Quantification of serum 1-84 parathyroid hormone in patients with hyperparathyroidism by immunocapture in situ digestion liquid chromatography-tandem mass spectrometry.
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. A methodology 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 ((1)SVSEIQLMHNLGK(13)).


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 <or=1% to <or=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.
Kumar
Vivek
V
Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN 55905, USA.
Barnidge
David R
DR
Chen
Li-Sheng
LS
Twentyman
Jolaine M
JM
Cradic
Kendall W
KW
Grebe
Stefan K
SK
Singh
Ravinder J
RJ
D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D006961:Hyperparathyroidism / Q000097:blood* / Q000175:diagnosis; D007118:Immunoassay / Q000379:methods*; D057230:Limit of Detection; D016014:Linear Models; D010281:Parathyroid Hormone / Q000097:blood*; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*
D010281:Parathyroid Hormone