Expert Insight: Overcoming key challenges in LC-MS quantitation to improve workflow efficiency

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Mark Roberts, Principal Product Manager, and Kate Wearden, Principal Product Marketing Manager, Waters Corporation

Quantitation is the act of determining a numerical value for the quantity of an analyte in a sample and quantitative mass spectrometry is the analytical process that answers the important question of 'How much is in my sample?'. 

Performing quantitative mass spectrometry analyses enables scientists to solve scientific challenges in a wide range of industries including food, environmental, pharmaceutical, biopharmaceutical, chemical and health sciences. In this on-demand SelectScience^® webinar Mark Roberts, Principal Product Manager, and Kate Wearden, Principal Product Marketing Manager, Waters Corporation, talk about the proven quantitative mass spectrometry workflow solutions Waters provides to deliver a fully integrated quantitative workflow, through simple, easy-to-use hardware and software.

Key learning objectives covered in this webinar are:

• Learn about Waters’ most current software and hardware developments for LC-MS quantitation
• Discover how Waters supports your application success at every stage of the quantitative workflow
• Futureproof your lab with quantitative capabilities that ensure your methods last a lifetime

Read on for highlights from the Q&A discussion with Mark Roberts, Kate Wearden, Peter Hancock, Eimear McCall, and Alex Hooper of Waters Corporation, or register to watch the full webinar on demand>>

Q: How can you overcome matrix effects if they cannot be removed by changes to the extraction or chromatography methods?

PH: Matrix-matched standards and stable isotope internal standards were both mentioned during the webinar. With matrix-matched standards, the blank matrix is taken through the extraction, so the calibration curve is constructed with any sort of matrix effects incorporated into that. It is a very good approach to take but does require very well-characterized blank matrix. You also end up asking yourself the question of whether one apple matrix could be used for all apples? Or could it be used for oranges? You have a real choice over what that well-characterized blank matrix is.

Stable isotope labelled standards are the best way to overcome matrix effects and will always be recommended for analysis with a small number of compounds. The problem here is that for a true large multi-residue method, the cost of keeping those standards and purchasing those standards becomes a consideration. There is a third way, which is linked to matrix-matched standards and that is to use standard addition. This gets around needing blank matrix like you would with traditional matrix-matched standards. Here, the calibration standards are added to the actual sample and the concentration is back-calculated from the X-axis intercept. This is an effective way to confirm any incurred residues, but each sample will need multiple injections. There is a time implication for using that method.

Q: Will Quanpedia transitions work on any TQ-system?

PH: We have spent a long time curating and testing the conditions that have been used across multiple Waters platforms so we know that the majority of the Quanpedia transitions will work well across our instrument range. We would always suggest that these are used as a starting point for any method development. It is only when you are not meeting the sensitivity requirements for your method that we would recommend that some simple tuning is done using one of the tools that have been described in the webinar and use that to optimize any transitions. You need to remember that specific transitions might be needed for particular contaminant/commodity combinations. If you have got a matrix with certain background interferences, it is possible that you might need another set of transitions to get the necessary selectivity of your method.

Q: Are there any tools that can be used to develop a method for peptide quantification?

MR: We do have some new tools and we know that the analysis of peptides is a hot topic for a lot of our customers. We are pleased to say we have a new MassLynx-Skyline interface which is a wizard that helps you to work through the automated method development workflow for MRMs for peptides and digested protein bioanalysis. It is available for free on the Waters Marketplace, which is marketplace.waters.com. 

Q: I am studying metabolomics and interested in developing quantitative methods. Is there any help that Waters can provide with this?

MR: Yes. We have many ready-made methods within Quanpedia. We have a Targeted Omics Method Library specifically, for metabolomics, lipidomics and other omics. We are focusing on these types of analytes. We have a whole webpage around this: it is on the Waters’ website, waters.com/targetedomics. On this page there is a green button that says “download methods” and it will take you to a registration form. You can get the information that you need for the Quanpedia library of those methods to build your own methods for your type of study.

Q: Would the new integration between fixed times be the best integration method to employ routinely across my methods?

MR: It depends on the type of work that you are doing. If you have very good, uniform peak shapes, then the Apex peak tracking, which is the normal algorithm that is used in TargetLynx, is still the preferred way to integrate those peaks and get good quantitation. We now have this new method where you can integrate between two fixed time points so if you are not getting good peak shapes, or you are doing flow injection analysis, that might be where you would employ this. It's also useful when the peak shape changes with intensity. Especially when you want to integrate down to low levels of detection that might not be expected to have nice Gaussian peaks. The other situation it's useful for is for reliably splitting co-eluting (m shaped) peaks. Often this is a problem as the splitting changes as the peak intensity changes (which massively changes the calculated concentrations). Obviously you need to have good, reliable time on your chromatography to use this method. It is probably not for every routine analysis, but it is a nice usable extra that we can put in to help you if you are not getting nice Gaussian peaks.

Q: Looking at the QuanOptimize process, is there an easy way to export the data from this into a Quanpedia database? Also, is there a way of checking retention time of the analyte that has just been infused on a standard method if you are just increasing the number of analytes in an existing method?

EM: Yes. One of the benefits of QuanOptimize is that it is fully integrated within the MassLynx workflow. We have tried to streamline that process, and within one window, Application Manager. When you optimize your compound, using QuanOptimize, you can then open the Quanpedia tool within MassLynx, and import the transitions that have been optimized. So, Quanpedia is not just a transition database, it is a method management tool. We can bring in compounds optimized using QuanOptimize or transitions in Excel databases, we can also import them using those tools. The other benefit, with Quanpedia being the method management tool, is we can then use it to update the retention times, as asked in the second part of this question. Within Quanpedia, we can use the “update retention time” functionality to check the retention time – it is all there within the MassLynx window.

Explore practical guidance on some of the common challenges discussed in the webinar, including chromatographic retention and matrix, in these short videos >>