Powdered Forskohlii Extract
DEFINITION
Powdered Forskohlii Extract is prepared from Forskohlii using suitable solvents such as methanol, ethyl acetate, hexane or a mixture of these solvents. The ratio of plant material to extract is between 65:1 and 35:1. It contains NLT 90.0% and NMT 110.0% of the labeled amount of forskolin, calculated on the dried basis. It contains suitable added substances as carriers.
IDENTIFICATION
•  A. Thin-Layer Chromatographic Identification Test 201
Standard solution A:  50 µg/mL of USP Forskolin RS in acetonitrile. Sonicate for about 10 min.
Standard solution B:  5 mg/mL of USP Powdered Forskohlii Extract RS in acetonitrile. Sonicate for about 15 min, centrifuge, and use the supernatant.
Sample solution:  5 mg/mL of Powdered Forskohlii Extract in acetonitrile. Sonicate for about 15 min, centrifuge, and use the supernatant.
Adsorbent:  Chromatographic silica gel with an average particle size of 10–15 µm (TLC plates)
Application volume:  10 µL, as 4-mm bands
Developing solvent system:  A mixture of toluene and ethyl acetate (85:15)
Spray reagent:  A mixture of 5% vanillin in glacial acetic acid and 10% sulfuric acid in water (1:1)
Analysis 
Samples:  Standard solution A, Standard solution B, and Sample solution
Apply the samples as bands to a suitable thin-layer chromatographic plate (see Chromatography 621). Use saturated chamber. Develop the chromatograms until the solvent front has moved up about 90% of the plate. Remove the plate from the chamber, dry, spray with the Spray reagent, heat for 5–10 min at 105, and examine under visible light.
Acceptance criteria:  The Sample solution exhibits a violet zone due to forskolin at an RF value of approximately 0.3, corresponding in color and RF to that from Standard solution A; and a minor violet zone, a pink zone, and a brick red zone at RF values of approximately 0.1, 0.62, and 0.69, due to isoforskolin, 1,9-dideoxyforskolin, and crocetindialdehyde, respectively. Zones detected from the Sample solution correspond in position and color to zones from Standard solution B. Other minor zones may be observed from the Sample solution and Standard solution B.
•  B. The Sample solution from the test for Content of Forskolin shows a main peak at a retention time corresponding to that of forskolin from Standard solution A. Identify other diterpene peaks in the Sample solution by comparison with Standard solution B and the reference chromatogram provided with the lot of USP Powdered Forskohlii Extract RS being used. The Sample solution shows an additional peak corresponding to isoforskolin.
COMPOSITION
•  Content of Forskolin
Solution A:  Use filtered and degassed acetonitrile.
Solution B:  Use filtered and degassed water.
Standard solution A:  Sonicate a quantity of USP Forskolin RS in acetonitrile to obtain a solution having a known concentration of about 1.0 mg/mL.
Standard solution B:  5 mg/mL of USP Powdered Forskohlii Extract RS in acetonitrile. Sonicate for about 15 min, centrifuge, and use the supernatant. Before injection, pass through a membrane filter having a 0.45-µm or finer pore size.
Sample solution:  Transfer an amount of Powdered Forskohlii Extract equivalent to about 25 mg of forskolin to a 25-mL volumetric flask, and add 15 mL of acetonitrile. Sonicate and heat in a water bath for about 10 min, cool, dilute with acetonitrile to volume, and mix. Before injection, filter through a membrane filter having a 0.45-µm or finer pore size, discarding the first 5 mL of the filtrate.
System suitability solution:  A mixture of Standard solution A and 0.01% toluene in acetonitrile (1:1)
Mobile phase:  See the gradient table below.
Time
(min)
Solution A
(%)
Solution B
(%)
0 45 55
25 45 55
28 95 5
35 95 5
36 45 55
45 45 55
Chromatographic system 
Mode:  LC
Detector:  UV 220 nm
Column:  4.6-mm × 25-cm; 5-µm, 100
Column temperature:  30 ± 2
Flow rate:  1.8 mL/min
Injection size:  20 µL
System suitability 
Samples:  Standard solution A, Standard solution B, and System suitability solution
[Note—The relative retention times for isoforskolin and forskolin are 0.51 and 1.00, respectively. ]
Suitability requirements:  The chromatogram from Standard solution B is similar to the reference chromatogram provided with the lot of USP Powdered Forskohlii Extract RS being used.
Relative standard deviation:  NMT 2% determined from the forskolin peak in repeated injections, Standard solution A
Resolution:  NLT 1.5 between the forskolin and toluene peaks, System suitability solution
Analysis 
Samples:  Standard solution A, Standard solution B, and Sample solution
Using the chromatogram of Standard solution A, Standard solution B, and the reference chromatogram provided with the lot of USP Powdered Forskohlii Extract RS being used, identify the retention times of the peaks corresponding to isoforskolin and forskolin.
Calculate the percentage of forskolin in the portion of Powdered Forskohlii Extract taken:
Result = (rU/rS) × (CS/CU) × 100
rU== peak response of forskolin from the Sample solution
rS== peak response of forskolin from Standard solution A
CS== concentration of USP Forskolin RS in Standard solution A (mg/mL)
CU== concentration of Powdered Forskohlii Extract in the Sample solution (mg/mL)
Acceptance criteria:  NLT 90.0% and NMT 110.0% of the labeled amount of forskolin on the dried basis
IMPURITIES
Inorganic Impurities 
•  Heavy Metals, Method III 231: NMT 20 ppm
Organic Impurities 
SPECIFIC TESTS
•  Loss on Drying 731: Dry 1.0 g of Powdered Forskohlii Extract at 105 for 3 h: it loses NMT 5.0% of its weight.
•  Microbial Enumeration Tests 2021: The total aerobic microbial count does not exceed 104 cfu/g. The total combined yeasts and molds 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.
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.
•  Other Requirements: It meets the requirements of the test for Residual Solvents under Botanical Extracts 565.
•  USP Reference Standards 11
USP Forskolin RS Click to View Structure
USP Powdered Forskohlii 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 1301
Pharmacopeial Forum: Volume No. 36(3) Page 698