Powdered Bilberry Extract
DEFINITION
Powdered Bilberry Extract is prepared from the ripe fruits of Vaccinium myrtillus L. (Fam. Ericaceae) using suitable solvents such as alcohol, methanol, or water or mixtures of these solvents. The ratio of the starting plant material to Powdered Extract is between 153:1 and 76:1. It contains NLT 36.0% of anthocyanosides, calculated as cyanidin-3-O-glucoside chloride, and NMT 1.0% of anthocyanidins, calculated as cyanidin chloride; both are calculated on the anhydrous basis.
IDENTIFICATION
• A. Thin-Layer Chromatographic Identification Test
Standard solution:
4 mg/mL of USP Powdered Bilberry Extract RS in methanol. Centrifuge, and use the clear supernatant.
Sample solution:
4 mg/mL of Powdered Bilberry Extract in methanol. Centrifuge, and use the clear supernatant.
Adsorbent:
Use suitable thin-layer chromatographic plates coated with a layer of cellulose.
Application volume:
10 µL
Developing solvent system A:
Glacial acetic acid, hydrochloric acid, and water (15:3:82)
Developing solvent system B:
Glacial acetic acid and water (6:4)
Analysis
Samples:
Standard solution and Sample solution
Use a saturated chamber. Develop the chromatograms using Developing solvent system A, and dry the plate with the aid of a current of warm air. Develop the chromatograms in the same direction using Developing solvent system B. Examine the plate under visible light.
Acceptance criteria:
The chromatogram of the Sample solution exhibits three main red bands with RF values of approximately 0.55, 0.65, and 0.70 that are similar in position and color to the corresponding main bands in the chromatogram of the Standard solution.
• B.
The retention times of the anthocyanoside peaks in the chromatogram of the Sample solution correspond to those in the chromatogram of Standard solution C, as obtained in the test for Content of Anthocyanosides and Anthocyanidins. The peaks due to delphinidin-3-O-galactoside chloride and delphinidin-3-O-glucoside chloride are the most intense peaks and are of similar intensity, and each is more intense than the peak due to cyanidin-3-O-glucoside chloride. The peaks due to cyanidin-3-O-galactoside chloride, delphinidin-3-O-arabinoside chloride, and cyanidin-3-O-glucoside chloride are of similar intensity. Each of the remaining anthocyanoside peaks is of lower intensity than the peak due to cyanidin-3-O-glucoside chloride.
COMPOSITION
• Content of Anthocyanosides and Anthocyanidins
Solvent:
Methanol and hydrochloric acid (49:1)
Diluent:
85% phosphoric acid and water (1:9)
Solution A:
Formic acid and water (1:9)
Solution B:
Acetonitrile, methanol, formic acid, and water (45:45:20:80)
Mobile phase:
See Table 1.
Table 1
| Time (min) |
Solution A (%) |
Solution B (%) |
|---|---|---|
| 0 | 93 | 7 |
| 35 | 75 | 25 |
| 45 | 35 | 65 |
| 46 | 0 | 100 |
| 50 | 0 | 100 |
| 51 | 93 | 7 |
| 60 | 93 | 7 |
Standard stock solution A:
0.4 mg/mL of USP Cyanidin-3-O-glucoside Chloride RS in Solvent. [Note—Dissolve using sonication. ]
Standard solution A:
0.08 mg/mL of USP Cyanidin-3-O-glucoside Chloride RS from Standard stock solution A in Diluent. [Note—This solution is stable for 48 h at 4
. ]
. ]
Standard stock solution B:
0.5 mg/mL of USP Cyanidin Chloride RS in Solvent. [Note—Dissolve using sonication. ]
Standard solution B:
0.01 mg/mL of USP Cyanidin Chloride RS from Standard stock solution B in Diluent. [Note—This solution is stable for 36 h at 4 ]
]
Standard solution C:
Transfer 125 mg of USP Powdered Bilberry Extract RS to a 100-mL volumetric flask, add 25 mL of Solvent, sonicate to dissolve, and dilute with Diluent to volume. [Note—This solution is stable for 48 h at 4. ]
. ]
Sample solution:
Transfer 125 mg of Powdered Bilberry Extract to a 100-mL volumetric flask, add 25 mL of Solvent, sonicate to dissolve, and dilute with Diluent to volume.
Chromatographic system
[Note—Use deactivated silanized HPLC vials. ]
Mode:
LC
Detector:
UV-Vis 535 nm
Column:
4.6-mm × 25-cm; 5-µm packing L1
Temperature
Refrigerated autosampler:
4
Column:
30 ± 1
Flow rate:
1 mL/min
Injection size:
10 µL
System suitability
Samples:
Standard solution A and Standard solution C
Suitability requirements
Chromatogram similarity:
The chromatogram from Standard solution C is similar to the Reference Chromatogram provided with USP Powdered Bilberry Extract RS.
Resolution:
NLT 0.8 between the delphinidin-3-O-arabinoside, malvidin-3-O-galactoside, and petunidin-3-O-arabinose peaks and NLT 1.0 for other components, Standard solution C
Tailing factor range:
0.8–2.0 for the cyanidin-3-O-glucoside chloride peak, Standard solution A
Relative standard deviation:
NMT 2.0% for cyanidin-3-O-glucoside chloride peak in repeated injections, Standard solution A
Analysis
Samples:
Standard solution A, Standard solution B, Standard solution C, and Sample solution
Using the chromatogram of Standard solution C and the Reference Chromatogram, identify the retention times of the peaks corresponding to the different anthocyanosides and anthocyanidins. The approximate relative retention times, relative to cyanidin-3-O-glucoside chloride, are provided for the anthocyanosides in Table 2 and for the anthocyanidins in Table 3.
Separately calculate the percentages of each anthocyanoside (see Table 2) in the portion of Powdered Extract taken:
Result = (rU/rS) × (CS/CU) × 100
| rU | = | = peak response of each of the anthocyanosides in the Sample solution |
| rS | = | = peak response of cyanidin-3-O-glucoside chloride in Standard solution A |
| CS | = | = concentration of USP Cyanidin-3-O-glucoside Chloride RS in Standard solution A (mg/mL) |
| CU | = | = concentration of Powdered Extract in the Sample solution (mg/mL) |
Table 2
| Analyte | Relative Retention Time |
|---|---|
| Delphinidin-3-O-galactoside chloride | 0.61 |
| Delphinidin-3-O-glucoside chloride | 0.73 |
| Cyanidin-3-O-galactoside chloride | 0.84 |
| Delphinidin-3-O-arabinoside chloride | 0.86 |
| Cyanidin-3-O-glucoside chloride | 1.00 |
| Petunidin-3-O-galactoside chloride | 1.08 |
| Cyanidin-3-O-arabinoside chloride | 1.11 |
| Petunidin-3-O-glucoside chloride | 1.24 |
| Peonidin-3-O-galactoside chloride | 1.36 |
| Petunidin-3-O-arabinoside chloride | 1.39 |
| Peonidin-3-O-glucoside chloride | 1.55 |
| Malvidin-3-O-galactoside chloride | 1.58 |
| Peonidin-3-O-arabinoside chloride | 1.67 |
| Malvidin-3-O-glucoside chloride | 1.76 |
| Malvidin-3-O-arabinoside chloride | 1.91 |
Separately calculate the percentages of anthocyanidins (see Table 3) in the portion of Powdered Extract taken:
Result = (rU/rS) × (CS/CU) × 100
| rU | = | = peak response of each of the anthocyanidins in the Sample solution |
| rS | = | = peak response of cyanidin chloride in Standard solution B |
| CS | = | = concentration of USP Cyanidin Chloride RS in Standard solution B (mg/mL) |
| CU | = | = concentration of Powdered Extract in the Sample solution (mg/mL) |
Table 3
| Analyte | Relative Retention Time |
|---|---|
| Delphinidin chloride | 1.28 |
| Cyanidin chloride | 1.82 |
| Petunidin chloride | 2.08 |
| Peonidin chloride | 2.27 |
| Malvidin chloride | 2.30 |
Acceptance criteria
Sum of all anthocyanosides:
NLT 36.0% on the anhydrous basis
Sum of all anthocyanidins:
NMT 1.0% on the anhydrous basis
CONTAMINANTS
• Heavy Metals, Method II 
231
:
NMT 20 ppm
231:
NMT 20 ppm
• Articles of Botanical Origin, General Method for Pesticide Residues Analysis 561:
Meets the requirements
561:
Meets the requirements
• Microbial Enumeration Tests 2021
2021
Total aerobic microbial count:
NMT 104 cfu/g
Total combined yeasts and molds count:
NMT 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.
2022:
It meets the requirements of the tests for absence of Salmonella species and Escherichia coli.
SPECIFIC TESTS
• Acid Insoluble Fraction:
Sample:
5 g of Powdered Extract finely ground
Analysis:
Transfer about 1 g to a 500-mL flask, add 200 mL of 0.1 N hydrochloric acid, and shake vigorously for 2 h. Pass the solution through a previously tared sintered-glass filter, wash the flask with 30 mL of 0.1 N hydrochloric acid, and transfer the washings to the filter. Wash the filter with 30 mL of 0.1 N hydrochloric acid in 5-mL portions. Dry the filter for 3 h at 105, cool in a desiccator, and weigh. Calculate the percentage of the acid insoluble fraction.
, cool in a desiccator, and weigh. Calculate the percentage of the acid insoluble fraction.
Acceptance criteria:
NMT 5%
• Water Determination, Method Ia 921:
NMT 4.5%, determined on 0.5 g
921:
NMT 4.5%, determined on 0.5 g
• Residue on Ignition 281:
NMT 3.0%, determined on 1.0 g
281:
NMT 3.0%, determined on 1.0 g
• Botanical Extracts, Residual Solvents 565:
Meets the requirements
565:
Meets the requirements
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.
565.
• USP Reference Standards 11
11
USP Powdered Bilberry Extract RS
USP Cyanidin Chloride RS 
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USP Cyanidin-3-O-glucoside Chloride 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 1203
Pharmacopeial Forum: Volume No. 33(4) Page 685