Dr. Tina Settineri: Hi, everyone. My name's Tina Settineri, and I'm Director of HPLC Products at Exigent, part of ABSCIEX. And today, we're gonna talk about enabling improved sensitivity, faster separations and smaller sample volumes with the new ekspert™ Micro LC 200 System.

So, the ekspert™ Micro LC 200 System for LCMS is a new system ideally suited for Microflow UHPLC. It enables fast high throughput analysis and is very simple and easy to use. It comes in two flow rate range systems, either 5 to 50 microliters a minute or 20 to 200 microliters a minute. The system is designed and plumbed very specifically to give the absolute maximum chromatographic performance within those two flow rate regimes.

It is ideal for high throughput and highly sensitive LCMS applications, including the MPK, peptide quantitation [sp] and applied market applications.

So, why do people want to do micro LC in the first place when you think about either comparing that to either high flow analytical LC or manifold LC? Well, with micro LC, you can in general increase your throughput and sensitivity relative to high flow analytical flow LC and increase your throughput for sure versus manual LC. And that comes in combination with a lower total cost of ownership and operation versus both analytical LCs and manual LCs because the system is a much simpler design.

And then, certainly versus high flow LC, you're gonna save a lot of money in solvent or model save cost. So, that adds to your lower total cost of ownership versus high flow LC.

So, summarizing that here, you can increase your throughput with faster, high quality separations due to the very low dead volume design of the system, you could increase your sensitivity with higher or equal sensitivity versus high flow and with smaller ID columns and smaller injection volumes. And we'll talk about that in more detail with some examples.

And then, as I mentioned, you can reduce your total cost of ownership versus standard HPLC or UHPLC because the system is not a system pump design or anything like that that has parts that wear out and require changes and updates every quarter or so. It's much simpler. And then, again, you're gonna reduce your mobile phase cost because you're flowing at micro flow rates instead of high flow rates.

And then, with our new improvements versus our previous micro LC, you could reduce your total sample consumption. And we'll talk about that in more detail.

So, first, a comment around sensitivity - this is a very simple diagram representing how sensitivity and peak concentrations increase or get higher as you go to smaller column diameters. So, on the left side on the top, you see it says highest sensitivity and highest peak concentrations, and on the bottom there, you'll see with the different size columns and flow rates used, you're gonna be using significantly smaller columns and lower flow rates to get that highest sensitivity versus standard analytical flow LC where you're gonna be flowing usually at 500 microliters a minute or higher with a 2.1 millimeter diameter column or larger.

That enables your greatest column loading but not necessarily your highest sensitivity, especially depending on the interface to your mass spectrometer. So, sensitivity in column loading [unintelligible] scale with the square of the column diameter.

So, to go into a little bit more detail, reiterate again why people are interested in doing micro LC, so in terms of increasing your throughput, this system allows 10 to 50 times smaller delay volumes versus high flow LC systems because of the way the system's plumbed and designed specifically for micro flow LC. And so, that enables you to do faster gradients because your inject times can be quite fast.

Increasing your sensitivity--now, you get equal or higher sensitivity with smaller injection volumes on this system versus high flow LC systems. And typically, we see about a four times improvement in sensitivity with a range of either, you know, equivalency to analytical LC, or in some cases, we've seen up to a 20 times improvement in sensitivity simply going to micro LC and very small diameter columns such as these .3 millimeter columns that flow at 10 microliters a minute versus analytical flow LC.

And then, in terms of reducing your cost of ownership versus standard HPLC or UHPLC, we have a simplified pump design that lowers your operational cost. It's basically like a large syringe pump that this is pushing continuously at a very accurate slow rate versus a piston pump, which is always moving back and forth and has many more parts that can wear out over time.

Because you're flowing at micro LC flow rates, you have less money spent on mobile phase solvents and any waste disposal that might be involved or storage costs, for example, ahead of time. And then, because you're flowing a lot less liquid into the mass spectrometer, you have typically longer integrals between MS cleanings because you're not spraying large volumes of solution into the mass spectrometer.

And then, finally, in terms of reducing your sample consumption, you have generally lower injection volumes required for micro LC compared to standard LC. And so, if sample quantity is an issue, you're typically using less sample.

And then, places where that happens is in incurred sample reanalysis, sometimes if you have to rerun your bioanalytical samples and you might be running out of sample in that case, that becomes less problematic since you are injecting smaller amounts. For your sample prep and extraction, you typically don't have to extract as much sample, and so that can save you money. And then, often, by reinjecting say a tenth of your concentration of a sample versus what you might originally inject, you can perform a wider range of PK studies with a given sample set because of the capability of the system to inject very small sample volumes.

Okay. So, what have we done to make the ekspert™ Micro LC 200 System? So, we started with our previous version of the system, which is called the Express HT Ultra, which is our fast micro flow UHPLC system, and we've added a new auto sampler with dynamic load and wash capability called DLW, which enables fast washing and very low or almost completely no carryover for most samples. And then, we've mounted the valve in a slightly different way to have the syringe from the auto sampler inject directly into the valve, which enables very low waste sample transfer and enables very small volume injections.

So, here's just a quick example of how micro LC compares versus a standard LC for a method transfer and why you might want to choose to do micro flow LC. So, this is an example from a customer who had the micro LC system and compared running a standard method that they'd already had developed on a standard LC that runs at 1.2 mils a minute injecting 11 microliters on a 10 microliter loop, so essentially a 10 microliter injection, and running a standard at one nanogram per mil in crashed plasma, so a typical DNPK bioanalysis sample.

And then, they wanted to transfer that to micro LC, and so they--as shown on the bottom of this slide, the micro LC system, they injected half the amount of sample, so same concentration from their crashed plasma, but they injected five microliters into a five microliter loop and ran that same standard and obtained about four or five times the signal intensity.

So, with half the volume injected, they're getting better signal. And so, this is what micro LC provides you, because in this case, we went from flowing at 1.2 mils a minute to 40 microliters a minute and then a scaled column of course at the same time to provide you that increased signal intensity of up to six-fold in this case.

So, just to go over some features specific to the ekspert™ Micro LC 200 System, as I mentioned at the beginning, we configure the system into two different versions, 5 to 50 microliters a minute or 20 to 200 microliters a minute. It's compatible with .3, .5 and 1.0 millimeter ID columns. The pump design is our patented splitless [sp] pneumatic pumps with feedback on our micro flow control that enables very, very accurate and precise control of the flow rate and enables a maximum column dock pressure of 10,000 PSI.

And we've added this updated auto sampler with dynamic load and wash for minimal carryover. And coupled with that, of course, is a high throughput auto sampler. So, it can be cooled and you have options for up to six sample trays, which can be 96 well [sp] or 384 well plates as well as vials and enables very high throughput analysis.

And the unique feature of this system is the gradient delay volume is only one to three microliters depending on exactly how you've got things plumbed. So, it's extremely small. Most other analytical LCs have delay volumes in the, you know, 50 to 200 microliter range.

So, even though they might say in their specifications that they could--they can actually flow the system at 10 microliters a minute, ideally, you would never do that for any type of typical analysis, because if the delay volume is say 50 microliters and you're flowing at 10 microliters a minute, that means you're gonna be five minutes behind from injection before the peak even comes out. And then, your gradient that's programmed, if you don't do a very, very slow gradient, you're gonna be behind essentially in the gradient and have an inaccurate gradient profile.

So, with the micro LC system, again, where we want to flow typically anywhere from five to 200 microliters a minute, the gradient delay is well suited to match with that for high throughput analysis.

And then, there's a column you can use on the system, comes with the system, goes up to 80 degrees C. And our programmable injection capability is enabled for injections in the nanoliter range, which is quite unique. And we've injected on the system as low as 15 nanoliters up to 10 microliters with a 10 microliter loop without requiring a change. And I'll talk a minute in a slide about more specifics on our programmable injection capabilities.

In terms of the DLW specifications on the bottom right here, typically, it's less than .003 percent, which is quite good.

Okay, as mentioned, this is the programmable injection that recalled metered or time sliced to enable these very small but very accurate nano volume injections, which can be used for micro LC. That's also used on our nano LC system.

And essentially, it's doing it by time. Because we can control the flow rate of our system very precisely, we're actually determining a injection volume based on a, like an injection time. And it's easily programmable in the software. And as you can see from these examples showing peak area versus injected volume, you know, either up to one microliter on the top, so zero to 1,000 nanoliters as well as zero to 10 microliters on the bottom with very good R square values here. It's a very precise method for doing a whole range of small volume injections.

So, here we have the graphic of how the syringe outlet is connected to the valve from the auto sampler into the valve of the pump. And then, that pump is connected to a wash station to allow washing of the system to prevent any carryover.

So, what's new in this design is that there's no sample contact with the syringe whatsoever. And the sample goes into this holding loop that's in the middle of the graphic here. And this enables no carryover then of the sample touching the syringe.

And then, having this syringe plumbed directly into the value allows almost zero sample loss on injection so that you really only use one microliter or maybe 1.1 microliter to inject one microliter of sample. And then, we still have the capability to then wash through the loop to clear out any leftover sample, and again, prevent carryover.

So, here's an example showing these very small injections and with a real sample [unintelligible] quantitative experiment where we show increasing sample not injected and then the peak area obtained from that on the mass spectrometer. This is 10 nanograms per mil of Verapamil in a 53 second cycle time to inject time for the analysis.

It's shown here with a gradient--it's a 30 second gradient using a half millimeter by 50 millimeter halo column. And you can see in the table, as you inject larger sample volumes, the RSDs [sp] on those peak area measurements are all below 3 percent except for the last one. It just goes over 3 percent, so very small RSDs on triplicate measurements of these samples. And even the smallest volume injection of .25 has only a .6 percent RSD on the bottom left.

So, this slide shows an example of very small volume sample pickup and sample use, and again, measuring the accuracy here with peak area RSDs. So, with a very small amount of sample to start with, this is 750 nanograms per microliter of paraben sample and looking at, in fact, by UV here, the readout, the area versus injection volume, again, injecting one microliter, then two microliters up to five microliters with RSDs all less than 2 percent.

And we only use less than 10 microliters of sample to inject all of these samples, so all from the same vial. So, no overfill required to inject these very small volumes.

The one thing to point out, what’s critical for LCMS at these micro LC flow rates is to minimize any post column dispersion before you get into the mass spectrometer. And so, we're doing that by these new design hybrid electrodes for the lowest flow rates.

So, we have two different sizes that we've created, a 25 micron ID and a 50 micron ID. And they're made primarily of peak fill [sp] with a small stainless steel [sp] so that you can minimize any potential service interactions that might occur with an all stainless steel stip. And these just plop simply into the turbo resource. And as you can see from the graphic on the bottom left, using the narrower electrodes spraying into the mass spectrometer, you get significantly narrower peaks, and that results in higher counts in the mass spectrometer. So, a little bit more detail on these electrodes shown here - again, there's two different sizes. It shows you the peak fill with the stainless steel tip. And again, those just replace the electrode that goes in the standard turbo resource. You just screw it right in and there's no real optimization or adjustments required. The tip just has to stick out the end of the probe very slightly.

The wire there is for grounding onto the source. And as you can see, these are designed for flow rates of either five to 20 microliters a minute for the smallest electrode or 20 to 200 microliters a minute for the larger electrode. And they're very easy to install.

Okay. So now, we're gonna talk about some more applications using our new system. This slide here shows an example of real DNPK analysis done at a customer site where they were looking at transferring a method from a traditional two millimeter column at 500 microliters a minute for LCMS DNPK samples in biological matrices to a one millimeter column where they would flow at 150 microliters a minute to perform micro LC, which require less sample and save them a lot in both solvent and time.

So, in this case, they needed to scale the injection, because if you are typically doing a cross plasma cycle, it's in all organic. And so, 10 microliters of that into a flow of say 500 microliters a minute on a two millimeter column is okay. But, with micro LC, we tend to want to inject smaller volumes to make sure we can retain these samples on the smaller ID columns.

So, in this case, they scaled the injection down to a one microliter injection but essentially got the same signal to noise overall. And it's showing this one example of a single peak here on the right, but the details are summarized in this table on the left looking at both Haloperidol and Vesperon peak area and retention time with a 250 injection analysis provided very good standard deviation and RSDs.

And one thing people think about or worry about with--as they go to smaller ID columns and lower flow rates is robustness for DNPK analysis. So, here's an example where we actually did some DNPK samples down at 10 microliters a minute and injected repeatedly and, you know, did gradients repeatedly for over five days with 750 or more injections. And the graphic here is showing you the area response ratio between two of the peaks, Amipramine [sp] and Clomipramine. These were standard spiked [unintelligible] plasma for the study. So, it was a real matrix type sample. And you can see, it remained very constant across the entire run with an average response ratio equal to 1.3 between the two samples with a standard deviation of only 0.05 between these two. And then, for the entire analysis with more compounds involved, it was a standard deviation of 3.8 percent. And this was done with a .3 millimeter ID column at 10 microliters a minute. And on the bottom right here, it shows just an example of one of those peaks and the--so, there's the signal intensity in the mass spec from day one to day five and how it really did not change, how there was no loss in signal intensity or chromatographic performance observed across the 750 plus replicate injections.

Okay. For peptide quantitation workflows, micro LC offers the capability to flow at much higher flow rates relative to nano LC and allows a significant improvement in throughput because of that. So, typically, with nano LC, with real quantitative samples, you're often doing a trap in a looped type workflow, and that takes time.

Very typically, and shown in this example, it's a 75 minute run time for this nano LC workflow for something like a 10 to 15 minute trap step and then a 60 minute run time for the run the gradient plus to re-equilibrate the column.

So, this example compared to the same sample run on the ekspert™ Micro LC System at 10 microliters a minute instead of 300 nanoliters a minute now enables equivalent chromatography, but at a much higher throughput. So, you can see the length of the time for the micro LC analysis is eight minutes per run with no carryover to worry about.

So, an entire curve where you ran 33 samples with 11 data points per sample plus six blanks takes about six hours or less than six hours, and compared to doing that with nano LC where you have a 75 minute total run time, where the entire curve takes about 50 hours, that gives you an eight times improvement in throughput to go from nano LC to micro LC.

So, now you do have to inject more sample. So, in this case, there's something between five and ten times more sample injected for the two comparative experiments. But, if you have enough sample to inject and throughput is your primary need, this is a great alternative.

So, again, this was run on a QTRAP 4500 system, either in nano flow mode or micro flow mode and using a .3 millimeter column for micro LC and a 75 micron ID column for nano LC.

Okay, one last example is more classic applied markets sample. This is a sample from a class of what we call pharmaceutical and personal care product. So, that's abbreviated as PPCP. And these are measured often in environmental samples such as water.

And of a typical high flow method, here's one at 450 microliters a minute with a two millimeter ID column. And we had a customer interested in looking at moving this to a micro flow method. In this case, we're looking at 23 compounds in these water samples.

In the micro flow method, we used a .3 millimeter ID column run at 15 microliters a minute. And so, you can see several changes here.

So, first of all, we injected less sample both in volume and concentration to go from the analytical or high flow method, which is shown on the top of this chromatogram versus the micro flow method shown on the bottom. Then, you can see the gradient time was significantly reduced. So, it went from about a 15 minute gradient to a five and a half minute gradient.

And you can't see it in the detail, but we still did have resolution of three key estrogens that were part of this analysis. So, that was what kind of set the limitations for even going--providing a faster gradient.

In addition, with the four times less sample injected on the column, the peak response increased by a factor of two. So, we injected less volume, less total sample, but yet we still got increased sensitivity on the mass spectrometer.

And this is shown on the next slide in more detail, looking at a number of these compounds, area ratio, peak height ratio and signal to noise ratio comparing micro flow LC divided by high flow LC. And so, you can see, in every case, the area, height and signal to noise increased from--with micro LC versus the traditional high flow method.

So, what I didn't mention in the pervious slide is this is a high flow LC, not a UHPLC method. So, it's a very old style LC method that certainly could be improved at high flow, as well. But, in this case, the customer was interested in running at micro flow LC rates.

So, again, the high flow analysis involved the injection of four times more sample on column versus the low flow method. And yet, we got--and coupled with that, we got a four times increase in signal to noise overall in general for all these compounds.

So, last point to consider is saving money on your mobile phase cost to increase your ROI with regards to purchasing instrument is up to 95 percent less mobile phase required for micro LC. And so, this table basically does a very simple analysis to think about what your cost, just for Acetanitral [sp] might be with an analytical flow system running at 560 micro liters a minute versus running different flow rate regimes on the micro LC.

So, in any case, you're gonna be saving thousands of dollars in Acetanitral cost per year. And of course, this was based on one set of parameters which are listed on the very bottom here for what the typical cost is for, you know, a case of Acetanitral, and assuming that you're at a certain throughput running 768 samples per day run five days per week at 52 weeks per year.

So, clearly, depending on what your requirements are on your throughput, that's gonna dictate how much money you'll save in mobile phases. But, certainly, it's easily in the thousands of dollars per year.

Okay. In summary, hopefully, we've learned that the ekspert™ Micro LC System for LCMS has a number of new features that enable faster separations and smaller sample volumes.

So, it's a micro flow UHPLC system ideally suited for micro LCMS. It provides fast high throughput analysis. It's easy to use. It's ideal for high throughput and highly sensitive LCMS applications including DMBK, peptide quantitation and applied market applications and especially ideal for sample limited applications where you have really zero sample loss on your injections. So, something like a dry blood spot analysis or something with incurred sample requirements has big advantages here.

It allows you to increase your throughput, either when going from high flow to micro flow or certainly nano flow to micro flow, increase your sensitivity with equal or higher sensitivity versus high flow analyses with smaller ID columns and smaller injection volumes. And you can increase your total cost of ownership versus standard HPLC or UHPLC and reduce your sample consumption.

So, thank you, everyone, for your time, and hopefully, we've provided some relevant information on why you might want to do micro LC with our new ekspert™ Micro LC 200.