A microfluidic chip coupled to a liquid chromatography-mass spectrometry (LC-MS) system is a powerful tool to determine components of interest in tracing biological samples. However, producing a reusable and robust chip having chromatographic separation for LC-MS systems remains analytically challenging due to the high back pressure generally occurring when microfluidic channels are packed particles. In this study, a one-piece and high-pressure-resistant polymeric microfluidic chip was fabricated by commercial stereolithographic 3D printing with an inner diameter of 1 mm, packed with 5 µm diameter fluorophenyl propyl particles, and coupled to a regular LC-MS. The maximum pressure occurred up to 134 bar with a variation of 1 bar when using 70% methanol at a flow rate of 0.035 mL min-1. Ten drugs and two drug metabolites were analyzed in 15 min under continuous pressure change by LC-MS. The limit of detection was between 2 and 20 ng mL-1 in human urine. The coefficient of determination (R2) was > 0.991. The shift of retention time of each peak was maintained at < 2%. After a chip was eluted with a high organic solvent composition (90% methanol/water and 90% acetonitrile/water mixture) and a high back pressure for continuously 45 h, the cross-sectional areas were investigated and results showed that the microfluidic chip remained intact. The overall experiment results showed that this platform had significant reproducibility, robustness and stability without contamination, demonstrating that this 3D printing microfluidic chip coupled with LC-MS is an efficient and precise tool for bioanalysis.
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