11/9/2020 USP-NF Black Pepper

Printed on: Mon Nov 09 2020, 10:54:52 am

Printed by: Shruti Kharidia
O cial Status: Currently O cial on 09-Nov-2020
O cial Date: O cial as of 1-Dec-2015
Document Type: DIETARY SUPPLEMENTS
DocId: 1_GUID-8464E168-543F-44FE-8AF7-A78C6FC03DED_1_en-US
Printed from: https://online.uspnf.com/uspnf/document/1_GUID-8464E168-543F-44FE-8AF7-A78C6FC03DED_1_en-US
© 2020 USPC

Black Pepper
DEFINITION
Black Pepper consists of the dried fully developed unripe fruits of Piper nigrum L. (Fam. Piperaceae). It contains NLT 2.5% of piperine,
calculated on the dried basis.

IDENTIFICATION
• A. Black Pepper meets the requirements under Speci c Tests, Botanical Characteristics.
• B. HPTLC FOR ARTICLES OF BOTANICAL ORIGIN 〈203〉
Standard solution A: 0.9 mg/mL of USP Piperine RS in methanol
Standard solution B: 2.0 mg/mL of borneol in methanol
Standard solution C: 5 mg/mL of USP Powdered Black Pepper Extract RS in methanol. Sonicate for about 10 min, centrifuge, and use
the supernatant.
Sample solution: Sonicate for 10 min about 0.5 g of Black Pepper, nely powdered, in 5 mL of methanol, centrifuge, and use the
supernatant.
Chromatographic system
Adsorbent: Chromatographic silica gel mixture with an average particle size of 5 µm (HPTLC plates)
Application volume: 15 µL of Standard solution C, 7 µL of the Sample solution, and 3 µL of the Standard solution A and Standard
solution B, as bands
Developing solvent system: Hexanes and ethyl acetate (5:3)
Derivatization reagent: A mixture of 17 mL of ice-cooled methanol, 2 mL of acetic acid, 1 mL of sulfuric acid, and 0.1 mL of
anisaldehyde, mixed in this order
Analysis
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Samples: Standard solution A, Standard solution B, Standard solution C, and Sample solution
Apply the Samples as bands. Use a saturated chamber, and condition the plate to a relative humidity of about 33% using a suitable
device. Develop until the solvent front has moved up about 7 cm from the lower edge of the plate. Remove the plate from the
chamber, dry, and examine under UV light at 254 nm. Treat with the Derivatization reagent, heat for 5 min at 100°, and examine
under white light.
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Acceptance criteria: Under UV 254 nm, the chromatogram of the Sample solution exhibits an intense quenching band at RF of about
0.15 corresponding to the piperine band in the chromatogram of Standard solution A, a quenching band at RF of about 0.02, and
three quenching bands located between RF of about 0.3 and 0.5. Under white light, the derivatized chromatogram of the Sample
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solution exhibits main bands similar in position and color to the main bands in the chromatogram of Standard solution C. These
bands include a dark green band of the same color and RF as the piperine band in Standard solution A (RF of about 0.15), a weak
violet band at RF of about 0.47 below the position of the band due to borneol in Standard solution B, and a greenish band in the lower
part of the chromatogram at RF of about 0.07. Other minor bands may be observed in the Sample solution and Standard solution C
chromatograms. No blue bands are detected in the chromatogram of the Sample solution at RF of about 0.10 and 0.58 (distinction
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from long pepper).

• C. HPLC
Analysis: Proceed as directed in the test for Content of Piperine.
Acceptance criteria: The chromatogram of the Sample solution obtained at 343 nm exhibits a major peak at the retention time
corresponding to piperine. Identify other piperamide peaks in the Sample solution chromatogram by comparison with the
chromatogram of Standard solution B and the reference chromatogram provided with the lot of USP Powdered Black Pepper Extract
RS being used. The Sample solution chromatogram shows an additional peak corresponding to piperyline. The chromatogram of the
Sample solution obtained at 270 nm does not exhibit a peak due to (2E,4E)-N-isobutyldecadienamide at a relative retention time of
1.14 to the piperine peak (distinction from long pepper).

COMPOSITION
• CONTENT OF PIPERINE
Solution A: Dissolve 0.14 g of anhydrous potassium dihydrogen phosphate in 900 mL of water, and add 0.5 mL of phosphoric acid.
Dilute with water to 1000 mL, mix, lter, and degas.
Solution B: Acetonitrile
Mobile phase: See Table 1.

Table 1

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Time (min) Solution A (%) Solution B (%)

0 95 5

18 55 45

25 20 80

28 20 80

35 55 45

40 95 5

45 95 5

[NOTE—Proceed under subdued light or using low-actinic glassware.]

Standard solution A: 0.1 mg/mL of USP Piperine RS in methanol
Standard solution B: Sonicate a portion of USP Powdered Black Pepper Extract RS in methanol to obtain a solution having a
concentration of about 0.5 mg/mL. Before injection, pass through a membrane lter of 0.45-µm or ner pore size, discarding the rst
few mL of the ltrate.
Sample solution: Transfer about 2.0 g of Black Pepper, nely powdered and accurately weighed, to a 250-mL ask tted with a re ux
condenser. Add 50 mL of methanol, re ux on a water bath for about 20 min, allow to settle, and decant the supernatant. Repeat until
the last extract is colorless. Combine the extracts, concentrate under vacuum, and adjust the volume to 100 mL using methanol.
Before injection, pass through a membrane lter of 0.45-µm or ner pore size, discarding the rst few mL of the ltrate.
Chromatographic system
(See Chromatography 〈621〉, System Suitability.)
Mode: LC
Detector: UV 343 nm and 270 nm
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Column: 4.6-mm × 25-cm; 5-µm, 100 Å packing L1
Flow rate: 1.5 mL/min
Injection volume: 20 µL
System suitability
Samples: Standard solution A and Standard solution B
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Suitability requirements
Chromatogram similarity: The chromatogram obtained from Standard solution B is similar to the reference chromatogram
provided with the lot of USP Powdered Black Pepper Extract RS being used.
Tailing factor: NMT 1.5 for the piperine peak, Standard solution A
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Relative standard deviation: NMT 2.5% determined from the piperine peak in replicate injections, Standard solution A
Analysis
Samples: Standard solution A, Standard solution B, and Sample solution
[NOTE—Standard solution A, Standard solution B, and the Sample solution are stable for 6 h at room temperature.]
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Using the chromatograms of Standard solution A, Standard solution B, and the reference chromatogram provided with the lot of
USP Powdered Black Pepper Extract RS being used, identify the retention times of the peaks corresponding to piperine and
piperyline in the Sample solution chromatogram.
Calculate the percentage of piperine in the portion of Black Pepper taken:

Result = (rU/rS) × CS × (V/W) × 100

rU = peak area of piperine from the Sample solution chromatogram at 343 nm

rS = peak area of piperine from the Standard solution A chromatogram at 343 nm

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

V = nal volume of the Sample solution (mL)

W = weight of Black Pepper used to prepare the Sample solution (mg)

Acceptance criteria: NLT 2.5% on the dried basis

CONTAMINANTS
• ARTICLES OF BOTANICAL ORIGIN, Limits of Elemental Impurities〈561〉: Meets the requirements
• ARTICLES OF BOTANICAL ORIGIN, Pesticide Residue Analysis〈561〉: Meets the requirements

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• MICROBIAL ENUMERATION TESTS 〈2021〉: 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 bacterial count does not exceed 103 cfu/g.
• ABSENCE OF SPECIFIED MICROORGANISMS 〈2022〉: Meets the requirements of the tests for absence of Salmonella species and Escherichia coli

SPECIFIC TESTS
• BOTANICAL CHARACTERISTICS
Macroscopic: Fruit is an indehiscent, one-seeded berry, globose, ovoid to oblong, 3.5–6 mm in diameter, hard; surface is greyish-black
to brownish-black, rough, with raised reticulate wrinkles, shows remains of sessile stigma on the tip and a basal scar showing point
of attachment to the axis; characteristic aromatic odor; characteristic pungent taste; seed white and hollow.
Microscopic
Transverse cut: Circular in outline with corrugated margin; shows outer narrow brownish pericarp; a seed coat encircling the wide
central whitish perisperm, hollow in center; a narrow vertically running channel connecting the hollow center of the fruit to a small
endosperm adherent to the remains of the stigma; a small embryo is present in the endosperm; a conical short projection at the
base showing point of attachment to the axis.
Transverse section: Shows a well-differentiated pericarp, testa and perisperm; pericarp consists of a layer of epicarp, a wide
mesocarp, and a layer of endocarp; the epicarp layer is covered with thick cuticle containing a few stomata and small prisms of
calcium oxalate; mesocarp composed of 2–3 layers of parenchyma cells showing groups of rectangular to circular ligni ed
sclereids, a broad zone (12–15 layers) of tangentially running parenchyma cells containing starch grains and showing isolated
oval oil cells, a broad zone (10–15 layers) of compactly arranged parenchyma cells smaller than those of the outer zone and
showing groups of brovascular bundles, 1–2 layers of tangentially running oil cells, and 2–3 layers of thick-walled parenchyma
cells; endocarp is composed of one layer of three-sided thickened pitted stone cells (beaker-shape cells); testa is composed of
one layer of cells lled with brown pigments; perisperm is very wide, composed of cells full of starch grains, some aleurone grains,
and oil cells.
• ARTICLES OF BOTANICAL ORIGIN, Foreign Organic Matter〈561〉: NMT 2.0%
• ARTICLES OF BOTANICAL ORIGIN, Alcohol-Soluble Extractives, Method 1〈561〉: NLT 10.0%
• ARTICLES OF BOTANICAL ORIGIN, Water-Soluble Extractives, Method 1〈561〉: NLT 9.0%
• LOSS ON DRYING 〈731〉
Sample: 1.0 g of nely powdered Black Pepper
Analysis: Dry the Sample at 105° for 2 h.
Acceptance criteria: NMT 12.0%
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• ARTICLES OF BOTANICAL ORIGIN, Total Ash〈561〉
Sample: 1.0 g of nely powdered Black Pepper
Acceptance criteria: NMT 5.0%
• ARTICLES OF BOTANICAL ORIGIN, Acid-Insoluble Ash〈561〉
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Sample: 1.0 g of nely powdered Black Pepper

Acceptance criteria: NMT 1.0%

ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in well-closed containers, protected from light and moisture, and store at room temperature.
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• LABELING: The label states the Latin binomial and, following the o cial name, the part of the plant contained in the article.
• USP REFERENCE STANDARDS 〈11〉
USP Piperine RS
USP Powdered Black Pepper Extract RS
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Auxiliary Information- Please check for your question in the FAQs before contacting USP.

Topic/Question Contact Expert Committee

BLACK PEPPER Anton Bzhelyansky BDSHM2020 Botanical Dietary

Scienti c Liaison Supplements and Herbal Medicines

Chromatographic Database Information: Chromatographic Database

Most Recently Appeared In:

Pharmacopeial Forum: Volume No. 40(5)

Page Information:

USP43-NF38 - 4825
USP42-NF37 - 4781
USP41-NF36 - 4483

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