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Powdered Phyllanthus amarus
DEFINITION
Powdered Phyllanthus amarus is Phyllanthus amarus reduced to a powder or very fine powder. It contains NLT 0.25% of lignans, calculated as the sum of phyllanthin and hypophyllanthin, on the dried basis.
IDENTIFICATION
•  A. Powdered Phyllanthus amarus meets the requirements in Specific Tests for Botanic Characteristics.
•  B. Thin-Layer Chromatographic Identification Test 201
Standard solution A:  0.1 mg/mL of USP Phyllanthin RS in methanol
Standard solution B:  10 mg/mL of USP Powdered Phyllanthus amarus Extract RS in methanol. Sonicate for about 10 min, centrifuge, and use the supernatant.
Sample solution:  Sonicate about 0.5 g of Powdered Phyllanthus amarus in 5 mL of methanol for 10 min, centrifuge, and use the supernatant.
Adsorbent:  Chromatographic silica gel with an average particle size of 10–15 µm (TLC plates) or with an average particle size of 5 µm (HPTLC plates)
Application volume:  10 µL (TLC plates) or 4 µL (HPTLC plates)
Developing solvent system:  Hexane and ethyl acetate (2:1)
Spray reagent:  A solution of 10% sulfuric acid in methanol. [Note—Prepare fresh. ]
Analysis 
Samples:  Standard solution A, Standard solution B, and Sample solution
Apply the samples as bands to a suitable thin-layer chromatographic plate (see Chromatography 621). Use a saturated chamber. Develop the chromatograms until the solvent front has moved up about three-fourths of the plate. Remove the plate from the chamber, dry, spray with the Spray reagent, heat for 3 min at 120, and examine under visible light.
Acceptance criteria:  The chromatogram of the Sample solution exhibits a blue band in the lower third of the plate due to phyllanthin, corresponding in color and RF to that in the chromatogram of Standard solution A; a violet band due to hypophyllanthin at an RF higher than that of phyllanthin; a blue band at an RF higher than that of hypophyllanthin; and an additional violet band in the upper third of the plate. Bands detected in the chromatogram of the Sample solution correspond in position and color to bands in the chromatogram of Standard solution B. Other minor bands may be observed in the chromatograms of the Sample solution and Standard solution B.
•  C. HPLC: The chromatogram of the Sample solution obtained in the test for Content of Lignans shows a peak at a retention time corresponding to that of phyllanthin in the chromatogram of Standard solution A. Identify other lignan peaks in the chromatogram of the Sample solution by comparison with the chromatogram of Standard solution B and the reference chromatogram provided with the lot of USP Powdered Phyllanthus amarus Extract RS being used. The chromatogram of the Sample solution shows an additional peak corresponding to hypophyllanthin.
COMPOSITION
•  Content of Lignans
Solution A:  Dissolve 0.14 g of potassium dihydrogen phosphate in 900 mL of water, add 0.5 mL of phosphoric acid, dilute with water to 1000 mL, mix, filter, and degas.
Mobile phase:  Acetonitrile and Solution A (4:6)
Standard solution A:  0.1 mg/mL of USP Phyllanthin RS in methanol
Standard solution B:  Sonicate a portion of USP Powdered Phyllanthus amarus Extract RS in methanol to obtain a solution having a concentration of about 5.0 mg/mL. Before injection, pass through a membrane filter of 0.45-µm or finer pore size, discarding the first few mL of the filtrate.
Sample solution:  Transfer about 3.0 g of Powdered Phyllanthus amarus, accurately weighed, to a 250-mL flask fitted with a reflux condenser. Add 50 mL of methanol, reflux in a water bath for about 20 min, leave to settle, and decant the supernatant. Repeat until the last extract is colorless. Combine the extracts, concentrate under vacuum, and adjust the volume with methanol to 100 mL. Before injection, pass through a membrane filter of 0.45-µm or finer pore size, discarding the first few mL of the filtrate.
Chromatographic system 
Mode:  LC
Detector:  UV 230 nm
Column:  4.6-mm × 25-cm; 5-µm packing L1
Flow rate:  1.5 mL/min
Injection size:  10 µL
System suitability 
Samples:  Standard solution A and Standard solution B
Suitability requirements 
Chromatogram similarity:  The chromatogram from Standard solution B is similar to the reference chromatogram provided with the lot of USP Powdered Phyllanthus amarus Extract RS being used.
Resolution:  NLT 1.5 between the phyllanthin and hypophyllanthin peaks, Standard solution B
Tailing factor:  NMT 1.5 for the phyllanthin peak, Standard solution A
Relative standard deviation:  NMT 2.0% determined from the phyllanthin peak in repeated injections, Standard solution A
Analysis 
Samples:  Standard solution A, Standard solution B, and Sample solution. [Note—Standard solution A, Standard solution B, and the Sample solution are stable for 48 h at room temperature. ]
Using the chromatograms of Standard solution A, Standard solution B, and the reference chromatogram provided with the lot of USP Powdered Phyllanthus amarus Extract RS being used, identify the retention times of the peaks corresponding to phyllanthin and hypophyllanthin.
Separately calculate the percentages of phyllanthin and hypophyllanthin in the portion of Powdered Phyllanthus amarus taken:
Result = (rU/rS) × CS × (V/W) × F × 100
rU== peak response of the analyte from the Sample solution
rS== peak response of phyllanthin from Standard solution A
CS== concentration of USP Phyllanthin RS in Standard solution A (mg/mL)
V== volume of the Sample solution (mL)
W== weight of Powdered Phyllanthus amarus taken to prepare the Sample solution (mg)
F== conversion factors for the analytes: 1.00 for phyllanthin; 0.75 for hypophyllanthin
Acceptance criteria:  Add the percentages of phyllanthin and hypophyllanthin: NLT 0.25% is found on the dried basis.
CONTAMINANTS
•  Heavy Metals, Method III 231: NMT 20 ppm
•  Microbial Enumeration Tests 2021: The total aerobic bacterial count does not exceed 105 cfu/g, the total combined molds and yeasts count does not exceed 103 cfu/g, and the bile-tolerant Gram-negative bacteria does not exceed 103 cfu/g.
•  Absence of Specified Microorganisms 2022: Meets the requirements of the tests for absence of Salmonella species and Escherichia coli
SPECIFIC TESTS
•  Botanic Characteristics Greenish to greenish-brown in color; taste bitter. Under a microscope, fragments of epidermal cells of the leaves with wavy walls, showing anisocytic and paracytic stomata; parenchyma cells, some showing clusters of calcium oxalate crystals; narrow fibers from the stem; pitted vessels of the stem; fragments of the epicarp of the fruits showing anomocytic stomata
•  Loss on Drying 731: Dry 1.0 g of Powdered Phyllanthus amarus at 105 for 2 h: it loses NMT 12.0% of its weight.
•  Articles of Botanical Origin, Total Ash 561: NMT 8.0%, determined on 1.0 g of Powdered Phyllanthus amarus
•  Articles of Botanical Origin, Acid-Insoluble Ash 561: NMT 5.0%, determined on 1.0 g of Powdered Phyllanthus amarus
ADDITIONAL REQUIREMENTS
•  Packaging and Storage: Preserve in well-closed containers, protected from light and moisture, and store at room temperature.
•  Labeling: The label states the Latin binomial and, following the official name, the parts of the plant contained in the article.
•  USP Reference Standards 11
USP Phyllanthin RS
USP Powdered Phyllanthus amarus Extract RS
USP35
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Topic/Question Contact Expert Committee
Monograph Maged H. Sharaf, Ph.D.
Principal Scientific Liaison
1-301-816-8318
(DS2010) Monographs - Dietary Supplements
2021 Radhakrishna S Tirumalai, Ph.D.
Principal Scientific Liaison
1-301-816-8339
(GCM2010) General Chapters - Microbiology
2022 Radhakrishna S Tirumalai, Ph.D.
Principal Scientific Liaison
1-301-816-8339
(GCM2010) General Chapters - Microbiology
Reference Standards RS Technical Services
1-301-816-8129
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