Trigonella foenum-graecum Seed 4-Hydroxyisoleucine Powdered Extract

Trigonella foenum-graecum Seed 4-Hydroxyisoleucine Dry Extract

Proposed For Comment Version 0.2

Trigonella foenum-graecum Seed 4-Hydroxyisoleucine Powdered Extract


 

DEFINITION

The article is a fraction of an extract of the dried ripe seeds of Trigonella foenum-graecum L. (Family Fabaceae). The extract is prepared using hydroalcoholic mixtures then further enriched in 4-hydroxyisoleucine using suitable means. It contains NLT 90.0% and NMT 110.0% of the labeled amount of 4-hydroxyisoleucine, calculated on the anhydrous basis.

 

POTENTIAL CONFOUNDING MATERIALS

None known

 

CONSTITUENTS OF INTEREST

Amino acids: 4-Hydroxyisoleucine, 4-hydroxyisoleucine lactone, arginine, histidine, and lysine

 

IDENTIFICATION

Thin-Layer ChromatographyAmino Acids Profile

Standard solution A: 0.5 mg/mL of USP 4-Hydroxyisoleucine RS in methanol

Standard solution B: 50 mg/mL of USP Trigonella foenum-graecum Seed Powdered Extract RS in methanol. Sonicate for 10 min, centrifuge, and use the supernatant.

Sample solution: 2.5 mg/mL of Trigonella foenum-graecum Seed 4-Hydroxyisoleucine Powdered Extract in methanol. Sonicate for 10 min, centrifuge, and use the supernatant.

Chromatographic system

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

Adsorbent: Chromatographic silica gel mixture with an average particle size of 5 µm (HPTLC plates)

Application volume: 2 µL each of Standard solution A and Standard solution B, and 4 µL of Sample solution, as 8-mm bands

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

Temperature: 25°

Developing solvent system: n-Butanol, acetic acid, and water (7:2:1)

Developing distance: 6 cm

Derivatization reagent: Ninhydrin reagent created by combining 0.3 g of ninhydrin, 95 mL of isopropanol, and 5 mL of glacial acetic acid.

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 for 2 min at 100º–105°, and examine under visible light and UV light at 366 nm.

System suitability: Under visible light, the chromatogram of the Standard solution B exhibits, in the lower half, five brown bands in the following order of increasing RF: a minor band, the most intense band at an RF corresponding to the 4-hydroxyisoleucine band in the chromatogram of Standard solution A, and three less intense bands.

Under UV light at 366 nm, the chromatogram of Standard solution B exhibits, in the lower half, four bands in the following order of increasing RF: a minor dark brown band, the most intense band as a dark brown band at an RF corresponding to the 4-hydroxyisoleucine band in the chromatogram of Standard solution A, and two less intense purple bands. The chromatogram of Standard solution B exhibits, in the upper half, three lemon-yellow bands, the one with the lowest RF  appears diffuse and, in cases, may appear resolved into two bands.

Acceptance criteria: Under visible light, the chromatogram of the Sample solution exhibits, in the lower half, the most intense band as a brown band at an RF  corresponding to the 4-hydroxyisoleucine band in the chromatogram of Standard solution A, and four additional brown bands corresponding to similar bands in the chromatogram of Standard solution B: a minor band at an RF  below that of the 4-hydroxyisoleucine, and three less intense bands above the 4-hydroxyisoleucine band.

Under UV light at 366 nm, the chromatogram of the Sample solution exhibits, in the lower half, the most intense band as a dark brown band at an RFcorresponding to the 4-hydroxyisoleucine band in the chromatogram of Standard solution A, and three additional bands corresponding to similar bands in the chromatogram of Standard solution B: a minor dark brown band at an RF below that of the 4-hydroxyisoleucine, and two less intense purple bands above the 4-hydroxyisoleucine band. The chromatogram of the Sample solution exhibits, in the upper half, three yellow bands corresponding to similar bands in the chromatogram of Standard solution B.

 

ASSAY

• Content of 4-Hydroxyisoleucine

Solution A: 0.1% Phosphoric acid in water (v/v)

Solution B: Acetonitrile

Mobile phase: See Table 1.

 

Table 1

Time
(min)

Solution A
(%)

Solution B
(%)

0

80

20

20

40

60

 

Reagent: A mixture of acetonitrile, water, and triethylamine (40:12:8)

Diluent: Methanol and water (1:1) 

Standard solution: Transfer about 2.0 mg of USP 4-Hydroxyisoleucine RS, accurately weighed, to a 100-mL volumetric flask, dissolve in 10 mL of Reagent, add 500 µL of phenyl isothiocyanate, and shake for 5 min. Add 60 mL of methanol, complete to volume with water, and mix.

Sample stock solution: Transfer about 60 mg of Trigonella foenum-graecum Seed 4-Hydroxyisoleucine Powdered Extract, accurately weighed, to a 10-mL volumetric flask, add 8 mL of Diluent, sonicate to dissolve, cool, complete to volume with Diluent, mix, and filter.

Sample solution: Transfer 2.0 mL of Sample stock solution to a 100-mL volumetric flask, add 10 mL of Reagent and 500 µL of phenyl isothiocyanate, and shake for 5 min.  Add 60 mL of methanol, complete to volume with water, and mix. 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

(See Chromatography <621>, System Suitability.)

Mode: LC

Detector: UV 254 nm

Column: 4.6-mm × 15-cm; 5-µm packing L1 (Similar to Zorbax Eclipse XD-C18, Luna C18(2) and Cosmosil C18-MS-II)

Flow rate: 1.5 mL/min

Injection volume: 20 µL

System suitability

Sample: Standard solution

Suitability requirements

Tailing factor: NMT 2.0 for the 4-hydroxyisoleucine peak, Standard solution

Relative standard deviation: NMT 2.0%, determined from the 4-hydroxyisoleucine peak in repeated injections, Standard solution

Analysis

Samples: Standard solution and Sample solution

Using the chromatogram of Standard solution, identify the retention time of the peak corresponding to 4-hydroxyisoleucine in the Sample solution chromatogram.

Calculate the percentage of 4-hydroxyisoleucine in the portion of Trigonella foenum-graecum Seed 4-Hydroxyisoleucine Powdered Extract taken:

 

Result = (rU/rS) × (CS/CU) × d × 100

 

rU    = peak area of 4-hydroxyisoleucine from the Sample solution

r   = peak area of 4-hydroxyisoleucine from Standard solution

CS   = concentration of 4-hydroxyisoleucine in Standard solution (mg/mL)

CU   = concentration of Trigonella foenum-graecum Seed 4-Hydroxyisoleucine Powdered Extract in the Sample stock solution (mg/mL)

d      = dilution factor to prepare the Sample solution from the Sample stock solution

 

Calculate the percentage of the labeled amount of 4-hydroxyisoleucine in the Extract:

 

Result = (P/L) × 100

 

P    = content of 4-hydroxyisoleucine as determined above (%)

L    = labeled amount of 4-hydroxyisoleucine (%)

Acceptance criteria: 90.0%–110.0% of the labeled amount of 4-hydroxyisoleucine, on the anhydrous basis

 

CONTAMINANTS

• Elemental Impurities—Procedures <233>

Acceptance criteria

Arsenic: NMT 2.0 µg/g

Cadmium: NMT 1.0 µg/g

Lead: NMT 10.0 µg/g

Mercury: NMT 1.0 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 104 cfu/g and the total combined molds and yeasts count does not exceed 102 cfu/g.

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

 

SPECIFIC TESTS

• Water Determination, Method Ia <921>: NMT 6.0%

 

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, following the official name, the part of the plant from which the article was derived. It meets other labeling requirements under Botanical Extract <565>.

• USP Reference Standards <11>

USP 4-Hydroxyisoleucine RS

USP Trigonella foenum-graecum Seed Powdered Extract RS

The commenting period for this monograph has expired.