Bacopa consists of the dried stems and leaves of Bacopa monnieri (L.) Pennell (Fam. Scrophulariaceae). 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.
• A. Bacopa meets the requirements for 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 1520 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 1015 µm (TLC plates)
Application volume: 15 µL, as 510 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)
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 510 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.
• C. HPLC Identification Test: The Sample solution from the test for Content of Triterpene Glycosides shows a main peak at the 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. The Sample solution shows additional peaks corresponding to bacopaside I, bacopaside II, the jujubogenin isomer of bacopasaponin C, and bacopasaponin C.
• 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.
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 1520 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 Bacopa, finely powdered, 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.
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
Samples: Standard solution A and Standard solution B
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
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.
Separately calculate the percentages of bacopaside I, bacoside A3, bacopaside II, the jujubogenin isomer of bacopasaponin C, and bacopasaponin C in the portion of Bacopa taken:
Result = (rU/rS) × CS × (V/W) × F × 100
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, calculated on the dried basis.
• Heavy Metals, Method III 231: NMT 20 ppm
• Procedure 1: Articles of Botanical Origin, Foreign Organic Matters 561: NMT 2.0%
• Procedure 2: Articles of Botanical Origin, General Method for Pesticide Residues Analysis 561: Meets the requirements
• Botanic Characteristics
Macroscopic: Stem is creeping, succulent, glabrous, soft, obtuse-angular; with long internodes, rooting at nodes; devoid of leaves toward the base; branches ascending. Leaves are simple, sessile or short petiolate, opposite, succulent, 12 mm thick; oblong-obovate or spatulate, margin entire or rarely dentate, apex rounded, midrib indistinct, 0.62.5 cm long, 38 mm wide; the upper surface is green, the lower surface is green and dotted. Pharmacopeial article is yellowish in color; consists of dry mixtures of broken leaves and stems, with majority of leaves detached; mild and hay-like odor, and very bitter taste.
Transverse section of stems: Epidermal layer; a wide cortex composed of thin-wall parenchyma cells and large intercellular spaces; xylem vessels radially arranged, uniseriate medullary rays; pith composed of thin-wall, round or isodiametric cells with distinct intercellular spaces. Resin canals and pericyclic sclereids are absent.
Transverse section of leaves: Shows a more or less isobilateral structure; epidermis with glandular hair and stomata; upper surface has more hairs and less stomata than the lower surface; mesophyll composed of spongy tissue, a few prisms of calcium oxalate, and vascular bundles are present.
• Loss on Drying 731: Dry 1.0 g of finely 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 finely 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
• 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 Powdered Bacopa Extract RS
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USP35NF30 Page 1198Pharmacopeial Forum: Volume No. 36(3) Page 691