High sensitivity LC–MS/MS method for direct quantification of human parathyroid 1–34 (teriparatide) in human plasma

• First known LC–MS/MS method for teriparatide which reaches clinically relevant detection limits.
• Achieves an LLOQ of 15 pg/mL (3.6 fmol/mL) from 200 μL human plasma.
• Simple 2-step PPT/SPE 96-well extraction avoids affinity purification.
• Mean QC accuracy and precision (all points) were 97.5 and 5.7%, respectively.
• CV of matrix factors was 5%.

Teriparatide, the 1–34 fragment of human parathyroid hormone, is used to treat osteoporosis patients with a high risk of fracture by stimulating new bone formation. Routinely teriparatide is quantified using radioimmunoassay however the LC–MS/MS described here has the potential to achieve greater accuracy and precision, higher specificity, and is readily implemented in routine bioanalytical laboratories. Hence a complete method combining effective sample prep with appropriate LC separation and selected reaction monitoring (SRM) MS detection was developed to selectively separate teriparatide from closely related endogenous peptides and to reduce interferences. Samples were concentrated without evaporation, minimizing the risk of adsorptive losses. Chromatography was performed on a sub 2 μm particle charged surface hybrid column, which provided significantly higher peak capacity than a traditional C18 column when formic acid was used as the mobile phase modifier. Total LC cycle time was 6 min. An LOD of 15 pg/mL (3.6 fmol/mL) from 200 μL of human plasma was readily achieved and standard curves were accurate and precise from 15 pg/mL to 500 pg/mL. Mean QC accuracies ranged from 90% to 106%. Mean QC precision was better than 7%. The CV of matrix factors across 6 sources of human plasma was 5%. The assay presented here is the first LC–MS method which reaches clinically relevant detection limits for teriparatide