Drug Stability Assay Protocol in Frozen Plasma

This protocol is for an experiment to determine the stability of gemcitabine and its primary metabolite dFdU during storage in frozen plasma. Following the preparation of spiked plasma and the storage of those samples, as described in this protocol, the samples must be prepared and analyzed using a fully validated bioanalytical method. The choice of gemcitabine and dFdU concentrations and the sample volumes were based on gemcitabine pharmacokinetics and our assay method's inherent sensitivity and linear range. The choice of method for quantitation of these or any other analytes is up to the user. Still, full validation of the chosen method is required to ensure that any loss of analyte or lack of accuracy or precision reflects analyte stability in frozen plasma.

• Label 30 microcentrifuge tubes for plasma samples.
• Add 250 μL aliquots of plasma to each of the 30 microcentrifuge tubes.

Low analyte concentration sample set:

• Using a repeating pipettor set to deliver a 10 μL volume, spike each of the 15 microcentrifuge tubes labeled for the low analyte concentration with the 12.5 μg/mL analyte solution and mix thoroughly. This will result in a 0.50 μg/mL analyte concentration in the sample.
• Place the 3 control tubes for low concentration on ice until they are processed for analysis.
• Transfer the other low-concentration samples to the freezer.

High analyte concentration sample set:

• Using a repeating pipettor set to deliver a 10 μL volume, spike each of the 15 microcentrifuge tubes labeled for the high analyte concentration with the 250 μg/mL analyte solution and mix thoroughly. This will result in a 10 μg/mL analyte concentration in the sample.
• Place the 3 control tubes for high concentration on ice until they are processed for analysis.
• Transfer the other high-concentration samples to the freezer.

Sample analysis:

• Process and analyze the 0-month low and high concentration samples to determine gemcitabine and dFdU concentrations.
• Repeat step 9 with triplicate samples of low and high-concentration spiked plasma after 1, 3, 6, and 12 months of storage in the freezer.
• Determine the accuracy and precision of analyte measurements for each time point. Graphically and statistically examine the measured concentrations, using the samples immediately analyzed (i.e. without storage) to determine if degradation of analytes occurred in the frozen samples.

Creative Bioarray Relevant Recommendations

• At Creative Bioarray, our plasma stability assay can measure the stability of compounds in plasma, helping customers find unstable compound structures and screen prodrugs. We provide a comprehensive service for the metabolites in biological matrices in support of drug development research. This service can be provided as part of a full in vitro metabolism package or as a single assay. We also provide standard, cost-effective in vitro metabolism services, including drug metabolic stability services and drug-drug interaction services to support your drug development process.

• Suggested tube numbering: First number = analyte concentration (1 for 0.50 μg/mL, 2 for 10 μg /mL concentration in whole blood); second number = time in months sample was frozen before processing and analysis; third number = replicate sample of triplicates (1, 2, or 3).
• The storage times chosen in this basic protocol for the assessment of analyte stability in frozen plasma samples will determine that stability for a maximum of twelve months. The actual time points to be validated should be based on the anticipated maximum length of time for samples from the actual study to be stored before analysis.

Plasma Stability Assay

In Vitro Metabolism

For research use only. Not for any other purpose.