Clinical Applications and Benefits of In

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Solid phase extraction, or SPE, is a common sample preparation technique used to separate analytes of interest from matrices that often cause ion suppression. The most common formats of SPE on the market exist as cartridges, 96-well plates, on-line, and QuEChERS. Except for the last example, SPE was traditionally made in a fixed sorbent bed. This presentation will introduce a relatively new format, in-pipet tip dispersive SPE, DPX (Dispersive Pipette eXtraction).

DPX tips containing a recently developed polymeric HLB (Hydrophilic-Lipophilic Balanced) sorbent from MilliporeSigma were used in several clinical/toxicological applications across multiple biological matrices (urine and serum). Although the workflow using the DPX HLB tips can be completed manually, this webinar will highlight the full automatability of sample preparation using robotic liquid handlers. Advantages of this novel technology, including time and cost savings, will also be discussed.

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Hugh CramerScientistMilliporeSigma

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Hello everyone and welcome to lab managers Product Spotlight webinar series. My name is Mary Beth de Donna and I'll be moderating today's discussion, clinical applications and benefits of in pipette tip dispersive SPE. We like our webinars to be very interactive, so we encourage you to submit your questions to us at any point during today's webinar. Our speaker will address these questions during the question and answer session following his presentation. To ask a question or leave a comment, simply type your query into the q&a box located on the right hand side of your screen.

We will try to address as many questions as possible during our time together. But if we happen to run out of time, I will forward any unanswered questions to our speaker and he can respond to you directly if possible. I would like to remind you that this webinar recording will be available on demand shortly following this presentation. So please watch for an email from lab manager and how to access this free video once it's available.

I would also like to extend a special thank you to our sponsor milliporesigma. Their support allows lab manager to offer these webinars free of charge to our readers. With that I'd like to introduce our presenter for this webinar. Hugh Kramer has over 35 years experience working on analytical chemistry applications and products and milliporesigma a business of Merck KGaA, Darmstadt, Germany. He started his work as a GC chemist and he later supervise the QC area for several years before joining the applications area in HPLC. r&d. In recent years, his main focus has been the development of sample preparation HPLC and LC ms methods here thanks for joining us today.

The Life Science business of Merck KGaA Darmstadt, Germany operates as milliporesigma in the US and Canada.

In today's webinar, we'll cover the DP X intercept format and how it's used. Then we'll look at this appel swift HLB material. I'll show a short video displaying the tip in an automated process. I've also included three applications today. The first is opioid drugs from urine, then steroids from serum. And finally we'll look at THC CBD and their metabolites and this is also from serum the DP x in tip format. Today I'd like to share with you an innovative product launching soon the DP x in tip SPE. DP X stands for dispersive pipette extraction a patented technology. It differs from traditional solid phase extraction in that the absorbance not packed tightly between two frets, but rather is contained loosely within the tip. It works by aspirating and dispensing solutions with the tip. If we look at the picture, there's a fret at the bottom of the tip with the absorbent above.

Also note part of the way up there's a blue disperser that aids in the mixing. Here we show a an eight channel pipe pattern loaded with GPX tips ready to process and process a 96 well plate. This illustration shows a typical bind wash elude extraction process this common to SPE however, with the DP X tips, we use an aspirate dispense function, instead of flow through as with SPE. From left to right, we begin with conditioning. The conditioning solution is brought up and aspirate and dispensed. Then we move on to a bind step where we load the analyte onto this absorbent. The next step is a washed up where we wash away interference.

And finally, a strong solvent is used to elute the analyte of interest. The next few slides we're going to review the recommendations from our method development guide. First, conditioning is not always necessary with our HLB material. Sometimes it aids it's aids in recovery. It's analyte dependent. Now this conditioning step usually is five to 30% methanol. We use about 750 microliters if we're doing a one ml tip, usually one or two hours separations is adequate. To load the sample in the binding step, we usually use three or four aspirate dispense cycles. That's, that's usually adequate. If you have an aqueous non viscous sample, you can aspirate a little bit more air than the sample volume.

So if you have about a 300 microliters of sample, you can set your pipette or to about 400 microliters. And that extra 100 microliters of air will aid in the mixing. If you have the viscous sample like serum or oil fluid, not really a good idea to do that. So you just stick with the same volume as you have. We recommend only about 5% Organic in this step in your solution to make sure you get adequate binding. wash cycles used to remove interference, you can either use a single solution are a series of solutions in the process. It's common to use a 10% methanol solution aspirated just twice to do that. If you need to increase the get rid of more interference, you can increase the percent methanol, but that's all dependent on the polarity the analyte of interest.

A solution with high organic content is used to elute the analytes. That solution has to be water miscible because there may be a little leftover water from the previous step. Our datasheet includes a table that suggests elution volumes based on the mass of the sorbent in the tip. A suggestion to aspirate air as we did in the bind step to aid in recovery and also in recoveries can sometimes be improved by multiple illusion Alec Watts instead of just one large one. Our DPS HLB tips are available in different formats.

On the left side of the photo you can see the microevolution tip. The big advantage with the micro evolution tip is a smaller elution volume as low as 25 microliters. That tip is only available in the Hamilton format. On the right hand side you'll see the one ML type pipette tip. It's available in both the universal and Hamilton format. The Aleutian volumes for it ranges between three and 800 microliters.

One other comment you'll see you'll notice there's a legacy shaded portion or looks like a little the particles up in the tube on the one ml size. That's typical because the absorbent is loose in the tip. Differences between the DP x format and traditional SPE include solutions are aspirate and dispense the pipette not top loaded. That makes the function based on equilibration instead of flow rate. The loose sorbent allows for maximum surface area contact and increases speed. I list these as arguable benefits because we all know ESPYS a great technique. There are many ways to that are used in it. But we see greater contact and mixing speeds with the DP x hi analyte recovery high throughput, rapid extraction times high reproducibility and automation compatibility.

In the next section, we're going to review the material that we pack into these tips. In this case, it's the spell swift HLB material. Spell swift HLB material is a polymeric stationary phase. The ATL B stands for hydrophilic and lipophilic balanced. The structure contains both hydrophilic and lipo Vic philic functional groups that aid in the extraction of a broad range of compounds from aqueous samples. This table lists features and benefits of the HLB material.

Again, the broad range of analytes from POLAR NONPOLAR acidic to basic, the greater capacity here we feel like this leads to smaller bed weights and lower elution volumes. That helps out with the time savings in the sample processing. We list resistant overdrawing that's not applicable to the DP X cartridge format but some of the SPE some of the other SPE for are minutes that's important.

And we note our stringent production QC standards provide consistency and improve accuracy and precision. In the next slide, I'll show a video that demonstrates the ease at which the DB X tips can be automated. I will provide a little disclaimer here the video is played at twice its normal speed, so as not to bore you. And it shows a recovery of blue from green food coloring. There is no conditioning in this step. Here we see the robot picks up the GPX tips and their meat and he moves into the wells with the green food coloring. It draws them up and then just aspirates and dispenses it right back into those same wells.

You can see the blue has stayed with the support there at the bottom of the tip. We move over to the wash step I'm going to draw up a solution of 20% methanol and water dispense it and now you can see the state very well you can see the light blue at the bottom of the tip we move over to the Aleutian wills that are filled with 100% methanol, draw that up and lights move from the support on into the methanol and are dispensed straight back into that. That well for later analysis.

We begin the application section with a the extraction of opioid drugs from urine. This extraction was on 13 opioid drugs and internal standards from urine. They represent a wide range of log PS. Note the first three or what have a log p of less than one that's usually difficult for a CA team phase to retain. The cleanup was performed with the DPS HLB tips on Hamilton automated liquid sampler followed by LC ms ms. Analysis. When this particular study was done, our main interest was to look at tip to tip variability.

So we prepared a rather large 20 ML sample and then use that same sample across eight different pipette tips. I list here the sample preparation steps on the left that we did manually before we moved the samples onto a Hamilton automated liquid sampler to perform the extraction. First, we combined the urine, some phosphate buffer of beta kluk Your Honor day solution, the spiked analytes and their internal standards. This solution was then heated for 60 at 60 degrees for a couple of hours. And that allows the Bakel Beta Glucan Your Honor days enzyme to hydrolyze any glucuronide metabolites back to the parent drug.

The the tips were conditioned in this case, we use the 5% methanol solution, we aspirated dispense four times in the binding step we washed again with 5% methanol solution. And then the solution was performed with a 5% formic acid in methanol solution, and we aspirated and dispense two times to get good recoveries. The samples were then diluted with 100 and with 150 microliters of the solution was then diluted with 350 microliters of water before it was analyzed by LCMS. This slide shows the conditions used for the LCMS analysis of the samples.

The overlaid MRM signals on the right show the Santas Express phenol Hexcel column provides good resolution in less than six minutes. This slide shows the results of that extraction. We see recoveries for each analyte between 75 and 120% with an overall average around 96% With all RSDs general Less than 10% The table lists the the internal standard used with each analyte. Its recover corresponding recovery and percent RSD. This next application we're going to go over today is a steroid panel from serum. The work was performed by Madison Kilpatrick from GPX technologies, and James Ross from milliporesigma helped to assemble the information for us.

This method was developed to analyze various steroids fin serum certified reference materials serums will use from two different sources you tack and NIST total concentrations were determined. The microevolution tip was used in this case to go to very low levels and it was followed by LC ms ms analysis. This slide details the sample preparation and extraction processes used in the method. internal standard are added to the serum and incubated for one hour. Then a formic acid solution is added to dissociate the proteins and allowed to incubate for an additional 15 minutes. The sorbent in the tips was conditioned with a 20% solution of methanol and water.

The binding used five aspirate descent cycles to ensure good binding than to wash cycles were incorporated into this process. Initially, just 100% water is used to wash off the bulk proteins, then a second wash that included 20% methanol is used to clear the rest of the interference. Finally, the analytes of interest are eluted with 5050 methanol a cedar nitrile. The scatterplot presented here, so excellent overall correlation between the concentration values that were determined using the DPS steps and the values that were provided with the certified reference materials serums. The left hand graph is the uTec reference materials with the DP X results on the y axis and the U tack reference material results on the x axis. The right hand graph is the NIS material plot.

Here we have the detailed results from the NIS material only. I know the slides a little busy but I wanted to highlight the exceptional sensitivity and accuracy this method provides. This last application I consider a bonus. It doesn't use the ATL B material we've been demonstrating. Instead, this work uses our hybrid SPE material for the analysis of THC, CBD and their metabolites from serum. Normally cannabidiol and its metabolites would be a separate analysis from THC and its metabolites.

But because of structural similarities, we chose to develop a single method that can be used for both the method quantifies THC CBD and metabolites from serum protein precipitation and hybrid SPE cleanup was performed on Hamilton automated lipid liquid handler. And we use the internal standards. proteins and lipids are the most common matrix interference in blood and serum proteins and some lipids can be removed by organic solvent precipitation or a crash.

The remaining phospho lipids are a common source of ion suppression in LCMS analysis, the hybrid SPE materials he used to remove that matrix effect and it acts as a chemical filtration. In the diagram on the right, we see how the phospho lipid the phosphate group on the phospholipid interacts with the zirconium ion to filter it out. The following sample preparation was performed on a Hamilton liquid handler. First 300 microliters of Spike serum along with internal standard are placed in each well. Then we perform the protein precipitation by adding coal to Cedar nitrile with 1% formic acid.

The well plates are agitated at 1200 RPM for three minutes and allowed to settle from this 400 microliters of supernait and are transferred to new wells. They're then processed with the hybrid GPX tips, the tips are first conditioned with procedure nitrile with point 5% citric acid then the supernatant is loaded onto the tips and aspirate and dispense twice. That step filters out the phospho lipids, then the argument is dispensed into a new well for further processing with the LCMS.

The results from the sample preparation we see on the left hand side a bar a bar chart of the recoveries falling roughly between 80 and 20 120% for each analyte which is good. And then on the right side of a table of matrix cleanup for each analyte. The column titled serum extract is is what you would get after the foul protein precipitation only then the samples are further processed with the hybrid material.

So the difference in the matrix cleanup shows the improvement with the hybrid for this can cannabidiol snort much improvement but with the metabolites, it shows significant improvement from the 40% ion suppression down to 14% from 51% down to 16%. The TA TC is interesting in that you don't get ion suppression you get ion enhancement of that analyte and without one again, it's an improvement from the higher number to the lower where with the serum extract alone you get a enhancement of 207% yet with the after the hybrid cleanup is down to 166%. And for the other two analytes.

Again, we see improvement with the clean up with the DP X hybrid SPE material. To review today's presentation dispersive pipette extraction or DP X is a revolutionary patented technology and has the benefits of SPE internees use pipette tip. The absorbent is can loosely contained in the tip and low of elution volumes are a great benefit. The HLB D px tips are used for an extraction of broad range of compounds. It uses the binder washy loot process and provides good recoveries of analytes from both urine and serum matrix ease. The hybrid DPS tips are simple and generic way to remove gross levels of phospholipids it uses chemical filtration interferences not binding van lights of interest and it's an excellent clean up on a van lights from serum matrix. Thank you for joining today

All right, great. Thanks very much you for that wonderful presentation. So at this point, we are gonna move on to our question and answer session. again for those of you who may have joined us late, you can send in your questions by typing them into the q&a box located on the right hand side of your screen. I'd also like to let you know that the Chappelle swift HLB solid phase extraction tips discussed in this webinar will launch soon, along with the application notes presented. So please stay tuned to milliporesigma for more information.

If you leave a question or comment in the q&a box on the right hand side of the screen. We will have a record of your name and email address. So please leave a comment in the q&a box if you would like milliporesigma to contact you once this technology becomes available. Here. Thanks again for a great presentation. Let's jump right into the q&a here. Our first question here from the audience says what is the sample volume range I can use with the tips.

This the volume used is based on bed weight in the tip and we have different bed weights available. But for the small micro elution tip which had three micro three milligrams of bed weight, we would recommend in the range of 150 to 300 microliters. For the larger one ml tips. They can come with either 510 or 20 milligrams and material and There's a table in our product product data sheet that lists the volumes that we recommend. So with a five milligram, we would recommend you use less than 300 microliters of sample volume, the 10 milligram, less than 600 microliters. And with the with the large 20 milligram, you can go up to 800 microliters of sample volume.

Okay, great. Thanks very much. Let's move on to next question how to recoveries using these tips compared to SPE cartridges and 96? Well plates, well, you should get equivalent or or maybe a little better recoveries with a with a DP x, then you get in the traditional SPE or 96 well plates. And this probably has to do with the fact that it's based on an equilibration. The dispersal equal equilibration speed, and instead of the flow rate, not all SPE extractions are done at their optimum flow rate. So if you're, if you're off in that flow rate, you're gonna you probably would expect a little bit less than optimum recoveries.

All right, great. Thanks. Let's move on. We have a few more questions coming in. This one says How would I figure out what bed weight I need for my application?

Again, we'll we'll have a there will be a table with recommendations but with the HLB material, the capacity that materials about 10%. So if you know the, what's your expected recovery is or how much weight is is in your sample, you can you can work with that. But you always want to have a smaller bed weight as you can. Because the then you can use on a lower elution volume to be more sensitive. But again, it's based on the capacity of the polymeric materials about 10%. And you would generally try to stay with the smallest bed weight and smallest elution volumes as you can.

All right, great, thanks. Here's another question. This one says what is the upper limit of sample volume compatible with this technique?

Again, sort of go back to the first question that we had that had to do with sample volume range, the upper limit should probably be around that 800 Micro liter volume. And, again, that's that there's a relationship with the bed weight in that. Okay.

Okay, great. Thank you. A couple more questions here. This one says what is the mass of H lb polimer in the tip the mass that's the bed weight that we we list so on the on the micro illusion, it was three milligrams of mass in the tip for the that was for the steroid application, use three milligrams in that micro Galician illusion tip, the opioid application use five milligrams of bed weight, and the hybrid, I believe was 50 milligrams of bed weight in the hybrid clean up application.

Okay, great. Thanks. I think we have time for one more question here. This one says, Is there any variability in pipetting accuracy when using multi channel robotic arm technology?

Is there any variability in pipetting? Accuracy? Well, I would say that, if I'm interpreting this question, that's a great question. And if I'm interpreting it correctly, the across the tips, the variability is very low. And that's one of the things we we studied in that first application was that you know, we, we had the same sample and looked at it across the the eight tips and we saw very low variability.

Okay, great, thanks. We actually had another question come in if you've got the time for it. Have you attempted loading multiple Alex lots of sample for example, could you load 800 URLs of sample three times provided that of the sorbent can support that amount of analyte

I believe that you could. That's a real interesting question. I have not done that, that work. But that would be a nice experiment. And that's the good thing about these tips. It's it's such a friendly format, we're all used to pipetting. And these, all these small experiments can be run on the on the benchtop. Very quickly. So a study like that could be completed very quickly. Thanks for the question.

Okay, wonderful. Thanks so much. I'd like to give you all another reminder that this Chapelle swift HLB solid phase extraction tips discussed in this webinar will launch soon, along with the application notes presented. So please stay tuned to milliporesigma. For information, please leave a comment in the q&a box to your right if you would like milliporesigma to contact you once this technology becomes available, or you can reach out to them.

Hugh is kindly provided his email address and a QR code on the screen in front of you. So you can reach out for more information. So that does bring us to the end of this webinar. And again, I'd like to remind you that this webinar will be available on demand shortly following this presentation. Please watch your email for a message from lab manager once this video is available.

On behalf of lab manager. I'd like to thank you Kramer for all the hard work you put into his presentation. And I would like to thank all of you for taking time out of your busy schedules to join us today. Once again, thank you to our sponsor milliporesigma. Their support allows lab manager to keep these webinars free of charge to our readers. For more information on all of our upcoming or on demand webinars or to learn more about the latest tools and technologies for the laboratory, please visit our website at lab manager.com. We hope you can join us again. Thanks and have a great day.