Citrus reticulata Peel Dry Extract

Citrus reticulata Peel Dry Extract

Proposed For Comment Version 0.2

Citrus reticulata Peel Dry Extract


 

DEFINITION

The article is prepared from the dried ripe pericarp of Citrus reticulata Blanco (Family Rutaceae) collected in autumn or winter, by extraction with hydroalcoholic mixtures. It contains NLT 90.0% and NMT 110.0% of the labeled amount of total dihydroflavone glycosides, calculated as the sum of narirutin (C27H32O14), hesperidin (C28H34O15), and didymin (C28H34O14), on the anhydrous basis; NLT 90.0% and NMT 110.0% of the labeled amount of total polymethoxyflavones, calculated as the sum of nobiletin (C21H22O8), 3,5,6,7,8,3',4'- heptamethoxyflavone (C22H24O9), and tangeretin (C20H20O7), on the anhydrous basis. It may contain suitable added substances as carriers.

 

POTENTIAL CONFOUNDING MATERIALS

Dried ripe fruit of Citrus wilsonii Tanaka

Dried unripe pericarp of Citrus maxima (Burm.) Merr. (C. grandis (L.) Osbeck)

 

CONSTITUENTS OF INTEREST

Dihydroflavone glycoside: Narirutin, hesperidin, and didymin

Polymethoxylated flavone: Nobiletin, 3,5,6,7,8,3',4'-heptamethoxyflavone, and tangeretin

 

IDENTIFICATION

• A. HPTLC for Articles of Botanical Origin <203>

Standard solution A: 1.0 mg/mL of USP Hesperidin RS in methanol. Sonicate to dissolve.

Standard solution B: 50 mg/mL of USP Citrus reticulata Pericarp Dry Extract RS in methanol. Sonicate for 20 min, centrifuge, and use the supernatant.

Sample solution: 50 mg/mL of Citrus reticulata Pericarp Dry Extract in methanol. Sonicate for 20 min, centrifuge, and use the supernatant.

Chromatographic system

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

Application volume: Standard solution A, 10 µL; Standard solution B and Sample solution, 5 µL for each, as 10-mm bands

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

Developing solvent system: Ethyl acetate, formic acid, and water (100:15:13)

Developing distance: 8 cm

Derivatization reagent A: 10 mg/mL of 2-aminoethyl diphenylboriate in methanol

Derivatization reagent B: 50 mg/mL of polyethylene glycol 4000 in alcohol

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, remove the plate from the chamber, and dry the plate at 100° for 3 min. Treat the plate with Derivatization reagent A and dry for 5 min with a current of cool air. Immediately, treat the plate with Derivatization reagent B, dry for 5 min with a current of cool air, and examine under UV light at 366 nm.

System suitability: Standard solution A exhibits a dark green band due to hesperidin in the middle-third section. Standard solution B exhibits a band corresponding in RF and color to the band due to hesperidin in Standard solution A, a yellow-green band below hesperidin, a blue band below the yellow-green band, another blue band above hesperidin, and a faint band between hesperidin and the blue band above hesperidin. In the upper-third section, Standard solution B exhibits a bright blue band due to the co-elution of nobiletin with other components, and a green band above the bright blue band.

Acceptance criteria: The Sample solution exhibits a dark green band corresponding in RF and color to the band due to hesperidin in Standard solution A and Standard solution B, a yellow-green band below hesperidin, a blue band below the yellow-green band, another blue band above hesperidin, and a faint band between hesperidin and the blue band above hesperidin corresponding to the same bands in Standard solution B. There is no another dark green band due to naringin above the hesperidin band (distinction from the Citrus wilsonii Fruit, and the Citrus maxima Pericarp; these two plants do not contain hesperidin while containing naringin). In the upper-third section, the Sample solution exhibits a bright blue band and a green band above the bright blue band corresponding in RF and color to the same bands in Standard solution B. In the lower-third section, the Sample solution exhibits a couple of  yellow-green bands close to the starting position and a couple of faint bands above the yellow-green bands corresponding in RF  and color to the same bands in Standard solution B.

• B. HPLC

Analysis: Proceed as directed in the Assay for Content of Dihydroflavone Glycosides and Polymethoxy Flavones.

Acceptance criteria: The Sample solution exhibits the most intense peak at the retention time corresponding to hesperidin in Standard solution A; and peaks due to narirutin, didymin, nobiletin, 3,5,6,7,8,3',4'-heptamethoxyflavone, and tangeretin at retention times corresponding to the same constituents in Standard solution B. Sometimes, the peaks of nobiletin, 3,5,6,7,8,3',4'-heptamethoxyflavone, and tangeretin are much smaller, such that these peaks cannot be detected.

 

ASSAY

• Content of Dihydroflavone Glycosides and Polymethoxy Flavones

Solution A: 0.1% Formic acid in water

Solution B: Acetonitrile

Mobile phase: See Table 1.

Table 1

Time
(min)

Solution A
(%)

Solution B
(%)

Detection Wavelength
(nm)

0

85

15

283

8

81

19

283

10

81

19

283

17

60

40

330

28

56

44

330

 

[Note—The Standard solutions and the Sample solution are stable for 24 h at room temperature.]

Standard solution A: 0.40 mg/mL of USP Hesperidin RS in methanol

Standard solution B: 0.05 mg/mL of USP Nobiletin RS in methanol

Standard solution C: 5 mg/mL of USP Citrus reticulata Pericarp Dry Extract RS in methanol. Sonicate for 15 min, centrifuge, and pass through a suitable membrane filter of 0.22-μm pore size.

Sample solution: 5 mg/mL of Citrus reticulata Pericarp Dry Extract in methanol with a stoppered flask. Sonicate for 15 min, centrifuge, and pass through a suitable membrane filter of 0.22-μm pore size and discard the first portion of the filtrate.

Chromatographic system

(See Chromatography <621>, System Suitability.)

Mode: LC

Detector: UV 283 nm (0–17 min) and 330 nm (17–28 min)

Column: 4.6-mm × 5-cm; 1.8-μm, packing L1 (similar to Agilent Zorbax SB C18)

Column temperature: 25°

Flow rate: 0.7 mL/min

Injection volume: 2 µL

System suitability

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

Suitability requirements

Resolution: NLT 1.5 between the peaks of hesperidin and the small peak before it, Standard solution C

Tailing factor: NMT 1.5 for the hesperidin and nobiletin peaks, Standard solution A and Standard solution B

Relative standard deviation: NMT 2.0% for the hesperidin and nobiletin peaks, Standard solution A and Standard solution B

Chromatogram similarity: The chromatogram of Standard solution C is similar to the reference chromatogram provided with the lot of USP Citrus reticulata Pericarp Dry Extract RS being used.

Analysis

For dihydroflavone glycosides

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

Using the chromatograms of Standard solution A, Standard solution C, and the reference chromatogram provided with the lot of USP Citrus reticulata Pericarp Dry Extract RS being used, identify the retention times of the peaks corresponding to narirutin, hesperidin, and didymin in the Sample solution. [Note—See Table 2 for relative retention times.]

 

Table 2

Analyte

Approximate Relative
Retention Time

Conversion Factor

Narirutin

0.79

1.17

Hesperidin

1.00

1.00

Didymin

1.56

1.00

 

Separately calculate the percentages of narirutin, hesperidin, and didymin in the portion of Citrus reticulata Pericarp Dry Extract taken:

 

Result = (rU/rS) × CS × (V/W) × F × 100

 

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

rS      = peak area of hesperidin from Standard solution A

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

V     = volume of the Sample solution (mL)

W    = weight of Citrus reticulata Pericarp Dry Extract taken to prepare the Sample solution (mg)

F     = conversion factor for the analyte (see Table 2)

 

Calculate the content of total dihydroflavone glycosides as the sum of the percentages of narirutin, hesperidin, and didymin.

 

Calculate the percentage of the labeled amount of total dihydroflavone glycosides in the portion of Citrus reticulata Pericarp Dry Extract taken:

 

Result = (P/L) × 100

 

P    = content of total dihydroflavone glycosides, as determined above (%)

L    = labeled amount of total dihydroflavone glycosides (%)

 

For polymethoxylated flavones

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

Using the chromatograms of Standard solution B, Standard solution C, and the reference chromatogram provided with the lot of USP Citrus reticulata Pericarp Dry Extract RS being used, identify the retention times of the peaks corresponding to nobiletin, 3,5,6,7,8,3',4'-heptamethoxyflavone, and tangeretin in the Sample solution. [Note—See Table 3 for relative retention times.]

 

Table 3

Analyte

Approximate Relative
Retention Time

Conversion Factor

Nobiletin

1.00

1.00

3,5,6,7,8,3',4'-Heptamethoxyflavone

1.06

1.32

Tangeretin

1.12

0.88

 

 

Separately calculate the percentages of nobiletin, 3,5,6,7,8,3',4'-heptamethoxyflavone, and tangeretin in the portion of Citrus reticulata Pericarp Dry Extract taken:

 

Result = (rU/rS) × CS × (V/W) × F × 100

 

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

rS      = peak area of nobiletin from Standard solution B

CS    = concentration of USP Nobiletin RS in Standard solution B (mg/mL)

V     = volume of the Sample solution (mL)

W    = weight of Citrus reticulata Pericarp Dry Extract taken to prepare the Sample solution (mg)

F     = conversion factor for the analyte (see Table 3)

 

Calculate the content of total polymethoxylated flavones as the sum of the percentages of nobiletin, 3,5,6,7,8,3',4'-heptamethoxyflavone, and tangeretin.

 

Calculate the percentage of the labeled amount of total polymethoxylated flavones in the portion of Citrus reticulata Pericarp Dry Extract taken:

 

Result = (P/L) × 100

 

= content of total polymethoxylated flavones, as determined above (%)

= labeled amount of total polymethoxylated flavones (%)

 

Acceptance criteria

Total dihydroflavone glycosides: 90.0%–110.0% of the labeled amount on the anhydrous basis

Total polymethoxylated flavones: 90.0%–110.0% of the labeled amount on the anhydrous basis

 

CONTAMINANTS

• Elemental Impurities—Procedures <233>

Acceptance criteria

Arsenic: NMT 2.0 µg/g

Cadmium: NMT 0.5 µg/g

Lead: NMT 5.0 µg/g

Mercury: NMT 0.2 µg/g

• Botanical Extracts <565>, Preparations, General Pharmacopeial Requirements, Pesticide Residues: Meets the requirements

• Microbial Enumeration Tests <61>: The total aerobic bacterial count does not exceed 104 cfu/g, the total combined molds and yeasts count 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

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

• Articles of Botanical Origin <561>, Methods of Analysis, Total Ash: NMT 4.0%

 

ADDITIONAL REQUIREMENTS

• Packaging and Storage: Preserve in well-closed containers, protected from light and moisture, and store at controlled room temperature.

• Labeling: The label states the Latin binomial following the official name of the plant from which the article was prepared. It meets other labeling requirements in Botanical Extracts <565>.

• USP Reference Standards <11>

USP Citrus reticulata Pericarp Dry Extract RS

USP Hesperidin RS

USP Nobiletin RS

 

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Other Versions

Proposed For Development Version 0.1