Lab-in-a-Droplet: Utilizing Acoustic Levitation for Miniaturized Sample Preparation and LC-MS/MS, Exemplified by Measurement of Vitamin D in Serum.

Conventional sample preparation of biological matrices typically requires sample sizes ranging from micro- to milliliters. For difficult-to-obtain samples such as newborn blood or breast milk, collecting sufficient volumes can be challenging, thereby limiting the application of established in vitro assays. Performing the entire sample preparation step within a single levitated droplet (in stillo) of only a few microliters of the sample can address this challenge by reducing the required sample size while shortening sample preparation time and promoting green chemistry principles. In this proof-of-concept study, we demonstrate the feasibility of acoustic levitation as a miniaturization strategy, exemplified by the measurement of 25-hydroxyvitamin D in human serum samples. Preliminary experiments demonstrated that contactless handling of vitamin D samples via acoustic levitation was feasible, including liquid-liquid extractions with excellent recoveries. A [4 + 2]-Diels-Alder derivatization reaction for sensitivity improvement can be conducted in stillo within the levitated droplet. A direct comparison between in stillo and in vitro derivatization suggested a 60-fold increase in reaction rate and a corresponding reduction in analysis time. We successfully extracted and derivatized 25-hydroxyvitamin D from human serum under acoustic levitation conditions and subsequently detected the analyte with LC-MS/MS. Side-by-side evaluation indicated a 15.4% deviation between in stillo and in vitro results, with slightly lower response factors for the in stillo workflow, readily demonstrating the general feasibility of the miniaturization approach. Further optimization of droplet handling, including the injection and ejection processes, will undoubtedly improve the analytical figures of merit, indicating promising performance in future matrix and validation studies.