Biomaterial Database

Candidate reference method for total thyroxine in human serum: use of isotope-dilution liquid chromatography-mass spectrometry with electrospray ionization. 2002

Susan S-C Tai, and Lorna T Sniegoski, and Michael J Welch

Analytical Chemistry Division, National Institute of Standards and Technology, Gaithersburg, MD 20899-8392, USA. susan.tai@nist.gov

BACKGROUND There is a need for a critically evaluated reference method for thyroxine to provide an accuracy base to which routine methods can be traceable. We describe a candidate reference method involving isotope-dilution coupled with liquid chromatography/mass spectrometry. METHODS An isotopically labeled internal standard, thyroxine-d(5), was added to serum, followed by equilibration, protein precipitation, and ethyl acetate and solid-phase extractions to prepare samples for liquid chromatography-mass spectrometry electrospray ionization (LC/MS-ESI) analysis. For separation, a Zorbax Eclipse XDB-C(18) column was used with a mobile phase consisting of 1 mL/L acetic acid in acetonitrile-water (32:68 by volume) for positive ions and a Zorbax Extend-C(18) column with a mobile phase consisting of 2 mL/L ammonium hydroxide in methanol-water (32:68 by volume) for negative ions. [M + H](+) ions at m/z 778 and 783 for thyroxine and its labeled internal standard were monitored for positive ions and [M - H](-) ions at m/z 776 and 781 for negative ions. Samples of frozen serum pools were prepared and measured in three separate sets. RESULTS Within-set CVs were 0.2-1.0%. The correlation coefficients of all linear regression lines (measured intensity ratios vs mass ratios) were 0.999-1.000. Positive- and negative-ion measurements agreed with a mean difference of 0.45% at three concentrations (50, 110, and 168 microg/L). The detection limits (at a signal-to-noise ratio of approximately 3 to 5) were 30 and 20 pg for positive and negative ions, respectively. The results from the LC/MS-ESI method were within 1 SD of the composite means from many routine clinical methods, although it appears that the clinical method means may be biased high by 4-5 microg/L across the concentrations. Some routine clinical methods may be biased by up to 20% at low concentrations. CONCLUSIONS This well-characterized LC/MS-ESI method for total serum thyroxine with a theoretically sound approach, demonstrated good accuracy and precision, and low susceptibility to interferences qualifies as a candidate reference method. Use of this reference method as an accuracy base may reduce the apparent biases in routine methods along with the high interlaboratory imprecision.

UI	MeSH Term	Description	Entries
D011786	Quality Control	A system for verifying and maintaining a desired level of quality in a product or process by careful planning, use of proper equipment, continued inspection, and corrective action as required. (Random House Unabridged Dictionary, 2d ed)	Control, Quality,Controls, Quality,Quality Controls
D012015	Reference Standards	A basis of value established for the measure of quantity, weight, extent or quality, e.g. weight standards, standard solutions, methods, techniques, and procedures used in diagnosis and therapy.	Standard Preparations,Standards, Reference,Preparations, Standard,Standardization,Standards,Preparation, Standard,Reference Standard,Standard Preparation,Standard, Reference
D012016	Reference Values	The range or frequency distribution of a measurement in a population (of organisms, organs or things) that has not been selected for the presence of disease or abnormality.	Normal Range,Normal Values,Reference Ranges,Normal Ranges,Normal Value,Range, Normal,Range, Reference,Ranges, Normal,Ranges, Reference,Reference Range,Reference Value,Value, Normal,Value, Reference,Values, Normal,Values, Reference
D002853	Chromatography, Liquid	Chromatographic techniques in which the mobile phase is a liquid.	Liquid Chromatography
D006801	Humans	Members of the species Homo sapiens.	Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D012680	Sensitivity and Specificity	Binary classification measures to assess test results. Sensitivity or recall rate is the proportion of true positives. Specificity is the probability of correctly determining the absence of a condition. (From Last, Dictionary of Epidemiology, 2d ed)	Specificity,Sensitivity,Specificity and Sensitivity
D013974	Thyroxine	The major hormone derived from the thyroid gland. Thyroxine is synthesized via the iodination of tyrosines (MONOIODOTYROSINE) and the coupling of iodotyrosines (DIIODOTYROSINE) in the THYROGLOBULIN. Thyroxine is released from thyroglobulin by proteolysis and secreted into the blood. Thyroxine is peripherally deiodinated to form TRIIODOTHYRONINE which exerts a broad spectrum of stimulatory effects on cell metabolism.	L-Thyroxine,Levothyroxine,T4 Thyroid Hormone,3,5,3',5'-Tetraiodothyronine,Berlthyrox,Dexnon,Eferox,Eltroxin,Eltroxine,Euthyrox,Eutirox,L-3,5,3',5'-Tetraiodothyronine,L-Thyrox,L-Thyroxin Henning,L-Thyroxin beta,L-Thyroxine Roche,Levo-T,Levothroid,Levothyroid,Levothyroxin Deladande,Levothyroxin Delalande,Levothyroxine Sodium,Levoxine,Levoxyl,Lévothyrox,Novothyral,Novothyrox,O-(4-Hydroxy-3,5-diiodophenyl) 3,5-diiodo-L-tyrosine,O-(4-Hydroxy-3,5-diiodophenyl)-3,5-diiodotyrosine,Oroxine,Sodium Levothyroxine,Synthroid,Synthrox,Thevier,Thyrax,Thyroxin,Tiroidine,Tiroxina Leo,Unithroid,L Thyrox,L Thyroxin Henning,L Thyroxin beta,L Thyroxine,L Thyroxine Roche,Levo T,Thyroid Hormone, T4
D016014	Linear Models	Statistical models in which the value of a parameter for a given value of a factor is assumed to be equal to a + bx, where a and b are constants. The models predict a linear regression.	Linear Regression,Log-Linear Models,Models, Linear,Linear Model,Linear Regressions,Log Linear Models,Log-Linear Model,Model, Linear,Model, Log-Linear,Models, Log-Linear,Regression, Linear,Regressions, Linear
D021241	Spectrometry, Mass, Electrospray Ionization	A mass spectrometry technique used for analysis of nonvolatile compounds such as proteins and macromolecules. The technique involves preparing electrically charged droplets from analyte molecules dissolved in solvent. The electrically charged droplets enter a vacuum chamber where the solvent is evaporated. Evaporation of solvent reduces the droplet size, thereby increasing the coulombic repulsion within the droplet. As the charged droplets get smaller, the excess charge within them causes them to disintegrate and release analyte molecules. The volatilized analyte molecules are then analyzed by mass spectrometry.	ESI Mass Spectrometry,Electrospray Ionization Mass Spectrometry,Mass Spectrometry, ESI,Spectrometry, ESI Mass