A Fluidic Isolation-Assisted Homogeneous-Flow-Pressure Chip-Solid Phase Extraction-Mass Spectrometry System for Online Dynamic Monitoring of 25-Hydroxyvitamin D 3 Biotransformation in Cells.

It is well known that cell can response to various chemical and mechanical stimuli. Therefore, flow pressure variation induced by sample loading and elution should be small enough to ignore the physical impact on cells when we use a Chip-SPE-MS system for cells. However, most existent Chip-SPE-MS systems ignored the pressure alternation because it is extremely difficult to develop a homogeneous-flow-pressure hyphenated module. Herein, we developed an interesting fluidic isolation-assisted homogeneous-flow-pressure Chip-SPE-MS system and demonstrated that it is adequate for online high-throughput determination and quantification of the 25-hydroxyvitamin D ₃ (25(OH)D ₃ ) biotransformation in different cells. Briefly, the homogeneous ambient flow pressure is achieved by fluidic isolation between the cell culture channel and the SPE column, and an automatic sampling probe could accomplish the sample loading and dispensing to fulfill online pretreatment of the sample. Through this new system, the expression levels of 24,25-dihydroxyvitamin D ₃ (24,25(OH) ₂ D ₃ ) can be determined in real time with a detection limit of 2.54 nM. In addition, the results revealed that 25(OH)D ₃ metabolic activity differed significantly between normal L-02 cells and cancerous HepG2 cells. Treatment of L-02 cells with a high dose of 25(OH)D ₃ was found to increase significant formation of 24,25(OH) ₂ D ₃ , but this change was not apparent in HepG2 cells. The presented system promises to be a versatile tool for online accurate molecule biotransformation investigation and drug screening processes.