Powdered St. John's Wort Extract
DEFINITION
Powdered St. John's Wort Extract is prepared from comminuted St. John's Wort extracted with 80% methanol or other suitable solvents. It contains NLT 90.0% and NMT 110.0% of the labeled combined total of hypericin (C30H16O8) and pseudohypericin (C30H16O9) and NLT 90.0% and NMT 110.0% of hyperforin (C35H52O4), on the dried basis.
IDENTIFICATION
•  A. Thin-Layer Chromatographic Identification Test: (presence of hypericin, pseudohypericin, hyperoside, and rutin)
Standard solution:  50 mg/mL of USP Powdered St. John's Wort Extract RS in methanol. Shake well, and use the clear supernatant.
Sample solution:  50 mg/mL of Powdered Extract in methanol. Shake well, and use the clear supernatant.
Chromatographic system 
Adsorbent:  0.50-mm layer of chromatographic silica gel mixture
Application volume:  10 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 of diphenylborinic acid, ethanolamine ester in methanol
Spray reagent B:  50 mg/mL of polyethylene glycol 400 in alcohol
Analysis 
Samples:  Standard solution and Sample solution
Develop the chromatogram until the solvent front has moved NLT 18 cm, and dry the plate with the aid of a current of air. Spray the plate with Spray reagent A, then with Spray reagent B, and examine the plate under UV light at 365 nm.
Acceptance criteria:   The two red zones due to hypericin and pseudohypericin at RF values of about 0.85 and 0.80, respectively, in the chromatogram of the Sample solution, correspond in color and RF value to those in the chromatogram of the Standard solution; the two yellow zones due to hyperoside and rutin at RF values of about 0.50 and 0.35, respectively, in the chromatogram of the Sample solution, correspond in color and RF value to those in the chromatogram of the Standard solution. Other colored zones of varyious intensities may be observed in the chromatogram of the Sample solution.
•  B. Thin-Layer Chromatographic Identification Test: (presence of hyperforin)
Standard solution and Sample solution:  Proceed as directed in Identification test A.
Chromatographic system 
Adsorbent:  0.50-mm layer of chromatographic silica gel mixture
Application volume:  10 µL
Developing solvent system:  Solvent hexane and ethyl acetate (4:1)
Spray reagent:  Prepare a solution containing 0.38 g of ceric ammonium sulfate and 3.8 g of ammonium molybdate in 100 mL of 2 N sulfuric acid.
Analysis 
Samples:  Standard solution and Sample solution
Develop the chromatograms in a saturated chamber until the solvent front has moved NLT 18 cm, and dry the plate with the aid of a current of air. Spray the plate with Spray reagent, heat the plate at 140 for 15 min, and examine under UV light.
Acceptance criteria:  The blue zone due to hyperforin at an RF value of about 0.54 in the chromatogram of the Sample solution corresponds in color and RF value to that in the chromatogram of the Standard solution.
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:  1 mg/mL of Powdered Extract in a mixture of methanol and water (9:1). Sonicate to dissolve, pass 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 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, Standard solution A
Tailing factor:  NMT 1.5 for oxybenzone, Standard solution A
Relative standard deviation:  NMT 2.0%, Standard solution A
Analysis 
Samples:  Standard solution A and Sample solution
Measure the areas at 270 nm of the relevant peaks in the chromatogram of the Sample solution.
Calculate the percentage of the labeled amount of hypericin (C30H16O8) and pseudohypericin (C30H16O9) in the portion of Powdered St. John's Wort Extract taken:
Result = (SrUi /fi) × (1/rS) × (CS/CU) × 100
SrUi /fi== sum of the peak areas of hypericin and pseudohypericin from the Sample solution divided by their respective response factors relative to oxybenzone, 1.30 for hypericin and 1.24 for pseudohypericin
rS== peak area of oxybenzone from Standard solution A
CS== concentration of USP Oxybenzone RS in Standard solution A (mg/mL)
CU== nominal concentration of the total hypericins content in the Sample solution (mg/mL)
Acceptance criteria:  90.0%–110.0% 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 Powdered St. John's Wort Extract taken:
Result = (rU/rS) × (CS/CU) × 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 the Standard solution A (mg/mL)
CU== nominal concentration of the hyperforin in the Sample solution (mg/mL)
F== relative response factor for hyperforin relative to that of oxybenzone, 0.46
Acceptance criteria:  90.0%–110.0% on the dried basis
CONTAMINANTS
•  Heavy Metals, Method II 231: NMT 50 µg/g
•  Microbial Enumeration Tests 2021: The total bacterial count does not exceed 104 cfu/g, and the total combined molds and yeasts does not exceed 103 cfu/g.
•  Absence of Specified Microorganisms 2022: It meets the requirements of the tests for absence of Salmonella species and Escherichia coli.
SPECIFIC TESTS
•  Other Requirements: It meets the requirements in Botanical Extracts 565, Residue on Evaporation and Residual Solvents.
ADDITIONAL REQUIREMENTS
•  Packaging and Storage: Preserve in tight containers, protected from moisture and light.
•  Labeling: The label states the Latin binomial and, following the official name, the part of the plant from which the article was prepared. The label also indicates the content of hypericin, pseudohypericin, and hyperforin; the extracting solvent or solvent mixture used for preparation; and the ratio of the starting crude plant material to Powdered Extract. The label bears a statement indicating that “Rare cases of allergic reactions and photosensitivity have been reported with the use of St. John's Wort. St. John's Wort interacts with numerous medications. Check with your health care provider before using.”
•  USP Reference Standards 11
USP Oxybenzone RS Click to View Structure
USP Powdered St. John's Wort Extract RS
USP Rutin RS Click to View Structure
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
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2022 Radhakrishna S Tirumalai, Ph.D.
Principal Scientific Liaison
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