The evolution of mass spectrometry for endocrine medicine

Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is rapidly emerging as the technology of choice for measuring steroid hormones. This review will focus on the utility of clinical mass spectrometry for the assessment of endocrine disorders.

by Dr P. Monaghan, L. Owen, Prof. P. Trainer and B. Keevil

Mass spectrometry or immunoassay?
The technological armamentarium of the modern day clinical laboratory has been greatly enhanced by the introduction and continued evolution of liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodology. This technique is almost universally applicable to the measurement of small molecule compounds such as steroid hormones and is proving to be invaluable for the endocrinologist towards diagnosing and managing complex endocrine disorders [1]. Furthermore, LC-MS/MS is rapidly expanding for the application of quantitative peptide hormone and protein measurement. LC-MS/MS offers a number of considerable advantages over conventional immunoassay (IA) technology: greater analytical sensitivity and specificity, lack of susceptibility to interference from anti-reagent antibodies and cross-reacting compounds, multiplexing capability for steroid profiles, and low running costs for consumables in comparison to antibody-based reagents. However, like IA, LC-MS/MS is also vulnerable to interference that can compromise the analytical integrity of the method. The potential sources of analytical interference and inaccuracy to consider for IA and LC-MS/MS methodologies are summarized in Table 1.

Improved specificity: safer medical management of Cushing’s syndrome
The use of the 11β-hydroxylase inhibitor metyrapone generally has an adjunctive role in the medical management of Cushing’s syndrome with the aim of improving the medical status of patients prior to surgery or radiotherapy. Patients receiving adrenal-directed anti-steroidogenic drugs such as metyrapone require frequent clinical and biochemical monitoring to minimize the risk of treatment-induced hypoadrenalism.

Current clinical guidance advocates that metyrapone dose is titrated against serum cortisol concentration and some centres, including our own, assess normalization of cortisol production via the measurement a day curve with a mean serum cortisol target between 150–300 nmol/L. The monitoring of metyrapone therapy relies on the measurement of serum cortisol that by the vast majority of laboratories is performed by routine IA. However, metyrapone treatment causes altered steroid metabolism and therefore serum cortisol measurement is susceptible to positive interference when performed by IA due to cross-reactivity with precursor steroids such as 11-deoxycortisol (11DOC) that build up in the circulation as a result of the metyrapone blockade of the adrenal steroidogenic pathway.

Our group has recently quantified the level of positive interference in serum cortisol IA for patients receiving metyrapone therapy by employing a direct quantitative comparison with LC-MS/MS [2]. A modest correlation between plasma adrenocorticotropic hormone (ACTH) concentration and the extent of positive interference in the IA for serum cortisol was also observed as 90% of patients in our study had ACTH-driven Cushing’s syndrome [3]. Our study concluded that for patients receiving metyrapone therapy, cortisol analysis by LC-MS/MS mitigates the potential for erroneous clinical decisions concerning dose titration [Figure 1] and is likely to reduce the risk of unrecognized hypoadrenalism which may result in symptoms that mimic the side-effects of metyrapone treatment, or at worst be fatal.

Improved sensitivity: estradiol measurement
Progress in both LC-MS/MS and online sample preparation technology (pre-analytics) has advanced the analytical sensitivity of this methodology to the extent that for the measurement of many steroid hormones, modern MS applications have now transcended conventional IA methods in this regard. An example of this is the high sensitivity measurement of serum estradiol. External quality assurance data reveals that a wide range of concentrations can be obtained by immunoassay when measuring samples for estradiol at lower concentrations. Furthermore, a recent position statement from the Endocrine Society has stressed the need for better analytical methods to address the current poor performance of assays for measuring low concentrations of estradiol [4]. To this end, our group has developed a novel direct assay that is applicable to routine clinical use for the measurement of estradiol and estrone (therefore permitting calculation of total estrogen status) in male patients and patients on aromatase inhibitors [5]. This high sensitivity assay uses ammonium fluoride in the mobile phase to facilitate more efficient ionization and thereby increase analytical sensitivity. Additionally, an on-line solid phase extraction (OSM) system [Figure 2 (Waters, Manchester, UK)] allows a large volume of extract to be loaded and this coupled with a XEVO™TQS tandem mass spectrometer enables unprecedented analytical sensitivity to be achieved.

Conclusions and future prospects
LC-MS/MS is a very powerful tool which is enabling substantial innovations in the endocrine laboratory. Indeed, it is likely that the majority of emerging small molecules will be addressed by LC-MS/MS analysis. There are two keys areas in which future research and development for LC-MS/MS ought to be directed. Firstly, the utility of LC-MS/MS for the quantification of peptide hormones and proteins is already becoming a reality with published methods available for measurement of renin activity [6], parathyroid hormone [7] and insulin-like growth factor-1 [8] amongst others. These current methods require the skills of highly trained personnel in order to develop and run these assays, and it is hoped that continued innovation in this area will culminate in the development of rapid protein assays that are applicable to routine clinical use. Secondly, it seems feasible with existing technology to develop fully automated random-access LC-MS/MS analysers that will enable greater ease of use in non-specialist laboratory settings. However, the automation of mass spectrometry will not be achieved without a concerted effort from the in vitro diagnostics industry to fully realize the potential of LC-MS/MS across clinical medicine.

References
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3. Monaghan PJ, Owen LJ, Trainer PJ, Brabant G, Keevil BG, Darby D. Response to ‘Comparison of serum cortisol measurement by immunoassay and liquid chromatography-tandem mass spectrometry in patients receiving the 11β-hydroxylase inhibitor metyrapone’ by Halsall DJ and Gurnell M. Ann Clin Biochem 2012; 49: 204–205.
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The authors
Phillip J. Monaghan*1 PhD, Laura J. Owen2 MSc, Peter J Trainer3 MD, and Brian G Keevil2 MSc
1Department of Clinical Biochemistry, 3Department of Endocrinology, The Christie NHS Foundation Trust, Wilmslow Road, Manchester M20 4BX, UK.
2Department of Clinical Biochemistry, University Hospital of South Manchester, Southmoor Road, Manchester, M23 9LT, UK.

*Corresponding author
E-mail: Phillip.Monaghan@nhs.net