Intact IGF-I quantification on a TQ and HRMS system using simple SPE, without immuno-purification or digestion to achieve 5 ng/mL sensitivity
Insulin-like Growth Factor I (IGF-I), a 70 amino acid (7.6 kDa) peptide hormone, plays a significant role in mediating the effects of Growth Hormone (GH) and imbalance can lead to conditions such as acromegaly, dwarfism, and increased risk of cancer. While immunoassays have been used for IGF-I quantification, use of LC-MS has increased. Most of these LC-MS methods use immunoaffinity extraction, enzymatic digestion, nano-flow LC, or a combination of these. Here, we highlight a simplified sample preparation workflow using sample pretreatment, protein precipitation, and solid phase extraction (SPE) for the quantification of intact IGF-I from human serum using an analytical LC and a tandem quadrupole instrument. We further compare its performance characteristics to a targeted HRMS approach for quantification.
IGF-I containing plasma/serum samples (100 µL) were incubated with 100 µL of 0.6% Sodium Dodaceyl Sulphate (SDS) for 60 mins (37˚C). Acetonitrile containing 5% acetic acid (200 µL) was then added, samples vortexed, centrifuged, and resulting supernatant (300 µL) was diluted with 900 µL of 5% ammonium hydroxide solution. SPE of pretreated samples was performed using a mixed-mode anion exchange sorbent in the 96-well µElution format. LC-MS/MS analysis of the resulting supernatant was performed using a low dispersion LC, coupled to a TQ or QTof HRMS system. Chromatographic separation was achieved using a sub-2 µm charged surface hybrid C18 column, (2.1 mm x 50 mm), using a linear gradient ( 0.4 mL/min) with 0.1% formic acid in water and acetonitrile.
Using the described sample preparation strategy, accurate quantification of IGF-I from 5-1000 ng/mL was achieved on a TQ. Calibration curves were linear (1/x2 weighting) with r2 values >0.99 and mean accuracies between 99-102%. QC performance was excellent with accuracy ranges between 93-108% and CV’s < 10%, well within the acceptable FDA bioanalytical guidelines. In addition, this assay was reproducible and robust across multiple days.
Similar performance characteristics were observed for an analytical LC-HRMS system. IGF-I was accurately quantified from 10-1000 ng/mL using the targeted mode of the QToF system. Calibration curves were linear (1/x2 weighting) with r2 values >0.99 and mean accuracies between 98-103%. QC performance was excellent with accuracy ranges between 91-97% and CV’s < 10%.