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600-Å pore size. Condition column before use by washing

Garlic with 50 mL of methanol and with 50 mL of a mixture of
DEFINITION methanol and water (3:7). [NOTE—Do not allow the column
Garlic consists of the fresh or dried compound bulbs of to dry.]
Allium sativum L. (Fam. Liliaceae). It contains NLT 0.5% of Standard solution: 0.2 mg/mL each of USP β-Chlorogenin
alliin and NLT 0.2% of γ-glutamyl-(S)-allyl-L-cysteine, RS and USP Agigenin RS in methanol
calculated on the dried basis. Sample solution: Transfer about 10 g of freshly peeled
garlic cloves to a 37-mL homogenizing cup, and
IDENTIFICATION homogenize with 25 mL of methanol at the highest speed
• A. THIN-LAYER CHROMATOGRAPHY for 1 min. Centrifuge the mixture, and decant the
Standard solution A: 0.5 mg/mL of USP L-Methionine RS supernatant to a flask. Add 70 mL of water. Transfer to the
in a mixture of methanol and water (1:1) Extraction column, allow to drain, and discard the eluate.
Standard solution B: 0.5 mg/mL of USP Alliin RS in a Wash the column with 50 mL of a mixture of methanol and
mixture of methanol and water (1:1) water (3:7), allow the solvent mixture to drain, and discard
Sample solution: Cut a freeze-dried garlic bulb into small the eluate. Finally, elute the crude saponin fraction off the
pieces, transfer 1 g of the cut pieces to an extractor, and column with 20 mL of methanol, collect the eluate, and
extract with two 20-mL portions of a mixture of methanol evaporate to dryness. Dissolve the residue in 4 mL of a
and water (1:1), combining the extracts. Concentrate to a mixture of 8% sulfuric acid and alcohol (1:1), transfer the
small volume (about 5 mL), using a rotary evaporator. solution to a screw-capped test tube, and heat on a boiling
Chromatographic system water bath for 5 h. Cool the test tube, add 20 mL of water,
Adsorbent: 0.25-mm layer of chromatographic silica gel, and transfer the solution to a freshly conditioned Extraction
typically 20-cm long (TLC plates) column, allow to drain, and discard the eluate. Wash the
column with 30 mL of a mixture of methanol and water

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Application volume: 20 µL, applied separately as 10-mm
bands (7:3), and discard the eluate. Finally, elute the column with
Developing solvent system: Butyl alcohol, n-propyl 50 mL of methanol. Collect the eluate, evaporate it to
alcohol, glacial acetic acid, and water (3:1:1:1) dryness, and dissolve the residue in 0.5 mL of methanol.
Derivatization reagent: 0.2% Solution of ninhydrin in a Chromatographic system
mixture of butyl alcohol and 2 N acetic acid (19:1) Adsorbent: 0.25-mm layer of chromatographic silica gel,
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
ci typically 20-cm long (TLC plates)
Application volume: 20 µL, as 7-mm bands
Developing solvent system: Methylene chloride and
Develop the chromatograms until the solvent front has methanol (15:2)
moved up about three-fourths of the plate, in a saturated Derivatization reagent: Dissolve 0.5 mL of
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chamber. Remove the plate, and allow the solvent to 4-methoxybenzaldehyde and 0.5 mL of sulfuric acid in
evaporate. Spray with the Derivatization reagent, heat at sufficient alcohol to make 10 mL.
100°–105° for 10 min, and immediately examine the plate Analysis
under white light. Samples: Standard solution and Sample solution
Acceptance criteria: The chromatogram of the Sample Develop the chromatograms until the solvent front has
solution shows the following zones: a violet zone having an moved up about three-fourths of the plate, in a saturated
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RF value of about 0.89; a pink zone having an RF value of chamber. Remove the plate, and allow the solvent to
about 0.5 and corresponding in color and RF value to that evaporate. Spray the plate with Derivatization reagent,
obtained from the chromatogram of Standard solution A; a heat the plate at 100°–105° for 5 min, and examine the
pinkish zone having an RF value of about 0.43; a strong plate under white light.
orange zone having an RF value of about 0.38; a pinkish Acceptance criteria: The chromatogram of the Sample
violet zone having an RF value of about 0.3 and solution exhibits, among several yellowish and grayish
green spots, a grayish green spot at an RF value of about
corresponding in color and RF value to that of the
0.4, corresponding to the grayish green spot due to
chromatogram of Standard solution B; and additional
β-chlorogenin of the Standard solution. The chromatogram
pinkish orange zones situated very close to each other just
of the Sample solution does not exhibit a spot at an RF value
below the zone corresponding to alliin in the
chromatogram of Standard solution B. of about 0.2, corresponding to agigenin of the Standard
• B. solution.
Sample: About 10 g of garlic bulbs that have been cut into COMPOSITION
small pieces • CONTENT OF ALLIIN
Analysis: Transfer to a suitable flask. Add 10 mL of 1 N Allinase inhibitor solution: Dissolve 109 mg of
sodium hydroxide and 10 mL of water, heat the flask in carboxymethoxylamine hemihydrochloride in 100.0 mL of
boiling water for 10 min, cool, and filter. Add a few drops water.
of freshly prepared sodium nitroferricyanide TS to 2 mL of Solution A: Monobasic sodium phosphate 0.045 M in
the filtrate. water, adjusted with 0.2 M sodium hydroxide to a pH of
Acceptance criteria: The appearance of a red or orange-red 7.1
color indicates the presence of sulfur-containing Buffer: Monobasic sodium phosphate 0.05 M in water,
compounds in the Sample. adjusted with 0.2 M sodium hydroxide to a pH of 9.5
• C. The retention time of the major peak in the Sample Derivatization reagent: Dissolve 140 mg of
solution corresponds to that of one of the alliin diastereomer o-phthaldialdehyde in 5 mL of methanol, add 100 µL of
peaks in the Standard solution, as obtained in the test for t-butylthiol, and dilute with Buffer to 50 mL. [NOTE—This
Content of Alliin. reagent may occasionally become opaque during
• D. THIN-LAYER CHROMATOGRAPHY preparation. Store at room temperature, and use within
Extraction column: 1-cm × 5-cm solid-phase extraction 1 week.]
column containing styrene-divinylbenzene copolymer
packing with a 75- to 150-µm diameter and a 400- to

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Printed by: USP NF Official Date: Official as of 01-Dec-2015 Document Type: DIETARY SUPPLEMENTS @2024 USPC
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Mobile phase: Acetonitrile, 1,4-dioxane, tetrahydrofuran, with two 70-mL portions of a mixture of methanol and
and Solution A (25: 2.9: 2.2: 69.9) water (1:1), centrifuge, and transfer the supernatants to the
Standard solution: 0.05 mg/mL of USP Alliin RS in a mixture volumetric flask. Dilute the contents of the flask with a
of methanol and water (1:1) mixture of methanol and water (1:1) to volume.
Sample stock solution: Transfer about 10.0 g of freshly Chromatographic system
peeled garlic cloves, accurately weighed, to a 110-mL (See Chromatography á621ñ, System Suitability.)
homogenizing cup. Add 70.0 mL of Allinase inhibitor Mode: LC
solution, and blend at the highest speed for 30 s. Detector: UV 205 nm
Centrifuge, and decant the supernatant into a 100-mL Column: 4.6-mm × 15-cm; packing L1
volumetric flask. Mix the remaining solids in the cup with Flow rate: 0.8 mL/min
20 mL of Allinase inhibitor solution, centrifuge, and add the Injection volume: 10 µL
supernatant to the volumetric flask. Dilute the contents of System suitability
the flask with Allinase inhibitor solution to volume. Sample: Standard solution
Sample solution: Dilute a portion of the Sample stock Suitability requirements
solution 1 in 10 with a mixture of methanol and water (1:1). Relative standard deviation: NMT 2.0% for the
Chromatographic system γ-glutamyl-(S)-allyl-L-cysteine peak in repeated
(See Chromatography á621ñ, System Suitability.) injections
Mode: LC Analysis
Detector: UV 337 nm Samples: Standard solution and Sample solution
Column: 4-mm × 10-cm; packing L1 Calculate the percentage of γ-glutamyl-(S)-allyl-L-cysteine
Flow rate: 1 mL/min in the portion of Garlic taken:
Injection volume: 10 µL
Result = (rU/rS) × CS × (V/W) × 100

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System suitability
Sample: Standard solution
[NOTE—Alliin exhibits two major peaks representing its rU = peak response of γ-glutamyl-(S)-allyl-L-cysteine
diastereomers.] from the Sample solution
Suitability requirements rS = peak response of γ-glutamyl-(S)-allyl-L-cysteine
Relative standard deviation: NMT 2.0% for each of the from the Standard solution
major peaks, in repeated injections
Analysis
Samples: Standard solution and Sample solution
ci CS = concentration of USP
γ-Glutamyl-(S)-allyl-L-cysteine RS in the Standard
solution (mg/mL)
Using a syringe, transfer 0.1 mL of the Standard solution or V = volume of the Sample solution (mL)
Sample solution to separate septum-capped vials, and add W = weight of Garlic used to prepare the Sample
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0.5 mL of the Derivatization reagent to each vial. Allow a solution (mg)
reaction time of NLT 2 min before injection into the
chromatograph. Record the chromatograms, and Acceptance criteria: NLT 0.2% on the dried basis
measure the areas of the alliin diastereomer peaks. CONTAMINANTS
Calculate the percentage of alliin in the portion of Garlic • ARTICLES OF BOTANICAL ORIGIN, Limits of Elemental
taken: Impurities á561ñ: Meets the requirements
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• ARTICLES OF BOTANICAL ORIGIN, Pesticide Residue Analysis

Result = (rU/rS) × CS × (V/W) × D × 100
á561ñ: Meets the requirements
rU = peak area of alliin from the Sample solution SPECIFIC TESTS
rS = peak areas of alliin diastereomers from the • BOTANICAL CHARACTERISTICS
Standard solution Macroscopic: Subglobular compound bulbs, 3–5 cm in
CS = concentration of USP Alliin RS in the Standard width, consisting of 8–20 cloves, the whole surrounded by
solution (mg/mL) 2–5 layers of white scale leaves attached to a flattened,
V = volume of the Sample stock solution (mL) circular base; cloves ovoid and 3- to 4-sided, summit acute,
W = weight of Garlic used to prepare the Sample stock narrowed into a threadlike portion of fiber base, truncate,
solution (mg) each clove covered with a white scale leaf and a pinkish
D = dilution factor to prepare the Sample solution white epidermis, easily separated from the solid portion,
from the Sample stock solution, 10 consisting of two flaky scale leaves and two yellowish green
conduplicate foliage leaves
Acceptance criteria: NLT 0.5% on the dried basis Microscopic: The protective leaf contains an epidermis
• CONTENT OF γ-GLUTAMYL-(S)-ALLYL-L-CYSTEINE enclosing a mesophyll free from chlorophyll. The outer
Solution A: Dissolve 6.80 g of monobasic potassium epidermis consists of lignified sclereid cells of thick, pitted
phosphate in 900 mL of water, and adjust with phosphoric walls, elongated, covered with thin cuticle, long fibers up
acid to a pH of 2.6. Dilute with water to 1000.0 mL, to 500 µm in length and 30 µm in width.
and mix. The cortical cells are thick-walled, nonlignified, tending to
Mobile phase: Methanol and Solution A (3:17) collapse on maturity, isodiametric, and contain purple
Standard solution: 0.08 mg/mL of USP pigments. The vascular bundles consist of lignified spiral
γ-Glutamyl-(S)-allyl-L-cysteine RS in a mixture of methanol and annular vessels. The storage leaves show an outer
and water (1:1) epidermis of thin, delicate cells of variable shape, arranged
Sample solution: Transfer about 10 g of freshly peeled in somewhat irregular rows, 60 µm in length and 30 µm
garlic cloves, accurately weighed, to a 110-mL in width. Stomata are present on the outer epidermis only
homogenizing cup. Add 80 mL of a mixture of methanol at the extreme tip near the base of the foliage leaves.
and water (1:1), and homogenize at the highest speed for The mesophyll consists of swollen storage parenchyma cells
1 min. Centrifuge the mixture, and decant the supernatant filled with fine granular reserve material; scattered in the
into a 250-mL volumetric flask. Mix the remaining solids cortex are 20 laticiferous tubes, 500–1000 µm in length.

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Two series of vascular bundles consisting of narrow • LABELING: The label states the Latin binomial and, following
lignified spiral and annular vessels are arranged in the the official name, the part of the plant contained in the
mesophyll. article.
• ARTICLES OF BOTANICAL ORIGIN, Water Content á561ñ: NMT • USP REFERENCE STANDARDS á11ñ
65.0% for fresh bulbs, and NMT 7.0% for dried bulbs USP Agigenin RS
• ARTICLES OF BOTANICAL ORIGIN, Total Ash á561ñ: NMT USP Alliin RS
5.0% USP β-Chlorogenin RS
• ARTICLES OF BOTANICAL ORIGIN, Acid-Insoluble Ash á561ñ: USP γ-Glutamyl-(S)-allyl-L-cysteine RS
NMT 1.0% USP L-Methionine RS
ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Store in well-closed containers
in a cool, dry place, protected from light.

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