Mucuna pruriens Seed Powder

Mucuna pruriens Seed Powder

Proposed For Development Version 0.1

 

Mucuna pruriens Seed Powder

 


 

DEFINITION

The article consists of the dried seeds of Mucuna pruriens (L.) DC. (Family Fabaceae), reduced to a fine or very fine powder. It contains NLT 3.0% of levodopa on the dried basis.

 

POTENTIAL CONFOUNDING MATERIALS

None known

 

CONSTITUENTS OF INTEREST

Amino acid: Levodopa

Alkaloids: Mucunine, mucunadine, prurienidine, and prurieninine

 

IDENTIFICATION

• A. Botanical Characteristics

Macroscopic: Pale cream-colored powder

Microscopic: Shows fragments of testa with palisade-like cells thin-walled parenchyma, reticulate and pitted vessels, aleurone and starch grains small, simple, rounded to oval measuring 6–41 μm in diameter, but not over 45 μm in diameter.

• B. Thin-Layer Chromatography

Standard solution A: 0.5 mg/mL of USP Levodopa RS in 30% phosphoric acid

Standard solution B: Sonicate an amount of USP Mucuna pruriens Seed Powder RS with 1/5 the total volume of 30% phosphoric acid for 5 min. add 4/5 of the total volume of methanol and mix by vortex to prepare 20 mg/mL solution. Centrifuge and use the supernatant.

Sample solution: Mix 1 g of Mucuna pruriens Seed Powder with 10 mL of 30% phosphoric acid in a 50-mL centrifuge tube. Sonicate for 5 min, add 40 mL of methanol, and mix by vortex. Centrifuge and use the supernatant.

Chromatographic system

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

Adsorbent: Chromatographic silica gel F254 mixture with an average particle size of 5 µm

Application volume: 10 µL as 8-mm bands

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

Developing solvent system: Butanol, alcohol, acetic acid, and water (60:32:12:8)

Developing distance: 8 cm

Derivatization reagent: 30 mg of ninhydrininto 10 mL of butanol and 0.3 mL of glacial acetic acid.

Analysis

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

Apply the Samples as bands and dry in air. Develop in a saturated chamber (20 min with filter paper), remove the plate from the chamber, and dry in air. Treat the plate with the Derivatization reagent, heat at 100° for 5 min, and examine under white light.

System suitability: Under white light, the chromatogram of Standard solution B exhibits the most intense dark gray or black band with similar RF and color as the levodopa band in the chromatogram of Standard solution A. Three to four dark pink bands appear below the levodopa band and one strong and two weak dark pink bands appear above the levodopa band.

Acceptance criteria: Under white light, the chromatogram of the Sample solution exhibits the most intense dark gray or black band  in the middle of the chromatogram, corresponding in RF to the levodopa band in Standard solution A. The Sample solution exhibits additional bands corresponding to similar bands in Standard solution B, in particular, two weak pink bands: one above and one below the levodopa band.

• C. HPLC

Analysis: Proceed as directed in the test for Content of Levodopa.

Acceptance criteria: The Sample solution exhibits the most intense peak at a retention time corresponding to the peak due to levodopa in Standard solution A and Standard solution B.

 

ASSAY

• Content of Levodopa

Solution A: 1.36 g of monobasic potassium phosphate in 900 mL of water and adjust with phosphoric acid to a pH of 2.5.

Solution B: Methanol

Mobile phase: A mixture of Solution A and Solution B (92:8)

Standard solution: 0.2 mg/mL of USP Levodopa RS prepared as follows: in a volumetric flask, dissolve the USP Levodopa RS in a volume  of 30% phosphoric acid equivalent to 1/20 the flask’s nominal volume and dilute to volume with water.

System suitability solution: 25 mg/mL of USP Mucuna pruriens Seed Powder RS prepared as follows:  Transfer a suitable amount of USP Mucuna pruriens Seed Powder RS to a centrifuge tube. Sonicate for 5 min in a volume of 9 % phosphoric acid equivalent of about 1/20 of the final volume. Dilute with water to final volume, mix well and centrifuge. Before injection, pass through a polyethersulfone membrane filter of 0.45-µm or finer pore size.

Sample solution: Transfer about 2.5 g of Mucuna pruriens Seed Powder, accurately weighed, into a 100-mL centrifuge tube, and add 25 mL of 9% phosphoric acid. Sonicate for 5 min, centrifuge at 3000 rpm for 5 min, and transfer the supernatant to a 100-mL volumetric flask. Repeat the extraction three more times by adding 20 mL of 9% phosphoric acid, and transferring the supernatant to the same 100-mL volumetric flask; dilute with 9% phosphoric acid to volume, and mix. Before injection, pass through a polyethersulfone membrane filter of 0.45-µm or finer pore size and discard the first portion of the filtrate.

Chromatographic system

(See Chromatography <621>, System Suitability.)

Detector: UV 280 nm

Column: 4.6-mm × 25-cm; 5-µm packing L1 (similar to Merck kGaA Purospher Star RP-18)

Column temperature: 30°

Flow rate: 1.0 mL/min

Injection volume: 20 µL

System suitability

Samples: Standard solution and System suitability solution

Suitability requirements

Resolution: NLT 1.5 between levodopa and the peak before it, System suitability solution

Tailing factor: NMT 1.5 for the levodopa peak, Standard solution

Relative standard deviation: NMT 2.0% for the levodopa peak in repeated injections, Standard solution

Analysis

Samples: Standard solution A and Sample solution

Using the chromatograms of Standard solution and the reference chromatogram provided with the lot of USP Mucuna pruriens Seed Powder RS being used, identify the retention times of the peaks corresponding to the peaks due to levodopa in the Sample solution.

Calculate the percentage of levodopa in the portion of Mucuna pruriens Seed Powder taken:

 

Result = (rU/rS) × CS × (V/W) × 100

 

rU   = peak area of levodopa from the Sample solution

rS   = peak area of levodopa from Standard solution

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

V   = volume of the Sample solution (mL)

W   = weight of Mucuna pruriens Seed Powder taken to prepare the Sample solution (mg)

Acceptance criteria: NLT 3.0% of levodopa 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 <561>, Pesticide Residue Analysis: 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

• Articles of Botanical Origin <561>, Methods of Analysis, Foreign Organic Matter: NMT 1.0%

• Articles of Botanical Origin <561>, Methods of Analysis, Alcohol-Soluble ExtractivesMethod 1: NLT 3.0%

• Articles of Botanical Origin <561>, Methods of Analysis,  Water-Soluble ExtractivesMethod 2: NLT 23.0%

• Water Determination <921>Method II: NMT 8.0% 

• Articles of Botanical Origin <561>, Methods of Analysis, Total Ash:  NMT 5.0%

• Articles of Botanical Origin <561>, Methods of Analysis, Acid-Insoluble Ash: NMT 1.0%

 

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 Levodopa RS

USP Mucuna pruriens Seed Powder RS

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