Development and validation of a novel candidate reference procedure for the quantification of 3-nitro-L-tyrosine, 3-monoiodo-L-tyrosine and 3,5-diiodo-L-tyrosine in urine by isotope dilution and LC-ESI-MS/MS
Abstract
This work presents the development and validation of a candidate reference method for the simultaneous quantification of 3-nitro-L-tyrosine, 3-monoiodo-L-tyrosine, and 3,5-diiodo-L-tyrosine in urine. 13C and 15N labelled analogues of the three analytes were calibrated against natural abundance standards and used for the development of an isotope dilution quantification approach. Deuterated creatinine was also used to quantify in situ the creatinine content so that concentration of the target analytes relative to creatinine can be obtained in the same chromatogram. Liquid chromatography coupled to tandem mass spectrometry was used in the Selected Reaction Monitoring mode to measure isotopologue distributions from which concentration of the analytes are directly obtained without performing methodological calibrations. Chromatographic separation and mass spectrometric detection were optimized to achieve limits of quantification from 0.015 to 0.08 pmol center dot g-1. The full protocol was validated according to the Clinical and Laboratory Standards Institute (CLSI) guidelines, yielding satisfactory results. Interday variability (ranged from 0.8 to 2.5%) and intraday variability (from 0.7 to 4.2%) in levels of individuals with congenital hypothyroidism were evaluated. Accuracy was evaluated through recovery experiments in fortified urine obtaining 103 +/- 6, 97 +/- 4, 95 +/- 5% for 3-nitro-L-tyrosine, 3-monoiodo-L-tyrosine, and 3,5-diiodo-L-tyrosine, respectively. Linearity between 1.2 and 73.8, 1.8 and 102.3, and 0.6 and 35.8 pmol center dot g-1 for 3-NT, MIT, and DIT, respectively was obtained. Application to real samples was evaluated through the analysis of 1062 urine samples from pregnant women and their offspring. The candidate reference procedure demonstrated its robustness as a candidate reference procedure for the clinical investigation of thyroid hormone disorders and oxidative stress in the general population.
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BibTeX
@article{potesrodriguez2026development,
title = {{Development and validation of a novel candidate reference procedure for the quantification of 3-nitro-L-tyrosine, 3-monoiodo-L-tyrosine and 3,5-diiodo-L-tyrosine in urine by isotope dilution and LC-ESI-MS/MS}},
author = {Heli Potes-Rodriguez and Pablo Rodríguez-González and Juan Manuel Marchante-Gayon and Pouya Alikhani and Mònica Guxens and Anne-Claire Binter and Alireza Mani and Jose C Moreno and José Ignacio García‐alonso},
journal = {Microchemical Journal},
year = {2026},
month = aug,
doi = {10.1016/j.microc.2026.119391},
url = {https://hal.science/hal-05768611v1},
note = {CC BY-NC-ND 4.0},
}
RIS
TY - JOUR
TI - Development and validation of a novel candidate reference procedure for the quantification of 3-nitro-L-tyrosine, 3-monoiodo-L-tyrosine and 3,5-diiodo-L-tyrosine in urine by isotope dilution and LC-ESI-MS/MS
AU - Heli Potes-Rodriguez
AU - Pablo Rodríguez-González
AU - Juan Manuel Marchante-Gayon
AU - Pouya Alikhani
AU - Mònica Guxens
AU - Anne-Claire Binter
AU - Alireza Mani
AU - Jose C Moreno
AU - José Ignacio García‐alonso
PY - 2026
DA - 2026/08/13
JO - Microchemical Journal
DO - 10.1016/j.microc.2026.119391
UR - https://hal.science/hal-05768611v1
AB - This work presents the development and validation of a candidate reference method for the simultaneous quantification of 3-nitro-L-tyrosine, 3-monoiodo-L-tyrosine, and 3,5-diiodo-L-tyrosine in urine. 13C and 15N labelled analogues of the three analytes were calibrated against natural abundance standards and used for the development of an isotope dilution quantification approach. Deuterated creatinine was also used to quantify in situ the creatinine content so that concentration of the target analytes relative to creatinine can be obtained in the same chromatogram. Liquid chromatography coupled to tandem mass spectrometry was used in the Selected Reaction Monitoring mode to measure isotopologue distributions from which concentration of the analytes are directly obtained without performing methodological calibrations. Chromatographic separation and mass spectrometric detection were optimized to achieve limits of quantification from 0.015 to 0.08 pmol center dot g-1. The full protocol was validated according to the Clinical and Laboratory Standards Institute (CLSI) guidelines, yielding satisfactory results. Interday variability (ranged from 0.8 to 2.5%) and intraday variability (from 0.7 to 4.2%) in levels of individuals with congenital hypothyroidism were evaluated. Accuracy was evaluated through recovery experiments in fortified urine obtaining 103 +/- 6, 97 +/- 4, 95 +/- 5% for 3-nitro-L-tyrosine, 3-monoiodo-L-tyrosine, and 3,5-diiodo-L-tyrosine, respectively. Linearity between 1.2 and 73.8, 1.8 and 102.3, and 0.6 and 35.8 pmol center dot g-1 for 3-NT, MIT, and DIT, respectively was obtained. Application to real samples was evaluated through the analysis of 1062 urine samples from pregnant women and their offspring. The candidate reference procedure demonstrated its robustness as a candidate reference procedure for the clinical investigation of thyroid hormone disorders and oxidative stress in the general population.
SN - 0026-265X
SN - 1095-9149
ER -