Elsevier

Molecular and Cellular Endocrinology

Volume 458, 15 December 2017, Pages 44-56
Molecular and Cellular Endocrinology

Avoiding the pitfalls when quantifying thyroid hormones and their metabolites using mass spectrometric methods: The role of quality assurance

https://doi.org/10.1016/j.mce.2017.01.032Get rights and content

Highlights

  • Application of strict quality assurance is required in analytics of thyroid hormones and their metabolites.

  • Availability of certified reference materials would improve accuracy/precision evaluation.

  • Stringent selectivity criteria are required for confirmation of thyroid hormone metabolites in biological samples.

  • Rigorous documentation of recovery and matrix effects is necessary.

Abstract

This short review aims to assess the application of basic quality assurance (QA) principles in published thyroid hormone bioanalytical methods using mass spectrometry (MS). The use of tandem MS, in particular linked to liquid chromatography has become an essential bioanalytical tool for the thyroid hormone research community. Although basic research laboratories do not usually work within the constraints of a quality management system and regulated environment, all of the reviewed publications, to a lesser or greater extent, document the application of QA principles to the MS methods described. After a brief description of the history of MS in thyroid hormone analysis, the article reviews the application of QA to published bioanalytical methods from the perspective of selectivity, accuracy, precision, recovery, instrument calibration, matrix effects, sensitivity and sample stability. During the last decade the emphasis has shifted from developing methods for the determination of L-thyroxine (T4) and 3,3′,5-triiodo-L-thyronine (T3), present in blood serum/plasma in the 1–100 nM concentration range, to metabolites such as 3-iodo-L-thyronamine (3-T1AM), 3,5-diiodo-L-thyronine (3,5-T2) and 3,3’-diiodo-L-thyronine (3,3′-T2). These metabolites seem likely to be present in the low pM concentrations; consequently, QA parameters such as selectivity and sensitivity become more critical. The authors conclude that improvements, particularly in the areas of analyte selectivity, matrix effect measurement/documentation and analyte recovery would be beneficial.

Section snippets

Scope of the review

The use of mass spectrometry (MS) for the analysis of thyroid hormones.

(TH) and TH metabolites (THM) in biological samples has become widespread. MS methods for the determination of TH and THM in human (Siekmann, 1987, Thienpont et al., 1994, De Brabandere et al., 1998, Tai et al., 2002, Tai et al., 2004, Thienpont et al., 1999, Van Uytfanghe et al., 2004, Soukhova et al., 2004, Gu et al., 2007, Yue et al., 2008, Saba et al., 2010, DeBarber et al., 2008, Galli et al., 2012, Jonklaas et al., 2014

Background

THM are a group of low molecular mass iodine-containing hormonally active compounds derived from the amino acid L-tyrosine. The pro-hormone L-thyroxine (T4, Fig. 1A), the more biologically active 3,3′,5-Triiodo-L-thyronine (T3, Fig. 1B) and the di-iodometabolites 3,5-T2 and 3,3′-T2 (Fig. 1D and E respectively) belong to the thyronine family of compounds. The de-carboxylated, de-iodinated derivatives of T4, include 3-iodothyronamine (3-T1AM, Fig. 1C); these are known as thyronamines.

Endogenous

The history of mass spectrometry in thyroid hormone analysis

T4 and T3 are 99.97 and 99.7% respectively bound to carrier proteins in the circulation (Spencer, 2000). The availability of TH to cells is considered to be dependent on their free rather than protein-bound concentrations (Cheng et al., 2010), making the concentrations of free T3 and T4 of major diagnostic interest to clinicians. Hence, free T4 and T3 concentrations are clinically important parameters that are routinely measured on a high throughput daily basis in blood samples from adult

Quality assurance and method validation

QA is a term associated with analytical chemistry, but less so with basic research. Whilst it is unreasonable to expect thyroid hormone researchers to apply full quality management procedures to their laboratory environment, it is appropriate to expect an adequate standard of QA to be applied to published bioanalytical MS methods. What should such a standard include? The US Food and Drug Administration (FDA) guidelines for bioanalytical method validation (B.C. Committee, 2001) state that

Immunoassays

In the decades before the introduction of the LC-MS/MS methodology, the thyroid field depended on the use of IA for the quantification of TH (Wiersinga and Chopra, 1982, Chopra et al., 1972, Brown et al., 1970, Ekins, 1998). In contrast to LC-MS/MS, early IA did not have multi-analyte capability. The ELISA-based (enzyme-linked immunosorbent assay) methods in principle use one antibody to immobilize the antigen and a further antibody as a detection antibody. The latter antibody is labelled with

Summary

Tandem MS has proved to be an extremely valuable bioanalytical tool for THM, and its combination with liquid chromatography has become the method of choice for many laboratories. Although the validation of published LC-MS/MS methods has become a routine requirement, there is no generally accepted “golden rule” as to how a validation procedure should be defined, and there are clearly areas of QA that can be improved. In particular, the use of MRM quantitation and confirmation ions and their

Funding

This work is supported by grants from the Deutsche Forschungsgemeinschaft within the DFG-SPP 1629 ThyroidTransAct KO 922/17-1/2 to JK.

Conflict of interest statement

The authors declare that there are no competing interests.

Acknowledgements

We thank Mr. Ray Monk for expert technical assistance.

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