June 2017

Medical Mass Spectrometry Vol. 1 No. 1

Short Communication

Measurement of reverse triiodothyronine levels using liquid
chromatography-tandem mass spectrometry in the serum of 89 outpatients
112

3

1

Hideyuki Iwayama *, Kana Sugahara , Masaru Nakano , Minoru Fukayama , Akihisa Okumura
12

Department of Pediatrics, Aichi Medical University School of Medicine, Nagakute, Aichi, Japan
Department of Pediatrics and Neonatology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Aichi, Japan
3Advanced Medical Research Center, Aichi Medical University, Nagakute, Aichi, Japan
Abstract Background: Reverse triiodothyronine (rT3) has useful clinical applications, such as differentiating euthyroid
sick syndrome from hypothyroidism. However, rT3 measurement is unavailable in the clinical setting in Japan. Therefore,
we assessed the feasibility of using liquid chromatography‒tandem mass spectrometry (LC‒MS/MS) for measuring serumrT3 levels in outpatients.
Method: MS/MS detection was performed in the positive ionization mode on a tandem quadrupole mass spectrometer
13
equipped with an electrospray ionization interface. The precursor‒product ion pairs of rT3 (m/z 652 → 508) and C6-rT3(m/z 658 → 514) were selected. Quantiﬁcation was performed via selected reaction monitoring using peak areas. The serumfrom 89 patients (age, 8.1±8.1 years) who visited Aichi Medical University was collected. Patients diagnosed with thyroid
disease were excluded. rT3 was extracted from 100 μL of patient serum by liquid‒liquid extraction. C6-rT3 was used as13

the internal standard. Serum rT3 levels were also determined using a radioimmunoassay (RIA) kit.
Result: The lower limit of rT3 quantiﬁcation was 0.02 ng/mL. The relative standard deviation and relative error were ＜9%and ＜4%, respectively, indicating an acceptable level of reproducibility. Compared with RIA, LC‒MS/MS exhibited superior linearity (0.99872 versus 0.99999). rT3 levels in the patient serum samples were in the range of 0.038‒0.853 ng/mL.
Conclusion: The LC‒MS/MS technique reliably measured rT3 in the serum of 89 outpatients.
Key words: thyroid hormone, reverse T3, LC‒MS/MS, C-labeled internal standard, quantiﬁcation limit, feasibility studies
13Introduction1)

plantation . The most common hormonal pattern in ESS isa low T3 level with normal thyroxine and thyroid-stimulat-

Reverse triiodothyronine (rT3) is the inactive form ofthyroid hormone that binds to its receptor upside down.

ing hormone levels; this pattern resembles the ﬁndings in1)
hypothyroidism . rT3 is extremely useful for differentiat-

Despite its inertness, rT3 is used to diagnose euthyroid sick1)

syndrome (ESS) . ESS is the abnormal ﬁndings in thyroid

ing ESS from hypothyroidism because rT3 is typically high1)
in the former and low in the latter . However, rT3 measure-function tests that often occurs in critical conditions, suchment is unavailable in the clinical setting in Japan.

as inﬂammation, myocardial infarction, starvation, sepsis,burns, trauma, surgery, malignancy, and bone marrow trans-

Comparedwithimmunoassays,

suchradio-

asimmunoassay (RIA), mass spectrometry (MS) has the* Corresponding author

potential to measure the levels of multiple hormones during

Hideyuki Iwayama

a single analysis with improved speciﬁcity and sensitivity,

Department of Pediatrics, Aichi Medical University School ofMedicine, 1‒1 Yazakokarimata, Nagakute, Aichi 480‒1195,2‒5)

lower reagent cost, and a wide measurement range. Thisstudy aimed to validate the liquid chromatography‒tandem
MS (LC‒MS/MS) technique for measuring serum rT3 lev-Japan

Tel: 0561‒62‒3311, Fax: 0561‒63‒4835
E-mail: iwahide1976@gmail.com

els. The technique was applied to rT3 quantiﬁcation in theserum of 89 outpatients.

Received November 4, 2016. Accepted March 9, 2017.
DOI: 10.24508/mms.2017.06.003
10June 2017

Medical Mass Spectrometry Vol. 1 No. 1

SubjectsVelos Dual Pressure Linear Ion Trap LC‒MS/MS system
coupled to an Accela ultra performance liquid chromatogra-

centrations, we collected the serum from 89 patients withphy system (ThermoFisher, Waltham, MA, USA) in thepositive ion multiple-reaction monitoring mode. The Phe-various conditions who visited Aichi Medical University

nomenex Kinetex C18 2.6-μm column and Securi-

between June 18 and June 22, 2016. These patients (n＝89;age, 8.1±8.1 years; range, 1 month‒48 years) included fourtyGuard ™ ULTRA cartridge were used as the analyticaladults. Patients diagnosed with thyroid disease wereexcluded. All samples were stored at −80°
C and were ana-

the LC gradient were 50% A [0.1% formic acid in water (v/lyzed in a single batch at the completion of the study. In

gradually over 3 min till a composition of 20% A and 80%

accordance with the Ethical Guidelines for Medical and

B was obtained. MS/MS detection was performed in theHealth Research Involving Human Subjects, Chapter 5,

positive ionization mode on a tandem quadrupole massPart 12, informed consent was not obtained because thespectrometer equipped with an electrospray ionizationinterface. The precursor‒product ion pairs of rT3 (m/zMaterials and Methods
To select patients with a wide range of serum rT3 con-and guard columns, respectively. The initial conditions forv)] and 50% B (MeOH). The composition was changed

serum used in this research had been anonymized. The
study was approved by the ethics committee of Aichi Medi-

C6-rT3 (m/z 658 → 514) were selected.
The spray voltage was set at 4.0 kV, and the capillary tem652 → 508) andcal University (reference number 2015-H359).

13perature was set at 450°
C. The sheath gas ﬂow rate was 10Reagents and standards(arbitrary units). Quantiﬁcation was performed by selected

rT3 and its C-labeled internal standard ( C6-rT3) were13

13

reaction monitoring using peak areas.

purchased from IsoSciences (King of Prussia, PA, USA).

Sample preparation

According to the manufacturer s certiﬁcate of analysis, the13purity of C6-rT3 was determined to be 97% using

high-performance liquid chromatography. A Kinetex C18
2.6-μm column (00D-4462-AN) and SecurityGuard ™

13Serum samples (100 μL) from patients were spiked with
C6-rT3 as the internal standard. After vortexing, 400 μL of

2% NH4OH in EtOH (v/v) was added. The solutions wereULTRA cartridge (AJ0-9298) were obtained from Phenomenex (Torrance, CA, USA). Hydrophilic polyvi-vortexed and maintained at 4°
C for 1 h. The mixtures werethen centrifuged at 13000×g for 5 min. The supernatants
were decanted into new 2.0-mL tubes, and the pellets werenylidene ﬂuoride (PVDF) membrane (SLHVR04NL) waswashed by resuspending in 100 μL of 2% NH4OH in EtOH

purchased from Millipore (Billerica, MA, USA). An rT3
RIA kit (RVR-EW-125) was purchased from DIAsource(v/v). The supernatants were combined, and the solution
was evaporated by freeze-drying. Subsequently, the residue

ImmunoAssays SA (Louvain-La-Neuve, Belgium).
A 1-mg/mL stock solution of rT3 was prepared using

was reconstituted using 100 μL of 50% MeOH in waterwith 0.05% formic acid (v/v). After ﬁltering using a 4-mm

95% ethanol (EtOH) in water (v/v) containing 6 mM
13sodium hydroxide. C6-rT3 at 40 μg/mL concentration wassyringe ﬁlter and a hydrophilic PVDF membrane, a 15-μL
aliquot of the resulting sample was subjected to LC‒MS/prepared using 30% ammonium in methanol (MeOH; v/v).
The stock solutions were stored at −30°
C. Working solu-MS analysis.

tions were prepared by diluting the stock solutions withMethod validation

50% MeOH in water (v/v). Calibration standard solutions

The quantiﬁcation limit (lower limit of quantiﬁcation)

ranging from 0.02 to 5 ng/mL were prepared by spiking thedelipidized human serum with ultra-low hormones, steroids, and other analytes (MSG 3000, Golden West Biolog-

was determined by injecting the serum at the concentrationof rT3, ranging from 0.02 to 5 ng/mL, as serial diluted cali-ics, Inc., Temecula, CA, USA) with various amounts of6)
brators . Precision was obtained as the coefﬁcient of varia-

stock solution of rT3. A labeled internal standard mixture intion [relative standard deviation (RSD%)]. Accuracy wasserum was prepared at a ﬁnal concentration of 0.25 ng/mL.

obtained as the relative error (RE%), which was calculated
as follows: RE%＝[(measured amount − spiked amount)/spiked amount] × 100.

Instruments

The LC‒MS/MS technique was developed on a LTQ
11

June 2017

Medical Mass Spectrometry Vol. 1 No. 1RIA
Serum rT3 levels in the patients were also measured
using an rT3 RIA kit following the manufacturer s protocol.

Statistical analysis
rT3 levels were expressed as mean±standard deviation.
Data analysis and visualization were aided by the Daniel s
XL Toolbox add-in for Excel, version 7.1.4, by DanielKraus, Würzburg, Germany (www.xltoolbox.net).

Results and Discussion

The LC‒MS/MS technique for measuring serum rT3 lev-els was validated by evaluating a series of parameters, suchas the quantiﬁcation limit, linearity, precision, and accu-

Comparison of liquid chromatography‒tandem
mass spectrometry (LC‒MS/MS) and radioimmunoassay (RIA) for determining serum reverse
triiodothyronine (rT3) levels.

Fig. 1.

racy. The quantiﬁcation limit for rT3 was estimated to be
0.02 ng/mL with a signal-to-noise ratio of 10:1 under theLC‒MS/MS conditions used in the experiment. This limit

Arrows and boxes indicate values measured in patientswith various diseases. Open circles indicate values measured in patients with monocarboxylate transporter 8(MCT8) deﬁciency.

4)

was comparable with that obtained in a previous study .
The calibration curve for rT3 was generated using the following regression equation: y＝0.0593077x＋0.0002492,2
r ＝0.9999, in the range of 0.02‒5 ng/mL. The RSD% and
RE% values of the standards were 4.76% and 2.13% at
0.02 ng/mL, 8.46% and 3.78% at 0.05 ng/mL, 2.49% and1.11% at 0.2 ng/mL, 3.35% and 1.50% at 0.5 ng/mL, 1.92%
and 0.85% at 2.0 ng/mL, and 1.56% and 0.70% at 5.0 ng/mL, respectively. At concentrations＞0.02 ng/mL, all
RSD% and RE% values were ＜10%. The values were
acceptable at all concentrations.
The technique was then applied to rT3 quantiﬁcation in
the serum of 89 outpatients. Their serum rT3 levels weredetected in the range of 0.038‒0.853 ng/mL. Comparedwith RIA, LC‒MS/MS provided superior linearity [0.99872(RIA) versus 0.99999 (LC‒MS/MS); Fig. 1]. Moderate cor-Fig. 2. Relationship between the patient age and rT3 levelmeasured using liquid chromatography‒tandem
mass spectrometry (LC‒MS/MS).

relation was observed between LC‒MS/MS and RIA (r ,2The relationship between age and rT3 level measured
using LC‒MS/MS is shown in Fig. 2. The reference ranges

Arrows and boxes indicate values measured in patientswith various diseases. Open circles indicate values measured in patients with monocarboxylate transporter 8(MCT8) deﬁciency.

of serum rT3 levels measured using RIA have beenreported to be 0.6‒2.5 ng/mL (birth to 6 days), 0.09‒

nesses . On the contrary, patients with Fanconi syndrome

0.35 ng/mL (children older than 6 days), and 0.11‒0.32 ng/(n＝1) and epilepsy treated with carbamazepine (n＝1) had

0.7287; p＜0.001).

1)

7)

mL (adults) . These reference ranges for serum rT3 levels

low rT3 levels. Carbamazepine, which alters thyroxine andare comparable with our results.
Several patients with bilateral urethral stones (n＝1),

T3 metabolism due to increased hepatic metabolism, may1)
decrease rT3 levels . However, to the best of our knowl-

severe asthmatic bronchitis (n＝1), inﬂuenza (n＝2), andedge, the reason for decrease in rT3 levels in patients withcyclic vomiting (n＝1) had high rT3 levels. Elevated rT3

Fanconi syndrome is yet to be clariﬁed. A high school

was observed in most patients with acute or chronic ill-

handball player among the subjects also had a low rT3 level12June 2017

Medical Mass Spectrometry Vol. 1 No. 1References(0.056 ng/mL). Aerobic exercise with adequate caloric
8)

intake can reasonably decrease rT3 levels .

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inherited thyroid disease. MCT8 deﬁciency, also known as
Allan‒Herndon‒Dudley syndrome, is characterized by themedscape.com/article/118651-overview, updated: Oct 16,

dysfunction of the transporter protein for thyroid hormone2) Field HP: Tandem mass spectrometry in hormone mea-2016.
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mal range of rT3 levels . In our study, a 1-year-old patient

Inﬂuence of thyroidectomy on thyroxine metabolism andwith MCT8 deﬁciency had an rT3 level of 0.038 ng/mL,
which is clearly low. Another 10-year-old patient withturnover rate in rats. Endocrinol 210(1): 117‒123, 2011.
4) Sakai H, Nagao H, Sakurai M, Okumura T, Nagai Y, et al:

MCT8 deﬁciency had an rT3 level of 0.141 ng/mL, which-TriiodothyCorrelation between serum levels of 3,3′,5′is comparable with those in other children. Further studies

ronine and thyroid hormones measured by liquid chroma-should be performed to determine the relationship between

tography‒tandem mass spectrometry and immunoassay.

serum rT3 levels and illness.

PloS ONE 10(10): e0138864, 2015.

9)

Because the primary aim of this study was to examine

5) Yue B, Rockwood AL, Sandrock T, La ulu SL, Kushnir

the reliability of measuring serum rT3 levels using LC‒MS/MM, et al: Free thyroid hormones in serum by direct equi-MS in the clinical setting, we will investigate the normal
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children and the relationship between abnormal rT3 levels
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dated the LC‒MS/MS technique for measuring serum rT3

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levels. The technique reliably measured serum rT3 levels inin tissue using SPE UPLC-tandem MS. Biomed Chro-89 outpatients.

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2008.

Acknowledgements

8) Loucks AB, Heath EM: Induction of low-T3 syndrome inWe thank Divya Shaji for the English language review.
This work was supported by JSPS KAKENHI Grant Num-exercising women occurs at a threshold of energy avail-ber 16K19676, The Nitto Foundation, and Aichi Medical
University Aikei-Kai Foundation.

9) Dumitrescu AM, Fu J, Dempsey MA, Refetoff S:ability. Am J Physiol 266(3 Pt 2): R817‒823, 1994.
MCT8-speciﬁc thyroid hormone cell-membrane transporter deﬁciency. in Pagon RA, Adam MP, Ardinger HH(eds): GeneReviews [Internet]. 2013.

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