
25:57
So the result is something we call cross reactivity, which means that the reported value, let's say I'm measuring low levels of testosterone in a patient and my antibody and that assay may not be very specific.
26:09
I may measure a little bit of testosterone, but maybe I'm going to pick up a little bit of cortisone or estradiol along the way.
26:15
It's going to out compete the low level of testosterone that's present.
26:19
Well then I'm going to actually generate a result that overestimates the amount of target analyte because I'm not just detecting testosterone, I'm detecting one or more of these other things that may be present and slightly cross reacting with with my detection antibody.
26:35
So this isn't so much of an issue in high levels.
26:38
For instance, we measure testosterone by immunoassay and men all the time because they naturally have really elevated testosterone levels and so even if they bind low levels of some of the other hormones that may be present, that signal is still going to be majority, vast majority testosterone.
26:57
At very low levels.
26:58
The across reactivity becomes more problematic and we end up with more imprecision and inaccuracy in the reported result.
27:06
This is where again we can utilize that specificity by mass spectrometry to 1 achieve separation in the chromatographic space on the liquid chromatography and between some of these compounds based on some of the organic functional groups that are present on the steroid structures.
27:26
So we can separate these in time space and even if there was coalition of some of these molecules in the time space, these will all have slightly different intact masses.
27:39
And they will also have specific fragments that if I collide and break these molecules into individual pieces, I may have little spots on these molecules that vary significantly from steroid structure to steroid structure.
27:54
So just looking at this position on this five membered ring, we've got this carbonyl group here, a carbonyl and hydroxyl group, just a hydroxyl group, again just a hydroxyl group, but we'd have two different intact masses and then this carboxylic acid group or this amide.
28:13
So what this allows us to do is leverage the additional specificity of liquid chromatography, tandem mass spectrometry to be able to detect at very low levels, very specific and accurately the amount of the target analyte we would be interested in.
28:31
In this case it would most likely be testosterone.
28:35
Now we wouldn't necessarily always prefer a mass spectrometer in these cases.
28:42
And that you know, just by saying that a mass spectrometer can detect these things very reliably all the time.
28:49
That doesn't mean that we have to throw everything, you know every assay that we use for an amino acidate on the mass spectrometer.
28:58
And in some cases we can actually use that cross reactivity in the antibody or just the antibody's ability to detect very low levels as a substance to 1st enrich our samples.
29:10
So essentially give us another degree of reducing mixture complexity and then using the specificity and cell activity of mass spectrometry to get a more accurate measurement that way as well.
29:23
So we would utilize the antibodies more for boosting our sensitivity, but then we would leave all of the detection capability to the mass spectrometer.
29:31
And that's why again this technology being so versatile and being able to customize workflows before we even get to that system makes this a very, very powerful technique to deploy in the clinical lab.
29:48
So with that I'm going to wrap up this discussion.
29:50
Hopefully you've coming away from this presentation understanding at a high level what A a liquid chromatography mass spectrometry system looks like, the basic principles with which it operates, but also the power of analytical separation and and reduction of complexity and very complex biological samples that this technology brings to bear.
30:16
And I am happy to answer any questions she might have.
30:21
So Sarah, I will turn it back over to you.
30:27
Wow, thank you so much Doctor Collier for that information.
30:31
You know as a clinician that's been ordering labs for over a decade, I always find these conversations very humbling in that there is so much intricate detail that goes into the results of our our laboratory assessment.
30:44
So thank you so much for the that information.
30:47
A couple of questions that I did have after listening, you know, is it safe to say that LCMS is a superior technology to the immunoassay?
30:57
Should we be utilizing LCMS for all of our testing?
31:02
You know at this point there's still a lot of scenarios where immunoassays are the gold standard of a clinical measurement or there is just not the need for the additional specificity of the technology.
31:15
There are clearly some areas where mass spectrometry is going to be a superior approach to a tackling a a specific problem.
31:23
You know the hormone testing, testosterone testing, the example I gave is is one of the things that illustrate that point.
31:30
There's a lot of assays out there where the immuno assay is perfectly precise and accurate, well developed and standardized.
31:42
So no, I wouldn't necessarily say that everything that we ever perform should be done on a mass spectrometer.
31:48
As a scientist who was trained in that technology, I may be biased and say I wish that everything was done on that instrument, right.
31:55
But just practically there's there's not necessarily the the clinical need that everything be done with that technology.
32:03
But there are certainly very specific scenarios where we can leverage the mass spectrometry technology and all of that extra separation to to give us better answers and and better results and more reliable results for our patients.
32:16
Awesome.
32:16
Thank you.
32:17
I I appreciate that clarification.
32:19
So what are your top take home points for our listeners watchers today regarding the use of LCMS.
32:26
So the take home is this your blood, any biofluid that we're producing and and sending to the clinical lab, it's a very complex mixture.
32:36
There's thousands of things present and the best way to measure any of those things is to reduce that complexity.
32:42
And that is something that LCMS technology brings to the table.
32:46
The ability to separate in three or more different dimensions to get that complexity of that mixture to a minimum level.
32:54
And then the combination of the intact masses and the fragment masses giving us unambiguous detection of the the analyte of interest it.
33:07
It becomes something again that's very powerful, it's highly customizable and it's probably one of the best technologies that I would go to to develop a test to detect something novel or new in in the clinical space.
33:22
But otherwise you may be sitting there for years waiting for an assay to be developed by a traditional method that we could tackle in, you know, weeks, months, you know, less than a year using an LCMS type platform.
33:37
So it's just a very versatile technology.
33:40
It's it's something that is becoming more widely used in the clinical laboratory and it's something that's it.
33:46
It produces a lot of our cutting edge assays.
33:50
It sounds like there's so much more to come with LCMS.
33:53
We've really just scratched the surface on what we can use it from a clinical perspective.
33:57
So excited to see what's to come in the world of LCMS in the future for our audience.
34:03
Just know that we have experts that span the country.
34:06
In regards to helpful resources that can answer your questions.
34:11
Regarding methodology, you can connect with your local Quest account Executive or you can try reaching out to the client service number listed at the bottom of your report to connect you with resources available for you at Quest.
34:25
Thank you again, Doctor Collier.
34:26
It's been a pleasure.
34:28
The pleasure was online for additional education related to cardiovascular, metabolic, endocrine and Wellness health.
34:36
Please check out Quest Diagnostics Clinical Education Center for more webinars, podcasts in the most up to date publications and conference presentations.
34:45
Take care.
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