Accessed from 47.180.160.
190 by akpmtn3zv on Mon May 14 18:42:39 EDT 2018
4446 Asian Ginseng / Dietary Supplements
Mode: LC
Detector: UV 203 nm
Column
Guard: 4.6-mm × 2.0-cm; packing L1
Analytical: 4.6-mm × 15-cm; 3-µm packing L1
Column temperature: 25°
Flow rate: 1.5 mL/min
Injection size: 20 µL
System suitability
Sample: Standard solution
Suitability requirements
Chromatogram similarity: The Standard solution
chromatogram is similar to the Reference Chromato-
gram provided with the lot of USP Powdered Asian
Ginseng Extract RS being used.
Relative standard deviation: NMT 2.0%, determined
for the sum of the peak areas for the six major ginse-
nosides, in repeated injections
Analysis
Samples: Standard solution and Sample solution
Record the chromatograms, identify the peaks for the
ginsenosides by comparison with the Reference Chro-
matogram provided with the lot of USP Powdered
Asian Ginseng Extract RS being used, and measure
the peak areas for the six major ginsenosides.
Calculate the quantity, in mg, of each relevant ginse-
noside (Rg1, Re, Rb1, Rc, Rb2, and Rd) in the portion
of Tablets taken:
Result = 0.05 × (rU/rS) × CS × P
rU = peak areas for each relevant ginsenoside from
the Sample solution
rS = peak areas for each relevant ginsenoside from
the Standard solution
CS = concentration of USP Powdered Asian Ginseng
Extract RS in the Standard solution (mg/mL)
P = labeled amount, in percentage, of each
relevant ginsenoside in the USP Powdered
Asian Ginseng Extract RS lot being used
Calculate the content of total ginsenosides, T, in mg,
by adding the amounts of individual ginsenoside.
Calculate the percentage of Powdered Extract with
respect to the label claim:
Result = T × (AWT/W) × (100/LE) × (100/L)
T = content of total ginsenosides in the portion of
Tablets taken (mg)
AWT = average weight of Tablets (mg/Tablet)
W = weight of the portion of Tablets taken (mg)
LE = content of total ginsenosides in 100 mg of the
DS Monographs
Extract used to prepare the Tablets (mg)
L = amount of Extract per Tablet according to
label claim (mg/Tablet)
Acceptance criteria: 90.0%–110.0% of Powdered Ex-
tract, calculated as the sum of ginsenosides Rg1, Re,
Rb1, Rc, Rb2, and Rd
PERFORMANCE TESTS
• DISINTEGRATION AND DISSOLUTION OF DIETARY SUPPLEMENTS
〈2040〉: Meet the requirements for Disintegration
• WEIGHT VARIATION OF DIETARY SUPPLEMENTS 〈2091〉: Meet
the requirements
CONTAMINANTS
• MICROBIAL ENUMERATION TESTS 〈2021〉: The total aerobic
microbial count does not exceed 104 cfu/g, and the total
. lower RF is the least intense.
combined molds and yeasts count does not exceed 1000
cfu/g. Tablets meet the requirements of the tests for ab-
sence of Salmonella species and Escherichia coli.
ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in tight containers,
protected from light.
Official from May 1, 2018
Copyright (c) 2018 The United States Pharmacopeial Convention. All rights reserved.
USP 41
• LABELING: The label states the Latin binomial and, follow-
ing the official name, the article from which the Tablets
were prepared. The label also indicates the amount of
Powdered Extract, in mg/Tablet, and the content, in mg,
of ginsenosides per 100 mg of Powdered Extract.
• USP REFERENCE STANDARDS 〈11〉
USP Powdered Asian Ginseng Extract RS
Aspartic Acid—see Aspartic Acid General
Monographs
.
Astaxanthin Esters
Astaxanthin esters;
Astaxanthin fatty acid esters;
Fatty acid esters of (3S,3’S)-3,3′-dihydroxy-β,β-carotene-4,4′-
dione.
DEFINITION
Astaxanthin Esters is obtained by extraction with either
supercritical carbon dioxide or acetone from cultures of
Haematococcus pluvialis. It consists mainly of 3S,3′S stereo-
isomers of astaxanthin in the monoester, diester, and free
forms. The monoester form is the most abundant, fol-
lowed by the diester form. The free form is a minor com-
ponent. Suitable antioxidants may be added. It contains
NLT 5% of total astaxanthin, calculated as free astax-
anthin on the anhydrous basis.
IDENTIFICATION
• A. THIN-LAYER CHROMATOGRAPHY
Standard solution: 10 mg/mL of USP Astaxanthin Es-
ters from Haematococcus pluvialis RS in acetone
Sample solution: 10 mg/mL of Astaxanthin Esters in
acetone
Chromatographic system
(See Chromatography 〈621〉, Thin-Layer Chromato-
graphy.)
Adsorbent: 0.25-mm layer of chromatographic silica
gel mixture. Dry the adsorbent at 110° for 1 h before
use.
Application volume: 5 µL
Developing solvent system: Hexane and acetone
(70:30)
System suitability
Suitability requirement: The chromatogram from the
Standard solution exhibits three clearly separated
zones, with astaxanthin diester having the highest RF
value, followed by astaxanthin monoester (the most
intense) and free astaxanthin (the least intense).
Analysis
Samples: Standard solution and Sample solution
Develop the chromatogram in the Developing solvent
system until the solvent front has moved about three-
fourths of the length of the plate. Remove the plate
from the chamber, and dry in a current of air. Ex-
amine the plates under white light.
Acceptance criteria: The Sample solution exhibits three
main zones corresponding in RF value to those obtained
from the Standard solution. The zone in the middle
(monoester) is the most intense, and the zone with the
• B. HPLC: The Sample solution exhibits three major peaks
with the retention times corresponding to those of
13-cis-astaxanthin, all-trans-astaxanthin, and 9-cis-astax-
anthin peaks in the Standard solution, as obtained in the
test for Content of Total Astaxanthin.
Accessed from 47.180.160.190 by akpmtn3zv on Mon May 14 18:42:39 EDT 2018

USP 41 Dietary Supplements / Astaxanthin 4447

ASSAY inert gas at room temperature, add 3 mL of acetone,

• CONTENT OF TOTAL ASTAXANTHIN sonicate, and filter the mixture. The filtered solution is
[NOTE—Astaxanthin determined by this method is total the Sample solution.
astaxanthin, including the free astaxanthin, the Chromatographic system
monoester, and the diester.] (See Chromatography 〈621〉, System Suitability.)
Buffer solution: Dissolve 6.06 g of tris(hydroxymethyl) Mode: LC
aminomethane in 750 mL of water, adjust with 1 N Detector: 474 nm
hydrochloric acid to a pH of 7.0, and dilute with water Column: YMC Carotenoid, 4.6-mm × 25-cm, 5-µm
to 1000 mL. packing L62
Cholesterol esterase solution: 4 U/mL of cholesterol Flow rate: 1.0 mL/min
esterase1 in Buffer solution. Prepare fresh daily.
. Injection volume: 20 µL
Solution A: Methanol System suitability
Solution B: t-Butylmethylether Sample: Standard solution
Solution C: Phosphoric acid, 1% aqueous [NOTE—The approximate relative retention times for
Mobile phase: See Table 1. 13-cis-astaxanthin, all-trans-astaxanthin, 9-cis-astax-
anthin, and apocarotenal (trans-beta-apo-8’-carotenal)
Table 1 are listed in Table 2.]
Time Solution A Solution B Solution C
Table 2
(min) (%) (%) (%)
0 81 15 4 Relative Relative
15 66 30 4 Retention Response
Name of Compound Time Factor
23 16 80 4
13-cis-Astaxanthin 0.9 1.3
27 16 80 4
all-trans-Astaxanthin 1.0 1.0
27.1 81 15 4
9-cis-Astaxanthin 1.4 1.1
35 81 15 4
Apocarotenal (internal
—
Internal standard solution: 37.5 µg/mL of USP Apo- standard) 1.7
carotenal RS in acetone
Standard stock solution: Transfer 30 mg of USP Astax- Suitability requirements
anthin Esters from Haematococcus pluvialis RS to a Chromatogram similarity: The chromatogram from
100-mL volumetric flask. Dissolve in 30 mL of acetone, the Standard solution is similar to the Reference
shake by mechanical means, and dilute with acetone to Chromatogram provided with the USP Astaxanthin
volume. Esters from Haematococcus pluvialis RS being used.
Standard solution: Combine 2.0 mL of the Standard Resolution: NLT 2.0 between 13-cis-astaxanthin and
stock solution and 1.0 mL of the Internal standard solu- all-trans-astaxanthin
tion in a glass centrifuge tube. Add 3.0 mL of Choles- Relative standard deviation: NMT 2.0% for the all-
terol esterase solution to the tube, and mix gently by trans-astaxanthin peak
inversion. Place the tube in a block heater set to 37°, Analysis
and allow the reaction to continue for 45 min, gently Samples: Standard solution and Sample solution
and slowly inverting the tube every 10 min. After 45 Calculate the percentage of total astaxanthin content in
min, add 1 g of sodium sulfate and 2 mL of petroleum the portion of sample taken:
ether to the tube. Mix on a vortex mixer for 30 s, then
centrifuge at 3000 rpm for 3 min. Carefully transfer the Result = (RU/RS) × (CS/CU) × P
petroleum ether layer to a 10-mL glass centrifuge tube RU = [(1.3 × peak area of 13-cis-astaxanthin + peak
containing 1 g of anhydrous sodium sulfate. Be careful area of all-trans-astaxanthin + 1.1 × peak
to avoid pipetting the intermediate emulsive layer. area of 9-cis-astaxanthin)/peak area of the
Evaporate the petroleum ether layer using a vacuum or internal standard] from the Sample solution
a stream of inert gas at room temperature, add 3 mL of RS = [(1.3 × peak area of 13-cis-astaxanthin + peak
acetone, sonicate, and filter the mixture. The filtered area of all-trans-astaxanthin + 1.1 × peak
solution is the Standard solution.

Sample stock solution: Warm a quantity of the sample
in a water bath at 50°–60° for 30 min. Shake the sam-
ple well at 10-min intervals. After 30 min, transfer
30 mg of the sample to a 100-mL volumetric flask. Dis-
solve in 30 mL of acetone, shake by mechanical means,
and dilute with acetone to volume.
Sample solution: Combine 2.0 mL of the Sample stock
solution and 1.0 mL of the Internal standard solution in a
glass centrifuge tube. Add 3.0 mL of Cholesterol esterase
solution to the tube, and mix gently by inversion. Place
the tube in a block heater set to 37°, and allow the
reaction to continue for 45 min, gently and slowly in-
verting the tube every 10 min. After 45 min, add 1 g of
sodium sulfate and 2 mL of petroleum ether to the
tube. Mix on a vortex mixer for 30 s, then centrifuge at
3000 rpm for 3 min. Carefully transfer the petroleum
ether layer to a 10-mL glass centrifuge tube containing
1 g of anhydrous sodium sulfate. Be careful to avoid
pipetting the intermediate emulsive layer. Evaporate the
petroleum ether layer using a vacuum or a stream of
DS Monographs
area of 9-cis-astaxanthin)/peak area of the
internal standard] from the Standard solution
CS = concentration of USP Astaxanthin Esters from
Haematococcus pluvialis RS in the Standard
solution (mg/mL)
CU = concentration of the Sample solution (mg/mL)
P = labeled amount of total astaxanthin as free
astaxanthin in the USP Astaxanthin Esters
from Haematococcus pluvialis RS (%)
Acceptance criteria: NLT 5% of total astaxanthin, cal-
culated as free astaxanthin on the anhydrous basis
CONTAMINANTS
• ELEMENTAL IMPURITIES—PROCEDURES 〈233〉
Acceptance criteria
Arsenic: NMT 2.0 µg/g
Cadmium: NMT 1.0 µg/g
Lead: NMT 1.0 µg/g
Mercury: NMT 1.0 µg/g
• MICROBIAL ENUMERATION TESTS 〈2021〉: The total aerobic
bacterial count does not exceed 103 cfu/g, and the total
.

1Use Wako Pure Chemicals catalog no. 037-11221, available from www. combined molds and yeasts count does not exceed 102 .

cfu/g.
.

wakousa.com; Sigma catalog no. C9281, available from www.sigmaaldrich.

com; or equivalent.

Official from May 1, 2018

Copyright (c) 2018 The United States Pharmacopeial Convention. All rights reserved.
 Accessed from 47.180.160.190 by akpmtn3zv on Mon May 14 18:42:39 EDT 2018
4448 Astaxanthin / Dietary Supplements
• ABSENCE OF SPECIFIED MICROORGANISMS 〈2022〉: Meets the
requirements of the tests for absence of Salmonella spe-
cies and Escherichia coli
• PHEOPHORBIDE CONTENT
Solution A: 50 mg/mL of sodium sulfate
Solution B: Saturated solution of sodium sulfate
Sample stock solution: Transfer 100 mg of the sample
to a 10-mL test tube, add 10 mL of acetone, and dis-
solve with sonication. Quantitatively transfer this solu-
tion to a separatory funnel, rinsing the test tube three
times with 10-mL portions of acetone and adding the
rinsings to the funnel. Add 30 mL of ethyl ether to the
separatory funnel, followed by 50 mL of Solution A. Mix
the contents of the separatory funnel by shaking gently,
then draw off and discard the lower layer. Repeat wash-
ing with Solution A three times. Dehydrate the remain-
ing extract with anhydrous sodium sulfate, then transfer
the extract to a 50-mL volumetric flask, and dilute with
ethyl ether to volume.
Sample solution: Transfer 20 mL of the Sample stock
solution to a small beaker. Add 20 mL of 17% hydro-
chloric acid, and mix the solution vigorously. Transfer
the hydrochloric acid layer to a separatory funnel, and
repeat the extraction with a second 10-mL portion of
17% hydrochloric acid, adding the hydrochloric acid
layer to the separatory funnel. Add 150 mL of Solution
B, 20 mL of ethyl ether, and mix the contents of the
separatory funnel by shaking. Transfer the ethyl ether
layer to a 20-mL volumetric flask, and dilute with ethyl
ether to volume.
Instrumental conditions
(See Ultraviolet-Visible Spectroscopy 〈857〉.)
Analytical wavelength: 667 nm
Cell path: 1 cm
Blank: Ethyl ether
Analysis
Sample: Sample solution
Calculate the percentage of pheophorbide in the por-
tion of sample taken:
Result = A/(C × F)
A = absorbance of the Sample solution
C = concentration of the Sample solution (g/mL)
F = coefficient of extinction (E1%) of pure
.
pheophorbide in ethyl ether (100 mL · g −1 ·.
cm−1), 702
.
Acceptance criteria: NMT 0.02%
SPECIFIC TESTS
• WATER DETERMINATION, Method I 〈921〉: NMT 0.5%
DS Monographs
ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in well-closed
containers.
• USP REFERENCE STANDARDS 〈11〉
USP Astaxanthin Esters from Haematococcus pluvialis RS
USP Apocarotenal RS
trans-beta-Apo-8’-carotenal.
C30H40O
.
Astragalus Root
DEFINITION
Astragalus Root consists of the dried root of Astragalus mem-
branaceus var. mongholicus (Bunge) P.K.Hsiao or Astragalus
membranaceus (Fisch.) Bunge (Fam. Fabaceae). Astragalus
root is typically harvested from a 2- to 3-year-old plant in
early fall. It contains NLT 0.04% of cycloartane saponins
and NLT 0.03% of isoflavonoids calculated on the dried
basis.
Official from May 1, 2018
Copyright (c) 2018 The United States Pharmacopeial Convention. All rights reserved.
USP 41
IDENTIFICATION
• A. HPTLC FOR ARTICLES OF BOTANICAL ORIGIN 〈203〉
Standard solution A: 1 mg/mL of USP Astragaloside IV
RS in methanol
Standard solution B: 2 mg/mL of USP Daidzin RS and
1 mg/mL of USP Daidzein RS in methanol
Standard solution C: 50 mg/mL of USP Astragalus Root
Dry Extract RS in methanol. Sonicate for about 10 min,
centrifuge, and use the supernatant.
Sample solution: Heat 3 g of Astragalus Root, finely
powdered, in 50 mL of methanol for 50 min under re-
flux. Centrifuge, withdraw the supernatant, and evapo-
rate to dryness under reduced pressure. Dissolve the
residue in 1.0 mL of water. Transfer the resulting solu-
tion onto a 6-mL solid-phase extraction column con-
taining 500 mg of sorbent previously conditioned with
3 mL of methanol and 3 mL of water.1 Wash with .
15 mL of water followed by 15 mL of 30% methanol,
and discard the rinsate. Elute with 20 mL of methanol,
collect the eluate, evaporate to dryness under reduced
pressure, and dissolve the residue in 2 mL of methanol.
Chromatographic system
Adsorbent: Chromatographic silica gel with an aver-
age particle size of 5 µm (HPTLC plate)2 .
Application volume: 3 µL each of Standard solution A,
Standard solution B, Standard solution C, and Sample
solution as 8-mm bands
Relative humidity: Condition the plate to a relative
humidity of 33%.
Temperature: Ambient, not to exceed 30°
Developing solvent system: Ethyl acetate, methanol,
and water (100: 13.5: 10)
Developing distance: 6 cm
Derivatization reagent: 10% Sulfuric acid in metha-
nol. [NOTE—Prepare fresh. Slowly and gradually add
sulfuric acid to ice-cold methanol, and mix well.]
System suitability
Samples: Standard solution A, Standard solution B, and
Standard solution C
Suitability requirements
Chromatographic pattern: Under long-wave UV
light (365 nm), following derivatization, Standard so-
lution A exhibits an orange band in the middle of the
lower third of the plate due to astragaloside IV, with
a retardation factor (RF) of approximately 0.15. In
Standard solution B, daidzin and daidzein form bluish-
grey bands with RF of approximately 0.34 and 0.76,
respectively; the proximal band is sharper, while the
distal is somewhat diffuse. In Standard solution C, four
orange bands are seen in the lower third of the plate,
corresponding to astragalosides IV, III, II, and I with RF
of approximately 0.15, 0.18, 0.24, and 0.34, respec-
tively. The RF of the astragaloside I band approxi-
mates that of daidzin in Standard solution B. The up-
per two-thirds of the plate typically display a number
of bluish, greenish, and pinkish diffuse bands, one of
which corresponds to that of daidzein in Standard so-
lution B.
Analysis
Samples: Standard solution A, Standard solution B,
Standard solution C, and Sample solution
Apply the Samples as bands and dry in air. Develop in a
saturated chamber. Air-dry, treat with Derivatization re-
agent, heat for 5 min at 100°, and examine under
long-wave UV light (365 nm).
Acceptance criteria: Under long-wave UV light (365
nm), the Sample solution exhibits bands corresponding
in color and RF to similar bands from Standard solution
C. In the lower third of the chromatogram, a number
of orange bands are present; the most prominent ones
corresponding to astragalosides I and II, with RF of ap-
proximately 0.34 and 0.24, respectively. In the upper
1Suitable commercially available SPE columns are Bakerbond Octadecyl C18.
.
2Suitable commercially available plates are HPTLC Silica Gel 60 F254 from EMD
.
Millipore (e.g., part no. 1.05642.0001).