Chlorogenic acid content

REAGENTS & INSTRUMENT REQUIRED:

1.   High Performance Liquid Chromatography

2.   Water HPLC grade

3. Acetronitrile HPLC grade
4. Chlorogenic acid  working standard

PROCEDURE:

Standard Preparation :

Weigh accurately about 50 mg of working standard in 25 mL volumetric flask. Add about 15 mL of methanol. Ultrasonicate for 20 minutes and make up the volume with methanol.

Sample Preparation :

Weigh accurately about 50 mg of sample of extract into 25 mL volumetric flask. Add about 15mL of methanol. Ultrasonicate for 20 minutes and make up the volume with methanol.

Ensure that the concentration of active ingredient in sample solution is approximately similar to that of standard solution and filter through 0.45μm filter paper.

For Raw material: Take 10 gm of herb powder (20#) and reflux in methanol for 1hrs twice

Chromatographic Condition:

Mobile phase :              -A - 0.1 % Trifluoroacetic acid in water, B - Acetronitrile

Column                         -Phenomenex luna 5μ, C18 (250mm x 4.6mm)

Mode                            -Gradient

                                    15% to 22.5% B in 10 minutes, then increasing to 35 % B in 5 Minute.

Injection                       -20μL

Flow rate                      -1.0 mL/minute

Detector wave length   - 280 nm

Condition                     - Room temperature

Calculation:

                                         Area of sample   X  Concentration of std    X   purity of std

  % of Ursolic acid   =- --------------------------------------------------------------------------------------

                                            Area of standard   X  Concentration of sample 

Charantin content

REAGENTS & INSTRUMENT REQUIRED:

1. High Performance Liquid Chromatography
2. Water HPLC grade
3. Methanol LR grade
4. Charantin working standard.

PROCEDURE:

Standard Preparation :

Weigh accurately about 2.5mg of working standard in 10mL volumetric flask. Add about 5mL of Tetrahydrofuran, ultrasonicate till completely dissolved and make up the volume with Tetrahydrofuran.

Shake well and filter through 0.45µm filter paper.

Sample Preparation :

Weigh accurately about 100mg of sample of extract in 50mL volumetric flask. Add about 25mL of Tetrahydrofuran, ultrasonicate for five minutes and make up the volume with Tetrahydrofuran. Ensure that the concentration of active ingredient in sample solution is approximately similar to that of standard solution and Shake well and filter through 0.45µm filter paper.

Chromatographic Condition:

Mobile Phase               - Water (A) : Methanol (B) 

Conditions                    - 85% B increasing to 95% B over 15 minutes, then increasing to 100% B for                                                                                            

                                       5minutes.                                           

Column                        - Phenomenex luna 5µ, C18 (250mm X 4.6mm)

Mode                           – Gradient

Injection Volume         – 20µL

Flow rate                     – 1.0mL /minute

Detector wavelength   – 250 ± 50nm

Condition                    -  RT

Calculation:

                                             Area of sample  X  Concentration of std    X   purity of std

  % of  Charantin content =---------------------------------------------------------------------------------

                                                Area of standard    X   Concentration of sample 

Baccosides content

REAGENTS & INSTRUMENT REQUIRED:

1. High Performance Liquid Chromatography
2. Water HPLC grade
3. Acetronitrile HPLC grade
4. Orthophosphoric Acid
5. Methanol LR grade
6. Baccosides working Standard

PROCEDURE:

Standard Preparation :

Weigh accurately about 250mg of in working standard into a 25mL volumetric flask. Add 20mL methanol and sonicate for 15 minutes. Make up to volume with methanol and Filter through 0.45μm filter paper.

Sample Preparation :

Weigh accurately about 250mg of sample into a 25mL volumetric flask . Add about 20mL solvents. Ultrasonicate for 15 minutes cool and make up the volume with methanol. Ensure that the concentration of active ingredient in sample solution is approximately similar to that of standard solution and filter through 0.45μm filter paper.

For Rawmaterial:  Take 10 gm of Herb powder (20#) and reflux in methanol for 1hrs twice.

Chromatographic Condition:

Mobile Phase               - 0.1% Orthophosphoric acid : Acetronitrile  (65 : 35) filter through 0.45μ and degas.

Column                        - Phenomenex luna 5µ, C18 (250mm X 4.6mm)

Mode                           – Isocratic

Injection Volume         – 20 µL

Flow rate                     – 1.0/1.5 mL /minute

Detector wavelength   – 205 nm

Condition                     - RT

Calculation:

                                    Area of sample   X   Concentration of std    X   purity of std

  % of  Baccosides =------------------------------------------------------------------------------------

                                       Area of standard    X   Concentration of sample 

Asiaticoside content

REAGENTS & INSTRUMENT REQUIRED:

1. High Performance Liquid Chromatography

2. Water HPLC grade

3. Methanol LR grade

4 .Asiaticoside working standard

PROCEDURE:

Standard Preparation :

Weigh accurately about 25mg of in working standard into a 25mL volumetric flask. Add 20mL methanol. Ultrasonicate for 15 minutes. Cool and make up the volume with methanol. Shake well and filter through 0.45μm filter paper.

Sample Preparation :

Weigh accurately about 100mg of sample into a 100 mL volumetric flask.  Add about 20mL methanol. Ultrasonicate for 15 minutes. Cool and make up the volume with methanol. Ensure that the concentration of active ingredient in sample solution is approximately similar to that of std solution. Shake well and filter through 0.45μm filter paper.

For Raw material: Take 10 gm of herb powder(20#) and reflux in methanol for 1hrs. twice

Chromatographic Condition:

Mobile Phase               - Acetonitrile : Water [ 60 : 40 ]                                        

Column                        - Phenomenex luna 5µ, C18 (250mm X 4.6mm)

Mode                           – Isocratic

Injection Volume         – 20 µL

Flow rate                     – 1.0mL /minute

Detector wavelength   – 220nm

Condition                     - RT

Calculation:

                                        Area of sample  X Concentration of std    X   purity of std

  % of  Asiaticoside    =-----------------------------------------------------------------------------------

                                            Area of standard    X  Concentration of sample