The impact of different calibration matrices on the determination of insulin-like growth factor 1 by high-resolution-LC-MS in acromegalic and growth hormone deficient patients

Abstract

Objectives: Calibration is an important source of variability in liquid chromatography mass spectrometry (LC-MS) methods for insulin-like growth factor 1 (IGF-1). This study investigated the impact of different calibrator matrices on IGF-1 measurements by LC-MS. Moreover, the comparability of immunoassays and LC-MS was assessed.

Design & methods: Calibrators from 12.5 to 2009 ng/ml were prepared by spiking WHO international Standard (ID 02/254 NIBSC, UK) into the following matrices: native human plasma, fresh charcoal-treated human plasma (FCTHP), old charcoal-treated human plasma, deionized water, bovine serum albumin (BSA), and rat plasma (RP). A validated in-house LC-MS method was calibrated repeatedly with these calibrators. Then, serum samples from 197 growth hormone excess and deficiency patients were analysed with each calibration.

Results: The seven calibration curves had different slopes leading to markedly different patient results. The largest differences in IGF-1 concentration from the median (interquartile range) was observed with the calibrator in water and the calibrator in RP (336.4 [279.6-417.0] vs. 112.5 [71.2-171.2], p < 0.001). The smallest difference was observed with calibrators in FCTHP and BSA (141.8 [102.0-198.5] vs. 127.9 [86.9-186.0], p < 0.049). Compared to LC-MS with calibrators in FCTHP, immunoassays showed relevant proportional bias (range: -43% to -68%), constant bias (range: 22.84 to 57.29 ng/ml) and pronounced scatter. Comparing the immunoassays with each other revealed proportional bias of up to 24%.

Conclusions: The calibrator matrix is critical for the measurement of IGF-1 by LC-MS. Regardless of the calibrator matrix, LC-MS shows poor agreement with immunoassays. Also, the agreement between different immunoassays is variable.

Keywords: Hormone; Insulin-like Growth Factor 1 (IGF-1); Liquid Chromatography High-Resolution Mass Spectrometry (LC-HRMS); Online Solid Phase Extraction (OSPE); Peptide; Quantification; Serum.