Liquid chromatography combined with high-resolution mass spectrometry has become an important diagnostic tool for bioanalytical analysis. However, many protocols that come packaged with a new LC-MS/MS fail to meet expectations due to method columns having improper chromatographic selectivity or dimensions that don’t match the diagnostic needs. UHPLC methods, the gold standards for fast and high efficiency separations, are often not sufficiently robust to meet the demands of routine analyses in bio-matrices. This is due to the high tendency of those columns to clog, which shortens their lifetimes and causes frequent instrument downtime. This poster demonstrate the use of 2 μm high purity silica UHPLC columns with MS/MS detection for routine clinical analysis in urine, saliva and plasma samples. Calibration curves showed that the linear dynamic ranges of the methods were appropriate to measure these analytes in relevant clinical concentration intervals, and the LOQ levels were in the low ng/mL range. Buprenorphine and its main metabolite norbuprenorphine showed chromatographic retention times of 1.39 min and 1.20 min. Tramadol and its main metabolite N-desmethyl-tramadol eluted at 2.07 min and 2.09 min, respectively. Cortisol and its deuterated internal standard (D4-cortisol) eluted at 3.29 min and 3.27 min, respectively. Vitamin D’s (25-OH D2 and D3) and their deuterated internal standards (D3-labeled) eluted at 3.22 min and 3.24 min (D2).Vitamin D3 eluted at 2.92 and 2.90 min, respectively.