Quantification of recombinant human parathyroid hormone (rhPTH(1-84)) in human plasma by immunoassay: commercial kit evaluation and validation to support pharmacokinetic studies.

Immunoassays utilizing commercial kits designed for diagnostic use can be adapted and validated to meet Good Laboratory Practice (GLP) requirements to support pharmacokinetic (PK) studies. We illustrate in this paper a systematic approach for commercial kit evaluation and GLP-compliant method validation to establish selectivity, sensitivity, linearity, accuracy, precision and stability. Immunoassay kits for human parathyroid hormone (hPTH) quantification from three different vendors were assessed in a side-by-side comparison for their suitability for the PK analysis of recombinant humanPTH (rhPTH) in EDTA plasma. Two immunoradiometric (IRMA) assay kits and one immunoluminometric assay (ILMA) kit were evaluated. Since PTH is present as an endogenous component of human plasma, QC preparation in the biological matrix was handled differently than for a xenobiotic drug compound. The endogenous concentration of PTH was determined in plasma samples from 32 individual lots using the three kits. The lots with the lowest endogenous concentrations of PTH were selected, pooled to form the low QC and spiked with rhPTH to prepare the mid and high QCs. Four evaluation batches were run with each of the three commercial kits to evaluate reference standard linearity, and QC accuracy and precision. Selectivity against PTH peptide fragments PTH(7-84) and PTH(3-84) were assessed by cross-reactivity and accurate spike-recovery to the QC samples at two concentrations. One of the kits was chosen for full method validation because it had the lowest cross-reactivity against hPTH fragments (3-84) and (7-84), a wider dynamic range and the least total error. The accuracy and precision from six validation batches of the QCs were

Identification of carboxyl-terminal peptide fragments of parathyroid hormone in human plasma at low-picomolar levels by mass spectrometry.

For decades, researchers have tried to identify the primary structures of circulating carboxyl-terminal parathyroid hormone (C-PTH) peptide fragments that may be present at only picomolar levels in human plasma. Although immunoassays and radiosequencing techniques have provided valuable fragment characterizations, no analysis has successfully determined their exact primary structures. In this work, for the first time, four human C-PTH peptide fragments, hPTH(34-84), hPTH(37-84), hPTH(38-84), and hPTH(45-84), have been identified from human plasma using MS-based methods. C-PTH peptide fragments were isolated from plasma samples by immunoaffinity extraction. The eluate was analyzed by capillary LC fractionation followed by MALDI-TOF-MS or by on-line coupling of nano-LC with ESI-TOF-MS. Both the MALDI- and the ESI-based approaches were capable of detecting C-PTH peptide fragments in human plasma at <10 pmol/L. The MALDI-TOF approach was effective in preliminary searches for C-PTH peptide fragments, but the use of high laser power limited the resolution necessary for accurate C-PTH peptide identification. The high mass resolution (10,000) and accuracy (10 ppm) attained by the ESI-TOF approach enabled unambiguous identification of these peptides. The four C-PTH peptide fragments identified in plasma samples from patients with chronic renal insufficiency were also found in the plasma of healthy women receiving recombinant human PTH either by subcutaneous injection or by intravenous infusion. This newly developed analytical capability should greatly enhance the understanding of PTH metabolism and parathyroid gland function.