A brief update on mass spectrometry applications to routine clinical endocrinology

Highlights

• •Clinical Endocrinology has benefited tremendously from the analytical specificity of LC-MS/MS.
• •Endocrine metabolic profiling and targeted protein applications are the new frontier.
• •Quantitation with simultaneous clinical phenotype determination is possible in select circumstances.
• •LC-MS/MS permits rapid assay development for novel targets and ad hoc clinical investigations.

Abstract

Mass spectrometry in clinical laboratories has traditionally focussed on small molecule analysis making endocrinology applications a natural choice given the numerous diagnostic targets in the steroid family, many of which could be multiplexed. Over time, methods for lower abundance steroids were successfully translated meaning that almost all small molecule targets in clinical endocrinology could be performed using mass spectrometry. This has paved the way for standardization efforts which have ultimately forced the improvements in the immunoassay industry. More recently, however improvements in quantitative mass spectrometric protein workflows have permitted the translation of a number of specific protein targets into routine analysis. In addition to the benefits in analytical specificity, judicious selection of peptide targets has permitted simultaneous quantitation and phenotyping in some cases. Mass spectrometry continues to clarify previously unnoticed but significant analytical problems with commercial immunoassays and permits the investigation of interferences in individual patient cases on an ad hoc basis.

1. Introduction

The field of endocrinology has benefited from the analytical sensitivity and specificity of mass spectrometry since its incorporation into the clinical laboratory [1]. While LC-MS/MS applications to endocrinology were initially limited to higher abundance steroids, eventually the measurement of analytically difficult analytes such as dihydrotestosterone [2], estradiol [3], 1,25-dihydroxy-Vitamin D [4], and aldosterone [5] became achievable in routine laboratory environments. The specificity of mass spectrometry revealed that many automated immunoassays were not measuring steroid hormones accurately, particularly in the settings of deranged physiology. More recently, clinical mass spectrometrists have turned their attention toward endocrine peptide and protein targets with some exciting and unexpected results. LC-MS/MS has continued to benefit our field by facilitating standardization and harmonization efforts, allowing disease-specific metabolomic profiling and, in ad hoc ways, facilitated investigation of cases that would be otherwise confusing.

2. Progress in standardization and harmonization

Because analytical interference is in principle entirely avoidable using LC-MS/MS, the approach has afforded numerous candidate reference methods for steroid and peptide molecules. The availability of these methods has facilitated ongoing standardization efforts in testosterone and vitamin D, both improving the comparability of different mass spectrometric methods and those of immunoassay vendors. In turn, age-dependent reference interval studies that are costly to conduct, can benefit all labs by using LC-MS/MS as the reference point. For example, recently, 100-sample subsets of testosterone samples drawn from four major previously published cohort studies were sent for LC-MS/MS analysis using the Center for Disease Control reference method [6]. This allowed for harmonizing linear transformations to be applied to raw data produced by each of the respective studies and the generation of harmonized reference intervals from a 9054 subject pool of males aged 19 to 89 years. Perhaps unsurprisingly, this approach revealed that the reference intervals originally produced by the studies were dependent on the analytical method employed and could not have been adopted by labs using a different method. However, after transformation, decade-specific age-dependent centiles were produced for both non-obese and all-subject males taking advantage of the considerable power of the four studies.

However, it is important to emphasize that methodological traceability to standardized reference materials (SRMs) does not mean that an LC-MS/MS should be referred to a “reference method”. Candidate LC-MS/MS reference methods include a variety of sequential sample preparation techniques to ensure the analytical accuracy is not compromised by a variety of physiological and pathological clinical states [7], [8], [9]. This generally makes them too laborious for a high-throughput clinical environment. Additionally, in the domain of quantitative protein mass spectrometry assays, commutability issues with artificial calibrators prepared using SRMs may be very difficult to overcome [10].

3. Clarification of immunoassay discrepancies

Where harmonization and standardization has not yet been achieved, LC-MS/MS continues to reveal widespread problems with routine endocrine small molecule and protein assays. For example, the performance of routine cortisol assays against both a routine LC-MS/MS method and a candidate reference LC-MS/MS method revealed clinically significant biases in immunoassay analyses which for some platforms reached ~25 to 40% having obvious implications to their performance in ACTH-stimulation testing [11]. Interestingly, the observed biases differed by sex while in pregnancy, some assays under-recovered by ~100 to 200 nmol/L at a target concentration of 500 nmol/L. Finally, the presence of prednisolone was documented to induce up to $50\times$ over-recoveries for some popular assays.

Elucidation of immunoassay weaknesses is in no way limited to small molecules. For example, immunoglobulin G4 related disease (IgG4RD) is a chronic inflammatory condition associated with autoimmune pancreatitis, sialadenitis, Riedel’s thyroiditis, and hypophysitis, which can affect all organ systems. The condition is important to identify because the pancreatic inflammation can be mistaken for carcinoma on imaging and lead to unnecessary surgery whereas IgG4RD is very responsive to glucocorticoids. The traditional screening test is immunonephelometric measurement of IgG subclasses with a disproportionate elevation of IgG4 indicating probable disease. However, cases have been missed because of factitiously low IgG4 results caused by the antigen excess phenomenon. Recently, mass spectrometric approaches have been established for the IgG subclasses that are, by virtue of the approach, impervious to antigen-excess [12], [13]. However, this was not the only benefit. The LC-MS/MS approach has also demonstrated that the two popular commercial immunonephelometric reagents for IgG subclasses have significant cross-reactivity problems in their IgG1 and IgG2 methods. This led to factitious elevations of IgG2 in cases of IgG4RD and has even caused some to hypothesize a role for IgG2 as an early diagnostic marker of IgG4RD [14].

4. Metabolomic profiling of adrenal disease

The multiplexing ability of LC-MS/MS has produced steroid profiling strategies in application to adrenal disease. In particular, a 26-plex urinary steroid method on high resolution LC-MS platform has been recently published and has been used to profile patients with adrenocortical adenoma (ACA) as compared to adrenocortical carcinoma (ACC) and to pituitary versus adrenal Cushing Syndrome (CS) [15]. The approach seeks a diagnostic strategy to correctly classify patients who fall into diagnostic gray zones. Others are pursuing similar diagnostic strategies for ACC [16], the subclassification of Cushing Syndrome [17] from serum/plasma, and subtype classification of primary aldosteronism [18].

5. Assignment of clinical relevant phenotype

Mass spectrometry offers the unique ability to both quantify and phenotype proteins simultaneously. This becomes particularly clinically relevant when common polymorphisms are associated with disease states. For example, apolipoprotein E (ApoE) has three common alleles, ApoE$\epsilon 1$, ApoE$\epsilon 2$ and ApoE$\epsilon 3$, with ApoE$\epsilon 3$ representing the wild-type. Homozygosity for the ApoE$\epsilon 2$ allele along with a “second-hit” leads to Dysbetalipoproteinemia, a condition associated with increased risk of cardiovascular events. The presence of the ApoE$\epsilon 4$ allele confers increased risk of early- and late-onset Alzheimer’s dementia. A recent paper describes a method for simultaneous quantitation apolipoproteins A1, B100, C-I, C-II and C-III while qualitatively assigning ApoE phenotype [19]. Determination of a patient’s ApoE status is traditionally a gel-based or genetically determined but for this method is a simple added bonus.

Some studies have indicated that total plasma 25-hydroxy-vitamin D concentrations vary by race, with blacks and hispanics having lower values. This notion has been challenged using a mass spectrometric approach that identifies polymorphisms known to decrease the vitamin D binding globulin (VDBG) affinity for 25-hydroxy-vitamin D [20]. This approach has subsequently facilitated an investigation of cardiovascular risk by vitamin D status and ethnicity which showed that higher VDBG concentrations are strongly associated with coronary heart disease events in all ethnic and racial groups [21].

6. Elucidation of unusual cases

Finally, the flexibility of mass spectrometry allows labs to perform custom investigations around clinical case conundra. For example, a recent report described a 20-month old girl with classic salt-wasting congenital adrenal hyperplasia caused by 21-hydroxylase deficiency. The child developed growth deceleration, increased facial fat and body hair while receiving prescribed hydrocortisone obtained from a local compounding pharmacy. Investigation of the compounded material revealed that it contained $5-10\times$ the stated dose due to a compounding error [22].

In another case report, a nurse presented with clinical complaints consistent with Cushing Syndrome showing some but not all of the expected biochemical features. Over a year-long period her biochemistries were inconsistent with progression of Cushing Syndrome [23]. The investigation was thorough, even to the extent that she underwent an inferior petrosal sinus sampling. However, on one occasion a salivary sample from a midnight collection was analyzed using two different assay generations of the Roche Cobas e601 cortisol immunoassay for cortisol, known to have different cross-reactivities for synthetic glucocorticoids. Since the results obtained from two methods were widely discrepant, mass spectrometric analysis of the sample was undertaken revealing that it had been contaminated with prednisone. An subsequent unscheduled “surprise” salivary cortisol collection performed on the next clinic visit was entirely normal. After the patient was presented the data, she neither confirmed nor denied sample adulteration and did not return for follow-up visits. A diagnosis of Munchausen Syndrome was made. This manner of investigation is greatly facilitated by LC-MS/MS.

7. Future state

Automated immunoassay systems have been a mainstay for the delivery of clinical care in endocrinology since the early 1990s. Their dominance has largely remained unchallenged because of the convenience of random access and the high throughput afforded by the homogeneous assay format. As we have discussed, non-specificity, particularly for small molecule hormone targets, has always remained an Achilles heel of the immunoassay. However, fully automated mass spectrometry instrumentation promises to deliver the analytical quality of mass spectrometry with the convenience of automated immunoassay analyzers [24], with the expectation of some sacrifice of increased costs in exchange for convenience.

8. Conclusion

Clinical endocrinology has benefited tremendously from the advent of mass spectrometry into routine clinical laboratories and will continue to do so as clinical targets graduate from the single-plex or small-plex assays for small molecules to large-plex metabolic profiling assays and the analytically diverse and challenging domain of protein quantitation. Given the ease of use of immunoassay, it is not expected that mass spectrometry will be the analytical technique of choice for all endocrine small molecules, peptides and proteins, however, continued judicious application of mass spectrometry paves the way for assays that can simultaneously measure multiple related targets, have true analytical specificity, make phenotypic inferences where applicable and be used in creative ways to elucidate individual patient cases.

Conflicts of Interest

Dr. Holmes reports non-financial support from SCIEX, outside the submitted work.