A combined LC-MS/MS and LC-MS3 multi-method for the quantification of iodothyronines in human blood serum

3,5-T2—A Janus-Faced Thyroid Hormone Metabolite Exerts Both Canonical T3-Mimetic Endocrine and Intracrine Hepatic Action

“…This tight binding not only relates to their distribution proteins in blood, cells or tissues but especially also to surfaces of plastic material required during pre-analytical sample work and subsequent MS quantification. Recently, two groups reported for the first time 3,5-T2 serum concentrations determined by LC-MS. Lorenzini et al (75), observed a mean concentration range for 3,5-T2 in human serum of 78 ± 9 pM, while our own results (25) indicated that most human sera analyzed for 3,5-T2 have lower than 5 pM 3,5-T2 concentrations, which is the lower limit of quantification in our method. The fact that Lorenzini et al probably report higher LC-MS-based 3,5-T2 concentrations is due to the contamination of the 13 C 9-15 N -3,5-T2 internal standard, with "natural" unlabeled 3,5-T2.…”

“…This is in sharp contrast to concentrations reported for the other THM formed from either T3 via 5-deiodination at the tyrosyl-ring (3,3 ′ -T2), or from rT3 generated by phenolic ring deiodinaton (3,3 ′ -T2) or the rare T2 metabolite 3 ′ ,5 ′ -T2 generated from rT3 via tyrosylring deiodination. Reported serum concentrations covered a very narrow concentration range between 36 and 68 pM for 3,3 ′ -T2 (see Table 1) (23)(24)(25)(26)(27)(28)(29)(32)(33)(34). Authors discussed this point, but no clear explanation or hypothesis was put forward to explain this peculiar observation atypical for the usually highly precise immunoassays for THM.…”

“…Only one individual was exceptional in exhibiting rather pronounced variations of 3,5-T2 for unknown reasons (unpublished data, van Vliet and Köhrle). Unfortunately, no residual, sufficiently large serum sample volumes (additional 200 µL) were available to repeat those studies using the recently developed new LC-LIT-MS 3 analysis of THM (25).…”

“…However, various practical and technical issues related to above-mentioned challenges of TH physiology and pathophysiology hampered rapid progress and success in quantification of total and free THM concentrations. While meanwhile several laboratories developed procedures to accurately determine total T4 and total T3 in human plasma or serum and largely agree on concentrations ranges (25,(67)(68)(69)(70)(71)(72)(73), this consensus has not been achieved yet for most of the other TH metabolites, which occur at much lower concentrations. Analytical procedures typically work well with buffer solutions, in vitro reaction mixtures, as well as with samples of "simple" matrix composition, while reported concentrations in human or experimental animal serum, plasma, and tissue show wide variations and marked insufficiencies in terms of method validation, standardization, and quality assessment parameters [see (23,24,74)].…”

“…A surprising outcome of both our own and the Lorenzini study, however, is that MS-based analysis of 3,5-T2 concentration in sera of healthy individuals is significantly lower than concentrations reported by our above-mentioned CLIA based on monoclonal antibodies. At this point, no explanation for the divergent results is possible yet, but needs to be considered during future analysis and further improvement of analytical methods for concentrations determination of 3,5-T2 in human and animal serum (25). The method used by Richards et al involved a completely novel pre-analytical approach involving single step solid-phase delipidation, avoiding multiple use of plastic material during sample workup and employing silanized glass vessels during this procedure.…”