Abstract :
[en] INTRODUCTION Parathyroid hormone (PTH) is a common clinical marker whose quantification relies on immunoassays, giving variable results as batch, brand, or target epitope changes. Moreover, immunoassays may cross-react with PTH variants such as C-terminal fragments stemming from PTH catabolism. These issues make it difficult to compare results obtained in different laboratories. A reference quantification method is necessary to harmonize PTH assays, both sensitive and selective enough to detect PTH at low concentrations among a variety of closely related compounds.
OBJECTIVES In this study, our main goal was to reach a very high sensitivity (pg/mL range) for the analysis of PTH and its variants. Two variants were selected, namely 7-84 PTH as C-terminal fragment and 1-34 PTH as related peptide, but also as potential internal standard for future works.
METHODS To achieve our goal, we developed a sheathless CE-ESI-MS method for the separation of 1-34 PTH, 7-84 PTH, and 1-84 PTH. Fused silica and neutral-coated capillaries were investigated, as well as preconcentration methods such as transient isotachophoresis (t-ITP), field-amplified sample injection (FASI) and electrokinetic supercharging (EKS).
RESULTS The method for the separation of PTH and its variants was first developed using fused-silica capillary with UV detection. 1-84 PTH (full length), 7-84 PTH and 1-34 PTH were separated using an acidic background electrolyte containing acetonitrile to reduce peptide adsorption onto the capillary wall. Ammonium acetate was used as sample medium to improve sensitivity through t-ITP. The method was then transferred to a sheathless CE-ESI-MS instrument. CE-MS on fused silica capillary was limited to µg/mL levels. Indeed, despite the MS detection, only samples containing at least 10 µg/mL of 1-84 PTH, 7-84 PTH, and 1-34 PTH could be analyzed. The use of a neutral coating combined with FASI or EKS allowed a significant increase in sensitivity. Under these conditions, 1-84 PTH, 7-84 PTH and 1-34 PTH were detected at 100 ng/mL using FASI while 1-84 PTH and 1-34 PTH were detected at 100 pg/mL using EKS. The estimated LODs (S/N = 3) for the EKS method were 25 pg/mL for 1-84 PTH and 10 pg/mL for 1-34 PTH, while there was no signal anymore for 7-84 PTH at these levels.
CONCLUSION The developed sheathless CE-ESI-MS method has the potential to reach the low pg/mL range in biological samples after the optimization of the sample preparation method.
