Powdered Bacopa
DEFINITION
Powdered Bacopa is Bacopa reduced to a powder or very fine powder. It contains NLT 2.5% 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.
IDENTIFICATION
•  A. Powdered Bacopa meets the requirements under Specific Tests, Botanic Characteristics.
•  B. Thin-Layer Chromatographic Identification Test 201
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:  Use the Sample solution, prepared as directed in the test for Content of Triterpene Glycosides.
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 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.
•  C. 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 an accurately weighed quantity of USP Bacoside A3 RS in methanol to obtain a solution having a known 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 about 2.5 g of Powdered Bacopa, accurately weighed, to a 100-mL round-bottom flask fitted with a reflux condenser. Add 25 mL of methanol, reflux on a water bath for 10 min, cool to room temperature, and decant the supernatant. Repeat until the last extract is colorless. Combine the extracts, filter, concentrate under vacuum, and adjust the volume to 100 mL using methanol. 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 
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, 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, the jujubogenin isomer of bacopasaponin C, and bacopasaponin C in the portion of Powdered Bacopa taken:
Result = (rU/rS) × CS × (V/W) × F × 100
rU== peak response for each triterpene glycoside from the Sample solution
rS== peak response for bacoside A3 from Standard solution A
CS== concentration of USP Bacoside A3 RS in Standard solution A (mg/mL)
V== final volume of the Sample solution (mL)
W== weight of Powdered Bacopa used to prepare the Sample solution (mg)
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 2.5% is found on the dried basis.
IMPURITIES
Inorganic Impurities 
•  Heavy Metals, Method III 231: NMT 20 ppm
Organic Impurities 
SPECIFIC TESTS
•  Botanic Characteristics: Yellowish in color; mild and hay-like odor, and very bitter taste. Under a microscope, it shows fragments of upper and lower epidermal cells of the leaves in surface view, having sessile glandular trichomes with 4–8 cells and diacytic or anomocytic stomata; upper epidermis has more trichomes and less stomata than the lower epidermis; lower epidermis cells with sinuous anticlinal walls and at places striated cuticle; fragments of epidermal cells of the stem in surface view; parenchyma cells enclosing air cavities and some contain rosette and prismatic crystals of calcium oxalate; fragments of longitudinally cut annular and spiral vessels; fragments of cortical cells of the stem; and crystals of calcium oxalate.
•  Loss on Drying 731: Dry 1.0 g of Powdered Bacopa at 105 for 3 h: it loses NMT 12.0% of its weight.
•  Articles of Botanical Origin, Total Ash 561: NMT 18%, determined on 1.0 g of Powdered Bacopa
•  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 bacteria does not exceed 103 cfu/g.
•  Microbiological Procedures for Absence of Specified Microorganisms 2022: Meets the requirements of the tests for absence of Salmonella species and Escherichia coli
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 parts of the plant contained in the article.
•  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 1200
Pharmacopeial Forum: Volume No. 36(3) Page 692