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Rosemary
DEFINITION
Rosemary consists of the dried leaf of Rosmarinus officinalis L. (Fam. Lamiaceae). It contains NLT 2.0% of phenolic diterpenes, calculated as the sum of carnosic acid and carnosol, and NLT 1.2% (v/w) of volatile oil, both calculated on the dried basis.
IDENTIFICATION
•  A. 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 Rosemary, finely powdered, in 10 mL of methanol. 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 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. Develop the chromatograms in a saturated chamber, remove the plate from the chamber, heat for 3 min at 100, 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 the carnosic acid peak 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 Rosemary, finely powdered, 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 the Sample solution are stable for 12 h at room temperature. ]
Using the chromatograms of Standard solution A, identify the retention times 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 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 USP Carnosic Acid RS in Standard solution A (mg/mL)
V== volume of the Sample solution (mL)
W== weight of 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
•  Articles of Botanical Origin, Volatile Oil Determination 561: Use 25.0 g of coarsely powdered Rosemary. Distill for 3 h at a rate of 2–3 mL/min.
Acceptance criteria:  NLT 1.2% v/w of volatile oil 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
•  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:  The leaves are sessile, linear to linear lanceolate, needle-like, 1–4 cm long and 2–4 mm wide, with strongly revolute margins; apex obtuse, base tapering. The upper surface is dark green, somewhat pubescent when young, glabrous in older leaves, the lower surface is grayish-green and densely tomentose with a prominent midrib. Pharmacopeial article consists of dry, grayish-green to yellowish-green, brittle, leaves.
Microscopic 
Transverse section:  Upper epidermis, cells are tabular with slightly thickened walls and occasional pits, rare conical covering trichomes, stomata are absent; an underlying hypodermis composed of large, irregularly rounded to ovoid cells with thickened anticlinal walls, one to several layers of cell extending across the lamina at intervals, separating the one or two-layered palisade into large, crescent-shaped areas, each with a group of spongy mesophyll; lower epidermis, numerous stomata, masses of multicellular and characteristic, thin-walled, uniseriate, extensively branched covering trichomes, up to 300 µm in length, forming dense felted masses, with each branch arising from a slightly swollen joint and terminating in a single tapering cell; glandular trichomes of two types are present on both epidermis, more on the lower epidermis, 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.
•  Articles of Botanical Origin, Foreign Organic Matter 561: NMT 5% of stems; NMT 2.0% of other foreign matter
•  Loss on Drying 731
Sample:  1.0 g of finely 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 finely powdered Rosemary
Acceptance criteria:  NMT 9.0%
•  Articles of Botanical Origin, Acid-Insoluble Ash 561
Sample:  2–4 g of finely 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 contained in the article.
•  USP Reference Standards 11
USP Carnosic Acid RS Click to View Structure
USP Powdered Rosemary Hydrophilic Extract RS
USP Rosmarinic Acid RS Click to View Structure
USP38
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Monograph Anton Bzhelyansky, M.S.
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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
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Pharmacopeial Forum: Volume No. 39(1)