5. AOACSPDSMethods-2018AwardsV3

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AOAC Official Method 2018.08 Phenolic Compounds in Dietary Supplements and Dietary Ingredients Containing Echinacea HPLC-UV First Action 2018 Caution : Refer to Material Safety Data Sheets (MSDS) to ensure that safety guidelines are applied for all reagents used. Use appropriate personal protective equipment. A. Principle The method is suitable for the determination of major phenolic compounds in raw materials and select finished products containing E. purpurea , E. angustifolia , and E. pallida parts, including hard- shell capsules and tinctures. The phenolic compounds determined using the method are caftaric acid, chlorogenic acid, cynarin, echinacoside, and cichoric acid. Compounds are extracted from the matrixes with a mixture of methanol–water (60 + 40, v/v), using shaking or rotation, and then centrifuged, filtered, and analyzed by reversed-phase HPLC with a C18 column and UV detection at 330 nm. Quantitation is performed using a seven-point external calibration curve constructed from the analysis of mixed phenolic standard solutions. B. Apparatus Note : Equivalent apparatus may be substituted. All volumetric pipets and flasks are class A grade and must be calibrated before use. (a) HPLC system .—Equipped with a binary pump, refrigerated autosampler, and UV-diode-array detector. The gradient program is listed in Table 2018.08A . (b) HPLC operating conditions .— ( 1 ) Autosampler temperature. —5°C. ( 2 ) Column temperature. —25°C. ( 3 ) Detection wavelength. —330 nm. ( 4 ) Injection volume. —5 μL. ( 5 ) Mobile phase flow rate. —1.5 mL/min. ( 6 ) Mobile phase A (MPA). —0.1% o -Phosphoric acid in water (v/v). ( 7 ) Mobile phase B (MPB). —Acetonitrile. ( 8 ) Run time. —15 min. ( 9 ) Post time. —3 min. (c) HPLC column .—Cosmosil (Nacalai USA Inc., San Diego, CA, USA) C18, 5C18-AR-II, 4.5 × 150 mm or equivalent. (d) Analytical balance .—Readability, ±0.1 mg. (e) Centrifuge .—Capable of centrifuging 50 mL conical tubes at 5000 rpm at room temperature. (f) Vortex mixer .

C. Chemicals and Reagents Note : Chemicals from other suppliers meeting specifications may also be used. Acetonitrile and methanol are flammable organic solvents. Keep away from any sources of heat and open flames. Use a fume hood during solvent preparation. (a) Solvents .—Acetonitrile, methanol, and water; all HPLC grade. (b) o-Phosphoric acid (H 3 PO 4 ) .—≥85% ACS reagent grade or equivalent. (c) Extraction solvent .—Methanol–water (60 + 40, v/v). For every 1000 mL extraction solvent, mix 600 mL methanol with 400 mL water. (d) MPA .—Water containing 0.1% phosphoric acid. For every 1000 mL MPA, add 1 mL o -phosphoric acid into a 1 L volumetric flask containing ~900 mL water. Dilute to volume with water and mix well. (e) MPB .—100%Acetonitrile. For every 1000 mL MPB, aliquot 1000 mL acetonitrile. (f) Reference standards .—Caftaric acid, chlorogenic acid, Table 2018.08A. HPLC gradient program for analysis of phenolic compounds Time, min MPA, % MPB, % 0.0 90 10 13.0 78 22 14.0 60 40 14.5 60 40 18.0 90 10 ( a )  Preparation of calibration standard stock solutions .— Note : If not used immediately, all solutions should be stored at –20°C. When stored at 2–8°C, solutions are stable for at least 12 h. Stability of calibration standards has been previously determined (1). ( 1 ) Caftaric acid standard solution (1000 μg/mL) .—Using adjusted purity, calculate the amount of material required in 10 mL solution to make a 1000 μg/mL standard solution. Using an analytical balance, record exact weight and transfer amount to a clean 10 mL volumetric flask. Add approximately 5 mL extraction solvent. Swirl to mix and make up with the extraction solvent. Invert at least 10 times and transfer to a freezer-safe vessel. Store at –20°C, protected from light. Solution is stable for at least 3 weeks in this storage condition. ( 2 ) Chlorogenic acid standard solutions (1000 μg/mL) .—Using adjusted purity, calculate the amount of material required in 10mL to make a 1000 μg/mL standard solution. Using an analytical balance, record exact weight and transfer to a clean 10 mL volumetric flask. Add approximately 5 mL extraction solvent. Swirl to mix and make up with the extraction solvent. ( 3 ) Cynarin standard solution (1000 μg/mL) .—Using adjusted purity, calculate the amount of material required in 10 mL to make a 1000 μg/mL standard solution. Using an analytical balance, record the exact weight and transfer to a clean 10 mL volumetric flask. Add approximately 5 mL extraction solvent. Swirl to mix and make up with the extraction solvent. Invert at least 10 times and transfer to cynarin, echinacoside, and cichoric acid standards. D. Preparation of Standard and Test Solutions

(g) Conical tubes .—Polypropylene, 50 mL. (h) Volumetric flasks .—10 and 1000 mL. (i) Graduated cylinders .—10, 500, and 1000 mL. (j) Syringes .—3 mL.

(k) Syringe filters .—PTFE, 0.45 μm pore size. (l) Automatic pipets .—10, 100, 200, and 1000 μL. (m) LC vials .—2 mL, clear, amber, with Teflon-coated caps. (n) Wrist action shaker .—Variable speed. (o) Ultracentrifugal mill .—≤60 mesh. (p) Reagent bottles .—Clear or amber glass, 1000 and 2000 mL.

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