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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
stereoisomers of astaxanthin in the monoester, diester, and
free forms. The monoester form is the most abundant,
followed by the diester form. The free form is a minor
component. Suitable antioxidants may be added. It
contains NLT 5% of total astaxanthin, calculated as free
astaxanthin on the anhydrous basis.
IDENTIFICATION
• A. THIN-LAYER CHROMATOGRAPHY
Standard solution: 10 mg/mL of USP Astaxanthin Esters
from Haematococcus pluvialis RS in acetone
Sample solution: 10 mg/mL of Astaxanthin Esters in
acetone
Chromatographic system
(See Chromatography á621ñ, General Procedures, Thin-Layer
Chromatography.)
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)
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System suitability
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Sample: Standard solution
Suitability requirements: The chromatogram of 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).
O
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. Examine 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
lower RF is the least intense.
• 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-astaxanthin peaks in the Standard solution, as
obtained in the Assay for Content of Total Astaxanthin.
ASSAY
Change to read:
• CONTENT OF TOTAL ASTAXANTHIN
[NOTE—Astaxanthin determined by this method is total
astaxanthin, including the free astaxanthin, the
monoester, and the diester.]
Buffer solution: Dissolve 6.06 g of tris(hydroxymethyl)
aminomethane in 750 mL of water, adjust with 1 N
hydrochloric acid to a pH of 7.0, and dilute with water to
1000 mL.
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Cholesterol esterase solution: 4 units/mL of cholesterol
esterase1 in Buffer solution. Prepare fresh daily.
Solution A: Methanol
Solution B: t-Butylmethylether
Solution C: Phosphoric acid, 1% aqueous
Mobile phase: See Table 1.
Table 1
Time Solution A Solution B Solution C
(min) (%) (%) (%)
0 81 15 4
15 66 30 4
23 16 80 4
27 16 80 4
27.1 81 15 4
35 81 15 4
Internal standard solution: 37.5 µg/mL of USP
Apocarotenal RS in acetone
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Standard stock solution: Transfer 30 mg of USP
Astaxanthin Esters from Haematococcus pluvialis RS to a
100-mL volumetric flask. Dissolve in 30 mL of acetone,
shake by mechanical means, and dilute with acetone to
volume.
Standard solution: Combine 2.0 mL of the Standard 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 inverting 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, and 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 inert gas at room temperature. Add
3 mL of acetone, sonicate, and filter the mixture. The
filtered 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 sample well at
10-min intervals. After 30 min, transfer 30 mg of the sample
to a 100-mL volumetric flask. Dissolve 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 inverting 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, and 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 inert gas at room temperature. Add
3 mL of acetone, sonicate, and filter the mixture. The
filtered solution is the Sample solution.
1 Use
Wako Pure Chemicals catalog #037-11221, available from
www.wakousa.com; Sigma catalog #C9281, available from
www.sigmaaldrich.com; or equivalent.
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Chromatographic system Add the following:

(See Chromatography á621ñ, System Suitability.)
Mode: LC ▲
• LIMIT OF BENZO[a]PYRENE, BENZO[b]FLUORANTHENE,
Detector: UV 474 nm BENZO[a]ANTHRACENE, AND CHRYSENE
Column: ▲▲ (USP 1-Aug-2019)4.6-mm × 25-cm; 5-µm packing Internal standard stock solution: 10 µg/mL each of
L62 benzo[a]pyrene-d12-isooctane,2 benzo[b]
Flow rate: 1.0 mL/min fluoranthene-d12-isooctane,2 benzo[a]
Injection volume: 20 µL anthracene-d12-isooctane,2 and chrysene-d12-toluene-d82
System suitability in n-hexane
Sample: Standard solution Internal standard solution: 0.1 µg/mL each of benzo[a]
[NOTE—See Table 2 for approximate relative retention pyrene-d12-isooctane, benzo[b]fluoranthene-d12-isooctane,
times.] benzo[a]anthracene-d12-isooctane, and
Table 2
chrysene-d12-toluene-d8 in n-hexane, by serially diluting the
Internal standard stock solution with n-hexane
Relative Relative
Retention Response
Standard stock solution: 0.1 µg/mL each of benzo[a]
Name Time Factor pyrene-dichloromethane,3 benzo[b]
fluoranthene-acetone,3 benzo[a]
13-cis-Astaxanthin 0.9 1.3
anthracene-dichloromethane,3 and
all-trans-Astaxanthin 1.0 1.0 chrysene-dichloromethane3 in n-hexane
9-cis-Astaxanthin 1.4 1.1
Standard solution: 0.01 µg/mL each of benzo[a]
pyrene-dichloromethane, benzo[b]fluoranthene-acetone,
Apocarotenal (internal benzo[a]anthracene-dichloromethane, and

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—
standard) 1.7 chrysene-dichloromethane in n-hexane, by serially diluting
the Standard stock solution with n-hexane
Suitability requirements Working standard solutions: See Table 3.
Chromatogram similarity: The chromatogram of the
Standard solution is similar to the reference Table 3
chromatogram provided with the lot of USP
Astaxanthin Esters from Haematococcus pluvialis RS
being used.
ci Working
Volume Taken (mL) Final
Volume
after
Dilution
Resolution: NLT 2.0 between 13-cis-astaxanthin and Standard Standard Internal with
all-trans-astaxanthin Concentration Stock Standard Standard n-hexane
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Relative standard deviation: NMT 2.0% for (ng/mL) Solution Solution Solution (mL)
all-trans-astaxanthin 10 2 — 2 20
Analysis
Samples: Standard solution and Sample solution 5 1 — 2 20
Calculate the percentage of total astaxanthin content in the 2 — 4 2 20
portion of sample taken:
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1 — 2 2 20
Result = (RU/RS) × (CS/CU) × P 0.5 — 1 2 20

RU = ratio of [(1.3 × the peak area of

13-cis-astaxanthin) + the peak area of
all-trans-astaxanthin + (1.1 × the peak area of
9-cis-astaxanthin)] to the peak area of the
internal standard from the Sample solution
RS = ratio of [(1.3 × the peak area of
13-cis-astaxanthin) + the peak area of
all-trans-astaxanthin + (1.1 × the peak area of
9-cis-astaxanthin)] to the 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 USP Astaxanthin Esters from
Haematococcus pluvialis RS (%)
Acceptance criteria: NLT 5% of total astaxanthin,
calculated as free astaxanthin on the anhydrous basis
CONTAMINANTS
• ELEMENTAL IMPURITIES—PROCEDURES á233ñ
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
Sample solution: Transfer 1 g of Astaxanthin Esters to a
suitable refluxing flask. Add 100 µL of the Internal standard
solution, 10 mL of water, 5 g of potassium hydroxide, and
80 mL of ethanol. The mixture is refluxed at 90° for 1 h.
Cool, filter the solution through glass wool, and collect the
filtrate in a suitable separator. Add 100 mL each of water
and n-hexane to the separator, and shake for 10 min.
Transfer the upper layer (hexane extract) to a second
separator. Add 50 mL of n-hexane to the first separator and
repeat the extraction again. Combine the upper layer with
the extract in the second separator. Wash the combined
extracts twice, each with 50 mL of water. Pass the washed
extracts through anhydrous sodium sulfate to remove
water. Evaporate the extracts to dryness using a stream of
nitrogen. Dissolve the residue in 100 mL of n-hexane.
Transfer this solution to a separator. Add 50 mL of dimethyl
sulfoxide (DMSO) to the separator and shake it for 10 min.
Transfer the lower layer to a second separator. Repeat the
extraction with the solution in the first separator 2 more
times. Combine the second and third extracts with the first
extract in the second separator. To the combined extracts
2 Use
Supelco Certified Reference Material, available from
www.sigmaaldrich.com, or equivalent.
3 UsePolycyclic Aromatic Hydrocarbon (PAH) Standards and Standard
Mixtures, available from www.isotope.com/products/index.cfm, or
equivalent.

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add 150 mL of sodium chloride solution (1 in 75), mix, and
allow to cool. Add 50 mL of n-hexane to the extracts and
shake the separator for 10 min. Transfer the upper layer to a
third separator. Repeat the hexane extraction 2 more times.
Combine the second and third extracts with the first extract
in the third separator. Wash the combined extracts twice
with 150 mL of water each time. Pass the washed extracts
through anhydrous sodium sulfate to remove water.
Evaporate the extracts to dryness using a stream of
nitrogen. Dissolve the residue in 10 mL of n-hexane.
Quantitatively transfer this solution to a chromatographic
column (prepared by packing 5 g of silica gel in a brown
glass column and wash with 30 mL of n-hexane), wash the
column with 20 mL of n-hexane, and then elute the sample
with 90 mL of a mixture of n-hexane and acetone (99:1).
Evaporate the eluant to dryness with a stream of nitrogen,
and dissolve the residue in 1 mL of n-hexane. Quantitatively
transfer this solution to a molecular imprinted polymer
cartridge that has been prewashed with 1 mL of
cyclohexane. Wash the cartridge 3 times with 1 mL of
n-hexane each time. Elute the sample with 4 mL of ethyl
acetate. Evaporate the eluant to dryness with a stream of
nitrogen, and dissolve the residue in 1.0 mL of n-hexane.
• m/z 252 and 253: benzo[b]fluoranthene and benzo[a]
pyrene
• m/z 240: benzo[a]anthracene-d12 and chrysene-d12
• m/z 228 and 229: benzo[a]anthracene and chrysene
Construct a calibration curve for each benzo[a]pyrene,
benzo[b]fluoranthene, benzo[a]anthracene, and chrysene
standard by plotting the concentrations of the Working
standard solutions versus the ratios of the peak response of
the benzo[a]pyrene, benzo[b]fluoranthene, benzo[a]
anthracene, or chrysene standard to that of the internal
standard. Calculate the slope and y-intercept for each
standard calibration curve using linear regression analysis.
Separately calculate the content of benzo[a]pyrene,
benzo[b]fluoranthene, benzo[a]anthracene, and chrysene
in the portion of Astaxanthin Esters taken:
Result = [(RU − Y)/S] × (V/W)
RU = ratio of the peak response of benzo[a]pyrene,
benzo[b]fluoranthene, benzo[a]anthracene, or
chrysene to that of the internal standard from the
Sample solution

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Y = y-intercept of the calibration curve for benzo[a]
This is the Sample solution. pyrene, benzo[b]fluoranthene, benzo[a]
Chromatographic system anthracene, or chrysene from the Working
(See Chromatography á621ñ, System Suitability and Mass standard solutions
Spectrometry á736ñ.) S = slope of the calibration curve for benzo[a]pyrene,
Mode: GC
Detector: Mass spectrometer, electron energy 70 eV
Ionization method: Electron impact (EI)
Column: 0.15-mm × 15-m capillary; coated with a 0.1-µm
ci V
W
benzo[b]fluoranthene, benzo[a]anthracene, or
chrysene from the Working standard solutions
= total volume used to dissolve the sample (mL)
= weight of the sample (g)
film of phase G51
Temperatures Acceptance criteria
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Injection port: 320° Benzo[a]pyrene: NMT 2 ppb
Ion source: 300° Sum of benzo[a]pyrene, benzo[b]fluoranthene,
Transfer line: 320° benzo[a]anthracene, and chrysene: NMT
Column: See Table 4. 10 ppb▲ (USP 1-Aug-2019)
• MICROBIAL ENUMERATION TESTS á2021ñ: The total aerobic
Table 4 bacterial count does not exceed 103 cfu/g, and the total
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Hold Time combined molds and yeasts count does not exceed 102 cfu/
Initial Temperature Final at Final g.
Temperature Ramp Temperature Temperature
(°) (°/min) (°) (min) • ABSENCE OF SPECIFIED MICROORGANISMS á2022ñ, Test
Procedures, Test for Absence of Salmonella Species and Test
100 50 230 7 for Absence of Escherichia coli: Meets the requirements
230 50 280 7 • PHEOPHORBIDE CONTENT
Solution A: 50 mg/mL of sodium sulfate
280 30 350 10 Solution B: Saturated solution of sodium sulfate
Sample stock solution: Transfer 100 mg of the sample to a
Carrier gas: Helium 10-mL test tube, add 10 mL of acetone, and dissolve with
Flow rate: 1.2 mL/min sonication. Quantitatively transfer this solution to a
Injection volume: 2 µL separatory funnel, rinsing the test tube 3 times with 10-mL
Injection type: Pulsed splitless portions of acetone and adding the rinsings to the funnel.
Pulse pressure: 400 kPa Add 30 mL of ethyl ether to the separatory funnel, followed
Pulse time: 2 min by 50 mL of Solution A. Mix the contents of the separatory
Solvent delay: 9.2 min funnel by shaking gently, then draw off and discard the
System suitability lower layer. Repeat washing with Solution A 3 times.
Sample: Standard solution Dehydrate the remaining extract with anhydrous sodium
[NOTE—The relative retention times for benzo[a] sulfate, then transfer the extract to a 50-mL volumetric
anthracene, chrysene, benzo[b]fluoranthene, and flask, and dilute with ethyl ether to volume.
benzo[a]pyrene are about 1.00, 1.02, 1.23, and Sample solution: Transfer 20 mL of the Sample stock
1.35 min, respectively.] solution to a small beaker. Add 20 mL of 17%
Suitability requirements hydrochloric acid, and mix the solution vigorously. Transfer
Resolution: NLT 1.5 between benzo[a]anthracene and the hydrochloric acid layer to a separatory funnel, and
chrysene repeat the extraction with a second 10-mL portion of 17%
Analysis hydrochloric acid, adding the hydrochloric acid layer to the
Samples: Working standard solutions and Sample solution separatory funnel. Add 150 mL of Solution B, 20 mL of ethyl
Use the following monitoring ions for peak detection: ether, and mix the contents of the separatory funnel by
• m/z 254: benzo[b]fluoranthene-d12 and benzo[a] shaking. Transfer the ethyl ether layer to a 20-mL
pyrene-d12 volumetric flask, and dilute with ethyl ether to volume.

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Instrumental conditions Acceptance criteria: NMT 0.02%

(See Ultraviolet-Visible Spectroscopy á857ñ.)
Mode: UV-Vis SPECIFIC TESTS
Analytical wavelength: 667 nm • WATER DETERMINATION á921ñ, Method I: NMT 0.5%
Cell path: 1 cm ADDITIONAL REQUIREMENTS
Blank: Ethyl ether • PACKAGING AND STORAGE: Preserve in well-closed
Analysis containers.
Sample: Sample solution • USP REFERENCE STANDARDS á11ñ
Calculate the percentage of pheophorbide in the portion of USP Apocarotenal RS
sample taken: trans-beta-Apo-8′-carotenal.
C30H40O
Result = A/(C × F) USP Astaxanthin Esters from Haematococcus pluvialis RS
A = absorbance of the Sample solution
C = concentration of the Sample solution (g/mL)
F = coefficient of extinction (ε1%) of pure
pheophorbide in ethyl ether (100 mL · g−1 · cm−1),
702

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