The first exploration of pseudo-protein-precipitation: High-throughput pretreatment for serum vitamin A/D/E/K...

The accurate quantification of fat-soluble vitamins (A, D, E, K) in serum is essential for clinical diagnostics. However, traditional methods involving protein precipitation (PP) workflows, which require vortex mixing and centrifugation, pose significant challenges for automation. Current automated platforms are often complex, costly, and limited in throughput. This study presents an innovative strategy called "Pseudo-Protein-Precipitation combined with Cold-Induced Phase Separation (PPP + CIPS)" to streamline pretreatment and improve high-throughput compatibility.

The PPP + CIPS approach utilizes alkalized acetonitrile to form a semi-homogeneous serum suspension, facilitating in-situ vitamin extraction through CIPS. Eliminating vortexing and centrifugation for the very first time, this new method can be efficiently executed using 96-well plates and multi-channel pipettes. The whole protocol significantly reduced pretreatment time to approximately one-third of that required by PP. Validation results indicated excellent linearity, accuracy (86.5-113.2 %), precision (CV <10.5 %), and minimal matrix effects (88.8-112.7 %). Clinical comparisons (n = 384) demonstrated strong agreement with certified methods, with 94 % of retinol, 93 % of 25OHD3, 91 % of α-tocopherol, and 89 % of K1 falling within ±20 % limits.

The PPP + CIPS strategy represents a transformative advancement for high-throughput clinical testing. Its compatibility with 96-well formats and static workflows positions it as a foundational element for next-generation automated LC-MS/MS platforms, effectively addressing current bottlenecks in clinical mass spectrometry.