Validation of a high-throughput liquid chromatography - Tandem mass spectrometry method for urinary cortisol and cortisone

Robert L. Taylor, Dwaine Machacek, Ravinder Jit Singh

Research output: Contribution to journalArticle

171 Citations (Scopus)

Abstract

Background: Urinary free cortisol and cortisone measurements are useful in evaluation of Cushing syndrome, apparent mineralocorticoid excess, congenital adrenal hyperplasia, and adrenal insufficiency. To reduce analytical interference, improve accuracy, and shorten the analysis time, we developed a liquid chromatography - tandem mass spectrometry (LC-MS/MS) method for urinary cortisol and cortisone. Methods: We added 190 pmol (70 ng) of stable isotope cortisol-9,11,12,12-d4 to 0.5 mL of urine as an internal standard before extraction. The urine was extracted with 4.5 mL of methylene chloride, washed, and dried, and 10 μL of the reconstituted extract was injected Onto a reversed-phase C18 column and analyzed using a tandem mass spectrometer operating in the positive mode. Results: Multiple calibration curves for urinary cortisol and cortisone exhibited consistent linearity and reproducibility in the range 7-828 nmol/L (0.25-30 μg/dL). Interassay CVs were 7.3-16% for mean concentrations of 6-726 nmol/L (0.2-26.3 μg/dL) for cortisol and cortisone. The detection limit was 6 nmol/L (0.2 μg/dL). Recovery of cortisol and cortisone added to urine was 97-123%. The regression equation for the LC-MS/MS (y) and HPLC (x) method for cortisol was: y = 1.11x + 0.03 μg cortisol/24 h (r2 = 0.992; n = 99). The regression equation for the LC-MS/MS (y) and immunoassay (x) methods for cortisol was: y = 0.66x - 12.1 μg cortisol/24 h (r2 = 0.67; n = 99). Conclusion: The sensitivity and specificity of the LC-MS/MS method for urinary free cortisol and cortisone offer advantages over routine immunoassays or chromatographic methods because of elimination of drug interferences, high throughput, and short chromatographic run time.

Original languageEnglish (US)
Pages (from-to)1511-1519
Number of pages9
JournalClinical Chemistry
Volume48
Issue number9
StatePublished - Sep 2002

Fingerprint

Cortisone
Liquid chromatography
Tandem Mass Spectrometry
Liquid Chromatography
Mass spectrometry
Hydrocortisone
Throughput
Urine
Immunoassay
Congenital Adrenal Hyperplasia
Adrenal Insufficiency
Mineralocorticoids
Cushing Syndrome
Methylene Chloride
Mass spectrometers
Isotopes
Calibration
Limit of Detection
High Pressure Liquid Chromatography
Sensitivity and Specificity

ASJC Scopus subject areas

  • Clinical Biochemistry

Cite this

Validation of a high-throughput liquid chromatography - Tandem mass spectrometry method for urinary cortisol and cortisone. / Taylor, Robert L.; Machacek, Dwaine; Singh, Ravinder Jit.

In: Clinical Chemistry, Vol. 48, No. 9, 09.2002, p. 1511-1519.

Research output: Contribution to journalArticle

@article{a7d3a6b3b17c4d9cbadcb7fe12108e1f,
title = "Validation of a high-throughput liquid chromatography - Tandem mass spectrometry method for urinary cortisol and cortisone",
abstract = "Background: Urinary free cortisol and cortisone measurements are useful in evaluation of Cushing syndrome, apparent mineralocorticoid excess, congenital adrenal hyperplasia, and adrenal insufficiency. To reduce analytical interference, improve accuracy, and shorten the analysis time, we developed a liquid chromatography - tandem mass spectrometry (LC-MS/MS) method for urinary cortisol and cortisone. Methods: We added 190 pmol (70 ng) of stable isotope cortisol-9,11,12,12-d4 to 0.5 mL of urine as an internal standard before extraction. The urine was extracted with 4.5 mL of methylene chloride, washed, and dried, and 10 μL of the reconstituted extract was injected Onto a reversed-phase C18 column and analyzed using a tandem mass spectrometer operating in the positive mode. Results: Multiple calibration curves for urinary cortisol and cortisone exhibited consistent linearity and reproducibility in the range 7-828 nmol/L (0.25-30 μg/dL). Interassay CVs were 7.3-16{\%} for mean concentrations of 6-726 nmol/L (0.2-26.3 μg/dL) for cortisol and cortisone. The detection limit was 6 nmol/L (0.2 μg/dL). Recovery of cortisol and cortisone added to urine was 97-123{\%}. The regression equation for the LC-MS/MS (y) and HPLC (x) method for cortisol was: y = 1.11x + 0.03 μg cortisol/24 h (r2 = 0.992; n = 99). The regression equation for the LC-MS/MS (y) and immunoassay (x) methods for cortisol was: y = 0.66x - 12.1 μg cortisol/24 h (r2 = 0.67; n = 99). Conclusion: The sensitivity and specificity of the LC-MS/MS method for urinary free cortisol and cortisone offer advantages over routine immunoassays or chromatographic methods because of elimination of drug interferences, high throughput, and short chromatographic run time.",
author = "Taylor, {Robert L.} and Dwaine Machacek and Singh, {Ravinder Jit}",
year = "2002",
month = "9",
language = "English (US)",
volume = "48",
pages = "1511--1519",
journal = "Clinical Chemistry",
issn = "0009-9147",
publisher = "American Association for Clinical Chemistry Inc.",
number = "9",

}

TY - JOUR

T1 - Validation of a high-throughput liquid chromatography - Tandem mass spectrometry method for urinary cortisol and cortisone

AU - Taylor, Robert L.

AU - Machacek, Dwaine

AU - Singh, Ravinder Jit

PY - 2002/9

Y1 - 2002/9

N2 - Background: Urinary free cortisol and cortisone measurements are useful in evaluation of Cushing syndrome, apparent mineralocorticoid excess, congenital adrenal hyperplasia, and adrenal insufficiency. To reduce analytical interference, improve accuracy, and shorten the analysis time, we developed a liquid chromatography - tandem mass spectrometry (LC-MS/MS) method for urinary cortisol and cortisone. Methods: We added 190 pmol (70 ng) of stable isotope cortisol-9,11,12,12-d4 to 0.5 mL of urine as an internal standard before extraction. The urine was extracted with 4.5 mL of methylene chloride, washed, and dried, and 10 μL of the reconstituted extract was injected Onto a reversed-phase C18 column and analyzed using a tandem mass spectrometer operating in the positive mode. Results: Multiple calibration curves for urinary cortisol and cortisone exhibited consistent linearity and reproducibility in the range 7-828 nmol/L (0.25-30 μg/dL). Interassay CVs were 7.3-16% for mean concentrations of 6-726 nmol/L (0.2-26.3 μg/dL) for cortisol and cortisone. The detection limit was 6 nmol/L (0.2 μg/dL). Recovery of cortisol and cortisone added to urine was 97-123%. The regression equation for the LC-MS/MS (y) and HPLC (x) method for cortisol was: y = 1.11x + 0.03 μg cortisol/24 h (r2 = 0.992; n = 99). The regression equation for the LC-MS/MS (y) and immunoassay (x) methods for cortisol was: y = 0.66x - 12.1 μg cortisol/24 h (r2 = 0.67; n = 99). Conclusion: The sensitivity and specificity of the LC-MS/MS method for urinary free cortisol and cortisone offer advantages over routine immunoassays or chromatographic methods because of elimination of drug interferences, high throughput, and short chromatographic run time.

AB - Background: Urinary free cortisol and cortisone measurements are useful in evaluation of Cushing syndrome, apparent mineralocorticoid excess, congenital adrenal hyperplasia, and adrenal insufficiency. To reduce analytical interference, improve accuracy, and shorten the analysis time, we developed a liquid chromatography - tandem mass spectrometry (LC-MS/MS) method for urinary cortisol and cortisone. Methods: We added 190 pmol (70 ng) of stable isotope cortisol-9,11,12,12-d4 to 0.5 mL of urine as an internal standard before extraction. The urine was extracted with 4.5 mL of methylene chloride, washed, and dried, and 10 μL of the reconstituted extract was injected Onto a reversed-phase C18 column and analyzed using a tandem mass spectrometer operating in the positive mode. Results: Multiple calibration curves for urinary cortisol and cortisone exhibited consistent linearity and reproducibility in the range 7-828 nmol/L (0.25-30 μg/dL). Interassay CVs were 7.3-16% for mean concentrations of 6-726 nmol/L (0.2-26.3 μg/dL) for cortisol and cortisone. The detection limit was 6 nmol/L (0.2 μg/dL). Recovery of cortisol and cortisone added to urine was 97-123%. The regression equation for the LC-MS/MS (y) and HPLC (x) method for cortisol was: y = 1.11x + 0.03 μg cortisol/24 h (r2 = 0.992; n = 99). The regression equation for the LC-MS/MS (y) and immunoassay (x) methods for cortisol was: y = 0.66x - 12.1 μg cortisol/24 h (r2 = 0.67; n = 99). Conclusion: The sensitivity and specificity of the LC-MS/MS method for urinary free cortisol and cortisone offer advantages over routine immunoassays or chromatographic methods because of elimination of drug interferences, high throughput, and short chromatographic run time.

UR - http://www.scopus.com/inward/record.url?scp=0036721539&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0036721539&partnerID=8YFLogxK

M3 - Article

C2 - 12194928

AN - SCOPUS:0036721539

VL - 48

SP - 1511

EP - 1519

JO - Clinical Chemistry

JF - Clinical Chemistry

SN - 0009-9147

IS - 9

ER -