Sphaeranthus indicus Aerial Parts Dry Extract

Sphaeranthus indicus Aerial Parts Dry Extract

Proposed For Comment Version 0.2

Sphaeranthus indicus Aerial Parts Dry Extract

 


 

DEFINITION

The article consists of aerial parts of Sphaeranthus indicus L. (Family: Asteraceae) by extraction with a hydroalcoholic mixture. It contains NLT 90% and NMT 110% of the labeled amount of sphaeranthanolide, calculated on the dried basis.

 

POTENTIAL CONFOUNDING MATERIALS

None known

 

CONSTITUENTS OF INTEREST

Sesquiterpene glycoside: Sphaeranthanolide

Sterol glycoside: 7-hydroxyfrullanolide

 

IDENTIFICATION

• A. Thin-Layer Chromatography

Standard solution A: 1 mg/mL of USP Sphaeranthanolide RS in methanol

Standard solution B: Dissolve 0.1 g of USP Sphaeranthus indicus Aerial Parts Dry Extract RS in 10 mL of methanol with sonication for 10 min. Centrifuge and use the supernatant.

Sample solution: Mix 0.1 g of Dry Extract with 10 mL of methanol. Sonicate for 10 min, centrifuge, and use the supernatant.

Chromatographic system

(See Chromatography <621>, Thin-Layer Chromatography.)

Adsorbent: Use a suitable chromatographic material with an average particle size of 5 µm (HPTLC plates).

Application volume: 5 µL each of Standard solution AStandard solution B, and Sample solution, as 8-mm bands

Relative humidity: Condition the plate to a relative humidity of about 33% using a suitable device.

Developing solvent system: Dichloromethane and methanol (70: 9.2)

Developing distance: 7 cm

Derivatization reagent: Sulfuric acid reagent created by carefully adding 20 mL of sulfuric acid to 180 mL of ice-cooled methanol.

Analysis

Samples: Standard solution A, Standard solution B, and Sample solution

Apply the Samples as bands to a suitable HPTLC plate and dry in air. Develop the chromatograms in a saturated chamber, remove the plate from the chamber, and dry. Treat the plate with Derivatization reagent by dipping, heat for 5 min at 105°, and examine under visible light.

System suitability: Under visible light, the chromatogram of Standard solution B exhibits a dark brown band right above the origin in position and color similar to the sphaeranthanolide band in the chromatogram of Standard solution A. Six additional bands appear with increasing order of RF: a broad dark brown band near the origin, a light purple band near one half of the lower half, a purple band right above the half, a strong blue band above the purple band, a blue/purple band, and a strong brown band near the front.

Acceptance criteria: Under visible light, the chromatogram of Sample solution exhibits a band due to sphaeranthanolide corresponding in color and RF to the band in the chromatogram of Standard solution A. The following bands, with increasing RF, correspond to the similar bands in the chromatogram of Standard solution B: a broad dark brown band near the origin, a light purple band in two thirds of the lower half, a purple band right above the half, a strong blue band, a blue/purple band, and a strong brown band near the front.

• B. HPLC

Analysis: Proceed as directed in the Assay for Content of Sphaeranthanolide.

Acceptance criteria: The chromatogram of the Sample solution exhibits a peak at the retention time corresponding to the peak due to sphaeranthanolide in Standard solution B.

 

ASSAY

• Content of Sphaeranthanolide

Solution A: Acetonitrile

Solution B: Water

Mobile phase: See Table 1.

 

Table 1

Time
(min)

Solution A
(%)

Solution B
(%)

0

5

95

20

100

0

25

100

0

27

5

95

30

5

95

 

Standard solution A: 0.01 mg/mL of USP Sphaeranthanolide RS in methanol

Standard solution B: Dissolve 0.25 g of USP Sphaeranthus indicus Aerial Parts Dry Extract RS in 20 mL of methanol by shaking for 2 h at 60°. Cool to room temperature, pass through filter paper into a 50-mL volumetric flask, and fill with methanol. Dilute with methanol to obtain a solution with a concentration of about 0.5 mg/mL. Pass the solution through a membrane filter of 0.45-µm pore size, discarding the first few mL of the filtrate.

Sample solution: Transfer about 0.25 g of Dry Extract to a flask, add 20 mL of methanol, and shake for 2 h at 60°. Cool to room temperature and pass through filter paper into a 50-mL volumetric flask. Wash the flask and the residue on the filter with methanol, dilute with the washings, and mix. Transfer 1 mL of the solution to a 10-mL volumetric flask and dilute with methanol to obtain a solution with a concentration of about 5 mg/mL. Pass the solution through a membrane filter of 0.45-μm pore size, discarding the first few mL of the filtrate.

Chromatographic system

(See Chromatography <621>, System Suitability.)

Detector: UV 206 nm

Column: 4.6-mm × 25-cm; 5-µm packing L1 (similar to Zorbax XDB C18)

Flow rate: 1.0 mL/min

Injection volume: 10 µL

System suitability

Samples: Standard solution A and Standard solution B

Suitability requirements

Chromatogram similarity: The chromatogram of Standard solution B is similar to the reference chromatogram provided with the lot of USP Sphaeranthus indicus Aerial Parts Dry Extract RS being used.

Tailing factor: NMT 2.0 for the sphaeranthanolide peak, Standard solution A

Relative standard deviation: NMT 2.0%, Standard solution A

Analysis

Samples: Standard solution A, Standard solution B, and Sample solution

Calculate the percentage of sphaeranthanolide in the portion of Sphaeranthus indicus Aerial Parts Dry Extract taken:

 

Result = (rU/rS) × (CS/CU) × 100

 

rU   = peak area for sphaeranthanolide from the Sample solution

rS   = peak area for sphaeranthanolide from Standard solution A

CS  = concentration of the USP Sphaeranthanolide RS in Standard solution A (mg/mL)

CU  = concentration of Dry Extract in the Sample solution (mg/mL)

 

Calculate the percentate of the labeled amount of sphaeranthanolide in the Dry Extract:

 

Result = (P/L) × 100

 

P   = content of sphaeranthanolide as determined above (%)

L   = labeled amount of sphaeranthanolide (%)

Acceptance criteria: 90%–110% on the dried basis

 

CONTAMINANTS

• Elemental Impurities—Procedures <233>

Acceptance criteria

Arsenic: NMT 2.0 µg/g

Cadmium: NMT 0.5 µg/g

Lead: NMT 5 µg/g

Mercury: NMT 0.2 µg/g

• Articles of Botanical Origin, General Method for Pesticide Residues Analysis <561>: Meets the requirements

• Microbial Enumeration Tests <61>: 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.

• Tests for Specified Microorganisms <62>: Meets the requirements of the tests for the absence of Salmonella species and Escherichia coli

 

SPECIFIC TESTS

• Loss on Drying <731>

Sample: 1 g of Sphaeranthus indicus Aerial Parts Dry Extract

Analysis: Dry the Sample at 105° for 2 h.

Acceptance criteria: NMT 20%

 

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 the part(s) of the plant contained in the article.

• USP Reference Standards <11>

USP Sphaeranthanolide RS

USP Sphaeranthus indicus Aerial Parts Dry Extract RS

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