Methods for simultaneous quantification of thyroid hormones and metabolites thereof by mass spectrometry
The invention provides methods for simultaneously detecting or simultaneously quantifying any combination of thyroxine (T 4 ), triiodothyronine (T 3 ), 3,3′-diiodo-L-thyronine (3,3′-T 2 ), 3-iodothyronamine (T 1 AM), and, optionally, reverse T 3 (rT 3 ) in a sample obtained from a human. The method involves a simple, sensitive, accurate, and specific isotope dilution tandem mass spectrometry method for the simultaneous quantification of any combination of T 4 , T 3 , 3,3′-T 2 , T 1 AM, and, optionally, rT 3 in a sample obtained from a human, e.g., in human plasma or serum samples. This assay is far more sensitive than previously described assays for thyronamines and allows quantitation of T 1 AM in human plasma or serum, including from healthy controls.
1. A method of simultaneously assaying thyroxine (T 4 ), triiodothyronine (T 3 ), 3,3′-diiodo-L-thyronine (3,3′-T 2 ), and 3-iodothyronamine (T 1 AM), comprising:
a) providing a test sample, wherein the test sample comprises T 4 , T 3 , 3,3′-T 2 , and T 1 AM;
b) providing a calibration sample, wherein the calibration sample comprises known quantities of reference T 4 , T 3 , 3,3′-T 2 , and T 1 AM;
c) combining the test sample with the calibration sample;
d) determining by mass spectrometry the quantity of T 4 , T 3 , 3,3′-T 2 , and T 1 AM in the test sample and the quantity of the reference T 4 , T 3 , 3,3′-T 2 , and T 1 AM; and
e) calibrating the quantity of the T 4 , T 3 , 3,3′-T 2 , and T 1 AM in the test sample against the known and determined quantities of the reference T 4 , T 3 , 3,3′-T 2 , and T 1 AM in the calibration sample.
2. The method of claim 1 , wherein each of the reference T 4 , T 3 , 3,3′-T 2 , and T 1 AM is differentially labeled with one or more mass spectrometrically distinct groups, such that each of the test sample T 4 , T 3 , 3,3′-T 2 , and T 1 AM and each of the reference T 4 , T 3 , 3,3′-T 2 , and T 1 AM can be distinguished by mass spectrometry.
3. The method of claim 2 , wherein each of the reference T 4 , T 3 , 3,3′-T 2 , and T 1 AM is radioisotopically labeled.
4. The method of claim 3 , wherein the reference T 4 is deuterium-labeled T 4 .
5. The method of claim 3 , wherein the reference T 3 is 13 C-labeled T 3 .
6. The method of claim 3 , wherein the reference 3,3′-T 2 is 13 C-labeled 3,3′-T 2 .
7. The method of claim 3 , wherein the reference T 1 AM is deuterium-labeled T 1 AM.
8. The method of claim 1 , further comprising separating components of the combined sample of step (c) by liquid chromatography prior to step (d).
9. The method of claim 1 , wherein the test sample is selected from the group consisting of blood, serum, plasma, amniotic fluid, and cerebrospinal fluid.
10. The method of claim 1 , wherein the test sample further comprises reverse T 3 (rT 3 ); the calibration sample further comprises a known quantity of reference rT 3 ; the determining further comprises determining by mass spectrometry the quantity of rT 3 in the test sample and the quantity of the reference rT 3 ; and the calibrating further comprises calibrating the quantity of the rT 3 in the test sample against the known and determined quantity of the reference rT 3 in the calibration sample.
11. The method of claim 1 , wherein the detection limit for T 1 AM in the test sample is 2.5 pg/mL (7.0 pmol/L).
12. The method of claim 1 , wherein the quantitation limit for T 1 AM in the test sample is 4.0 pg/mL (11.3 pmol/L).
13. The method of claim 1 , wherein the detection limit for 3,3′-T 2 in the test sample is 2.5 pg/mL (4.8 pmol/L).
14. The method of claim 1 , wherein the quantitation limit for 3,3′-T 2 in the test sample is 4.0 pg/mL (7.6 pmol/L).
15. The method of claim 1 , wherein the detection limit for T 3 in the test sample is 1.5 pg/mL (2.3 pmol/L).
16. The method of claim 1 , wherein the quantitation limit for T 3 in the test sample is 3.0 pg/mL (4.6 pmol/L).
17. The method of claim 1 , wherein the detection limit for T 4 in the test sample is 1.0 pg/mL (1.3 pmol/L).
18. The method of claim 1 , wherein the quantitation limit for T 4 in the test sample is 1.8 pg/mL (2.3 pmol/L).