Analyte Stability Issues during Sample Preparation: Recommendations for Best Practices

Analyte Stability Issues during Sample Preparation: Recommendations for the Best Practices

AOAC Virtual Annual Meeting and Expo, September 2020

Ensuring analyte stability under various environmental conditions is critical for creation of an accurate and precise quantitative method. Known environmental conditions causing instability can be intentionally avoided during method development. It’s very important to comprehensively understand the compound stability before developing a quantitative method.

Recommendations for best practices

Assess the stability of analytes under a variety of environmental conditions. Three main environmental conditions are pH, light exposure and temperature. The assessment should use extreme conditions. Apply appropriate pH, light and temperature conditions during sample preparation to prevent instability issue.

Different pH levels are recommended for best practices. 5% FA is recommended to have a pH of around 1. Water pH is recommended to be between 6 and 7. A solution of 5% NH4OH is recommended to have a pH of 11. In our slides from the presentation we also discuss  the recommended best practices for light exposure and temperatures is discussed.

In our stability experiment design, we tested three different types of solutions- acidic, neutral, and basic, in different conditions. The conditions were Light Protection at room temperature, Elevated Light at room temperature, and Light Protection at 60 degrees Celsius.

EXAmple: Vitamin b12 Stability Study

USP and AOAC have a compendia method only for B12 raw material, while finished goods need a sensitive and fast method. Vitamin B12 supplements are typically derived from two sources: Methylcobalamin(mB12) and Cyanocobalamin (cB12). mB12 is a better source, but its product has stability issues. Vitamin mB12 improves bioavailability, has better retention in tissues, and contains no toxic cyanide. We also ran a comparison in our presentation slides of the stability difference in light, temperature, and pH between cB12 and mB12 (see presentation slides below for graphs). For these samples, the samples were stored in a black bag. They were prepared in the amber container under yellow light and extracted in neutral solvent. The sample is then sonicated in ice-water and stored at 1-8 oC.

Example: CBD Stability Study

In 2018, AOAC published a method to quantify 10 cannabinoids (CBDs) with a range of 0.500-10.0 µg/mL in cannabis. The standard solutions are expensive, but only have 3 days' stability indicated in the AOAC method. It’s very important to understand the stability information for these cannabinoids. In our presentation, we also included chart studies for delta-9, CBD, THCA, and CBDA at acidic, basic, and neutral temperatures (see presentation slides below for graphs). The sample preparation is in an amber container under a yellow light. The optimized solvent is acidic. The sample is sonicated at room temperature for 15 minutes. In our presentation slides, we include a chart illustrating the stability achieved after these boundaries are set for the sample.

Conclusion

With the best practices, the analyte stability could be comprehensively evaluated. The information on analyte stability can provide the optimized storage conditions for the original sample and the extracted sample. The information on analyte stability can provide the optimized sample preparation conditions. The quantitative method is more accurate and precise in comprehensively understanding the analyte stability.