Heat-induced plasma protein gelation strategy for efficient extraction of aminoglycosides from human plasma

Renewed use of aminoglycosides (AGs) against multidrug-resistant Gram-negative infections has increased the need for reliable therapeutic drug monitoring, but their high polarity can make conventional organic-solvent protein precipitation challenging. We developed an organic-solvent-free pretreatment based on heat-induced plasma protein gelation and investigated factors governing protein removal and gel-network formation.

Plasma was mixed with 0.07% (v/v) formic acid at a 1:2 ratio and heated at 95 °C for 7 min. Protein removal reached 99.3%, and mean extraction recoveries of the target analytes were 94.3-108.7%, exceeding those obtained with methanol or acetonitrile precipitation. Total protein concentration strongly affected gelation, with apparent 10-90% response-transition ranges (EC10-EC90) of 8.79-11.35 g/L for plasma and 10.52-11.48 g/L for serum. Fibrinogen facilitated interconnected porous gel-network formation more effectively than albumin. The LC-MS/MS method showed R² values of 0.9956-0.9999. Calibration ranges were 0.500-100 mg/L for amikacin, netilmicin, tobramycin, isepamicin, and etimicin, with component-specific ranges spanning 0.069-30.0 mg/L for gentamicin. Corresponding LLOQs were 0.500 and 0.069-0.150 mg/L, respectively. QC accuracy was 85.5-113.6% with precision ≤13.7%, while matrix-effect assessment across six plasma sources yielded accuracy of 85.2-112.8% and precision ≤14.3%.

Heat-induced plasma protein gelation enables efficient protein removal without organic-solvent precipitation. The method combines high analyte recovery with a simple, solvent-free workflow and identifies total protein concentration as a major determinant of gelation, with fibrinogen facilitating network formation. This strategy provides a promising approach for quantitative AG analysis and supports further evaluation for therapeutic drug-monitoring applications.