Vitex negundo Leaf Dry Extract

Vitex negundo Leaf Dry Extract

Final Authorized Version 1.0

Vitex negundo Leaf Dry Extract


 

DEFINITION

The article is prepared from the dried leaves of Vitex negundo L. (Family Lamiaceae) by extraction with a hydroalcoholic mixture. It contains NLT 90.0% and NMT 110.0% of the labeled amount of iridoid glycosides calculated as the sum of agnuside and negundoside on the dried basis.

 

POTENTIAL CONFOUNDING MATERIALS

Vitex agnus-castus L.

Vitex rotundifolia L.f

Vitex trifolia L.

 

CONSTITUENTS OF INTEREST

Iridoid glycosides: Negundoside, nishindaside, and agnuside

 

IDENTIFICATION

• A. Thin-Layer Chromatography

Standard solution A: 0.5 mg/mL of USP Negundoside RS in methanol

Standard solution B: 50 mg/mL of USP Vitex negundo Leaf Dry Extract RS in methanol. Sonicate for 10 min, centrifuge, and use the supernatant.

Sample solution: Vitex negundo Leaf Dry Extract at the concentration equivalent to 0.5 mg/mL of negundoside according to the label claim. Sonicate if necessary.

Chromatographic system

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

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

Application volume: 10 µL each of Standard solution A, Standard 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: Ethyl acetate, acetic acid, and water (80:10:5)

Developing distance: 6 cm

Derivatization reagent: Anisaldehyde-sulfuric acid reagent

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 with Derivatization reagent, heat at 100° for 3 min, and examine under visible light.

System suitability: Under visible light, Standard solution B exhibits six bands in the lower half and about five purple bands in the upper half. The bands in the lower half include the following bands, with increasing RF: two weak gray-brown, one pink, one dark-brown, one strong pink, and one pink. The strong pink band RF corresponds to the band due to negundoside in Standard solution A

Acceptance criteria: Under visible light, the Sample solution exhibits a strong pink band in the lower half that corresponds to the band due to negundoside in Standard solution A, and a dark-brown band below the strong pink band due to agnuside. The following bands, with increasing RF, correspond to similar bands in the lower half of Standard solution B: two weak gray-brown, one pink, one dark-brown, one strong pink, and one pink.

• B. HPLC

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

Acceptance criteria: The Sample solution exhibits peaks at retention times corresponding to the peaks due to negundoside and agnuside in Standard solution B.

 

ASSAY

• Content of Iridoid Glycosides

Solution A: Dissolve 0.14 g of potassium phosphate monobasic in 900 mL of water, add 0.5 mL of o-phosphoric acid, complete to 1 L with water, and mix.

Solution B: Acetonitrile

Mobile phase: See Table 1

 

Table 1

Time
(min)

Solution A
(%)

Solution B
(%)

0.01

90

10

10

85

15

20

80

20

23

80

20

25

85

15

30

90

10

35

90

10

 

Standard solution A: 1.0 mg/mL of USP Negundoside RS in methanol

Standard solution B: Heat 2 g of USP Vitex negundo Leaf Dry Extract RS in 75 mL of methanol at 70° in a water bath for 10 min, centrifuge, and use the supernatant.

Sample solution: Vitex negundo Leaf Dry Extract in methanol at the concentration equivalent to 1.0 mg/mL of negundoside according to the label claim. Sonicate if necessary. Before injection, pass through a membrane filter of 0.45-µm or finer pore size. Discard the first few mL of the filtrate.

Chromatographic system

(See Chromatography <621>, System Suitability.)

Detector: UV 254 nm

Column: 4.6-mm × 25-cm; L1 (similar to Hibar 250-4.6 HPLC Column, Lichrospher 100 RP-18)

Flow rate: 1.5 mL/min

Injection volume: 20 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 Vitex negundo Leaf Dry Extract RS being used.

Resolution: 17.6 between negunsodie and agnuside peaks, Standard solution B

Tailing factor: NMT 1.5, Standard solution A

Relative standard deviation: NMT 2.0%, Standard solution A

Analysis

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

Using the chromatograms of Standard solution A, Standard solution B, and the reference chromatogram provided with the lot of USP Vitex negundo Leaf Dry Extract RS being used, identify the retention times of the peaks corresponding to negundoside and agnuside. The approximate relative retention times of the peaks for negundoside and agnuside are 0.7 and 1.0, respectively.

Separately calculate the percentage of negundoside and agnuside in the portion of Vitex negundo Leaf Leaf Dry Extract taken:

 

Result = (rU/rS) × (CS/CU) × 100

 

rU    = peak area of the relevant analyte in the Sample solution

rS    = peak area of negundoside in Standard solution A

CS   = concentration of negundoside in Standard solution A (mg/mL)

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

Add the percentages of agnuside and negundoside.

 

Calculate the percentage of the labeled amount of iridoid glycosides in the Dry Extract:

 

Result = (P/L) × 100

 

P    = content of negundoside and agnuside as determined above (%)

L     = labeled amount of iridoid glycosides: negundoside and agnuside (%)

Acceptance criteria: 90.0%–110.0% on the dried basis

 

CONTAMINANTS

Elemental Impurities—Procedures <233>

Acceptance criteria

Arsenic: NMT 2.0 µg/g

Cadmium: NMT 1.0 µg/g

Lead: NMT 5.0 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: 1.0 g of Vitex negundo Leaf Dry Extract

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

Acceptance criteria: NMT 5%

 

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 parts of the plant contained in the article.

USP Reference Standards <11>

USP Negundoside RS

USP Vitex negundo Leaf Dry Extract RS

 

Other Versions

Proposed For Comment Version 0.2
Proposed For Development Version 0.1