AOAC Official Method 2018.08 Table  2018.08A.
  HPLC gradient program for analysis of
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.
(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.
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
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 (H3PO4).—≥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,
cynarin, echinacoside, and cichoric acid standards.
D. Preparation of Standard and Test Solutions
(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 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.
(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
© 2018 AOAC INTERNATIONAL
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
Phenolic compound 1 2 3 4
Caftaric acid 150 100 50
Chlorogenic acid 30 20 10
Cynarin 30 20 10
Echinacoside 300 200 100
Cichoric acid 200 100 50
(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 r2 for each phenolic standard. Visually inspect
Calibration standard
5 6 7
25 10 5 1
5 2 1 0.2
5 2 1 0.2
50 20 10 2
25 10 5 1
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each calibration curve to determine whether the points fall along
a straight line. The calculated r2 should be ≥0.995 for each analyte.
(d) Test sample analysis.—Make single 5 μL injections of each
test solution.
F. Calculations
The content of each phenolic compound in solid samples (mg/g)
is determined using the following formula:
where x = content of phenolic compound in the test sample in
milligrams per gram, P0 = observed peak area of the phenolic
compound, b0 = y-intercept of the calibration curve for phenolic
standard, b1 = slope of the calibration curve for phenolic standard,
V = final volume of test sample preparation (mL), W = initial
sample weight of test sample (mg), and D = dilution factor of
sample preparation.
Percent (w/w) can be calculated from milligrams per gram (mg)
as follows:
The content of each phenolic compound in liquid samples
(μg/mL) is determined using the following formula:
where x = concentration of phenolic compound in test sample
(μg/mL), P0 = observed peak area of phenolic compound, b0 =
y-intercept of the calibration curve for phenolic compound, b1 =
slope of the calibration curve for phenolic compound, V1 = final
volume of the test sample preparation (mL), V0 = initial sample
volume of the test sample (mL), and D = dilution factor of sample
preparation.
References: (1) Anal. Bioanl. Chem. 397, 1883(2010)
J. AOAC Int. (future issue) (First Action)
AOAC SMPR 2017.015
J. AOAC Int. 101, 315(2018)
DOI: http://dx.doi.org/10.5740/jaoacint.
SMPR2017.015
Posted: October 2018
© 2018 AOAC INTERNATIONAL