TITLE

TESTING PROCEDURE
ALD/QCTP-12

DOCUMENT NO.

PAGE NO

1 of 2

REVISION NO

00

DATE

Mar 2015

PREPARED BY

MR

APPROVED BY

C.E.O

Test Name: to determine the Azadirachtin content in given sample

Principle :
Azadirachtin in the sample is dissolve in methanol and separated by means of liquid
chromatography. Azadirachtin is detected by measuring the absorbance at 215 nm.
Apparatus : HPLC
HPLC unit equipped with UV detector and printer Integrator and is operated under the
following suggestive parameters. These parameters may be varied as per available facilities
provided standardization is done.
Column

C-18, 25 cm x 4.6 mm 5 particle size (waters ODS2)

Flow rate

1 ml/min

Detector

UV

Wave length

215 nm

Mobile phase
Retention time

Acetonitrile : Water (35:65) v/v

Azadirachtin-A 10-12 min or 16 min

Azadirachtin-B 11-13 min or 17 min

Injection volume

20 l .

Reagents & glassware

Acetonitrile

HPLC grade

Water

HPLC grade

Volumetric flask

10 & 100 ml capacity

Pipettes

micropippete

Preparation of standard solutions:

Preparation of working standard solution:
Weigh accurately 10 mg of standard Azadirachtin of known purity in 10 ml volumetric flask
& dissolve in Mobile phase. Pipette out 1 ml of this stock solution in to 100ml volumetric
flask and male up the volume with mobile phase
Preparation of test sample :
Weigh accurately 3.3gms of sample(equivalent to 10 mg of a content ) into a 100 ml
volumetric flask and add above 70 ml of Mobile phase, shake for 5 minutes and make up to

TESTING PROCEDURE

TITLE
DOCUMENT NO.

ALD/QCTP-12

PAGE NO

1 of 2

REVISION NO

00

DATE

Mar 2015

PREPARED BY

MR

APPROVED BY

C.E.O

the mark. Pipette out 1 ml of this stock solution in to 100ml volumetric flask and make up
the volume with mobile phase.
Estimation:
Inject 20 l of standard Azadirachtin and sample solution respectively to get area
reproducibility for two consecutive injections; the area of two consecutive injection should
not vary more than 2%. From the HPLC chromatograph calculate % of Azadirachtin in the
sample.
Calculation :
Azadirachtin content % by mass

A1

M2 x P

A2

M1

A1

= Peak area of Azadirachtin in sample solution injection

A2

= Peak area of Azadirachtin in reference standard injection

M1

= Mass in gms , of sample taken for the test

M2

= Mass in gms , of the reference standard Azadirachtin

= Purity of reference standard Azadirachtin

or
Area of test sample

standard conc x standard purity

Area of working standard

Tolerance: this method has tolerance up to 5-8%

sample conc