5. AOACSPDSMethods-2018AwardsV3

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( a ) For dry extracts in capsules, contents of at least 10 capsules must be combined and mixed with a spatula before weighing to ensure homogeneity. ( b ) Using an analytical balance, weigh out approximately 0.125 ± 0.005 g solid material into a 50 mL conical tube. Record exact weight. ( c ) Using a volumetric pipet, accurately add 25 mL extraction solvent to the tube. ( d ) Shake the tube on a wrist action shaker for 30 min at room temperature. ( e ) Centrifuge the tube at 5000 rpm for 5 min. ( f ) Filter approximately 1 mL sample through a 0.45 μm Teflon filter into a glass HPLC vial. Cap and analyze the sample as per HPLC operating conditions. ( 3 ) Tinctures. — Note: Method is not suitable for glycerite tinctures. ( a ) Invert tincture vessel several times to thoroughly mix sample. ( b ) Using a 1 mL volumetric pipet, dispense 1 mL tincture into a 50 mL conical tube. ( c ) Using a volumetric pipet, accurately add 24 mL extraction solvent to the tube. ( d ) Shake the tube on a wrist action shaker for 30 min at room temperature. ( e ) Centrifuge the tube at 5000 rpm for 5 min. ( f ) Filter approximately 1 mL sample through a 0.45 μm Teflon filter into a glass HPLC vial. ( g ) Cap and analyze the sample as per HPLC operating conditions. E. Determination (a)  System suitability test .—Equilibrate the HPLC system with mobile phases for at least 10 min until stable baseline is obtained. Make an injection of a sample blank and analyze as per HPLC operating conditions. Ensure that no artifact peaks appear in the resulting chromatogram. Make five replicate 5 μL injections of Calibration Standard 4 and analyze as per HPLC operating conditions. RSD of peak areas for each of the five phenolic compounds must be ≤5.0%. (b) Elution order and retention time determination .—For each phenolic compound, prepare individual 100 μg/mL standard solutions by adding 100 μL of the appropriate 1000 μg/mL stock standard solution to 900 μL extraction solvent and mix well. Transfer each solution to a glass HPLC vial and analyze as per HPLC operating conditions. Note retention times for each of the phenolic compounds. (c) Calibration .—Make single 5 μL injections of each mixed standard solution. Use simple linear regression to calculate slope, y -intercept, and r 2 for each phenolic standard. Visually inspect

a freezer-safe vessel. Store at –20°C, protected from light. Solution is stable for at least 3 weeks in this storage condition. ( 4 ) Echinacoside 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 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 a freezer-safe vessel. Store at –20°C, protected from light. Solution is stable for at least 3 weeks in this storage condition. ( 5 ) Cichoric acid standard solution (1000 μg/mL) .—Using adjusted purity, calculate the amount of material required in 10 mL to make 1000 ppm standard. 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. 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. (b)  Calibration curve standard solutions.— Seven mixed- calibration standard solutions from the individual stock solutions described above. To prepare calibration standard 1, transfer 1.5 mL caftaric acid standard solution, 0.3 mL chlorogenic acid standard solution, 0.3 mL cynarin standard solution, 3.0 mL echinacoside standard solution, and 2.0 mL cichoric acid standard solution into a 10 mL volumetric flask. Make up the flask to volume using extraction solvent and mix well. Prepare the other six calibration standards by mixing appropriate amounts of each stock solution and performing the appropriate serial dilutions with extraction solvent. Concentration levels of each standard in each of the calibration standards are described in Table 2018.08B . (c) Preparation of test solutions.— ( 1 ) Dry powdered raw materials and powdered extracts.— ( a ) Grind sample material to 60 mesh and homogenize. ( b ) Using an analytical balance, weigh out approximately 0.125 ± 0.005 g solid material into a 50 mL centrifuge tube. Record exact weight. ( c ) Using a volumetric pipet, accurately add 25 mL extraction solvent to the tube. ( d ) Shake the tube on a wrist action shaker for 30 min at room temperature. ( e ) Centrifuge the tube at 5000 rpm for 5 min. ( f ) Filter approximately 1 mL sample through a 0.45 μm Teflon membrane filter into a glass HPLC vial. Cap and analyze the sample as per HPLC operating conditions. ( 2 ) Capsules. —

Table 2018.08B. Approximate concentrations of phenolic compounds at each calibration standard Approximate concn, µg/mL Calibration standard Phenolic compound 1 2 3 4 5 6

7 1

Caftaric acid

150

100

50 10 10

25

10

5 1 1

Chlorogenic acid

30 30

20 20

5 5

2 2

0.2 0.2

Cynarin

Echinacoside Cichoric acid

300 200

200 100

100

50 25

20 10

10

2 1

50

5

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