Protocol for Formulation Preparation for Drug Quantitation by HPLC-UV/Vis
GUIDELINE
This protocol describes the processing of nano-formulation 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 formulation quantitation. The mobile phase used for separation and the wavelength used for detection are compound-specific.
METHODS
- Pour 10 mL HPLC grade methanol from a stock bottle into a 50 mL conical tube.
- Dilute formulations 100-fold in HPLC grade methanol by adding 10 μL sample to 990 μL fresh HPLC grade methanol in a 1.5 ml microcentrifuge tube.
- Sonicate samples for 8 minutes in a water bath sonicator; place a small ice pack into the water bath to avoid high temperatures from sonication.
- Vortex each sample for 10 seconds at the highest speed to ensure it is well-mixed.
- Centrifuge samples at 20,000 rcf for 10 minutes at 4°C.
- In a clean microcentrifuge tube, dilute sample supernatant either: a. 10-fold (100 μL sample into 900 μL fresh HPLC grade methanol) or b. 100-fold (10 μL sample into 990 μL fresh HPLC grade methanol).
- Vortex 10 seconds at the highest speed to mix thoroughly.
- Pipet 80 μL of sample into glass maximum recovery HPLC vial.
- Carefully cap each vial.
- Flick the vial with a finger to make sure there is no air bubble at the bottom of the vial.
- Place the vial into the HPLC autosampler tray for analysis.
Creative Bioarray Relevant Recommendations
- Creative Bioarray provides physicochemical characterization assays to help customers accurately evaluate compounds' physicochemical properties such as solubility and stability, reducing drug development difficulties.
NOTES
- Choose the appropriate solvents and their ratios to achieve optimal separation of the target compound. Ensure compatibility with the detection method; UV-absorbing solvents should be avoided.
- Dilution is based on the target drug concentration range (middle of the standard range of 0.048-50 μg/mL) and expected drug concentration in formulation based upon drug and polymer amounts added to the formulation mixture before manufacture.
- Determine the injection volume that will provide optimal sensitivity without exceeding column capacity.
