Molecular and Cellular Endocrinology xxx (2017) 1e13 Contents lists available at ScienceDirect Molecular and Cellular Endocrinology journal homepage: www.elsevier.com/locate/mce Avoiding the pitfalls when quantifying thyroid hormones and their metabolites using mass spectrometric methods: The role of quality assurance * Keith Richards, Eddy Rijntjes, Daniel Rathmann, Josef Kohrle€ Institut für Experimentelle Endokrinologie, Charite-Universitatsmedizin€ Berlin, Berlin, Germany article info abstract Article history: This short review aims to assess the application of basic quality assurance (QA) principles in published Received 18 November 2016 thyroid hormone bioanalytical methods using mass spectrometry (MS). The use of tandem MS, in Received in revised form particular linked to liquid chromatography has become an essential bioanalytical tool for the thyroid 20 January 2017 hormone research community. Although basic research laboratories do not usually work within the Accepted 20 January 2017 constraints of a quality management system and regulated environment, all of the reviewed publications, Available online xxx 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 Keywords: Thyroid hormone metabolites application of QA to published bioanalytical methods from the perspective of selectivity, accuracy, Tandem mass spectrometry precision, recovery, instrument calibration, matrix effects, sensitivity and sample stability. During the last 3-Iodothyronamine decade the emphasis has shifted from developing methods for the determination of L-thyroxine (T4) and 0 Iodothyronine 3,3 ,5-triiodo-L-thyronine (T3), present in blood serum/plasma in the 1e100 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- 0 thyronine (3,3 -T2). These metabolites seem likely to be present in the low pM concentrations; conse- quently, 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/docu- mentation and analyte recovery would be beneficial. © 2017 Elsevier B.V. All rights reserved. Contents 1. Scope of the review . ................................................. 00 2. Background . ................................................. 00 3. The history of mass spectrometry in thyroid hormone analysis . ..................... 00 4. Quality assurance and method validation . .................................... 00 4.1. Selectivity . ....................... 00 4.2. Accuracy, precision and recovery . ....................... 00 4.3. Instrument calibration . ....................... 00 4.4. Matrix effects and the use of ISTD . ....................... 00 4.5. Sensitivity . ....................... 00 4.6. Stability . ....................... 00 0 Abbreviations: CLIA, Chemiluminescence immunoassays; 3,5-T2, 3,5-Diiodo-L-thyronine; 3,3 -T2, 3,3‘-Diiodo-L-thyronine; ELISA, Enzyme-linked immunosorbent assay; FDA, Food and Drug Administration; GC-MS, Gas chromatography-mass spectrometry; IA, Immunoassay; 3-T1AM, 3-Iodothyronamine; ID, Isotope dilution; LC, Liquid chromatography; LC-MS, Liquid chromatography mass spectrometry; LC-MS/MS, Liquid chromatography tandem mass spectrometry; LLOD, Lower limit of detection; LLOQ, Lower limit of quantification; m/z, Mass/charge ratio; MS, Mass spectrometry; QA, Quality assurance; TH, Thyroid hormones; THM, Thyroid hormones and metabolites; T4,L- 0 0 3 Thyroxine, 3,3’,5,5 -Tetraiodo-L-thyronine; T3, 3,3 ,5-Triiodo-L-thyronine; SRM, Selected reaction monitoring; MRM, Multiple reaction monitoring; MS , Triple MS; ISTD, À À Internal standard; SPE, Solid phase extraction; nM, nanomolar (nmolL 1); pM, picomolar (pmolL 1). * Corresponding author. Charite-Universit atsmedizin€ Berlin, Institut für Experimentelle Endokrinologie, Augustenburger Platz 1, 13353 Berlin, Germany. E-mail address: [email protected] (J. Kohrle).€ http://dx.doi.org/10.1016/j.mce.2017.01.032 0303-7207/© 2017 Elsevier B.V. All rights reserved. Please cite this article in press as: Richards, K., et al., Avoiding the pitfalls when quantifying thyroid hormones and their metabolites using mass spectrometric methods: The role of quality assurance, Molecular and Cellular Endocrinology (2017), http://dx.doi.org/10.1016/j.mce.2017.01.032 2 K. Richards et al. / Molecular and Cellular Endocrinology xxx (2017) 1e13 4.7. Equilibration . .................................................. 00 5. Immunoassays . ..................... 00 6. Summary .................................................................................................... ..................... 00 Funding...................................................................................................... ..................... 00 Conflict of interest statement . ..................... 00 Acknowledgements . .................................................. 00 References . .................................................. 00 1. Scope of the review review are equally applicable to all biological matrices. The use of mass spectrometry (MS) for the analysis of thyroid 2. Background hormones. (TH) and TH metabolites (THM) in biological samples has THM are a group of low molecular mass iodine-containing become widespread. MS methods for the determination of TH and hormonally active compounds derived from the amino acid L- THM in human (Siekmann, 1987; Thienpont et al., 1994; De tyrosine. The pro-hormone L-thyroxine (T4, Fig. 1A), the more Brabandere et al., 1998; Tai et al., 2002, 2004; Thienpont et al., 0 biologically active 3,3 ,5-Triiodo-L-thyronine (T3, Fig. 1B) and the 1999; Van Uytfanghe et al., 2004; Soukhova et al., 2004; Gu et al., 0 di-iodometabolites 3,5-T2 and 3,3 -T2 (Fig. 1D and E respectively) 2007; Yue et al., 2008; Saba et al., 2010; DeBarber et al., 2008; belong to the thyronine family of compounds. The de-carboxylated, Galli et al., 2012; Jonklaas et al., 2014; Kunisue et al., 2011a; de-iodinated derivatives of T4, include 3-iodothyronamine (3- Wang and Stapleton, 2010; Kahric-Janicic et al., 2007; Ackermans T1AM, Fig. 1C); these are known as thyronamines. et al., 2010) and animal (Saba et al., 2010; DeBarber et al., 2008; Endogenous TH are found in tissues, blood and other body fluids Kunisue et al., 2011a; Wang and Stapleton, 2010; Ackermans and their accurate measurement in these biological matrices is a et al., 2010; Hackenmueller and Scanlan, 2012; Nagao et al., 2011; key requirement for both clinical diagnostic and research purposes. Kunisue et al., 2011b; Kiebooms et al., 2014; Hansen et al., 2016) TH and THM may exist in both free and protein-bound forms in blood serum/plasma, animal tissues (Saba et al., 2010; Ackermans biological matrices and it is desirable to have available bioanalytical et al., 2010, 2012; Kunisue et al., 2011c; Kunisue et al., 2010), cell methods to determine both free and total (i.e. free plus bound) culture lysates (Saba et al., 2010; Piehl et al., 2008) and superna- concentrations in both blood and tissues. Among low molecular tants (Rathmann et al., 2015), saliva (Higashi et al., 2011), and urine mass hormones the avid and extremely high affinity binding of TH (Aqai et al., 2012; Fan et al., 2013) have been published. Over the and THM to several binding proteins in the blood and various last 25 years the availability of affordable, high sensitivity benchtop subcellular compartments is a unique challenge to every diagnostic tandem mass spectrometers, in particular those coupled to liquid approach intending their quantification (Refetoff, 2000; Roy et al., chromatography (LC-MS/MS) and as a consequence of the manu- 2012; Little, 2016). facturer's constant search for new markets and hence new appli- cations, the thyroid research community has access to this additional powerful tool to compliment traditional immunoassay 3. The history of mass spectrometry in thyroid hormone (IA)-based methods. The move of LC-MS/MS into new application analysis areas brings with it not only a new bioanalytical approach and gains in productivity, but also potential pitfalls. These pitfalls are perhaps T4 and T3 are 99.97 and 99.7% respectively bound to carrier more familiar to the early adopters of the technique, who tended to proteins in the circulation (Spencer, 2000). The availability of TH to be active in application areas subject to quality management sys- cells is considered to be dependent on their free rather than tems and accredited/regulated environments. The need for quality protein-bound concentrations (Cheng et al., 2010), making the assurance (QA), including method validation has been recognised concentrations of free T3 and T4 of major diagnostic interest to and taken on board by the majority of TH research groups pub- clinicians. Hence, free T4 and T3 concentrations are clinically lishing bioanalytical MS methods; however, the implementation of important parameters that are routinely measured on a high QA and the interpretation of data is variable. The limited availability throughput daily basis in blood samples from adult patients. The of certified reference materials and isotopically labelled internal accurate, precise determination of total versus free T4 and T3 in standard (ISTD) compounds and the lack of proficiency testing are blood serum present quite different challenges related to their particular limitations.