Development of a SPE LC-MS/MS Method for the Bioanalytical Quantification of Salmon Calcitonin from Human Serum

Importance of the Topic

Salmon calcitonin is a synthetic 32-amino-acid peptide used to treat bone and calcium disorders. Its rapid absorption and elimination result in circulating levels in the low picogram per milliliter range, posing analytical challenges. Accurate quantification is critical for pharmacokinetic studies, dose optimization, and safety monitoring. Compared with ligand-binding assays, LC-MS/MS offers superior specificity, a wider dynamic range, reduced cross-reactivity, and faster method development.

Study Objectives and Overview

The primary goal was to develop and validate a sensitive, selective, and high-throughput LC-MS/MS method for quantifying salmon calcitonin in human serum using minimal sample volume. Specific aims included:

• Achieving a lower limit of quantification of 25 pg/mL from a 100 µL serum sample.
• Implementing a simple, mixed-mode μElution solid-phase extraction workflow.
• Utilizing UPLC separation and triple quadrupole detection for rapid and robust analysis.

Methodology and Instrumentation

A two-step sample preparation combined protein precipitation (1:1 acetonitrile dilution) and mixed-mode cation exchange μElution SPE (Oasis WCX). Following washes with ammonium hydroxide and acetonitrile, analytes were eluted with 1% trifluoroacetic acid in acetonitrile/water. Chromatography employed an ACQUITY UPLC I-Class system equipped with a CORTECS UPLC C18+ column (1.6 µm, 2.1×50 mm) and a 5-minute gradient using 0.1% formic acid mobile phases. Detection was performed on a Xevo TQ-XS triple quadrupole in ESI+ mode with MRM transitions 687.5→830.3 (quantifier) and 859.2→1106.7 (qualifier). Data acquisition and processing used MassLynx and TargetLynx.

Key Results and Discussion

• Mass Spectrometry: The 5+ (m/z 687.5) and 4+ (m/z 859.2) precursors produced high-m/z b/y fragments, enhancing selectivity and minimizing background noise.
• Chromatography: The CORTECS C18+ column delivered peak widths under 4 seconds and a tenfold improvement in signal-to-noise compared with conventional fully porous phases.
• Sample Preparation: Combined precipitation and μElution SPE achieved >90% extraction recovery with matrix effects below 15%, and total processing time under 2 hours.
• Performance Metrics: Calibration curves were linear (r²>0.99) from 25–1500 pg/mL. Intra- and inter-day accuracy (85–115%) and precision (CV<15%) met bioanalytical validation criteria.

Benefits and Practical Applications

• Requires only 100 µL serum and minimal preparative steps for high sample throughput.
• No enzymatic digestion or extensive sample cleanup reduces variability and development time.
• High sensitivity and specificity support pharmacokinetic, clinical, and quality control laboratories.
• Approach is adaptable to multiplex assays for other peptide therapeutics and metabolites.

Future Trends and Opportunities

Anticipated developments include expansion to multiplexed peptide panels, automation of μElution workflows, integration with high-resolution mass spectrometry for comprehensive metabolite profiling, and adoption of microflow or nanoflow LC to further reduce sample volume and enhance throughput.

Conclusion

The described SPE LC-MS/MS method combines streamlined μElution sample preparation, sub-2-µm core-shell chromatography, and optimized MRM detection to achieve high-sensitivity quantification of salmon calcitonin at low picogram levels using minimal serum volume. The approach offers robustness, speed, and specificity suitable for pharmacokinetic and clinical bioanalysis.

References

1. Zeng K., Geerlof-Vidavisky I., Gucinski A., Jiang X., Boyne M. Liquid Chromatography–High Resolution Mass Spectrometry for Peptide Drug Quality Control. AAPS J. 2015;17(3).
2. Novartis. Miacalcin (Calcitonin–salmon) Prescribing Information. United States; Oct 2009.
3. Li Y., Hackman M., Wang C. Quantitation of Polypeptides (Glucagon and Salmon Calcitonin) in Plasma by High-Resolution Triple Quadrupole MS. Bioanalysis. 2012;4(6):685–691.
4. Salmon calcitonin sequence and structure. DrugBank. 2018.