| Abstract: | Parathyroid hormone (PTH) is a common clinical marker whose quantification relies on immunoassays, giving variable results as batch, brand, or target epitope changes. Sheathless CE‐ESI‐MS, combining CE resolution power and low‐flow ESI sensitivity, was applied to the analysis of PTH in its native conformation in the presence of related forms. Fused silica and neutral‐coated capillaries were investigated, as well as preconcentration methods such as transient isotachophoresis, field‐amplified sample injection (FASI), and electrokinetic supercharging (EKS). The method for the separation of PTH and its variants was first developed using fused‐silica capillary with UV detection. An acidic BGE was used to separate 1–84 PTH (full length), 7–84 PTH, and 1–34 PTH. Acetonitrile was added to the BGE to reduce peptide adsorption onto the capillary wall and transient isotachophoresis was used as analyte preconcentration method. The method was then transferred to a sheathless CE‐ESI‐MS instrument. When using a fused silica capillary, CE‐MS was limited to μg/mL levels. 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 concentrations in the low ng/mL (FASI) or pg/mL (EKS) range. |
