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Powdered Rosemary

DEFINITION

Powdered Rosemary is Rosemary reduced to powder or very fine powder. It contains NLT 2.0% of phenolic diterpenes, calculated as the sum of carnosic acid and carnosol, on the dried basis.

IDENTIFICATION

• A. Powdered Rosemary meets the requirements for Specific Tests, Botanic Characteristics.

• B. Thin-Layer Chromatography

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

Standard solution B: 100 mg/mL of USP Powdered Rosemary Hydrophilic Extract RS in methanol

Sample solution: Sonicate for 10 min about 1 g of Powdered Rosemary in 10 mL of methanol. Centrifuge, and use 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 of Standard solution A, 4 µL of Standard solution B, and 8 µL of the 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: A mixture of ethyl acetate, formic acid, and water (15:1:1)

Developing distance: 6 cm

Derivatization reagent A: 5-mg/mL solution of 2-aminoethyl diphenylborinate in ethyl acetate

Derivatization reagent B: 50-mg/mL solution of polyethylene glycol 400 in dichloromethane

Analysis

Samples: Standard solution A, Standard solution B, and Sample solution. Apply the samples as bands to a suitable high performance thin-layer chromatographic plate (see Chromatography

621

), and dry in air. Develop the chromatograms in a saturated chamber, remove the plate from the chamber, heat at 100

for 3 min, derivatize the plate while still warm with Derivatization reagent A, dry in air, then derivatize with Derivatization reagent B, dry in air, and examine under UV light at 366 nm.

System suitability: The chromatogram of Standard solution B exhibits, in the upper-third section, a blue fluorescent band corresponding to the band due to rosmarinic acid in the chromatogram of Standard solution A, and a less intense band, right above the band due to rosmarinic acid, corresponding to caffeic acid. The bands due to rosmarinic acid and caffeic acid are clearly separated.

Acceptance criteria: The chromatogram of the Sample solution exhibits the following main bands similar in positions and colors to the corresponding bands in the chromatogram of Standard solution B. A blue fluorescent band at an RF corresponding to the rosmarinic acid band in Standard solution A; a band at an RF corresponding to the caffeic acid; a green band at about the middle of the chromatogram and an intense orange band in the lower third section of the chromatogram; below the green band, the Sample solution chromatogram exhibits a pattern of characteristically colored bands of low intensity (distinction from sage leaf, thyme leaf, holy basil leaf, basil leaf, and oregano leaf).

The Sample solution chromatogram does not show a pair of major orange bands in the lower-third section of the chromatogram (distinction from sage leaf and thyme leaf), a light blue band at an RF of about one-third of the chromatogram (distinction from holy basil leaf), an orange band in the lower-third section of the chromatogram and a light blue band at about two-thirds of the chromatogram (distinction from basil leaf), or a red band right above the band due to caffeic acid in the upper-third section of the chromatogram (distinction from thyme leaf and oregano leaf.) [Note—This red band is different from the red band, at the solvent front, present in Rosemary and other leaves due to chlorophyll.

]

• C. HPLC

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

Acceptance criteria: The chromatogram of the Sample solution exhibits the major peak at the retention time corresponding to carnosic acid in the chromatogram of Standard solution A and an additional peak corresponding to carnosol at a relative retention time of about 0.64 compared to that of the carnosic acid peak.

COMPOSITION

• Content of Phenolic Diterpenes

Mobile phase: A mixture of acetonitrile and a solution of 0.5% phosphoric acid in water (65:35)

[Note—Proceed under subdued light.

]

Solvent: 0.5% phosphoric acid in methanol

Standard solution A: [Note—Use clear glassware.

] 0.2 mg/mL of USP Carnosic Acid RS in Solvent. Sonicate to dissolve if necessary.

Sample solution: 1.0 g of Powdered Rosemary in 50 mL of methanol. Sonicate for 3 h. Before injection, pass through a membrane filter of 0.45-µL or finer pore size, discarding the first few mL of the filtrate.

Chromatographic system

(See Chromatography

621

, System Suitability.)

Mode: LC

Detector: UV, 230 nm

Column: 4.6-mm × 25-cm; 5-µm packing L1

Column temperature: 25 ± 1

Flow rate: 1.5 mL/min

Injection volume: 5 µL

System suitability

Sample: Standard solution A

Suitability requirements

Tailing factor: Between 0.90 and 1.30 for the carnosic acid peak, Standard solution A

Relative standard deviation: NMT 2% determined from the carnosic acid peak in repeated injections, Standard solution A

Analysis

Samples: Standard solution A and Sample solution

[Note—Standard solution A and Sample solution are stable for 12 h at room temperature.

]

Using the chromatograms of Standard solution A, identify the retention time of the peaks corresponding to carnosic acid and carnosol in the Sample solution chromatogram. [Note—The approximate relative retention times for the carnosol and carnosic acid peaks are 0.64 and 1.00, respectively.

]

Calculate the percentages of carnosic acid and carnosol in the portion of Powdered Rosemary taken:

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

rU	=	= peak area of the relevant analyte in the Sample solution chromatogram
rS	=	= peak area for carnosic acid in the Standard solution A chromatogram
CS	=	= concentration of carnosic acid in Standard solution A (mg/mL)
V	=	= volume of the Sample solution (mL)
W	=	= weight of Powdered Rosemary taken to prepare the Sample solution (mg)
F	=	= conversion factor (1.00 for carnosic acid and 0.92 for carnosol)

Add the percentages of carnosic acid and carnosol.

Acceptance criteria: NLT 2.0% on the dried basis

CONTAMINANTS

• Elemental Impurities—Procedures

233

Acceptance criteria

Arsenic: NMT 1.0 µg/g

Cadmium: NMT 0.5 µg/g

Lead: NMT 5.0 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—Nutritional and Dietary Supplements

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 bacteria do not exceed 103 cfu/g.

• Microbiological Procedures for Absence of Specified Microorganisms—Nutritional and Dietary Supplements

2022

: Meets the requirements of the tests for absence of Salmonella species and Escherichia coli

SPECIFIC TESTS

• Botanic Characteristics

Macroscopic: Grayish-green to yellowish-green powder

Microscopic: It shows fragments of upper epidermal cell, polygonal to irregular, with slightly thickened walls and occasional pits, stomata absent; lower epidermal cells, with straight or slightly sinuous walls, numerous diacytic stomata, masses of multicellular and characteristic, thin-walled, uniseriate, extensively branched covering trichomes, up to 300 µm in length, with each branch arising from a slightly swollen joint and terminating in a single tapering cell; glandular trichomes of two types, the majority with a short, unicellular stalk and a radiate head composed of eight cells, less abundant with a unicellular stalk and a spherical, unicellular or bicellular head; glandular trichomes in surface view appear as a disk with eight cells; hypodermis cells with distinctly beaded walls; fibers; vascular tissue.

• Loss on Drying

731

Sample: 1.0 g of Powdered Rosemary

Analysis: Dry at 105

for 2 h.

Acceptance criteria: NMT 10%

• Articles of Botanical Origin, Total Ash

561

Sample: 2–4 g of Powdered Rosemary

Acceptance criteria: NMT 9.0%

• Articles of Botanical Origin, Acid-Insoluble Ash

561

Sample: 2–4 g of Powdered Rosemary

Acceptance criteria: NMT 1.5%

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, following the official name, the part of the plant from which the article was obtained.

• USP Reference Standards

USP Carnosic Acid RS

USP Powdered Rosemary Hydrophilic Extract RS

USP Rosmarinic Acid RS

USP38

Auxiliary Information— Please check for your question in the FAQs before contacting USP.

Topic/Question	Contact	Expert Committee
Monograph	Anton Bzhelyansky, M.S. Scientific Liaison (301) 230-6303	(DS2010) Monographs - Dietary Supplements and Herbal Medicines
2021	Radhakrishna S Tirumalai, Ph.D. Principal Scientific Liaison (301) 816-8339	(GCM2010) General Chapters - Microbiology
2022	Radhakrishna S Tirumalai, Ph.D. Principal Scientific Liaison (301) 816-8339	(GCM2010) General Chapters - Microbiology
Reference Standards	RS Technical Services 1-301-816-8129 rstech@usp.org

USP38–NF33 Page 6203

Pharmacopeial Forum: Volume No. 39(1)