In this study, a novel and high-throughput liquid chromatography-tandem mass spectrometric (LC-MS-MS) assay was developed and validated for simultaneous determination of two glycopeptides (vancomycin, teicoplanin) and two small molecule compounds (meropenem, voriconazole) in human plasma. Only 50 μL of human plasma is used to quantify these four drugs simultaneously at clinical concentration levels. After a relative simple protein precipitation, the supernatant was then diluted with mobile phase acetonitrile: 0.1% formic acid (5:95, v/v) to avoid solvent effect and reduce the matrix effect. Then, the target compounds were separated on an Agilent Zorbax SB-C18 column (4.6 × 50 mm, 2.7 μm). All the target compounds were detected by positive ion mode. The teicoplanin concentration was determined as the sum of six components (A2-1, A2-2, A2-3, A2-4, A2-5 and A3-1). The method was linear in the concentration range 0.3-30 μg/mL for meropenem; 1-100 μg/mL for teicoplanin and vancomycin; 0.3-10 μg/mL for voriconazole. The lower limit of quantitation (LLOQ) of meropenem and voriconazole were 0.30 μg/mL; and the LLOQ of teicoplanin and vancomycin were 1.0 μg/mL. The intra- and inter-day accuracies were <9.67% and 13.0%, and the precisions were <14.5% at all tested concentrations. The entire analysis time for the four drugs was only 5 min for each sample. The currently developed assay was successfully applied for the therapeutic drug monitoring of 85 patients administered with standard drug treatments, demonstrating its high-throughput clinical usage for the therapeutic drug monitoring.

Phosphopeptide enrichment with high selectivity and detection sensitivity is essential for phosphoproteomic studies and remains a long-standing challenge. In this study, new immobilized metal affinity chromatography nanocomposite adsorbents with a phosphonate-functionalized ionic liquid (PFIL) as a surface modifier are successfully prepared via a reaction sequence of amination, quaternization, phosphonate hydrolyzation, and metal immobilization. Taking advantages of integrated features of a flexible and strong tripodal phosphonate chelator, a hydrophilic ionic liquid linker, a large surface area, and the size-exclusion effect, the resulting nanocomposite G@mSiO 2 -PFIL-Ti 4+ exhibits excellent detection sensitivity to enrich phosphorylated peptides from a tryptic β-casein digest (0.15 fmol), and superior enrichment selectivity to capture phosphorylated peptides from a digest mixture of β-casein and bovine serum albumin (a molar ratio of 1:10,000). Strong immobilization of tripodal chelation to metal ions endows the nanocomposite adsorbent with high tolerance to experimental conditions, and thus excellent reusability of the adsorbent has been achieved without remarkable loss of enrichment efficiency for 10 cycles. Due to the excellent size-exclusion effect, high enrichment specificity of G@mSiO 2 -PFIL-Ti 4+ to phosphopeptides has been observed and 23 endogenous phosphopeptides have been captured from human saliva. In addition, 924 phosphopeptides (enrichment specificity, 56.1%) have been identified from the tryptic digest of mouse brain lysate. Particularly, six of 975 phosphorylation sites were Alzheimer's disease-related hyperphosphorylation sites within tau protein. These results demonstrate that G@mSiO 2 -PFIL-Ti 4+ nanocomposite affinity materials show great application potential for a proteomic study of complicated biological samples.

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