Internal calibration as an emerging approach for endogenous analyte quantification: Application to steroids

Area of Science:

• Analytical Chemistry
• Biochemistry
• Clinical Diagnostics

Background:

• Triple quadrupole mass spectrometry is standard for quantification.
• Traditional calibration curves are labor-intensive and error-prone.
• A need exists for more efficient and accurate calibration methods.

Purpose of the Study:

• To develop and validate an innovative internal calibration (IC) approach.
• To utilize a one-standard calibration with stable isotope-labeled (SIL) standards.
• To assess the method's performance for quantifying endogenous steroids in human serum.

Main Methods:

• Developed a novel internal calibration (IC) method using stable isotope-labeled (SIL) standards.
• Evaluated analyte-to-SIL response factor (RF) stability, SIL purity, and instrumental reproducibility.
• Applied the method to six endogenous steroids and tested on quality control and human serum samples.

Main Results:

• Consistent response factors (RFs) were observed across different LC-MS platforms.
• The IC method demonstrated satisfactory precision (1.3%-12.4%) and trueness (77.5%-107.0%).
• Results from the IC method closely matched conventional external calibration, with minimal bias.

Conclusions:

• The developed internal calibration approach offers a robust and accurate alternative for mass spectrometry quantification.
• This method simplifies the calibration process, reducing time and potential analytical errors.
• Naturally occurring isotopes in SIL standards can further enhance accuracy for low-concentration analytes.