Protocol for Preparing HPLC Standards for Drug Quantification

High-performance liquid chromatography (HPLC) is a cornerstone technique in pharmaceutical analysis for quantifying drugs and their metabolites. The use of HPLC standards is crucial for achieving accurate and precise results. This protocol describes the preparation of drug stock solutions to prepare standards for the quantitation of drugs using reversed-phase HPLC or ultraperformance liquid chromatography (UPLC) with ultraviolet/Visible wavelength (UV/Vis) detection.

For 1.0 mg/mL ultimate stock

• Weigh out 2-7 mg drug powder with balance on the 4^th floor using an antistatic micro spatula into an 8 mL amber glass vial.
• Carefully add enough HPLC-grade methanol to the vial to create a 1.0 mg/mL ultimate stock.
• Cap the vial and vortex for 10 seconds at high speed to mix and ensure all drug powder is dissolved in the methanol.
• Place in the sonicating bath for 1 minute to help dissolve any remaining drug then vortex 10 seconds at high speed to mix.
• Label vial with: drug name; 1 mg/ml in methanol; date; your initials.

For 200 ug/mL working stock

• Carefully add 800 μL HPLC grade Methanol to a 4 mL glass amber vial.
• Carefully add 200 μL of 1.0 mg/mL drug stock to the methanol in the 4 mL vial.
• Pipette the solution up and down several times to rinse the tip after adding to methanol.
• Cap and vortex for 10 seconds at high speed to mix.
• Label vial with: drug name; 200 μg/ml in methanol; date; your initials.
• Tightly wrap the caps of both vials with parafilm to ensure they are tightly sealed; store each at -80°C.

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• Saturate the pipette tip thoroughly by pipetting up and down twice before adding to the vial to avoid dripping.
• Ensure all glassware and instruments are clean to avoid contamination.
• Maintain consistent temperature and solvent composition.
• Document all procedures and calculations meticulously for reproducibility.

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