Bioanalytical method development and validation of highly selective and sensitive LC-MS/MS method for determination of teriparatide (parathyroid hormone fragment 1-34) in human serum through direct detection of intact teriparatide molecule.

Teriparatide is a novel recombinant peptide fragment of the first 1-34 amino acids of human parathyroid recommended for treatment of osteoporosis. Therapeutic proteins and peptides are routinely estimated using ligand binding assay formats however LC-MS/MS technique which is routinely used in bioanalysis of small molecules has now gained importance in large molecule bioanalysis for the advantages it can offer over LBAs in terms of improved accuracy, selectivity and anti-body free method development. This paper presents a sensitive bioanalytical method for determination of teriparatide in human serum using ultra performance liquid chromatography aligned with tandem mass spectrometric detection. Teriparatide was isolated from human serum using solid phase extraction. The intact peptide was separated on a chromatograph and the multiply charged ion (+7) was detected using a mass spectrometer. The total run time was 4.0 min. The internal standard used was rat PTH 1-34 fragment. The mass transitions of m/z 589.3 > 656.3 for teriparatide and m/z 677.4 > 778.6 for internal standard were used for MS/MS detection. The sample extraction involved a solid phase extraction method followed by concentration of the eluent by evaporation and subsequent reconstitution. The non-specific binding effect caused by the adherence of the peptides/proteins to the vials/tube walls was significantly reduced by using BSA solution as blocking agent. The method has been validated over a linear range of 15.07-913.3 pg/mL with a correlation coefficient ≥ 0.99. The precision (%RSD) was 6.36 to 10.85 and accuracy was within 96.71% to 100.88%. A two-treatment, two-period, cross over study was conducted to establish bioequivalence between test and reference formulation (20 mcg/80 mL - solution for injection) and the method was successfully applied to quantify teriparatide in serum samples of this clinical study and about 1220 human serum samples were analyzed to determine teriparatide. This method is a promising anti-body free LC-MS/MS based methodology for estimation of teriparatide in human serum and may be applied as starting method for other such peptide molecules.

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