Add the following:
Rosemary Leaf Dry Aqueous Extract
DEFINITION
Rosemary Leaf Dry Aqueous Extract is prepared from Rosemary by extraction with water. It contains NLT 90% and NMT 110% of the labeled amount of rosmarinic acid, on the dried basis. It may contain suitable added substances as carriers.
IDENTIFICATION
•  A. 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:  100 mg/mL of Rosemary Leaf Dry Aqueous Extract in methanol
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 the Standard solution A, and 4 µL of the Standard solution B and 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, 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 shows, 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. ]
•  B. HPLC
Analysis:  Proceed as directed in the test for Content of Rosmarinic Acid.
Acceptance criteria:  The chromatogram of the Sample solution exhibits the major peak at the retention time corresponding to the rosmarinic acid peak in the chromatogram of Standard solution A. It also shows a less intense peak corresponding to the luteolin-3-O-glucuronide peak in the chromatogram of Standard solution B and at a relative retention time of about 0.8 compared to the rosmarinic acid peak.
COMPOSITION
•  Content of Rosmarinic Acid
Mobile phase:  A mixture of acetonitrile and a solution of 0.1% trifluoroacetic acid in water (32:68)
Solvent:  Methanol and water (1:1)
Standard solution A:  0.3 mg/mL of USP Rosmarinic Acid RS in Solvent. Sonicate to dissolve if necessary.
Standard solution B:  10 mg/mL of USP Powdered Rosemary Hydrophilic Extract RS in Solvent. Sonicate to dissolve if necessary. Before injection, pass through a membrane filter of 0.45-µL or finer pore size, discarding the first few mL of the filtrate.
Sample solution:  Sonicate for 5 min an amount of Rosemary Leaf Dry Aqueous Extract equivalent to 15 mg of rosmarinic acid in 50 mL of Solvent. 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, 328 nm
Column:  4.6-mm × 25-cm; 5-µm packing L1
Column temperature:  20 ± 1
Flow rate:  1.0 mL/min
Injection volume:  5 µL
System suitability 
Samples:  Standard solution A and Standard solution B
Suitability requirements 
Chromatogram similarity:  The chromatogram from Standard solution B is similar to the reference chromatogram provided with the lot of USP Powdered Rosemary Hydrophilic Extract RS being used.
Tailing factor:  Between 0.90 and 1.30 for the rosmarinic acid peak, Standard solution A
Relative standard deviation:  NMT 2% determined from the rosmarinic acid peak in repeated injections, Standard solution A
Analysis 
Samples:  Standard solution A, Standard solution B, and Sample solution
Using the chromatograms of Standard solution A, Standard solution B, and the reference chromatogram provided with the lot of USP Powdered Rosemary Hydrophilic Extract RS being used, identify the retention time of the peak corresponding to rosmarinic acid in the Sample solution chromatogram.
Calculate the percentage of rosmarinic acid in the portion of Rosemary Leaf Dry Aqueous Extract taken:
P = (rU/rS) × (CS/CU) × 100

rU== peak area for rosmarinic acid in the Sample solution chromatogram
rS== peak area for rosmarinic acid in Standard solution A chromatogram
CS== concentration of rosmarinic acid in Standard solution A (mg/mL)
CU== concentration of Dry Aqueous Extract in the Sample solution (mg/mL)
Calculate the percentage of the labeled amount of rosmarinic acid in the portion of Rosemary Leaf Dry Aqueous Extract taken:
Result = (P/L) × 100

P== percentage of rosmarinic acid as determined above (%)
L== labeled amount of rosmarinic acid (%)
Acceptance criteria:  90%–110% 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 104 cfu/g, the total combined molds and yeasts count does 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
•  Loss on Drying 731
Sample:  1.0 g of Rosemary Leaf Dry Aqueous Extract
Analysis:  Dry at 105 for 2 h.
Acceptance criteria:  NMT 8%
ADDITIONAL REQUIREMENTS
•  Packaging and Storage: Preserve in well-closed containers, protected from light, moisture, and oxygen; store at controlled 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 in Botanical Extracts 565.
•  USP Reference Standards 11
USP Powdered Rosemary Hydrophilic Extract RS
USP Rosmarinic Acid RS Click to View Structure
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 6201
Pharmacopeial Forum: Volume No. 39(1)