Quantification of serum 1-84 parathyroid hormone in patients with hyperparathyroidism by immunocapture in situ digestion liquid chromatography-tandem mass spectrometry

Vivek Kumar, David R. Barnidge, Li Sheng Chen, Jolaine M. Twentyman, Kendall W. Cradic, Stefan K. Grebe, Ravinder Jit Singh

Research output: Contribution to journalArticle

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Abstract

BACKGROUND: Immunoassays specific for 1-84 parathyroid hormone (PTH) reportedly reflect the bioactivity of PTH; however, PTH immunoassays can be susceptible to interference by cross-reacting PTH fragments. In addition, these assays currently lack standardization. Amethodology using immunocapture purification with liquid chromatography-tandem mass spectrometry (LC-MS/MS) detection, along with a stable isotope-labeled internal standard, may help address these issues. METHODS: We isolated 1-84 PTH from 1 mL serum by immunocapture on a 6.5-mm polystyrene bead. The immobilized PTH was digested in situ and analyzed by LC-MS/MS. For quantification, we used the selected reaction monitoring response from the N-terminal tryptic peptide 1-13 PTH (1SVSEIQLMHNLGK13). RESULTS: The linear range of the assay was 39.1-4560 ng/L, and the limit of detection and limit of quantification were 14.5 ng/L and 39.1 ng/L, respectively. The intraassay CVs ranged from 6% to 11%, and the interassay CVs ranged from 7% to 17%. Interference by PTH fragments 1-44 PTH, 7-84 PTH, 43-68 PTH, 52-84 PTH, 64-84 PTH, and PTH-related protein (PTHrP) was ≤1% to ≤0.001%.Method comparison of LC-MS/MS vs the Roche Cobas® immunoassay yielded Deming fit of LC-MS/MS = 1.01x immunoassay - 13.21. The mean bias by Bland-Altman plot was -9.4%. CONCLUSIONS: In patients with hyperparathyroidism, the immunocapture in situ digestion LC-MS/MS method can provide accurate and precise PTH results compared with immunoassay.

Original languageEnglish (US)
Pages (from-to)306-313
Number of pages8
JournalClinical Chemistry
Volume56
Issue number2
DOIs
StatePublished - Feb 1 2010

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Hyperparathyroidism
Liquid chromatography
Tandem Mass Spectrometry
Parathyroid Hormone
Liquid Chromatography
Mass spectrometry
Digestion
Serum
Immunoassay
Assays
Parathyroid Hormone-Related Protein
Polystyrenes
Bioactivity
Isotopes
Standardization
Purification
Limit of Detection

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Biochemistry, medical

Cite this

Quantification of serum 1-84 parathyroid hormone in patients with hyperparathyroidism by immunocapture in situ digestion liquid chromatography-tandem mass spectrometry. / Kumar, Vivek; Barnidge, David R.; Chen, Li Sheng; Twentyman, Jolaine M.; Cradic, Kendall W.; Grebe, Stefan K.; Singh, Ravinder Jit.

In: Clinical Chemistry, Vol. 56, No. 2, 01.02.2010, p. 306-313.

Research output: Contribution to journalArticle

Kumar, Vivek ; Barnidge, David R. ; Chen, Li Sheng ; Twentyman, Jolaine M. ; Cradic, Kendall W. ; Grebe, Stefan K. ; Singh, Ravinder Jit. / Quantification of serum 1-84 parathyroid hormone in patients with hyperparathyroidism by immunocapture in situ digestion liquid chromatography-tandem mass spectrometry. In: Clinical Chemistry. 2010 ; Vol. 56, No. 2. pp. 306-313.
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abstract = "BACKGROUND: Immunoassays specific for 1-84 parathyroid hormone (PTH) reportedly reflect the bioactivity of PTH; however, PTH immunoassays can be susceptible to interference by cross-reacting PTH fragments. In addition, these assays currently lack standardization. Amethodology using immunocapture purification with liquid chromatography-tandem mass spectrometry (LC-MS/MS) detection, along with a stable isotope-labeled internal standard, may help address these issues. METHODS: We isolated 1-84 PTH from 1 mL serum by immunocapture on a 6.5-mm polystyrene bead. The immobilized PTH was digested in situ and analyzed by LC-MS/MS. For quantification, we used the selected reaction monitoring response from the N-terminal tryptic peptide 1-13 PTH (1SVSEIQLMHNLGK13). RESULTS: The linear range of the assay was 39.1-4560 ng/L, and the limit of detection and limit of quantification were 14.5 ng/L and 39.1 ng/L, respectively. The intraassay CVs ranged from 6{\%} to 11{\%}, and the interassay CVs ranged from 7{\%} to 17{\%}. Interference by PTH fragments 1-44 PTH, 7-84 PTH, 43-68 PTH, 52-84 PTH, 64-84 PTH, and PTH-related protein (PTHrP) was ≤1{\%} to ≤0.001{\%}.Method comparison of LC-MS/MS vs the Roche Cobas{\circledR} immunoassay yielded Deming fit of LC-MS/MS = 1.01x immunoassay - 13.21. The mean bias by Bland-Altman plot was -9.4{\%}. CONCLUSIONS: In patients with hyperparathyroidism, the immunocapture in situ digestion LC-MS/MS method can provide accurate and precise PTH results compared with immunoassay.",
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T1 - Quantification of serum 1-84 parathyroid hormone in patients with hyperparathyroidism by immunocapture in situ digestion liquid chromatography-tandem mass spectrometry

AU - Kumar, Vivek

AU - Barnidge, David R.

AU - Chen, Li Sheng

AU - Twentyman, Jolaine M.

AU - Cradic, Kendall W.

AU - Grebe, Stefan K.

AU - Singh, Ravinder Jit

PY - 2010/2/1

Y1 - 2010/2/1

N2 - BACKGROUND: Immunoassays specific for 1-84 parathyroid hormone (PTH) reportedly reflect the bioactivity of PTH; however, PTH immunoassays can be susceptible to interference by cross-reacting PTH fragments. In addition, these assays currently lack standardization. Amethodology using immunocapture purification with liquid chromatography-tandem mass spectrometry (LC-MS/MS) detection, along with a stable isotope-labeled internal standard, may help address these issues. METHODS: We isolated 1-84 PTH from 1 mL serum by immunocapture on a 6.5-mm polystyrene bead. The immobilized PTH was digested in situ and analyzed by LC-MS/MS. For quantification, we used the selected reaction monitoring response from the N-terminal tryptic peptide 1-13 PTH (1SVSEIQLMHNLGK13). RESULTS: The linear range of the assay was 39.1-4560 ng/L, and the limit of detection and limit of quantification were 14.5 ng/L and 39.1 ng/L, respectively. The intraassay CVs ranged from 6% to 11%, and the interassay CVs ranged from 7% to 17%. Interference by PTH fragments 1-44 PTH, 7-84 PTH, 43-68 PTH, 52-84 PTH, 64-84 PTH, and PTH-related protein (PTHrP) was ≤1% to ≤0.001%.Method comparison of LC-MS/MS vs the Roche Cobas® immunoassay yielded Deming fit of LC-MS/MS = 1.01x immunoassay - 13.21. The mean bias by Bland-Altman plot was -9.4%. CONCLUSIONS: In patients with hyperparathyroidism, the immunocapture in situ digestion LC-MS/MS method can provide accurate and precise PTH results compared with immunoassay.

AB - BACKGROUND: Immunoassays specific for 1-84 parathyroid hormone (PTH) reportedly reflect the bioactivity of PTH; however, PTH immunoassays can be susceptible to interference by cross-reacting PTH fragments. In addition, these assays currently lack standardization. Amethodology using immunocapture purification with liquid chromatography-tandem mass spectrometry (LC-MS/MS) detection, along with a stable isotope-labeled internal standard, may help address these issues. METHODS: We isolated 1-84 PTH from 1 mL serum by immunocapture on a 6.5-mm polystyrene bead. The immobilized PTH was digested in situ and analyzed by LC-MS/MS. For quantification, we used the selected reaction monitoring response from the N-terminal tryptic peptide 1-13 PTH (1SVSEIQLMHNLGK13). RESULTS: The linear range of the assay was 39.1-4560 ng/L, and the limit of detection and limit of quantification were 14.5 ng/L and 39.1 ng/L, respectively. The intraassay CVs ranged from 6% to 11%, and the interassay CVs ranged from 7% to 17%. Interference by PTH fragments 1-44 PTH, 7-84 PTH, 43-68 PTH, 52-84 PTH, 64-84 PTH, and PTH-related protein (PTHrP) was ≤1% to ≤0.001%.Method comparison of LC-MS/MS vs the Roche Cobas® immunoassay yielded Deming fit of LC-MS/MS = 1.01x immunoassay - 13.21. The mean bias by Bland-Altman plot was -9.4%. CONCLUSIONS: In patients with hyperparathyroidism, the immunocapture in situ digestion LC-MS/MS method can provide accurate and precise PTH results compared with immunoassay.

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