Imark microplate absorbance reader 168 1130

The Sunahara method was followed to extract total rifamycins in urine.^{19 (link)} Clinical information and serum results were not available to technicians performing the urine spectrophotometric assay. For a working volume of 500 μL of urine, 250 μL of phosphate buffer (pH 7, Sigma-Aldrich) and 500 μL of isoamyl alcohol (Sigma-Aldrich) were added. Samples were vortexed for 20s and centrifuged at 13000 rpm for 5 min at room temperature. Next, 100μL of the aqueous phase was removed and added to a 96-well plate, and the optical density was measured at 475 nm using a spectrophotometer (Bio-Rad, iMark Microplate Absorbance Reader #1681130). A seven-point calibration curve was constructed with 1000 and 31.2μg/mL as upper and lower limits of calibration. We have previously documented interday and intraday agreement, as well as the stability of the assay in various environmental conditions including prolonged light exposure and differing urine protein and pH measurements.^{15 (link)} The amount of rifampin excreted in urine over the following intervals was calculated: 0–4, 4–8, 0–8, 8–24 and 0–24 hours.