HA-C@silica sorbent for simultaneous extraction and clean-up of steroids in human plasma followed by HPLC-MS/MS multiclass determination.

Aim and novelty of this work are the development of a simple and straightforward analytical procedure for multiclass determination of steroid hormones in human plasma. The method entails a single pre-treatment step based on solid-phase extraction using a recently proposed sorbent phase (HA-C@silica). This is easily prepared with good reproducibility via pyrolysis of humic acids onto silica, and not yet tested in biological fluids. It proved to be advantageous as it showed poor affinity for the protein matrix constituents while quantitatively extracting and pre-concentrating the target analytes. Indeed, as demonstrated in bovine serum albumin solution, up to ca. 90% protein is not retained by the sorbent, similarly to the behaviour of restricted access carbon nanotubes, tested for comparison. The high albumin exclusion allowed a satisfactory clean-up avoiding protein precipitation and centrifugation before extraction. The extraction procedure, optimized by a chemometric approach (2 experimental design) in BSA solution, provided quantitative recovery (76-119%, n = 3) for all steroids working with 1:8-diluted plasma (2 mL) and 100 mg HA-C@silica. Before analytes elution by 1 mL methanol-acetonitrile (1:1, v/v), selective washings (2% v/v formic acid and 30% v/v methanol) were applied to remove the small fraction of retained proteins, thus obtaining very clean SPE extracts to be analyzed by HPLC-ESI-MS/MS. This allowed identification/quantification (MRM mode) at few ng mL by a single chromatographic run. The procedure was verified in blank-certified foetal bovine serum (spikes 10-100 ng mL), obtaining good recovery and suitable inter-day precision (RSDs < 15%, n = 3). The analytical method, applied to real plasma samples analysis, is appealing in terms of sample throughput, extraction efficiency and clean-up.