Garlic Delayed-Release Tablets
DEFINITION
Garlic Delayed-Release Tablets are prepared from Powdered Garlic or Powdered Garlic Extract and contain NLT 90.0% and NMT 140.0% of the labeled amount of alliin (C6H11NO3S) and NLT 90.0% and NMT 140.0% of the labeled amount of potential allicin (C6H10OS2).
IDENTIFICATION
• A. Thin-Layer Chromatographic Identification Test
Standard solution A:
0.5 mg/mL of USP l-Methionine RS
Standard solution B:
0.5 mg/mL of USP Alliin RS, in a mixture of methanol and water (1:1)
Sample solution:
Transfer an amount of pulverized Tablets, equivalent to 30 mg of alliin, to a 100-mL volumetric flask. Add 70 mL of a mixture of methanol and water (1:1), shake, and centrifuge. Concentrate to a small volume (about 5 mL) using a rotary evaporator.
Chromatographic system
Application volume:
20 µL, applied separately as 10-mm bands
Developing solvent system:
Butyl alcohol, n-propyl alcohol, glacial acetic acid, and water (3:1:1:1)
Spray reagent:
2 mg/mL of ninhydrin, in a mixture of butyl alcohol and 2 N acetic acid (19:1)
Analysis
Samples:
Standard solutions and Sample solution
Proceed as directed in the chapter. Spray with the Spray reagent, heat at 100
–105 for 10 min, and immediately examine the plate.
–105 for 10 min, and immediately examine the plate.
Acceptance criteria:
The chromatogram of the Sample solution shows the following orange and pinkish violet zones: a violet zone having an RF value of 0.89; a pink zone having an RF value of 0.5 and corresponding in color and RF value to that of the chromatogram of Standard solution A; a pinkish zone having an RF value of 0.43; a strong orange zone having an RF value of 0.38; a pinkish violet zone having an RF value of 0.3 and corresponding in color and RF value to that of the chromatogram of Standard solution B; and additional pinkish orange zones situated very close to each other, just below the zone attributed to alliin in the chromatogram of Standard solution B.
• B. HPLC Identification Test
Analysis:
Proceed as directed in the test for Content of Alliin.
Acceptance criteria:
The Sample solution exibits a peak for alliin corresponding to one of the diasteroisomer pairs of peaks in the Standard solution.
STRENGTH
• Content of Alliin
Alliinase inhibitor solution:
Dissolve 109 mg of carboxymethoxylamine hemihydrochloride in 100.0 mL of water.
Solution A:
Dissolve 1.24 g of monobasic sodium phosphate in 100 mL of water, adjust with 0.2 M sodium hydroxide to a pH of 7.1, and dilute with water to 200.0 mL.
Buffer:
Dissolve 1.38 g of monobasic sodium phosphate in 100 mL of water, adjust with 0.2 M sodium hydroxide to a pH of 9.5, and dilute with water to 200.0 mL.
Derivatization reagent:
Dissolve 140 mg of o-phthaldialdehyde in 5 mL of methanol, add 100 µL of t-butylthiol, and dilute with Buffer to 50 mL. [Note—This reagent may occasionally become opaque during preparation. Store at room temperature, and use within 1 week. ]
Mobile phase:
Acetonitrile, 1,4-dioxane, tetrahydrofuran, and Solution A (25: 2.9: 2.2: 69.9)
Standard solution:
0.05 mg/mL of USP Alliin RS in a mixture of methanol and water (1:1). Use a syringe to transfer 0.1 mL of this solution to a septum-capped vial, and add 0.5 mL of the Derivatization reagent. Allow a reaction time of NLT 2 min before injection into the chromatograph.
Sample solution:
Pulverize a counted number of Tablets, equivalent to 50 mg of alliin, with a mortar and pestle. Transfer a quantity of powder equivalent to 5 mg of alliin to a 100-mL volumetric flask. Add 70 mL of Alliinase inhibitor solution, and shake for 1 min. Dilute with Alliinase inhibitor solution to volume. Use a volumetric syringe to transfer 0.1 mL of this solution to a septum-capped vial, and add 0.5 mL of the Derivatization reagent. Allow a reaction time of NLT 2 min before injection into the chromatograph.
Chromatographic system
Mode:
LC
Detector:
UV 337 nm
Column:
4-mm × 10-cm; packing L1
Flow rate:
1 mL/min
[Note—Alliin exhibits two major peaks, representing its diastereomers. ]
Injection size:
10 µL
System suitability
Sample:
Standard solution
Suitability requirements
Relative standard deviation:
NMT 2.0% for each of the major peaks
Analysis
Samples:
Standard solution and Sample solution
[Note—Record the chromatograms, and measure the areas of the responses of the alliin diastereomer peaks. ]
Calculate the percentage of alliin in the portion of Tablets taken:
Result = (rU/rS) × (CS/CU) × 100
| rU | = | = peak area for alliin from the Sample solution |
| rS | = | = sum of the peak area for alliin diastereomers from the Standard solution |
| CS | = | = concentration of USP Alliin RS in the Standard solution (µg/mL) |
| CU | = | = nominal concentration of alliin in the Sample solution (µg/mL) |
Acceptance criteria:
90%–140.0%
• Content of Potential Allicin
Alliinase inhibitor solution:
Dissolve 109 mg of carboxymethoxylamine hemihydrochloride in 100.0 mL of water.
Crude alliinase solution:
Homogenize 5 g of raw garlic cloves with 25 mL of water. Filter, and extract three times with 50 mL of tert-butyl methyl ether. Discard the organic phase, and remove the residual solvent from the aqueous phase by rotary evaporation in vacuum for 5 min. Filter, and store frozen in small vials. [Note—This solution is stable for 6 months when stored as directed. Thaw at room temperature just before use. ]
Mobile phase:
Methanol and water (3:2)
Standard stock solution:
50 µg/mL of USP Alliin RS
Standard solution:
Transfer 1.0 mL of the Standard stock solution to a 5-mL volumetric flask containing 100 µL of Crude alliinase solution, and allow to stand for 5 min at room temperature. Dilute with water to volume, and pass through a filter having a 0.45-µm or finer pore size.
Sample solution:
Transfer an equivalent to 5 mg of potential allicin, from finely powdered Tablets (NLT 20), to a 200-mL volumetric flask, and add 25 mL of water. Incubate at room temperature for exactly 30 min. Stop the enzymatic reaction by diluting with Alliinase inhibitor solution to volume. Centrifuge a portion of this solution, transfer 1.0 mL of the supernatant to a 5-mL volumetric flask, and dilute with water to volume.
Blank solution:
100 µL of Crude alliinase solution diluted with water to 1 mL
Chromatographic system
Mode:
LC
Detector:
UV 240 nm
Column:
4.6-mm × 25-cm; packing L1
Flow rate:
1 mL/min
Injection size:
100 µL
System suitability
Samples:
Standard solution, Sample solution, and Blank solution
[Note—The allicin peak is identified by comparing the chromatograms of the Blank solution and the Standard solution. ]
Suitability requirements
Resolution:
NLT 2.0 between the allicin peak and the preceding peak at a relative retention time of 0.80 (allyl methyl thiosulfinates), Sample solution
Relative standard deviation:
NMT 2.0%
Analysis
Samples:
Standard solution, Sample solution, and Blank solution
Calculate the percentage of potential allicin in the portion of Tablets taken:
Result = (rU/rS) × (CS/CU) × (Mr1/Mr2) × 100
| rU | = | = peak area of allicin, corrected by the response of the Blank solution, from the Sample solution |
| rS | = | = peak area of allicin, corrected by the response of the Blank solution, from the Standard solution |
| CS | = | = concentration of USP Alliin RS in the Standard solution (µg/mL) |
| CU | = | = nominal concentration of potential allicin in the Sample solution (µg/mL) |
| Mr1 | = | = molecular weight of allicin, 162.26 |
| Mr2 | = | = twice the molecular weight of alliin, 354.42 |
Acceptance criteria:
90.0%–140.0%
PERFORMANCE TESTS
• Allicin Release:
Proceed as directed in Dissolution 
711
for Method A in Apparatus 1 and Apparatus 2, Delayed-Release Dosage Forms. Place a number of Tablets, equivalent to 5 mg of potential allicin, in each vessel.
711 for Method A in Apparatus 1 and Apparatus 2, Delayed-Release Dosage Forms. Place a number of Tablets, equivalent to 5 mg of potential allicin, in each vessel.
Apparatus 2:
100 rpm
Time:
60 min for the Buffer stage
Crude alliinase solution, Mobile phase, Blank solution, and Chromatographic system:
Proceed as directed in the test for Content of Potential Allicin.
Standard stock solution:
50 µg/mL of USP Alliin RS
Standard solution:
Transfer 1.0 mL of the Standard stock solution to a 5-mL volumetric flask containing 100 µL of Crude alliinase solution, and allow to stand for 5 min at room temperature. Dilute with water to volume, and pass through a membrane filter having a 0.45-µm or finer pore size.
Sample solution:
Transfer 1.0 mL of the solution under test to a test tube containing 50 µL of 0.21 M carboxymethoxylamine hemihydrochloride solution.
[Note—The solution must be transferred immediately upon removal from the dissolution vessel to inhibit the alliinase enzyme. ]
Injection size:
100 µL
Analysis
[Note—Do not perform the allicin determination in the Acid stage. ]
Samples:
Standard solution and Sample solution
Calculate the percentage of potential allicin released in the Buffer stage:
Result = (rU/rS) × (CS × D × V/L) × (Mr1/Mr2) × 100
| rU | = | = peak area of allicin from the Sample solution |
| rS | = | = peak area of allicin from the Standard solution |
| CS | = | = concentration of USP Alliin RS in the Standard solution (µg/mL) |
| D | = | = dilution factor for the Sample solution, 1.050 (1 mL of the Sample solution + 50 µL of 0.21 M carboxymethoxylamine hemihydrochloride solution) |
| V | = | = volume of final medium, 1000 mL |
| L | = | = labeled amount of potential allicin (µg/Tablet) |
| Mr1 | = | = molecular weight of allicin, 162.26 |
| Mr2 | = | = twice the molecular weight of alliin, 354.42 |
Tolerances:
It meets the requirements of Acceptance Table 4 in Dissolution 711. [Note—Q is the percentage of the labeled amount of potential allicin released only in the Buffer stage. ]
711. [Note—Q is the percentage of the labeled amount of potential allicin released only in the Buffer stage. ]
• Weight Variation 2091:
Meet the requirements
2091:
Meet the requirements
SPECIFIC TESTS
• Alliinase Activity
Alliinase inhibitor solution, Solution A, Buffer, Derivatization reagent, Mobile phase, Standard solution, and Chromatographic system:
Proceed as directed in the test for Content of Alliin.
Sample solution:
Transfer an equivalent to 5 mg of alliin, from finely powdered Tablets (NLT 20), to a 100-mL volumetric flask, and add 25 mL of water. Incubate at room temperature for exactly 5 min. Stop the enzymatic reaction by diluting with Alliinase inhibitor solution to volume. Centrifuge a portion of this solution, and use a volumetric syringe to transfer 0.1 mL of the supernatant to a septum-capped vial. Add 0.5 mL of the Derivatization reagent, and allow a reaction time of NLT 2 min before injection into the chromatograph.
Analysis
Samples:
Standard solution and Sample solution
Proceed as directed in the test for Content of Alliin.
Acceptance criteria:
The area of the alliin peak from the Sample solution is NMT 1% of the area of the alliin peak from the Standard solution.
ADDITIONAL REQUIREMENTS
• Packaging and Storage:
Preserve in tight containers.
• Labeling:
The label states the Latin binomial and, following the official name, the article from which the Tablets were prepared. Label it to indicate the amount of total alliin, in µg/Tablet, and the amount of potential allicin, in µg/Tablet.
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Auxiliary Information—
Please check for your question in the FAQs before contacting USP.
| Topic/Question | Contact | Expert Committee |
|---|---|---|
| Monograph | Natalia Davydova
Scientific Liaison 1-301-816-8328 |
(DS2010) Monographs - Dietary Supplements |
| Reference Standards | RS Technical Services 1-301-816-8129 rstech@usp.org |
USP35–NF30 Page 1315
Pharmacopeial Forum: Volume No. 31(1) Page 159