Lab-in-syringe automated sample preparation including deproteination, analyte extraction, and solvent evaporation coupled online to HPLC for the determination of water-soluble vitamins in plant-based milk alternatives

Solvent-assisted matrix precipitation, followed by centrifugation, supernatant subtraction, solvent evaporation, and reconstitution, is a commonly used yet laborious procedure for preparing complex sample matrices. Its automation has not yet been tested using flow approaches, despite the potential to save time and consumables and yield higher procedural reproducibility. To tackle this challenging issue, we have developed a Lab-In-Syringe-automated methodology coupled online to HPLC for the determination of water-soluble vitamins B1, B2, B3, B6, B9, and B12 in plant-based milk alternatives. Salt-assisted homogeneous liquid-liquid extraction, automated using an automatic syringe pump, was used to simultaneously denature proteins and separate the precipitable sample matrix from oat, soy, hazelnut, almond, and coconut milk substitutes. The organic phase was passed into a secondary, coupled automatic syringe pump that used preheated air for solvent evaporation. To the best of our knowledge, this is the first report on combining fully automated matrix precipitation and solvent evaporation in flow techniques. This way, we achieved a significant improvement in analyte concentration compared to earlier reported in-syringe deproteination, where significant extract dilution had been required to achieve compatibility with HPLC. Highlighted features include a sub-milliliter sample volume, a total processing time of only 9 min, including intermediate system cleaning, and online coupled HPLC-UV analysis of the extract in parallel with sample processing, yielding a sample throughput of 5/h. Adequate analytical performance for routine analysis was achieved with quantitative protein elimination, LOQs of 0.5-2.7 µg/L, and RSDs of 1.1-2.6 %, < 5 % omitting an internal standard. Analysis of milk samples, both genuine and spiked, yielded an average recovery of 92 ± 11 %.