Robert MacNeill: Multiple analytes, metabolites and mayhem

In this installment of Robert MacNeill’s (Envigo) column, Robert discusses the perils and pitfalls of cramming as many analytes as possible, particularly metabolites, into a regulated bioanalytical LC-MS method.

Robert MacNeill received his Bachelor’s degree with Honors in Chemistry from Heriot Watt University then his MSc in Analytical Chemistry from the University of Huddersfield, both in the United Kingdom. Robert is also a Chartered Chemist and Member of the Royal Society of Chemistry. With 20 years of experience in all aspects of quantitative bioanalytical LC–MS/MS method development, 11 of these years heading method development activities within HLS/Envigo, and a regular author and peer reviewer for the journal Bioanalysis, Robert is a recognized expert and innovator in the field.

In his current role Robert coordinates all LC–MS/MS method development and associated training, takes the lead in keeping abreast of innovation and technological development in the industry, leads in-house research projects and performs technical writing for the purpose of producing publications.

I recall several occasions in which various characters in the industry have remarked that they prefer protein precipitation as a means of sample extraction prior to LC-SRM. The grounds for this point of view are along the lines of ‘everything is still in there’; ‘you won’t lose any metabolites’; and ‘there is no need to worry about recovery’. A more recent reason I hear, which I am entirely attuned to, is to do with state of the art instruments often giving so much sensitivity that only a tiny volume of extract need be injected, which bestows positive implications for minimizing matrix effects.

Prior to the main focus of this piece, I would like to address what I believe to be a misunderstanding, which is the aspect of recovery here. Recovery can indeed be lost in protein precipitation. Usually the technique is done using organic solvent like acetonitrile or methanol, adding two or three volumes per volume of plasma or serum. The organic content drives the aggregation and subsequent precipitation of larger proteins like albumin, mainly through disruption of the water-rich layer around them. If you happen to be analyzing for a protein like this, it will clearly have precipitated and you need to take the pellet for analysis, indeed pellet digestion is an established technique as part of therapeutic mAb analysis.

Focusing on the realm of small molecules and peptides, a compound polar enough to be insoluble in the resultant mixture will not be efficiently extracted, and the more water-soluble it is, the worse the recovery will be, in addition to being less precise and reproducible. Similarly, for protein precipitation methods that use the likes of trichloroacetic acid in aqueous solution to effect the aggregation and precipitation, compounds that have issues solubilizing in the resultant acidic aqueous conditions will have issues with recovery.

The underlying mechanism of protein precipitation just described makes it clear that, from an extraction selectivity perspective, it only eliminates proteins large enough to take on globular form and possess a water-rich outer layer, weakly bound to the hydrophilic outer regions of the protein. This brings to consideration the most compelling of the above reasons for being drawn to protein precipitation. That is to do with recovering the metabolite content, and yes, there is value in having an extract of an incurred sample that still contains a full complement of metabolites as well as the analyte content. This is especially if the likes of TOF-MS is being used, perhaps in a semi-qualitative sense, the nature of which is continuously all-scanning hence all these data are opportunely collected and with accurate mass, allowing easy identification.

For the quantitative bioanalytical scientist, however, using such a non-selective extraction method also has a down side. The extraction method must be made non-selective in accordance with the number of analytes, where accordingly more interferences must be dealt with, yet the method as a whole must perform ruggedly. The less selective an extraction, the better the LC-MS side needs to be to deal with the potential host of interferences that are present in the extract. Metabolites can certainly qualify as interferences; in fact they hover near the top of the danger list. The best quantitative methods have extracts so clean they are akin to solution rather than extract. All interferences, including proteins, salts, and lipids, are completely eliminated, and all individual analytes with their internal standards are completely recovered. That is the ideal, the goal that can never quite be achieved. The more analytes that a method includes, and the more they vary physicochemically, the further from this ideal we find ourselves.

Metabolites are a great example of compounds related to a parent analyte that may have emphatically variable physicochemical properties from both the parent compound and each other. They are by far most often the reason for multiple analyte assays associated with programs involving a single therapeutic candidate, but there are also plenty of instances of multiple analyte methods involving simply different drugs or drug candidates. Metabolites can engage in quite sinister phenomena. Phase 1 metabolites like N-oxides, for instance, can at least partially revert back to the parent in the heat of an ion source, creating an extraneous peak in the ion channel of the parent compound, or co-elute if the chromatography is inadequate.

Conjugates, Phase II metabolites, can also show reactivity, especially when the conjugate link is prone to hydrolysis, like ester-linked glucuronides. In the presence of methanol, trans-esterification of such conjugates has been known to occur where the resultant product is the parent. This obviously takes away all reliability from the analysis of incurred samples. This kind of behavior from metabolites comes in addition to posing the usual interferent-type risks to do with competition for ionic release into the gas phase, a.k.a. competition for ionization, when co-eluting with an analyte, whether or not they are visible in any given ion channel.

Multiple analyte assays are a big part of life and a nice but potentially frustrating challenge, as is quantification of many metabolites. There needs to be good reason to undertake them, that is to say a fit-for-purpose argument. In quantitative bioanalysis we must be prepared to have a harder time with establishing unquestionable method performance under these circumstances. For every additional analyte, particularly of different physicochemical properties each time, the extraction method will be inherently less selective in order to ensure decent analyte recovery, and the need for excellent discriminating power in the LC-MS will be all the more emphatic.

This article is part of Robert MacNeill’s (Envigo) quarterly column for Bioanalysis Zone which focuses on quantitative method design. You can read past installments of the column here. 

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