Powdered St. John's Wort
DEFINITION
Powdered St. John's Wort is St. John's Wort reduced to a fine or a very fine powder. It contains NLT 0.6% of hyperforin (C35H52O4) and NLT 0.04% of hypericin (C30H16O8) and pseudohypericin (C30H16O9) combined, on the dried basis.
IDENTIFICATION
• A. Thin-Layer Chromatographic Identification Test
Standard solution:
0.5 mg/mL of USP Hyperoside RS in methanol
Sample solution:
Shake 10 g of Powdered St. John's Wort in 100 mL of methanol for 15 min, and filter.
Chromatographic system
Adsorbent:
0.50-mm layer of chromatographic silica gel mixture
Application volume:
20 µL
Developing solvent system:
Upper layer of a mixture of ethyl acetate, glacial acetic acid, formic acid, and water (10: 1.1: 1.1: 2.6)
Spray reagent A:
10-mg/mL solution of 2-aminoethyl diphenylborinate in methanol
Spray reagent B:
50-mg/mL solution of polyethylene glycol 400 in alcohol
Analysis
Samples:
Standard solution and Sample solution
Apply the Samples as bands and allow to dry. Develop and allow the plate to air-dry. Spray with Spray reagent A and allow the plate to air-dry. Immediately after, spray the plate with Spray reagent B, and allow the plate to air-dry. Examine the plate under UV light at 365 nm.
Acceptance criteria:
The chromatogram of the Sample solution exhibits several zones having a yellowish-orange fluorescence, one of which, appearing at an RF value of about 0.5, corresponds in RF value and intensity to a similar zone in the chromatogram of the Standard solution. The chromatogram of the Sample solution exhibits also two zones of red fluorescence, one at an RF value of about 0.85 (presence of hypericin) and the other at an RF value of about 0.80 (presence of pseudohypericin), and two zones of higher blue fluorescence (presence of chlorogenic and neochlorogenic acids) located below the yellow to yellowish-orange hyperoside zone.
COMPOSITION
• Content of Hypericin and Pseudohypericin
[Note—Conduct all sample preparations with minimal exposure to subdued light, and use low-actinic glassware to protect solutions from light. ]
Solvent:
Methanol and acetone (1:1)
Solution A:
Phosphoric acid and water (3:997)
Solution B:
Acetonitrile
Solution C:
Methanol
Mobile phase:
See Table 1.
Table 1
| Time (min) |
Solution A (%) |
Solution B (%) |
Solution C (%) |
|---|---|---|---|
| 0 | 100 | 0 | 0 |
| 10 | 85 | 15 | 0 |
| 30 | 70 | 20 | 10 |
| 40 | 10 | 75 | 15 |
| 55 | 5 | 80 | 15 |
| 56 | 100 | 0 | 0 |
| 66 | 100 | 0 | 0 |
Standard solution A:
2.5 µg/mL of USP Oxybenzone RS in Solvent
Standard solution B:
1 mg/mL of USP Powdered St. John's Wort Extract RS in Solvent
Sample solution:
Weigh 10 g of Powdered St. John's Wort. Transfer 1 g to a round-bottom flask equipped with a condenser and protected from light, add 50 mL of Solvent and a magnetic stirring bar, and heat at 60
for 2 h while stirring. Cool to room temperature, and pass through filter paper into a 50-mL volumetric flask. Wash the flask and the residue on the filter with Solvent, and dilute with the washings to volume. Pass the solution through a polytef membrane filter having a 0.45-µm or finer pore size, and use the filtrate.
for 2 h while stirring. Cool to room temperature, and pass through filter paper into a 50-mL volumetric flask. Wash the flask and the residue on the filter with Solvent, and dilute with the washings to volume. Pass the solution through a polytef membrane filter having a 0.45-µm or finer pore size, and use the filtrate.
Chromatographic system
Mode:
LC
Detector:
UV 270 nm and Vis 588 nm
Columns
Guard:
Packing L1
Analytical:
4.6-mm × 25-cm; packing L1
Column temperature:
30
Flow rate:
1 mL/min
Injection size:
20 µL. [Note—First equilibrate the system with 100% Solution A. ]
System suitability
Samples:
Standard solution A (record the peak responses at 270 nm) and Standard solution B (record the peak responses at 270 nm and 588 nm)
Suitability requirements
Chromatogram similarity:
The chromatograms from Standard solution B are similar to the respective reference chromatograms provided with the lot of USP Powdered St. John's Wort Extract RS being used.
Column efficiency:
NLT 100,000 theoretical plates for oxybenzone
Tailing factor:
NMT 1.5 for oxybenzone
Relative standard deviation:
NMT 2.0%, Standard solution A
Analysis
Samples:
Standard solution A and Sample solution
Measure the areas of the relevant peaks at 270 nm in the chromatogram of the Sample solution.
Separately calculate the percentage of hypericin (C30H16O8) and pseudohypericin (C30H16O9) in the portion of Powdered St. John's Wort taken:
Result = (rU/rS) × CS × (V/W) × 1/F × 100
| rU | = | = peak area of the relevant analyte from the Sample solution |
| rS | = | = peak area of oxybenzone from Standard solution A |
| CS | = | = concentration of USP Oxybenzone RS in Standard solution A (mg/mL) |
| V | = | = volume of the Sample solution (mL) |
| W | = | = weight of Powdered St. John's Wort taken to prepare the Sample solution (mg) |
| F | = | = relative response factor for the relevant analyte relative to that of oxybenzone, 1.30 for hypericin and 1.24 for pseudohypericin |
Acceptance criteria:
NLT 0.04% on the dried basis
• Content of Hyperforin
Analysis:
Using the chromatograms obtained in the test for Content of Hypericin and Pseudohypericin, calculate the percentage of hyperforin (C35H52O4) in the portion of Powderd St. John's Wort taken:
Result = (rU/rS) × CS × (V/W) × 1/F × 100
| rU | = | = peak area of hyperforin from the Sample solution |
| rS | = | = peak area of oxybenzone from Standard solution A |
| CS | = | = concentration of USP Oxybenzone RS in Standard solution A (mg/mL) |
| V | = | = volume of the Sample solution (mL) |
| W | = | = weight of Powdered St. John's Wort taken to prepare the Sample solution (mg) |
| F | = | = response factor for hyperforin relative to that of oxybenzone, 0.46 |
Acceptance criteria:
NLT 0.6% on the dried basis
CONTAMINANTS
• Heavy Metals 
231
:
NMT 20 µg/g
231:
NMT 20 µg/g
• Articles of Botanical Origin, General Method for Pesticide Residues Analysis 561:
Meets the requirements
561:
Meets the requirements
• Microbial Enumeration Tests 2021:
The total bacterial count does not exceed 104 cfu/g, and the total combined molds and yeasts count does not exceed 102 cfu/g.
2021:
The total bacterial count does not exceed 104 cfu/g, and the total combined molds and yeasts count does not exceed 102 cfu/g.
• Absence of Specified Microorganisms 2022:
It meets the requirements of the tests for absence of Salmonella species and Escherichia coli and for absence of Staphylococcus aureus.
2022:
It meets the requirements of the tests for absence of Salmonella species and Escherichia coli and for absence of Staphylococcus aureus.
SPECIFIC TESTS
• Botanic Characteristics
Macroscopic:
Buff to greenish-brown powder with an aromatic and balsamic odor
Microscopic:
Elongated and polygonal epidermal cells with thickened and beaded anticlinal walls, some accompanied by paracytic (occasionally anomocytic) stomata; fragments of leaf and sepal with schizogenous oil and pigment glands; narrow, thin-walled, elongated epidermal cells with straight and wavy anticlinal walls from petal; narrow, lignified vessels with annular or reticulate thickening; tracheids and tracheidal vessels with lignified, pitted thickening; thick-walled lignified fibers with tapering apices and occasional oblique pits; rectangular, lignified, pitted parenchyma; fibrous layer of antheral wall; pollen grains, 20–25 µm in diameter, with smooth exine.
• Articles of Botanical Origin, Total Ash 561:
NMT 5.0%
561:
NMT 5.0%
• Articles of Botanical Origin, Water Content 561:
NMT 10.0%
561:
NMT 10.0%
ADDITIONAL REQUIREMENTS
• Packaging and Storage:
Store in tight containers, protected from light and moisture.
• Labeling:
The label states the Latin binomial and, following the official name, the parts of the plant source from which the article was derived.
• USP Reference Standards 11
11
USP Hyperoside RS
USP Oxybenzone RS 
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USP Powdered St. John's Wort Extract RS
USP Rutin RS
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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 1427