Salmon calcitonin is a cyclic, 32 amino acid synthetic polypeptide, commonly used in the treatment of osteoporosis. The ability to accurately quantify this peptide with high sensitivity and selectivity in support of drug research and development is of interest. Generally, cyclic peptides are difficult to analyze by LC-MS, as MS sensitivity is low due to poor transfer into the gas phase and poor fragmentation. Additionally, the pharmacokinetics of calcitonin is characterized by rapid absorption within 15 minutes and rapid elimination with a half-life of <20 minutes, resulting in low pg/mL circulating levels, making accurate quantification in serum particularly challenging.
Tandem quadrupole instruments are the gold standard for routine LC-MS/MS quantification of small molecules, peptides and digested proteins. As the need to quantify intact larger peptides and proteins increases, HRMS instruments are increasingly becoming an attractive orthogonal platform for quantitative laboratories.
Traditional LC-MS assays for calcitonin have involved time-consuming and laborious enzymatic digestion. This work provides a simple method for the quantification of salmon calcitonin from human serum. The method described herein uses UPLC and fast, selective mixed-mode solid phase extraction (SPE) in 96-well format to achieve limits of detection of 10 pg/mL and limits of quantification of 25 pg/mL with QC accuracies of within 15 % from 100 µL serum using a tandem quadrupole instrument. Additionally, we compare these performance characteristics to a targeted HRMS approach for quantification.
Calibrators and quality controls samples (QCs) of salmon calcitonin were prepared using commercially available human serum (25–1500 pg/mL). A protein precipitation (PPT) of the prepared samples (100 µL) was performed using acetonitrile containing 0.1% formic acid (100 µL). The resulting supernatant was diluted with a 4% phosphoric acid solution and extracted on an Oasis WCX µElution plate. LC-MS/MS quantification of the post-extract eluate was performed on a low dispersion ACQUITY UPLC I-Class LC, coupled to a Xevo TQ-XS tandem quadrupole mass spectrometer using positive electrospray ionization (ESI+) and multiple-reaction monitoring mode (MRM). Chromatographic separation was achieved using a sub-2 µm particle Waters CORTECS UPLC C18+ column (2.1 x 50m), at a flow rate of 0.4 mL/min using and a linear gradient with 0.1% formic acid in water and 0.1% formic acid in acetonitrile. Final injection volume was 20 µL. The different acquisition modes available on the HRMS system were evaluated and quantitation was performed using Tof MRM (precursor to precursor) mode.
In this work, we have a developed a complete sample preparation and LC-MS/MS workflow for the sensitive and accurate quantification of salmon calcitonin from human serum. Using analytical scale LC, tandem quadrupole MS and only 100 µL of extracted serum, a limit of detection of 10 pg/mL was achieved. Using 1/x weighted regression, calibration curves from 25-1500 pg/mL were linear (>0.99), with a mean accuracy across the calibration curve of 100.625 %. For all QC levels, accuracies were between 85-115% with % CVs ≤ 15%, indicating an accurate, precise and reproducible method.
For the HRMS system, a LLOQ of 50 pg/mL was achieved and % CV for all calibration curve and QC points was below 10%.
Use of a simple, yet selective SPE sample preparation strategy (without time-consuming digestion) combined with a fast UPLC analysis (4.5 minutes) and a highly sensitive tandem quadrupole mass spectrometer enabled a LLOQ of 25 pg/mL with 5x lower sample volumes compared to previously published methods. Quantitative performance of the HRMS system was comparable to the tandem quadrupole system, achieving LLOQ of 50 pg/mL.