Powdered Andrographis Extract
DEFINITION
Powdered Andrographis Extract is prepared from Andrographis by extraction with methanol or alcohol. The ratio of plant material to extract is between 15:1 and 10:1. It contains NLT 90.0% and NMT 110.0% of the labeled amount of diterpene lactones, calculated on the dried basis as the sum of andrographolide, neoandrographolide, 14-deoxy-11,12-didehydroandrographolide, and andrograpanin. The content of 14-deoxy-11,12-didehydroandrographolide is NMT 15% of the total diterpene lactones. It may contain suitable added substances as carriers.
IDENTIFICATION
•  A. Thin-Layer Chromatographic Identification Test 201
Standard solution 1:  Use Standard solution A, prepared as directed in the test for Content of Diterpene Lactones.
Standard solution 2:  Sonicate for 10–15 min a quantity of USP Powdered Andrographis Extract RS, equivalent to about 15 mg of diterpene lactones, in 25 mL of methanol. Centrifuge, and use the supernatant.
Sample solution:  Sonicate for 10–15 min a quantity of Powdered Andrographis Extract, equivalent to about 15 mg of diterpene lactones, in 25 mL of methanol. Centrifuge, and use the supernatant.
Adsorbent:  Chromatographic silica gel mixture with an average particle size of 10–15 µm (TLC plates)
Application volume:  10 µL, as 5–10 mm bands
Developing solvent system:  Chloroform, acetone, and toluene (2:2:1)
Spray reagent:  A mixture containing 1% vanillin in alcohol and 10% sulfuric acid in alcohol (1:1)
Analysis 
Samples:  Standard solution 1, Standard solution 2, and Sample solution
Use a saturated chamber. Develop until the solvent front has moved up about 90% of the plate. Remove the plate from the chamber. Dry, and spray with Spray reagent. Heat for 5–10 min at 100, and examine under visible light.
Acceptance criteria:  The Sample solution exhibits three main grayish blue zones with RF values of approximately 0.4, 0.6, and 0.8 that correspond in position and color to zones in Standard solution 2. Standard solution 1 exhibits a grayish blue zone due to andrographolide at an RF of about 0.4. The Sample solution exhibits a zone similar in color and RF value to that due to andrographolide in Standard solution 1.
•  B. The Sample solution in the test for Content of Diterpene Lactones shows a main peak at a retention time corresponding to that of andrographolide in Standard solution A. Identify other diterpene lactone peaks in the Sample solution by comparison with Standard solution B and the reference chromatogram provided with the lot of USP Powdered Andrographis Extract RS. The Sample solution shows additional peaks corresponding to neoandrographolide, 14-deoxy-11,12-didehydroandrographolide, and andrograpanin.
COMPOSITION
•  Content of Diterpene Lactones
Solution A:  Dissolve 0.14 g of 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.
Standard solution A:  Dissolve a weighed quantity of USP Andrographolide RS in methanol to obtain a solution having a known concentration of about 1.0 mg/mL. Transfer 5.0 mL of this solution to a 10-mL volumetric flask, dilute with acetonitrile to volume, and mix.
Standard solution B:  Transfer an amount of USP Powdered Andrographis Extract RS, equivalent to about 25 mg of diterpene lactones, to a 50-mL volumetric flask, add 25 mL of methanol, heat gently for 15–20 min, dilute with acetonitrile 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 a weighed amount of Powdered Andrographis Extract, equivalent to about 25 mg of diterpene lactones, to a 50-mL volumetric flask, add 25 mL of methanol, heat gently for 15–20 min, dilute with acetonitrile 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.
Mobile phase:  See the gradient table below.
Time
(min)		 Solution A
(%)		 Solution B
(%)
0 95 5
18 55 45
25 20 80
28 20 80
35 55 45
40 95 5
45 95 5
Chromatographic system 
(See Chromatography 621, System Suitability.)
Mode:  LC
Detector:  UV 223 nm
Column:  4.6-mm × 25-cm; 5-µm packing L1
Flow rate:  1.5 mL/min
Injection size:  20 µL
System suitability 
Samples:  Standard solution A and Standard solution B
Suitability requirements 
The chromatogram from Standard solution B is similar to the reference chromatogram provided with the lot of USP Powdered Andrographis Extract RS.
Column efficiency:  NLT 5000 theoretical plates, Standard solution A
Tailing factor:  NMT 1.5 for the andrographolide peak, Standard solution A
Relative standard deviation:  NMT 2.0%, determined from the andrographolide peak for replicate injections, Standard solution A
Resolution:  NLT 5 between the neoandrographolide and 14-deoxy-11,12-didehydroandrographolide peaks, Standard solution B
Analysis 
Samples:  Standard solution A, Standard solution B, and Sample solution
Using Standard solution A, Standard solution B, and the reference chromatogram provided with the lot of USP Powdered Andrographis Extract RS being used, identify the retention times of the peaks corresponding to different diterpene lactones. The approximate relative retention times of the different diterpene lactones are provided in the following table:
Analyte Relative
Retention Time
Andrographolide 1.00
Neoandrographolide 1.16
14-Deoxy-11,12-didehydroandrographolide 1.31
Andrograpanin 1.50
Separately calculate the percentages of andrographolide, neoandrographolide, 14-deoxy-11,12-didehydroandrographolide, and andrograpanin in the portion of Powdered Andrographis Extract taken:
Result = (rU/rS) × (CS/W) × 5F
rU== peak response for each diterpene lactone from the Sample solution
rS== peak response for andrographolide from Standard solution A
CS== concentration of USP Andrographolide RS in Standard solution A (mg/mL)
W== weight of Powdered Andrographis Extract taken to prepare the Sample solution (g)
F== conversion factor for each analyte (1.00 for andrographolide, 3.90 for neoandrographolide, 1.45 for 14-deoxy-11,12-didehydroandrographolide, and 2.65 for andrograpanin)
Acceptance criteria:  90.0%–110.0%, on the dried basis, of the labeled amount of diterpene lactones calculated as the sum of the percentages of andrographolide, neoandrographolide, 14-deoxy-11,12-didehydroandrographolide, and andrograpanin
IMPURITIES
Inorganic Impurities 
•  Heavy Metals, Method II 231: NMT 20 ppm
Organic Impurities 
SPECIFIC TESTS
•  Loss on Drying 731: Dry 2.0 g 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 prepared. It meets other labeling requirements under Botanical Extracts 565.
•  USP Reference Standards 11
USP Andrographolide RS
USP Powdered Andrographis 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 1190
Pharmacopeial Forum: Volume No. 35(5) Page 1186