Brian Rappold (Presenter)
Essential Testing
Bio: Brian Rappold is the Scientific Director at Essential Testing Laboratories in Collinsville, Il. He currently serves as the clinical chemistry chair for the American Society for Mass Spectrometry as well as on the scientific committees for endocrinology and small molecule analysis/toxicology for Mass Spectrometry: Applications to the Clinical Laboratory (MSACL). He has published scientific articles and presented his research at international conventions on topics including hydrophilic interaction liquid chromatography for bio-analysis, multiplexed mass spectrometric detection of amino acidopathies, and the origins and solutions of ion suppression in electrospray ionization. He is a recognized expert in the field of method development and validation of mass spectrometry assays for clinical diagnostic use, teaching courses on the topic at numerous scientific conferences.
Authorship: Brian Rappold
Essential Testing
| Short Abstract Ingrained in the idea of mass spectrometric analysis is the concept of selectivity, particularly in the use of tandem mass analysis using collisional dissociation. This view, however, is contradicted when thoroughly evaluated, and in some cases specific measurement can be hindered in a variety of mechanisms directly related to the process of ionization prior to mass filtering. This talk will utilize examples of both exogenous and endogenous compounds of clinical interest to illustrate why mass spectrometry not only benefits from adjunct tools such as liquid chromatography, but indeed requires them due to interferences generated from MS-related components (isotopes, isobars, in-source dissociation, etc). |
Long Abstract
Ingrained in the idea of mass spectrometric analysis is the concept of selectivity, particularly in the use of tandem mass analysis using collisional dissociation. This view, however, is contradicted when thoroughly evaluated, and in some cases specific measurement can be hindered in a variety of mechanisms directly related to the process of ionization prior to mass filtering. This talk will utilize examples of both exogenous and endogenous compounds of clinical interest to illustrate why mass spectrometry not only benefits from adjunct tools such as liquid chromatography, but indeed requires them due to interferences generated from MS-related components (isotopes, isobars, in-source dissociation, etc).
A mass spectrometer is believed to be an inherently selective tool in the measurement of small molecules. Indeed, it is often referred to as the “gold standard” measurement technique. It should be appreciated, however, that MS analysis only has the capability to provide specific analysis, and typically if an orthogonal technique is utilized prior to MS detection. This is also true in the use of collisional dissociation tandem mass spectrometry (MS/MS). This talk will discuss the mechanisms in which a mass spectrometer might be “fooled” into providing inaccurate results by a variety of MS-related, or in some cases, MS-created interfering species.
All experiments were performed on a Sciex API 5000 mass spectrometer. Chromatographic separations were performed on a Shimadzu LC-20XR HPLC system; LC reagents were of MS quality and purchased from Spectrum Chemicals. A single class of well characterized compounds, opioids, (opiates, their primary metabolites and associated opioids including heroin’s primary metabolite 6-monoacetlyl morphine or 6-MAM) was purchased from Cerilliant. Materials were infused and the 4 most abundant product ions were selected for MS/MS analysis. All compounds were separated chromatographically and individually injected. Neat materials were analyzed for the nature of contribution to other analytes within the class of molecules. Additionally, patients prescribed these compounds or using them illicitly were identified and assessed to determine reproducibility of the interferences generated from the neat solutions as well as samples from individuals who were determined to be on no prescribed exogenous substances. Drug-free patients were used to remove aberrant signals related general human biology and unrelated to the signals generated via opioids. Data was reduced to rationalize the pathway of possible interference generation when a material presented a signal for a discrete compound.
Certain contributions are easily recognized, such as that of isomers (i.e. morphine/hydromorphone) particularly when there are significant similarities in the fragmentation of the molecules within a class. All expected isomers and isobars were observed; observed differences between transition ratios (the comparison of the response of 2 different product ions from the same precursor, commonly used to assess peak purity) between isomers will be discussed during the talk.
Isotopes are a key component to MS analysis; internal standardization would be poor indeed without them. Isotopes also represent a challenge in MS specificity. For example, the contribution of hydrocodone to noroxycodone is produced by naturally occurring carbon-13 replacing carbon-12 at two different locations. Thus a molecule with sufficient carbons and a subtle negative mass difference from the analyte of interest may present as an interferent. This was also observed the opioids separated by 2 amu (i.e. codeine contribution to oxymorphone).
The above examples are not outside the scope of standard MS analysis and should be readily recognized as a challenge. They are, however, only the beginning of the complexity which can exist in MS measurement. For example, the opioids measured also exhibited a frequent occurrence of desaturation. Just as codeine can contribute to oxymorphone via 13C2, it was observed that in-source desaturation induced an oxymorphone-derived interferent for codeine. The relationship of these in-source events to previous studies related to charge remote in-source decay will be examined.
Desaturation is not the only in-source event which occurs during atmospheric pressure ionization. Facile cleavages, such as loss of H2O, carbon dioxide or a carboxylic acid are frequently observed. For the molecules assessed, none exhibited mass difference equivalent to the expected mass loss from such a facile cleavage occurring prior to mass analysis. These steps did provide possible mechanisms for the generation of novel interference routes when combined with other previously described events. For example, oxycodone is 16 AMU more massive than hydrocodone and a response was observed for hydrocodone’s 4 mass transitions at the retention time of oxycodone, thus indicating an interferent. One possible explanation is the loss of water in the source of the mass spectrometer for oxycodone molecules which contain 2C13. Further examples of these combined mechanisms will elucidate further the possibility of error in MS analysis.
When patient samples containing the drug(s) of interest, interfering signals were observed in coordination with the study of neat solutions. Additionally, the variety of metabolites produced during opioid metabolism also presented a host of interfering signals not observed in the drug-free samples. As an example, a patient prescribed a high dose of oxycodone yielded signals for the transitions associated with 6-MAM at 3 different retention times unrelated to that of 6-MAM itself across all 4 transitions. These 3 distinct peaks shared transition ratios with 6-MAM for 2 of the transition pairs. A review of oxycodone metabolism in humans will be presented to demonstrate the absence of any possible Q1 precursor for 6-MAM based on the above experimentation. This example and others will be offered as core reasons why the use of high quality chromatographic separations is key to high quality data analysis in MS workflows.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | no | |
| Board Member | no | |
| Stock | no | |
| Expenses | yes | MSACL |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | no |