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 D3 (25(OH)D3) 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
D3 (24,25(OH)2D3) can be determined
in real time with a detection limit of 2.54 nM. In addition, the results
revealed that 25(OH)D3 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)D3 was found to increase
significant formation of 24,25(OH)2D3, 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.