Sensitivity, Selectivity, and Speed | Solving Analytical Challenges in Endocrinology using LC-MS/MS
Hello, everyone. And welcome to today's live broadcast, "Sensitivity, Selectivity and Speed." Solving analytical challenges in endocrinology using LC-MS/MS for clinical research. Presented by Dominic Foley, Senior Scientist at Waters Corporation.
I'm Benjamin Dugas, the Senior Marketing Manager for clinical diagnostics at Waters Corporation. And I'll be your moderator for today's event. Today's educational web seminar is brought to you by LabRoots and sponsored by Waters Corporation. For more information on our clinical solution, please visit us at waters.com/clinical.
At Waters, we understand that clinical diagnostics is more than collecting data. It's making a difference in someone's life. This is why we provide clinical LC-MS/MS solutions that you can trust in every step of the workflow. Now let's get started.
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I'd like to now introduce our presenter, Dominic Foley at Waters Corporation. Dominic Foley is a Senior Scientist in our clinical scientific operations team working at Waters' mass spectrometry headquarters in Winslow, UK. Dominic is specialized in the development of LC-MS/MS clinical research applications with a particular focus on steroid hormone analysis.
Dominic has worked in separation sciences for 13 years following his ambition of improving the quality of health care. For a complete biography on Dominic, please visit the biography tab on the top of your screen. With that said, I'd like to hand it over to Dominic to start his presentation.
Dominic. Thank you, Ben. I would like to welcome everyone to this webinar today. Analytical sensitivity, selectivity, and speed are critical parameters in most analytical methods. The challenges we observe in endocrinology research can be solved by leveraging the sensitivity, selectivity, and speed of LC-MS/MS technologies.
Today, I want to provide you with an overview of how high sensitivity Tandem MS platforms can be used to meet the demands of a clinical research environment. In this presentation on sensitivity, selectivity, and speed-- solving analytical challenges in endocrinology using LC-MS/MS for clinical research. There are three main learning objectives today.
The first objective is to understand the analytical benefits of using LC-MS/MS for endocrine methods over more traditional platforms, including immunoassay. The second objective is to show how we can incorporate selective sample preparation workflows to improve analytical performance of the method using LC-MS/MS systems. The final objective is to demonstrate how offline automation can be leveraged to improve method performance and optimize efficiency in LC-MS/MS labs. First of all, I wanted to cover the first learning objective by discussing the benefits of using LC-MS/MS methods of over platforms, particularly in regards to analyzing endocrine analytes in clinical research. I will outline what is LC-MS/MS IVD systems and the capabilities of these systems based on analytical sensitivity requirements in analyzing endocrine analytes.
I will discuss examples of the benefits of using LC-MS/MS and endocrinology clinical research applications, which includes steroids hormones in dried blood spots, estrogens in serum, and plasma catecholamines/metanephrines. Finally, I will summarize the presentation to highlight those key learning objectives again. Now, we want to highlight the key benefits using LC-MS/MS technology in clinical research of steroid hormone. This slide shows a steroid pathway, which contains many structurally similar steroid species. Using more traditional platforms, such as amino acid to analyze samples, cross reactivity of steroids can occur due to the lack of specificity of the antibodies being used. This can lead to poor analytical precision and accuracy, particularly a lower physiological concentration.
In addition these platforms, although fast, cannot only analyze a single analyte at once leading to increased time and cost if multiple online test methods are required. The beauty of using mass spectrometry and its ability to mass detect is that greater selectivity can be achieved for the steroid hormones while also simultaneously analyzing more than one steroid hormone on one once, if required. As many steroid hormones are isomeric to each other, the use of Tandem MS can improve selectivity even further by fragmenting the analyte into product fragments thus providing detection through a transition or multiple reaction monitoring approach. However, Tandem MS alone isn't infallible. Even with this level of selectivity, isomeric interference can still be observed as many analytes may share the same MRN transitions. Those on analytes highlighted in the same course here are examples of this.
Higher resolution mass spectrometry would also suffer from the same problems due to interference from the steroid isomers. In addition, isotopomers are also detected using MS/MS. And these can also cause interference, particularly those with analytes only two daltons apart, as shown here. Therefore, to attain the levels of performance required, an additional separation technique is needed.
The technique we use is liquid chromatography. For many of those analytes highlighted in the previous slides that share the same MRM transitions, we are able to separate them based on their chemical properties. In these examples here, differences in the positioning of the hydroxyl groups in the isomers allow separation of the analyte using UPLC. Therefore, it is critical to use this orthogonal technique with tandem mass spectrometry to minimize interference in steroid analysis. Before I move on to some examples of LC-MS/MS technology being applied to endocrinology research applications, I want to briefly cover these specific systems being used for LC-MS/MS analysis in this presentation. Here is the family of the Waters' IVD systems for clinical research analysis.
The foundation of the IVD family is the ACQUITY UPLC I-Class Xevo TQ-D which is a reliable robust and has a proven track record of performance. The mid-tier ACQUITY UPLC I-Class - Xevo TQ-S micro provides excellent sensitivity in a compact system. And at the head of the family is the ACQUITY UPLC I-Class - Xevo TQ-XS system.
This has replaced the Xevo TQ-S system in the family. And it's the most analytically sensitive tandem mass spectrometer available. This slide illustrates how we would relay the performance of these IVD systems to the sensitivity requirements of the classes of endocrine analytes. Xevo TQ-D system is able to analyze a number of well characterized analytes such as cortisol, testosterone, and 25-hydroxychloroquine vitamin D. Xevo TQ-S micro system is able to analyze these in addition to more challenging analytes such as metanephrines, catecholamines, and aldosterone which require detection at lower physiological concentrations.
The Xevo TQ-XS is capable of analyzing all the analytes shown here, including estrogens, thyroglobulin, and the active metabolite 1.25-dihydroxy vitamin D. In order to meet the demands of more challenging applications, higher performance LC-MS/MS platforms are needed to meet the requirements of speed, analytical sensitivity, and selectivity. MRM and chromatographic selectivity can be applied in the same manner across all the ACUITY UPLC Xevo mass spectrometer systems.
However, a high end platform such as the Xevo TQ-S micro and Xevo TQ-XS are necessary to avoid the trade-offs between sample throughput or speed and analytical sensitivity requirements of a method. Now, I would like to cover examples of applications where we are striving for either analytical sensitivity or speed while maintaining selectivity of the methodology using LC-MS/MS. These examples include a range of endocrine analytes in either dried blood spots, serum, or plasma. Dried blood spots are an established micro sampling technique providing a low cost approach of collecting, shipping and analyzing samples for clinical research. Immunoassays are are used as a primary testing methodology for DBS samples and steroid hormone analysis.
Although rapid, this sometimes require a follow up for confirmation purposes. The focus for LC-MS/MS method for steroid analysis in dried blood spots will be a high throughput method similar to immunoassay. But with the ability to measure more steroid hormones in a selective manner. Faster analysis requires greater selectivity in sample preparation with automation of the extraction providing a robust and reproducible sample preparation workflow. The analytical sensitivity of the UPLC I-Class and Xevo TQ-S micro system will enable analysis down to relevant steroid hormone concentrations using 3 millimeter blood spot punches. The primary goal of this method was to examine 17-hydroxy progesterone, the 17-OHP, androstenedione, cortisol, 11-deoxycortisol, and 21-deoxycortisol which are impacted by the 21- and 11-hydroxylase enzymes in the steroid pathway.
A reduction in 21- hydroxylase enzyme leads to elevated 17-OHP, 21-deoxycortisol, and androstenedione. We've reduced cortisol. A reduction in 11-hydroxylase leads to similar changes to 21-hydroxylase deficiency. But 11-deoxycortisol is elevated as opposed to 21-deoxycortisol.
Examination of multiple steroid hormone concentrations improves our understanding of these pathways. These steroids are also non-polar making selective sample preparation strategies for optimum analytical sensitivity challenging. Therefore, introducing a method to remove matrix effects is a route forward. In this case using mixed-mode solid phase extraction or SPE. Oasis mixed-mode anion exchange was used for this analysis.
Previous steroid hormone applications have used this approach. And it was also adopted in this instance. Ion exchange occurs at high pH, trapping acidic interferences which have been previously shown to contribute to ion suppression and background noise on the system. Steroid hormones are retained by reversed pair interactions. And these are eluted with increased organic solvents which leads to interference trapped on the SPE chemistry. This is a summary of the methodology we adopted to meet the challenge of analytical speed and selectivity, but achieving the necessary analytical sensitivity, too.
Sample preparation was automated on the Tecan liquid handler with 3 millimeter blood spots being mixed in internal standard solution and diluted. SPE was performed using the Oasis MAX SPE plates. The sample loaded and washed, and steroid hormone diluted, and directly injected onto the system. Separation was performed using ACQUITY UPLC I-Class with this CORTECS C18 2.7 micron, 2.1 millimeter by 50 millimeter column with a VanGuard precolumn.
Mobile phases where ammonium fluoride in water and methanol with a flow rate of 1 milliliter per minute providing a rapid separation time and sample throughput. Tandem mass spectrometry was performed on the Xevo TQ-S micro system with MassLynx version 4.2. Utilizing all the technologies available to us, automation of this SPE protocol was performed in less than 90 minutes per plate allowing for extraction of four plates in a typical working day. A rapid 1.3 minute LC-MS method with a cycle time of less than 2.3 minutes was created
with the separation of the relevant isomeric steroids including 11-deoxycortisol, corticosterone, and 21-deoxycortisol. This allows for the analysis of 394 samples in less than 15 hours using LC-MS/MS. This slide shows the typical workflow you might expect during a typical day of analysis of four full plates of DBS samples. The entire workflow from start to finish for a single plate is less than six hours. four 96 well plates, this can be prepared and analyzed within 18 hours. Proof of concept experiments were performed for this method that included linearity, analytical sensitivity, and precision.
Calibration of androstenedione and 11-deoxycortisol was performed from 0.5 to 500 nanograms per milliliter. And 17-OHP, cortisol, and 21-deoxycortisol from 1 to 500 nanograms per milliliter. LoQs also shown in the chromatographs here are based on signal-to-noise at peak-to-peak 0.5 nanograms per milliliter for androstenedione and
11-deoxycortisol. And 1 nanogram per milliliter for 17-OHP, cortisol, and 21-deoxycortisol. In-house DBS samples were also prepared at 2, 5, 50, and 400 nanograms per milliliter with total precision and repeatability evaluated in five replicates over five occasions. Also precision and repeatability for the five steroid hormones was less than 9.3% demonstrating excellent reproducibility of the method across the concentration ranges. So in summary, we believe we have met the analytical challenges associated with this type of method.
obtained excellent analytical sensitivity in DBS, down to 0.5 nanograms per milliliter for the steroid hormones. And selective offline automated sample preparation can improve lab workflows and removes metrics interference providing greater sensitivity to the final extract. Rapid separation with a run time of less than 2.3 minutes
cycle time enables analysis of four plates in less than 15 hours making this method an ideal candidate for rapidly evaluating steroid hormones in dried blood spots for clinical research. Next, I would like to cover the analytical challenges associated with the analysis of estrogens in serum. The focus for this method is analytical sensitivity where guidelines have been issued that methods require reproducible measurement down to one picogram per milliliter or three picomoles per liter.
In order to meet this challenge, selected sample preparation is required using LLE or liquid liquid extraction, or SPE. And the top performing ACQUITY UPLC I-Class and Xevo TQ-XS system for best in class analytical sensitivity. Mobile phase modifiers can improve analytical sensitivity further. Ammonium fluoride additives in the mobile phase have been shown to provide significant improvements in analytical sensitivity for this method. Estrogens themselves are generated from the androgens androstenedione and testosterone through the action of aromatase. The efficacy of aromatase inhibitors can be studied in clinical research by examining the changes in estrogen concentrations through aromatase inhibitor action on the pathway.
Analysis of estrogens at such low concentrations can be problematic for less selective platforms such as immunoassay. Use of LC-MS/MS can help overcome such a challenge. Much like the other steroids previously discussed, the estrogens are non-polar making, again, sample preparation a challenge for optimum analytical sensitivity. In this instance with analytical sensitivity, the main challenge to the method analytical alternative sample preparation approaches were evaluated which included SPE and LLE with LLE using a combination of different extraction solvents for evaluation purposes. Typically for LC-MS/MS methods, optimum analytical sensitivity is achieved using more selective techniques such as SPE and LLE.
And in this instance, both techniques were evaluated. And LLE with hexane/ethyl acetate was found to provide the highest analytical sensitivity when considering both estradiol and estrone together. The addition of ammonium fluoride as an additive makes a significant difference in analytical sensitivity for the estrogens.
Compared to mobile phase with no additives shown in red, there is a greater than 5-fold increase in analytical sensitivity and electrospray ionization negative ion mode to both estradiol and estrone. This is the summary of the methodology we developed to meet the main challenge of analytical sensitivity with selectivity. Important aspects of the method are highlighted in blue. Sample preparation was performed by adding internal standards to 250 microliters of serum. LLE with hexane/ethyl acetate the mixture was added to the samples and shaken for five minutes with the estrogens partitioning into the organic phase. Samples were centrifuged and then 700 microliters supernatant was transferred to a 96-well plates with glass vials inserts.
This was evaporated and reconstituted. The glass vial insert in this instance reduced absorption during evaporation compared to polypropylene 96-well plates. Separation was performed using the ACQUITY UPLC I-Class with the CORTECS Phenyl, 2.7 micron, 2.1 millimeters by 50 millimeters ID column.
Mobile phases were ammonium fluoride in water methanol with a flow rate of 0.3 milliliter per minute providing separation of estradiol and estrone. And the mass spectrometry was performed on the Xevo TQ-XS system providing us with the best possible analytical sensitivity for this analysis. The key performance characteristic is shown here, which is analytical sensitivity.
The limits of quantification was 3 picogram per milliliter 11 picomoles per liter for estradiol which showed the signal- to- noise appeared to be greater than 10 on the CV of less than 20%. The limits of detection was 1 picogram per milliliter with a CV of less than 20%. But only signal-to-noise greater then five, but less than 10 to 1.
The chromatogram here illustrates an overview of this testing, which demonstrates we are able to differentiate between a blood serum sample and a sample of 1 picogram per milliliter. For over performance characteristics, in-house serum QCs were prepared at 10, 75, and 750 picograms per milliliter with total precision and repeatability evaluated in five replicates over five occasions. Total precision and repeatability for the estrogens was less than 4.8% demonstrating excellent reproducibility of the method. Comparisons were also performed against samples from the CDC Hormone Standardization Program and NEQAS scheme for estradiol. During regression and Altman Bland analysis demonstrated excellent agreements with a minimal bias within 4% for both sets of comparisons.
With only a statistically significant proportional bias noted for estradiol when compared to the CDC Hormone Standardization Program concentrations. Certified reference material was also evaluated in triplicate over three concentrations and these returned results within 4.9% of the nominal concentrations provided. In summary for this particular method, using a combination of selective liquid-liquid extraction sample preparation, ammonium fluoride in the mobile phase, and the Xevo TQ-XS system, we were able to detect 1 picogram per milliliter concentrations for both estradiol and estrone. Precision assessments demonstrated reproducibility of the method across the concentration range and excellent agreement was noted for external quality assessment schemes. And the final message I would like to cover today are the analytical challenges associated with the analysis of plasma metanephrines and catecholamines.
As with the estrogens, the focus for this method is analytical sensitivity where these compounds exist at low physiological concentrations. The polarity and stability of these analytes make sample preparation a challenge. A selective extraction is required that can remove these ionic, polar analytes from metrics of lower pH conditions due to the instability of higher pH. Chromatography also becomes problematic due to this polarity.
An analytical sensitive system is required to detect the lower concentrations. So in this case, we used the Xevo TQ-S micro mass spectrometry for this analysis. The pathways here show how these catecholamines and metanephrines are generated in the adrenal gland. The metanephrines are generated from the catecholamines through the action of catechol- or methyl transferase. Now that these are structurally similar species, which means that typical analytical methodologies for this analysis such as HPLC with electrochemical detection or immunoassay can suffer from interference.
As discussed previously, the use of LC-MS/MS can overcome these types of problems. This picture also shows a LogP which, as you may know, is a measure of the hydrophobicity of the molecule. We are dealing with molecules that have negative LogP values which indicates that the molecules are very polar amines and molecules are elute earlier in the typical reverse phase chromatography conditions with issues around peak shape and matrix effect. In addition, the nature of these analytes makes sample preparation more challenging for analytically sensitive analysis. We meet the challenge of sample preparation through the use of Oasis mixed-mode with cation exchange SPE.
Analytes are retained by a mixture of reversed-phase and ion exchange retention. But ion exchange mechanism is a requirement for improved recovery and extra cleanliness. The ion exchange retention mechanism of this SPE plate occurs at higher than pH five. These analytes are ionized at a lower pH typically below 8 meaning that the region of ion exchange using this chemistry is between pHs 5 and 8.
Therefore, the pH for maximum ion exchange needs to be tightly controlled using a buffer diluent. 100% organic wash can be formed to remove non-ionic species from the plate. And the analytes can be eluted in a low pH organic solution which deactivates the ion exchange mechanism on the plates by deionizing the chemistry on the sorbents. The use of this chemistry also helps us avoid the use of higher pH conditions that can cause analyte instability. In relation to the challenges with the chromatography, many chromatographic methods to these analytes involve the use of hydrophilic interaction, liquid chromatography separations otherwise known as HILIC which can be challenging to implement.
Maintaining the pH of the mobile phase and SPE elution solvent is critical for reproducible retention times. In addition, chromatography is very sensitive to water contents of the extracted sample, which is illustrated in the chromatograms here with fronting and poor baseline resolution as a result of residual water in the injection solvent. Longer equilibration times are also needed to replenish the water layer on which the separation relies upon. This doesn't necessarily fit into most laboratory workforce were reversed phase methods are typically used, which is why reversed-phase method would be preferable for the workflow, ease of use, and improved robustness.
This slide shows an outline of the methodology applied to this analysis. Again some of the critical points are highlighted in blue. Sample preparation was performed using 250 microliters plasma diluted with internal standard in ammonium acetate buffer. Samples were mixed and loaded directly onto an Oasis WCX MicroElution plates and washed prior to elution. The use of microelution technology helps minimize the volume in the elution step, thus minimizing the time required for evaporation. Samples were reconstituted prior to injection.
UPLC was performed using the ACQUITY UPLC I-Class FTN system with a HSS/PFP column for retention and separation of the analytes using reverse base chromatography conditions with mobile phases of formic acid, water, and acetonitrile. Detection was performed using Xevo TQ-S micro mass spectrometer. And now I want to show some of the performance characteristics associated with this method. We were able to develop a fast and selective chromatographic separation using a PFP stationary phase, which fits into their reversed phase workflows in most laboratories.
With all analytes separated within two minutes. Potential interferences such as metformin, midodrine, and L-dopa were all well separated. LLOQ experiments demonstrated analytical sensitivity limits between 1 and 10 picograms per milliliter for the analytes of interest making quantification of physiological levels possible.
The method was also shown to be linear for the analytes to a concentration of 20,000 picograms per milliliter. We also looked at precision performance, which was evaluated for the assessment of in-house QC samples prepared at 15, 30, 750, and 1,500 picograms per milliliter of plasma. Inter- and Intra-Batch Precision was examined over six replicates per occasion over three occasions with a CV of less than 15.9%
for all analytes across the concentration ranges with many of these on the 10% CV. In summary, for this particular method, a reversed phase chromatography method was employed to provide a rapid, robust separation based on resolution of interferences. An efficient sample preparation method using solid phase extraction provides extraction of polar analytes from plasma, lower pH, and a 96-well plates microelution format with the option to automate the method using a liquid handling system in the future. The ACQUITY UPLC I-Class Xevo TQ-S micro system enables detection of low physiological concentrations of catecholamines and metanephrines for clinical research providing better detection limits compared to entry level systems. In conclusion for this presentation, use of LC-MS/MS systems provide significant selectivity advantages over more traditional techniques used in clinical research. Use of higher end platforms such as the TQ-S micro and particularly the Xevo TQ-XS enables users to meet the analytical sensitivity, precision, accuracy, and speed requirements for the methods.
Selective sample preparation and chromatography separation helps users meet the unique method challenges such as removal of interferences, polar attention, and analytical sensitivity while leveraging automation of sample preparation particularly in high throughput environments which can improve lab efficiency and minimize operator error. And so before I finish this presentation today, I would like to thank my colleagues, Heather Brown, Robert Wardle, QianQian Li, and John Danaceau who have all been involved in generating the methods found in this presentation. I've also attached links relating to collateral for the methods and also the Waters' clinical landing page at www.waters.com/clinical where you can find further information on the Waters' clinical laboratory LC-MS workflow ranging from sample handling for leasing. And so I just would like to thank you for your attention today during this webinar.
And I would like to now open the floor to questions. Thank you. Thank you Dominic for this very informative presentation. Now, we'll start the live Q&A portion of this webinar. If you have a question you'd like to ask, please do so now.
Just click on the Ask a Question box located on the far left of your screen. We'll answer as many of your questions as we have time for. We have time for a few. So let's get started.
First question that came in Dominic is, have you looked into the impact of the hematocrit effect on the steroids dried blood spot method? Thank you, Ben. We are aware of the impact of the hematocrit effect in regards to dried blood spots. But, this in regards to the steroid methods was not investigated. We know that dried blood spots is widely adopted, so the focus was on that particular matrix.
And we are aware that oversample or microsampling techniques that could help negate this effect could well be adopted within this methodology, too. So they include VAMS or fixed-volume blood spots from capillary collection type devices could be used. But, we didn't go into that level of detail in terms of the hematocrit effect in this instance. Great. Thank you, Dominic. So another question that came in is, have you examined any micro or nano slow techniques for these methods.
That's OK. No not particularly. We haven't really looked at those techniques as our goal was to use a standard flow system as these are more widely adopted in clinical research labs. So the aim was to use a fairly standard UPLC setup. So any of the methods we do create will fit within the workflows of a typical lab.
So in this instance no we haven't looked at those techniques. OK great. Thanks. And for the sake of time, I'll take one more question. Can the dried blood spot method be transferred to plasma or serum based methods for steroid analysis? In answer to that question, yes. Theoretically the main aspects of the method can be transferred.
But, when doing that sort of transfer, protein binding particularly with the steroids can be an issue. So that's to be taken into consideration. Robustness and matrix effects also have to be considered which means that any minor changes to sample preparation and chromatography runtime might be required to mitigate the impact of those changes.
OK great. Thank you. I'd like to thank the audience for joining us today and for your interesting questions. The questions we do not have time for today and those were submitted during on demand period, we will address these by the speaker via the contact information you provided at the time of registration. We would like to thank Dominic Foley for his time today and his important research.
We'd also like to thank LabRoots in association with us here at Waters Corporation for underwriting today's educational webcast. This webcast can be viewed on demand. LabRoots will alert you via email when it's available for replay. We encourage you to share that email with your colleagues who may have missed today live event. At the close of the webinar, we want to direct everyone to the next Waters' webinar that's on June 25 at 11 AM Eastern time entitled, Mass Spectrometry Past and Present-- emerging technologies and strategies for quality management in today's clinical laboratory. So we hope to see you there and thank you again everyone.