Sphaeranthus indicus Aerial Parts Dry Extract

Sphaeranthus indicus Aerial Parts Dry Extract

Proposed For Development Version 0.1

Sphaeranthus indicus Aerial Parts Dry Extract

 


 

DEFINITION

 

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

 

POTENTIAL CONFOUNDING MATERIALS

None known

 

CONSTITUENTS OF INTEREST

Sesquiterpene glycoside: Sphaeranthanolide

 

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 supernatant.

Sample solution: Mix 0.1 g of Sphaeranthus indicus Aerial Parts Dry Extract with 10 mL of methanol. Sonicate for 10 min, centrifuge and use 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 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. 20 mL of sulfuric acid is carefully added to an ice-cooled mixture of 180 mL of methanol.

Analysis

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

Apply the samples as bands to a suitable high performance thin-layer chromatographic 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 the visible light, the chromatogram of Standard solution B exhibits a dark brown band right above the origin of chromatogram in position and color similar to sphaeranthanolide band in Standard solution A. Six additional bands appear in the chromatogram with increasing order of RF: a broad dark brown band near origin, a light purple band near one half of the lower half of the chromatogram, a purple band right above the half of the chromatogram, a strong blue band above the purple band, a blue/purple band, and  a strong brown band near front.

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

 

B. HPLC

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

Acceptance criteria: The chromatogram of the Sample solution exhibits peaks 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 in 20 mL of methanol by shaking for 2 hours at 60°. Cool to room temperature, and pass through filter paper into 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. Discard the first few mL of the filtrate.

Sample solution: Transfer about 0.25 g of Sphaeranthus indicus Aerial Parts Dry Extract to a flask, add 20 mL of methanol, and shake for 2 hours at 60°. Cool to room temperature, and pass through filter paper into 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 above solution to 10 mL volumetric flask and dilute with methanol to obtain a solution with a concentration of about 5 mg/mL. Pass solution through membrane filter of 0.45 μm pore size, discard the first few mL of the filtrate.

Chromatographic system

(See Chromatography <621>, System Suitability.)

Detector: UV 206 nm

Column: 4.6-mm × 25-cm; L1 (100 Å)

Flow rate: 1.0 mL/min

Injection volume: 10 µL

System suitability

Sample: 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 Sphaeranthus indicus Aerial Parts Dry Extract being used.

Tailing factor: NMT 2.0 for 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 the Standard solution A

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

CU  = concentration of Sphaeranthus indicus Aerial Parts Dry Extract in Sample solution (mg/mL)

 

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

 

Result = (P/L) × 100

 

P   = content of sphaeranthanolide as determined above (%)

L   = labeled amount of sphaeranthanolide (%)

 

Acceptance criteria: 90%-110% of the labeled amount of sphaeranthanolide, on 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 absence of Salmonella species and Escherichia coli

 

SPECIFIC TESTS

• Loss on Drying <731>:

Analysis: Dry 1 g Sphaeranthus indicus Aerial Parts Dry Extract 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 Sphaeranthus indicus Aerial Parts Dry Extract RS

USP Sphaeranthanolide RS

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