Protocol for Drug Quantitation by UV/Visible Wavelength Detection
GUIDELINE
This protocol describes the processing of cell extract samples for quantitation of drug using reversed-phase high-performance liquid chromatography (HPLC) with ultraviolet/Visible wavelength (UV/Vis) detection. This is a standard method of drug quantitation and has been used in our numerous publications for cell extract quantitation. The mobile phase used for separation and the wavelength used for detection are compound-specific.
METHODS
- If samples have been stored, remove samples from the -80°C freezer and let them warm to room temperature on the bench in a microcentrifuge rack.
- Pour 10-20 mL fresh HPLC-grade methanol from the bottle into a 50 mL conical tube.
- Add 200 μL HPLC grade methanol to each sample tube.
- Sonicate each sample for 2 seconds each using the sonication probe set at 20% amplitude.
- Between each sample: a. Rinse the probe with 70% ethanol using a squirt bottle into a waste beaker; b. Clean by immersion in 100% HPLC-grade methanol and sonication for 2-5 seconds; c. Wipe dry.
- After sonication, vortex each sample for 10 seconds at high speed to ensure each is well mixed.
- Centrifuge samples at 20,000 rcf for 10 minutes at 4°C.
- Prepare standard curve for UPLC.
- Pipet 75 μL of each standard and sample supernatant into a separate well of a 96-well plate; record what standard or sample is in each well.
- Carefully cover the plate with a 96-well plate cover; make sure to avoid any cross-contamination between samples.
- Place plate in UPLC-TUV sample manager for analysis.
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NOTES
- Ensure samples are well-mixed to achieve uniform concentration. If necessary, filter samples to remove particulates that could scatter light. Dilute samples to fall within the calibration range, if required.
- Prepare a series of standard solutions with known concentrations of the drug. Ensure that the calibration curve is linear over the expected concentration range. Perform multiple trials to confirm consistent results across different batches.
