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

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Analyte Stability Issues during Sample Preparation: Recommendations for the Best Practices Aihua Liu, Ph.D. Director, R&D Dyad Labs 1 134th Annual Meeting & Exposition | September 8 – 24, 2020 Outline Background Recommendations for the Best Practices Examples: Case I and Case II Conclusion 2 134th Annual Meeting & Exposition | September 8 – 24, 2020 Background 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. 3 134th Annual Meeting & Exposition | September 8 – 24, 2020 Recommendations for the 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. 4 134th Annual Meeting & Exposition | September 8 – 24, 2020 Recommendations for the Best Practices 5% NH4OH Different pH Levels Water 5% FA 5 134th Annual Meeting & Exposition | September 8 – 24, 2020 Recommendations for the Best Practices Different Temperatures o Ice-Water RT 60 C 6 134th Annual Meeting & Exposition | September 8 – 24, 2020 Recommendations for the Best Practices Different Light Exposures 300 Watts Light Protection Light Exposure 7 134th Annual Meeting & Exposition | September 8 – 24, 2020 Recommendations for the Best Practices Stability Experiment Design Sample Replicates Conditions Solution-Acidic 3 Light Protection, RT (Control) Solution-Acidic 3 Elevated Light, RT Solution-Acidic 3 Light Protection, 60 C Solution-Neutral 3 Light Protection, RT (Control) Solution-Neutral 3 Elevated Light, RT Solution-Neutral 3 Light Protection, 60 C Solution-Basic 3 Light Protection, RT (Control) Solution-Basic 3 Elevated Light, RT Solution-Basic 3 Light Protection, 60 C 8 134th Annual Meeting & Exposition | September 8 – 24, 2020 Examples: Case I---Vitamin B12 Stability Study USP and AOAC have compendia method only for B12 raw material, while finished goods needs 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. mB12 improves bioavailability mB12 has better retention in tissues mB12 contains no toxic cyanide mB12 cB12 9 134th Annual Meeting & Exposition | September 8 – 24, 2020 Examples: Case I---Vitamin B12 Stability Study Stability Study of mB12 2000000 1800000 1600000 1400000 1200000 1000000 800000 600000 400000 200000 0 1 2 3 4 5 6 7 8 9 Light sensitive Temperature sensitive pH sensitive 10 134th Annual Meeting & Exposition | September 8 – 24, 2020 Examples: Case I---Vitamin B12 Stability Study Stability Study of cB12 400000 350000 300000 250000 200000 150000 100000 50000 0 1 2 3 4 5 6 7 8 9 Light sensitive Temperature sensitive pH sensitive 11 134th Annual Meeting & Exposition | September 8 – 24, 2020 Examples: Case I---Vitamin B12 Stability Study 1. Sample is stored in black bag. 2. Sample is prepared in amber container under yellow light. 3. Sample is extracted in neutral solvent. 4. Sample is sonicated in ice-water and stored at 1-8 oC. 12 134th Annual Meeting & Exposition | September 8 – 24, 2020 Examples: Case II---CBD Stability Study In 2018, AOAC has published a method to quantify 10 cannabinoids (CBDs) with the range of 0.500-10.0 µg/mL in cannabis. The standard solutions are expensive, but only have 3 days stability indicated in AOAC method, . It’s very important to understand the stability information for these cannabinoids. Analyte and IS Abbreviation Cannabidivarinic acid CBDVA Cannabidivarin CBDV Cannabidiolic acid CBDA CBDVA CBDV CBDA THCA Tetrahydrocannabinolic Acid THCA Cannabigerolic acid CBGA Cannabigerol CBG Cannabidiol CBD Terahydrocannabivarin THCV CBGA CBG CBD THCV Tetrahydrocannabivarinic acid THCVA Cannabinol CBN Cannabinolic Acid CBNA Delta-9-Tetrahydrocannabinol delta9-THC Delta-8-Tetrahydrocannabinol delta8-THC THCVA CBN CBNA delta9-THC Cannabicyclol CBL Cannabichromene CBC Cannabichromenic Acid CBCA Cannabidiol-D3 (IS) CBD-d3 Cannabinol-D3 (IS) CBN-d3 delta8-THC CBL CBCA Delta-9-Tetrahydrocannabinol-D3 (IS) delta9-THC-d3 CBC 13 134th Annual Meeting & Exposition | September 8 – 24, 2020 Examples: Case II---CBD Stability Study delta-9 THC Stability Study CBD Stability Study 140000 300000 120000 250000 100000 200000 80000 150000 60000 40000 100000 20000 50000 0 0 THCA Stability Study CBDA Stability Study 70000 3.50E+05 60000 3.00E+05 50000 2.50E+05 40000 2.00E+05 30000 1.50E+05 20000 1.00E+05 10000 5.00E+04 0 0.00E+00 14 134th Annual Meeting & Exposition | September 8 – 24, 2020 Examples: Case II---CBD Stability Study Sample preparation is in amber container under yellow light. The optimized solvent is acidic. Sample is sonicated at room temperature for 15 mins. Concentration Storage Temperature Storage Container Stability (days) Standard (50.0 ug/mL) -20 oC Amber/Glass 149 Standard (20-2000 ng/mL) 1 oC-8 oC Amber/Glass 4 Extract 1 (20-2000 ng/mL) 1 oC-8 oC Amber/Glass 9 Extract 2 (20-2000 ng/mL) 1 oC-8 oC Amber/Glass 4 15 134th Annual Meeting & Exposition | September 8 – 24, 2020 Conclusions With the best practices the analyte stability could be comprehensively evaluated. The information of analyte stability can provide the optimized storage conditions for original sample and extracted sample. The information of analyte stability can provide the optimized sample preparation conditions. The quantitative method is more accurate and precise with comprehensively understanding the analyte stability 16 134th Annual Meeting & Exposition | September 8 – 24, 2020 Questions? Aihua Liu Ph.D, [email protected], 801-554-2835 17 134th Annual Meeting & Exposition | September 8 – 24, 2020.
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