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IS 14299 (1995): Neem Extract Concentrate Containing

Azadirachtin [FAD 1: Pesticides and Pesticides Residue
Analysis]

! $ ' +-
Satyanarayan Gangaram Pitroda
Invent a New India Using Knowledge

! > 0 B


BharthariNtiatakam
Knowledge is such a treasure which cannot be stolen
 Jf!yrf)Q Jf/rt2fJ
~ WRf ;fr1:r ~ em ~ - ~

Indian Standard
NEEM EXTRACT CONCENTRATE CONTAINING
AZADIRACHTIN - SPECIFICATION
I First Reprint JANUARY 1999)

ICS 65.100

\~.

C 8151995
BUREAU ()F INDIAN ST~NDARDS
M"ANAK BHAVAN. 9 BAHADUR SHAH ZAFAR MARG
., NEW DELHI 110002

PrIee Group 2
 AMENDMENT NO. 1 MARCH 2000
TO
IS 14299: 1995 NEEM EXTRACT CONCENTRATE
CONTAINING AZADIRACIITIN - SPECIFICATION
(Page 2, Annex A ) -Sul~titute the following for the existing text:
DETEIWINA1'ION OF AZADIRACHTIN CONTENT INNEEM
1""oRMUlATION EC
A-I I'JUNCII)LE
Azadirachtin in the sanlple is dissolved in methanol-water (90:10) and analyzed
on HPLC at 215 nm.
AZ AIIAJ~ArUS

A-Z.t Hlgb Iterfonnance Llquld Chromatography

HPLC unit equipped with ultraviolet (UV) detector and printer-plouer-cum-
integrator and is operated under the following suggestive parameters. These
parameters may be varied as per available facilities provided standardization is
done:

i) Column e-1S, 25 CIII x .4.6 mmStainless Steel

5 IJ Pa rtlcle size or equivalent
ii) Flow rate 1.0 Inl/loin approx.

iii) Detector Ultraviolet (UV) - 215 mm

iv) Mobile phase Acetonitrile:Water(35:6S) (vlv)
v) Retention time Azadirachtin - A 10-12minutes (aJlprox.)
A7..adirachtin- B 1113 minutes (approx.)
A-2.Z ~ticrosyringf, 25 J.ll capacity.
A-3 RF..AGE~rS

A-3.t Refenl1ce StundanJ - Working standard of known purity.

A3.z A&:etollitrile - HPLC Grade.
1
 Anlfnd NQ.l t.. IS 14199 : l"S

"\..3.3 Water - HPLCGlide.

1\-3.4 Methanol - HPLCGrade.
A..3.5 Volul1Ietrlc Flasks - 10 nIl, SO nIland 100ml.
/\..3.6 Itlpet*" - ~raduated, 2 011 and SOli.
A-4laREPARATION OF STANDARD SOLUTIONS
. A-4.1 Prepanltlo. or aererence"Standani Solutio.
Weigb Iceuntely tbe 2.0 mg working standard of known purity into SO mI
volumetne Oaskand dissolve in methanol: wlter (90:10). M.ke up to tbe mlrk
aDd suke well. Take 2 III of tbls IOlution with tbe belp of pipette into I syringe
prepiRd II per A-4.3. Elute it into 10 mI volumetric Disk witb repelted
washings with methanol: water (90:10). Make up to tbe mark,sbake well.
A4.z ~pantlon or SaID pie Solution
Weigb Iccufltely sample quantity 10 I. 10 contain 20 mg or Azadillcbtin in
50 ml volumetric flask and dissolve in methanol wiler (90:10). Shake weIland
keep it aside to separate the layell. Take 2 ml of this solution with tbe help of
pipette into a syringe prepared as per A-4.3. Elute it into 10 ml volumetric Dask
with repeated washings with metbanol : water (90:10). Make up to tbe Dlark and
. shake well.
A-4.3 Preparallon or Thin Colullln or-e18 Material InSyringe
Take a scp pak cartridge conlaining O.S g of CIS packing material and wet it
with solvent nlclhano) : water (90:10) before loading the sample solution.
Alternatively take a 5 .u' glass syringe of 10 mot internal diameter and insert a
small wad of silanised glass wool inside the syringe to the bottom. Add Ct8
powder (30..40 um) 10 rom, about 1 em beighl columa (approx 0.5 g), put
another wad of glass wool and wet witb solvent(nletbanol : water 90:10).
1\-5 .~~IMAl'I()N

"'.~.l tn.icct 20 JA.! of working slttndard and sample solution respectively to get
area reproducibllity for two consecutive injections. The area of two consecutive
ill.icctiol1s should not vary by more tban 2 percent. From the HPLC
(,hron,atogranl calculatepercentage of Azadirachtin in tbe sanlple as in A-S.Z.

2
 Amend No.1 to IS 141'9 : 1995

A-S.Z Calculation
A7..adirarhlin content, Ai x M2
percentby mass = x P
A2 )( Ml
wbere
Al = peakarea of Azadiracbtin insample solution (Aza A+B );
A2 II: peak area of Azadirachtin inworking standard (Aza A+8);
Ml = mass, in g. of tbe sampletaken for test;
M2 := mass, in g, of the working standard Azadirachtin; and
P = purity of workingstandard.
A5.3 Analytical Tolerance
Adopt a 8nalyti~.a1 tolerance after' analyzing tbe fortified Slioplea of oil
formulauons, if necessary (norlnally the tolerance level is 101S percent).

(FAD J )

Reprography Unit, BIS, NewDelhi, hklia

3
 AMENDMENT NO. 2 MAY 2002
TO
IS 142" : 1995 NEBM EXTRACT CONCENTRATE
CONTAINING AZADIRACHTIN - SPECIFICATION
( Page 2, Annex A, titl, ( I~. al,O AmendmDat No.1) ] - Substitute
'DETERMINATION OF AZADIRACHTlN CONTENT IN NEEM
EXTRACT CONCENTRATE' for 'DETERMINATJON OF
AZADIRACHTIN CONTENT IN NEEM FORMULATION Be
[ Page 2, Annex Atclaus A5.J ( s also AlMrulIMnI No.1)] - Delete.

(FAD I )
Repropapby Unit, BIS,New Delhi. IDdia
Pesticides Sectional Co nunittee, FAD 1

FOREWORD

This Indian Standard was adopted by the Bureau of Indian Standards after the draft finalized bythe Pesticides
Sectional Committee had been approved by tbe Food and Agriculture Division Council.

Neem botanically known as Azadirachta indica AJuss belongs to family meliaceae bas a great economic
importance from ancient time, Neem seed/kernel contains triterpenes which are normally-called Neem bitters.
The Neem bitters are known to contain a number of active derivatives including Azadirachtin which have
the insect-pest control properties.

Neem extracts containing Azadirachtin may be used for making fonnulations or applied directly in the field
crops with appropriate dilution for the control of pests of agricultural crops.

In the preparation of tbis standard, due consideration bas been given to the provisions of the Insecticides
Act, 1968 and Rules framed thereunder and the Standards of Weights and Measures (Packaged Commodities)
Rules, 1977. However, the standard is subject to the restrictions imposed under these wherever applicable.

For tbe purpose of deciding wbether a particular requirement of this standard is complied with, the final
value, observed or calculated, expressing tbe result of a test or analysis, shall be rounded off in accordance
with IS 2 : 1960 'Rules for rounding off numerical values (revised)'. The number of significant places retained
in the rounded off value should be the same as that of the specified value in this standard.
 IS 14299 : 1995

Indian Standard
NEEM EXTRACT CONCENTRATE CONTAINING
AZADIRACHTIN - SPECIFICATION

1 SCOPE 3 REQUIREMENTS

This standard prescribes the requirements and methods 3.1 ConsUtueDts

of sampling and test for Neem extract concentrate
containing Azadirachtin. 3.1.1 The material shall consist of Azadirachtin
content extracted from Neem kemelldepulped Neem
Z REFERENCES seeds.
The following Indian Standards are necessary adjuncts
to this standard : 3.1.% Description
IS No. Title The material shall be in the form of powder/granules
light to dark brown in colour with repulsive odour.
4684: 1975 Edible groundnut flour (expeller
pressed) (first revision) 3.% The material shall also conform to the
requirements given in Table 1.
6940: 1982 Methods of test for pesticides and
their fonnuJations (first revision)
4 PACKING
8190 (Part 1) : Requirements for packing of
1988 pesticides : Part 1 Solid pesticides The material shall be packed in cardboard cartons
(second revision) lined with HM HDPF./Polyethylene of thickness not
less than 0.062 mm, which shall be encased in
10946 : 1984 Methods of sampling for technical
composit containers IS per IS 11357:1985. For bulk
grade pesticides packing fibre board drums shall be used. It shall also
11357 : 1985 Composit containers for dry conform to the general requirements given in
products IS 8190 (Part 1) : 1988.

Table 1 Requirements for NEEM Extract ConceDtrate CODtalnlng Azadlrac:htin

, (Clause 3.2 )

81 Characteristic RequlnDleat Method 01 Test, Rei to

No. ___----'..A.'-----.. .
Annex of Clause of
This Standard IS 6940: 1982
(1) (2) (3) (4) (5)

i) Azadirachtin Nomanal value u A

content, percent declared OD lbe
by mass (m1m) container

Ii) Moisture content, 1.0 4

percent by mass
(mlm), Max

IU) Material insoluble 1.0 9

in acetone, percent
by mass (ID/m), Max

lv) Acidity IS ~SO4 or 0..5 11.3

alkalinity as NaOH,
percent by mass
(milD), MtI%

v) Anatoxin conteDt Nil B

1
IS 14299 : 1995
5 MARKING 5-Z DIS C,rtlfteatloD MarklIII
5.1 The cartons/containers shall be mar~ed legibly The product may also be marked with the Standard
and indelibly witb the following information: Mark.
a) Name of the material;
b) Name of manufacturer; S-Z.l The use of the Standard Mark is governed by
c) Batch number; the provisioDS of the Bureau ofilldia" Standards Act,
1986 and the Rules and Regulations made thereunder.
d) Date of maDufacture;
The details of coDditioDl under which the licence
e) Date of expiry; for the use of Standard Mark may be granted to
t) Net mass in kg; manufacturell or producers may be obtained from
g) Azadinchtin content, percent (mlm), nominal; the Bureau of Indian Standards:
h) Any other requirement as given under the
Insecticides Act, 1968 and Rules fnmed .6 SAMPLING
thereunder; and Stands,ds 01 Weights tutd
Measures (Pac,",ged Commodities) Rules, Representative samples oftbe material shall be drawn
1977. as prescribed in IS 10946:1984.

ANNEX A
[Table 1, 51 No. (i)]
DETERMINATION OF AZADIRACRTIN CONTENT

A-I PRINCIPLE A-3 REAGENTS

Azadirachtin in the sample is dissolved in acetonitrile A-3.t Reference Standard Azadlrachtln - of

and analysed by HPLC method at 217 nm, purity, 9S percent, minimum.

A-2 APPARATUS A-3.2 Acetonitrile - HPLC. grade.

A-2.1 11Igb Perfo.....ance Liquid Chromatograph A-3.3 Water - HPLC grade.

A HPLC unit equipped with ultraviolet (UV) detector A-3.4 Volumetric: Flask - 10 mI and 100 mi.
and printer-plotter-curn-integrator and is operated
A-3.5 Pipettes - graduated, 1 mI and 5 .mi..
under the following suggestive parameters. These .. _itl ...

parameters may be varied as per available facilities

provided standardization is done. A-4 PREPARATION OF STANDARD
SOLUflONS
i) Column C-18, 2S em x 4.6 nun SS,
A-4.1 Preparation of RefereDce Standard Soludoa
SfJ Particle size or equivalent
ii) Flow rate 0.8 mlIminute approx. Weigh accurately 1 mg of standard Azadirachtin of
iii) Detector Ultraviolet (UV) knownpurity into 10 ml volumetric flask and dissolve
iv) Wavelength 217 nm in acetonitrile and dilute up to the mark with
v) Mobile phase - Acetonitrile: Waler (28:72) acetonitrile. Pipette out 1 ml of this stock solution
, tvtv) into 100 mI volumetric Oaskand make up the volume
vi) Retention Azadirachtin - 3.6 minutes with .~tonitrile~'
time (approx)
A-4.2 Prep....ttoa of Sample Solution
A-Z.Z Micros)''''''' - 25. pi capacity,
Weigh .~urltely Simple equivalent to 1 JIll of I.i.
 Jb 14299 : 1995
content into a 10 mI volumetric Oask. Add about 7 A5.2 c..tIoa
mI of acetonitrile and sbake for S minutes and make
up the volume with acetonitrile. Pipette out 1 mI of Azadincbtin content,
solution into a 100 mI volumebic Disk and make percent by mass
up the volume with acetonitrile.
where
A5 ESTIMATION Al =: peak area of Azadiracbtin in sample
solution;
A.5.1Inject 20,ul of standard Azadirachtin and sample A2 :a peak area of Azadincbtin in reference
solution respectively to get aiea reproducibility for standard solution; "
two consecutive injections. The arel of two m1 mass, in g, of the sample taken for the test
consecutive injections should not vary by more than m2 mass, in " of the reference standard
2 percent. From the HPLC chromatogram calculate Azadinchtin; and
percentage of Azadirachtin in the sample as A5.2. P = purity of reference standard Azadinchtin. '

ANNEX 8
[Table 1, 51 No. (v)]
DETERMINATION OF AnATOXIN CONTENT

8-1 PRINCIPLE OF THE METHOD B-2 METHOD

8-1.1 The method involves extraction of aflatoxin
from Neem extract concentrate using chloroform as
solvent and subsequent clean up through silica gel
column using methanol or chloroform for defating.
TLC of the extract is done to estimate aflatoxin
content.
The method as given in Appendix J of IS 4684:1975
shall be followed except where the groundnut flour,
it should be used as Neem extract concentrate
contlining azadinchtin.
Burea. of I"'. Staaclardl
BIS is a statutory institution established under the Bureau' 01 Indian Sttuulards Act, 1986 to promote
harmonious development of the activities of standardization, marking and quality certification of goods. and
attending to,connected matters in the country.

CopyrlPt
BIS, bas the copyright of all i~ publicatioas, No part of these publications may be reproduced in any form
without the prior pennission in writing of BIS. This does not preclude the Cree use, in the course of
implementing the standard, of necessary details, such as symbols and sizes, type or grade designations.
Enquiriesrelating to copyright be addressed to the Director(Publication), BIS.

Rev.", ofIn diaD Slan4ards

Amendments are issued to standards as the need arises on the basis of comments. Standards are also reviewed
periodically; a standard along with amendments is reaffirmed when such review indicates that no changes are
needed; if the review indicates that changes are needed, it is taken up for revision. Users of Indian Standards
should ascertain that they are in possession of the latest amendments or edition by refening to thelatest issue
of 'BIS Handbook' and 'Standards MonthlyAdditions' .
This Indian Standard has been developed from Doc: No. FAD 1 ( 393 )

Amendments Issued Since PublkatioD

Amend No. Date of Issue Text Affected

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