Powdered Bacopa Extract
DEFINITION
Powdered Bacopa Extract is prepared from Bacopa by extraction with water, alcohol, methanol, or a mixture of these solvents. The ratio of plant material to extract is between 20:1 and 10:1. It contains NLT 90.0% and NMT 110.0% of the labeled amount of triterpene glycosides, calculated on the dried basis as the sum of bacopaside I, bacoside A3, bacopaside II, the jujubogenin isomer of bacopasaponin C, and bacopasaponin C. It may contain suitable added substances as carriers.
IDENTIFICATION
•  A. Thin-Layer Chromatographic Identification Test
Standard solution:  Transfer about 10 mg of USP Powdered Bacopa Extract RS to a 10-mL volumetric flask, and add about 8 mL of methanol. Sonicate and heat gently for 15–20 min, dilute with methanol to volume, mix, centrifuge, and use the supernatant.
Sample solution:  Sonicate for about 10 min an amount of Powdered Bacopa Extract equivalent to about 40 mg of triterpene glycosides in 10 mL of methanol, centrifuge, and use the supernatant.
Adsorbent:  Chromatographic silica gel mixture with an average particle size of 10–15 µm (TLC plates)
Application volume:  15 µL, as 5–10 mm bands
Developing solvent system:  Ethyl acetate, methanol, and water (7:2:1)
Spray reagent:  1% vanillin in alcohol and 10% sulfuric acid in alcohol (1:1)
Analysis 
Samples:  Standard solution and Sample solution
Apply the samples as bands to a suitable thin-layer chromatographic plate (see Chromatography 621). Use a saturated chamber. Develop the chromatograms until the solvent front has moved up about three-fourths of the plate. Remove the plate from the chamber, dry, spray with Spray reagent, heat for 5–10 min at about 70, and examine under visible light.
Acceptance criteria:  The Sample solution exhibits a main dark blue zone due to a mixture of bacoside A3, bacopaside II, the jujubogenin isomer of bacopasaponin C, and bacopasaponin C at an RF value of approximately 0.6 and a faint pink spot due to bacopaside I at an RF value of approximately 0.4, both of which correspond in position and color to zones in the chromatogram of the Standard solution. Other zones are observed for the Sample solution and Standard solution.
•  B. HPLC Identification Test: The Sample solution from the test for Content of Triterpene Glycosides shows a main peak at a retention time corresponding to that of bacoside A3 in the chromatogram of Standard solution A. Identify other triterpene glycoside peaks in the Sample solution by comparison with the chromatogram of Standard solution B and the reference chromatogram provided with the lot of USP Powdered Bacopa Extract RS being used. The Sample solution shows additional peaks corresponding to bacopaside I, bacopaside II, the jujubogenin isomer of bacopasaponin C, and bacopasaponin C.
COMPOSITION
•  Content of Triterpene Glycosides
Solution A:  Dissolve 0.14 g of anhydrous potassium dihydrogen phosphate in 900 mL of water, add 0.5 mL of phosphoric acid, dilute with water to 1000 mL, mix, filter, and degas.
Solution B:  Use filtered and degassed acetonitrile.
Mobile phase:  See the gradient table below.
Time
(min)		 Solution A
(%)		 Solution B
(%)
0 70 30
25 60 40
26 70 30
30 70 30
Standard solution A:  Sonicate a weighed quantity of USP Bacoside A3 RS in methanol to obtain a solution with a concentration of about 0.5 mg/mL.
Standard solution B:  Transfer about 10 mg of USP Powdered Bacopa Extract RS to a 10-mL volumetric flask, and add about 8 mL of methanol. Sonicate and heat gently for 15–20 min, dilute with methanol to volume, and mix. Before injection, pass through a membrane filter of 0.45-µm or finer pore size, discarding the first 5 mL of the filtrate.
Sample solution:  Transfer an amount of Powdered Bacopa Extract, equivalent to about 25 mg triterpene glycosides, to a 25-mL volumetric flask, and add 15 mL of methanol. Sonicate and heat gently for 15–20 min, dilute with methanol to volume, and mix. Before injection, pass through a membrane filter of 0.45-µm or finer pore size, discarding the first 5 mL of the filtrate.
Chromatographic system 
(See Chromatography 621, System Suitability.)
Mode:  LC
Detector:  UV 205 nm
Column:  4.6-mm × 25-cm; 5-µm, endcapped, base-deactivated packing L1
Column temperature:  27 ± 1
Flow rate:  1.5 mL/min
Injection size:  20 µL
System suitability 
Samples:  Standard solution A and Standard solution B
Suitability requirements 
Chromatographic similarity:  The chromatogram from Standard solution B is similar to the reference chromatogram provided with the lot of USP Powdered Bacopa Extract RS being used.
Resolution:  NLT 1.0 between the bacopaside II and bacoside A3 peaks, Standard solution B
Tailing factor:  NMT 1.5 for the bacoside A3 peak, Standard solution A
Relative standard deviation:  NMT 2% determined from the bacoside A3 peak for replicate injections, Standard solution A
Analysis 
Samples:  Standard solution A, Standard solution B, and Sample solution
Using the chromatograms of Standard solution A and Standard solution B and the reference chromatogram provided with the lot of USP Powdered Bacopa Extract RS being used, identify the retention times of the peaks corresponding to different triterpene glycosides. The approximate relative retention times of the different triterpene glycosides are provided in the following table.
Analyte Relative
Retention
Time
Bacopaside I 0.73
Bacoside A3 1.00
Bacopaside II 1.04
The jujubogenin isomer of bacopasaponin C 1.15
Bacopasaponin C 1.22
Separately calculate the percentages of bacopaside I, bacoside A3, bacopaside II, jujubogenin isomer of bacopasaponin C, and bacopasaponin C in the portion of Powdered Bacopa Extract taken:
Result = (rU/rS) × (CS/CU) × F × 100
rU== peak response for each triterpene glycoside from the Sample solution
rS== peak response for bacoside A3 in Standard solution A
CS== concentration of USP Bacoside A3 RS in Standard solution A (mg/mL)
CU== concentration of Powdered Bacopa Extract in the Sample solution (mg/mL)
F== conversion factor for each analyte: 1.00 for bacoside A3, 1.03 for bacopaside I, 0.81 for bacopaside II, 0.99 for the jujubogenin isomer of bacopasaponin C, and 0.75 for bacopasaponin C
Acceptance criteria:  Add the percentages of bacopaside I, bacoside A3, bacopaside II, the jujubogenin isomer of bacopasaponin C, and bacopasaponin C: NLT 90.0%–NMT 110.0% of the labeled amount of triterpene glycosides is found on the dried basis.
IMPURITIES
Inorganic Impurities 
•  Heavy Metals, Method III 231: NMT 20 ppm
SPECIFIC TESTS
•  Loss on Drying 731: Dry 1.0 g of Powdered Bacopa Extract at 105 for 3 h: it loses NMT 5% of its weight.
•  Microbial Enumeration Tests 2021: The total aerobic microbial 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 2022: It meets the requirements of the tests for absence of Salmonella species and Escherichia coli.
•  Other Requirements: It meets the requirements of the test for Residual Solvents under Botanical Extracts 565.
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 and, following the official name, the part of the plant from which the article was derived. It meets other labeling requirements under Botanical Extracts 565.
•  USP Reference Standards 11
USP Bacoside A3 RS
USP Powdered Bacopa Extract RS
Auxiliary Information— Please check for your question in the FAQs before contacting USP.
Topic/Question Contact Expert Committee
Monograph Maged H. Sharaf, Ph.D.
Principal Scientific Liaison
1-301-816-8318		 (DS2010) Monographs - Dietary Supplements
2021 Radhakrishna S Tirumalai, Ph.D.
Principal Scientific Liaison
1-301-816-8339		 (GCM2010) General Chapters - Microbiology
2022 Radhakrishna S Tirumalai, Ph.D.
Principal Scientific Liaison
1-301-816-8339		 (GCM2010) General Chapters - Microbiology
Reference Standards RS Technical Services
1-301-816-8129
rstech@usp.org
USP35–NF30 Page 1201
Pharmacopeial Forum: Volume No. 36(3) Page 694