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है”ह”ह

IS 14299 (1995): Neem Extract Concentrate ContainingAzadirachtin [FAD 1: Pesticides and Pesticides ResidueAnalysis]

Jf!yrf)Q Jf/rt2fJ

~ WRf ;fr1:r ~ em~ -~

Indian Standard

NEEM EXTRACT CONCENTRATE CONTAININGAZADIRACHTIN - SPECIFICATION

I First Reprint JANUARY 1999)

ICS 65.100

\~.

C 8151995

BUREAU ()F INDIAN ST~NDARDSM"ANAK BHAVAN. 9 BAHADUR SHAH ZAFAR MARG

., NEW DELHI 110002

PrIee Group 2

AMENDMENT NO. 1 MARCH 2000TO

IS 14299: 1995 NEEM EXTRACT CONCENTRATECONTAINING AZADIRACIITIN - SPECIFICATION(Page 2, Annex A ) -Sul~titute the following for theexisting text:

DETEIWINA1'ION OF AZADIRACHTIN CONTENT INNEEM1""oRMUlATION EC

A-I I'JUNCII)LE

Azadirachtin in the sanlple is dissolved in methanol-water (90:10) and analyzedon HPLCat 215 nm.

A·Z AI·I·AJ~A·rUS

A-Z.t Hlgb Iterfonnance Llquld Chromatography

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

e-1S, 25 CIII x .4.6 mmStainless Steel5 IJ Pa rtlcle size or equivalent

1.0 Inl/loin approx.

Ultraviolet (UV) - 215mm

Acetonitrile:Water(35:6S) (vlv)iv) Mobilephase

v) Retention time

ii) Flow rate

iii) Detector

i) Column

Azadirachtin - A 10-12minutes (aJlprox.)A7..adirachtin- B 11·13 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.

A·3.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 - 10nIl,SO nIland 100ml.

/\..3.6 Itlpet*" - ~raduated, 2 011 and SOli•.

A-4laREPARATIONOF STANDARD SOLUTIONS

. A-4.1 Prepanltlo. or aererence"Standani Solutio.

Weigb Iceuntely tbe 2.0 mg working standard of known purity into SO mIvolumetne Oaskand dissolve in methanol: wlter (90:10). M.ke up to tbe mlrkaDd suke well. Take 2 III of tblsIOlution with tbe belp of pipette intoI syringeprepiRd II per A-4.3. Elute it into 10 mI volumetric Disk witb repeltedwashingswith methanol: water(90:10). Makeup to tbe mark,sbake well.

A·4.z ~pantlon orSaIDpie Solution

Weigb Iccufltely sample quantity 10 I. 10contain 20 mg or Azadillcbtin in50 ml volumetric flask and dissolve in methanol wiler (90:10). Shake weIlandkeep it aside to separate the layell. Take 2 ml of this solution with tbe help ofpipette into a syringe prepared as per A-4.3. Elute it into 10 ml volumetric Daskwith repeated washings withmetbanol : 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 itwith solvent nlclhano) : water (90:10) before loading the sample solution.Alternatively take a 5 .u' glass syringe of 10 mot internal diameter and insert asmall wad of silanised glass wool inside the syringe to the bottom. Add Ct8powder (30..40 um) 10 rom, about 1 em beighl columa (approx 0.5 g), putanother wad of glass wooland 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 getarea reproducibllity for two consecutive injections. The area of two consecutiveill.icctiol1s should not vary by more tban 2 percent. From the HPLC(,·hron,atogranl calculatepercentage of Azadirachtin in tbesanlple as in A-S.Z.

2

Amend No.1 to IS 141'9 : 1995

A-S.Z Calculation

A7..adirarhlin content, Ai x M2percentby mass = x P

A2 )( Mlwbere

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 sampletakenfor test;

M2 := mass, in g,of the working standard Azadirachtin; and

P =purity of workingstandard.

A·5.3 Analytical Tolerance

Adopt a 8nalyti~.a1 tolerance after' analyzing tbe fortified Slioplea of oilformulauons, if necessary (norlnally the tolerance level is 10·1S percent).

(FAD J )

Reprography Unit, BIS, NewDelhi, hklia

3

AMENDMENT NO. 2 MAY 2002TO

IS 142" : 1995 NEBM EXTRACT CONCENTRATECONTAINING AZADIRACHTIN - SPECIFICATION( Page 2, Annex A, titl, ( I~. al,O AmendmDat No.1) ] - Substitute

'DETERMINATION OF AZADIRACHTlN CONTENT IN NEEMEXTRACT CONCENTRATE' for 'DETERMINATJON OFAZADIRACHTIN CONTENT INNEEM FORMULATION Be •

[ Page 2,Annex Atclaus« A·5.J ( s•• alsoAlMrulIMnINo.1)] - Delete.

(FAD I )Repropapby Unit, BIS,New Delhi. IDdia

Pesticides Sectional Conunittee, FAD 1

FOREWORD

This Indian Standard was adopted by the Bureau of Indian Standards after the draft finalized bythe PesticidesSectional 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 economicimportance 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 havethe insect-pest control properties.

Neem extracts containing Azadirachtin may be used for making fonnulations or applied directly in the fieldcrops 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 InsecticidesAct, 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 finalvalue, observed or calculated, expressing tbe result of a test or analysis, shall be rounded off in accordancewith IS 2 : 1960 'Rules for rounding off numerical values (revised)'. The number of significant places retainedin 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 CONTAININGAZADIRACHTIN - SPECIFICATION

1 SCOPE 3 REQUIREMENTS

This standard prescribes the requirements and methodsof sampling and test for Neem extract concentratecontaining Azadirachtin.

Z REFERENCES

3.1 ConsUtueDts

3.1.1 The material shall consist of Azadirachtincontent extracted from Neem kemelldepulped Neemseeds.

The following Indian Standards are necessary adjunctsto this standard :

4 PACKING

3.1.% Description

The material shall be in the form of powder/granuleslight to dark brown in colour with repulsive odour.

3.% The material shall also conform to therequirements given in Table 1.

The material shall be packed in cardboard cartonslined with HM HDPF./Polyethylene of thickness notless than 0.062 mm, which shall be encased incomposit containers IS per IS 11357:1985. For bulkpacking fibre board drums shall be used. It shall alsoconform to the general requirements given inIS 8190 (Part 1) : 1988.

TitleIS No.

4684: 1975

6940: 1982

Edible groundnut flour (expellerpressed) (first revision)

Methods of test for pesticides andtheir fonnuJations (first revision)

8190 (Part 1) : Requirements for packing of1988 pesticides : Part 1 Solid pesticides

(second revision)

10946 : 1984 Methods of sampling for technicalgrade pesticides

11357: 1985 Composit containers for dryproducts

Table 1 Requirements for NEEM Extract ConceDtrate CODtalnlng Azadlrac:htin, (Clause 3.2 )

81No.

Characteristic RequlnDleat Method 01 Test, Rei to___----'..A.'-----.....

(1) (2) (3)

Annex ofThis Standard

(4)

Clause ofIS 6940: 1982

(5)

i) Azadirachtincontent, percentby mass (m1m)

Nomanal value udeclared OD lbecontainer

A

Ii) Moisture content,percent by mass(mlm), Max

1.0 4

IU) Material insolublein acetone, percentby mass (ID/m), Max

1.0 9

lv) Acidity IS ~SO4 oralkalinity as NaOH,percent by mass(milD), MtI%

0..5 11.3

v) Anatoxin conteDt Nil B

1

IS 14299 : 1995

5 MARKING

5.1 The cartons/containers shall be mar~ed legiblyand indelibly witb the following information:

a) Name of the material;b) Name of manufacturer;c) Batch number;d) Date of maDufacture;e) Date of expiry;t) Net mass in kg;g) Azadinchtin content, percent (mlm), nominal;h) Any other requirement as given under the

Insecticides Act, 1968 and Rules fnmedthereunder; and Stands,ds 01 Weights tutdMeasures (Pac,",ged Commodities) Rules,1977.

5-Z DIS C,rtlfteatloD MarklIII

The product may also be marked with the StandardMark.

S-Z.l The use of the Standard Mark is governed bythe provisioDS of the Bureau ofilldia" Standards Act,1986 and theRules and Regulations made thereunder.The details of coDditioDl under which the licencefor the use of Standard Mark may be granted tomanufacturell or producers may be obtained fromthe Bureau of Indian Standards:

.6 SAMPLING

Representative samples oftbe material shall be drawnas prescribed in IS 10946:1984.

A-I PRINCIPLE

ANNEX A

[Table 1, 51 No. (i)]

DETERMINATION OF AZADIRACRTIN CONTENT

A-3 REAGENTS

Azadirachtin in the sample is dissolved in acetonitrileand analysed by HPLC method at 217 nm,

A-2 APPARATUS

A-2.1 11Igb Perfo.....ance Liquid Chromatograph

A HPLC unit equipped with ultraviolet (UV) detectorand printer-plotter-curn-integrator and is operatedunder the following suggestive parameters. Theseparameters may be varied as per available facilitiesprovided standardization is done.

A-3.t Reference Standard Azadlrachtln - ofpurity, 9S percent, minimum.

A-3.2 Acetonitrile - HPLC. grade.

A-3.3 Water - HPLC grade.

A-3.4 Volumetric: Flask - 10 mI and 100 mi.

A-3.5 Pipettes - graduated, 1 mI and 5 .mi.... _itl ...

A-4 PREPARATION OF STANDARDSOLUflONS

A-Z.Z Micros)''''''' - 25. pi capacity,

i) Column

ii) Flow rateiii) Detectoriv) Wavelengthv) Mobile phase -,

vi) Retentiontime

C-18, 2S em x 4.6 nun SS,SfJ Particle size or equivalent0.8 mlIminute approx.Ultraviolet (UV)217 nmAcetonitrile: Waler (28:72)tvtv)Azadirachtin - 3.6 minutes(approx)

A-4.1 Preparation ofRefereDce Standard Soludoa

Weigh accurately 1 mg of standard Azadirachtin ofknownpurity into 10ml volumetric flask and dissolvein acetonitrile and dilute up to the mark withacetonitrile. Pipette out 1 ml of this stock solutioninto 100 mI volumetric Oaskand make up the volumewith .~tonitrile~' ·

A-4.2 Prep....ttoa of Sample Solution

Weigh .~urltely Simple equivalent to 1 JIll of I.i.

content into a 10 mI volumetric Oask. Add about 7mIof acetonitrile and sbake for S minutes and makeup the volume with acetonitrile. Pipette out 1 mI ofsolution into a 100 mI volumebic Disk and makeup the volume with acetonitrile.

A·5 ESTIMATION

A.5.1Inject 20,ul ofstandard Azadirachtinand samplesolution respectively to get aiea reproducibility fortwo consecutive injections. The arel of twoconsecutive injections should not vary by more than2 percent. From the HPLC chromatogram calculatepercentage of Azadirachtin in the sample as A·5.2.

Jb 14299 : 1995

A·5.2 c..tIoaAzadincbtin content,

percent by mass

whereAl =: peak area of Azadiracbtin in sample

solution;A2 :a peak area of Azadincbtin in reference

standard solution; "m1 • mass, in g, of the sample taken for the testm2 • mass, in " of the reference standard

Azadinchtin; andP = purity of reference standard Azadinchtin. '

ANNEX 8

[Table 1, 51 No. (v)]

DETERMINATION OF AnATOXIN CONTENT

8-1 PRINCIPLE OF THE METHOD

8-1.1 The method involves extraction of aflatoxinfrom Neem extract concentrate using chloroform assolvent and subsequent clean up through silica gelcolumn using methanol or chloroform for defating.TLC of the extract is done to estimate aflatoxincontent.

B-2 METHOD

The method as given in Appendix J of IS 4684:1975shall be followed except where the groundnut flour,it should be used as Neem extract concentratecontlining azadinchtin.

Burea. ofI"'. Staaclardl

BIS is a statutory institution established under the Bureau' 01 Indian Sttuulards Act, 1986 to promoteharmonious development of the activities of standardization, marking and quality certification of goods. andattendingto,connected matters in thecountry.

CopyrlPt

BIS, bas the copyright of all i~ publicatioas, No part of these publications may be reproduced in any formwithout the prior pennission in writing of BIS. This does not preclude the Cree use, in the course ofimplementing the standard, of necessary details, such as symbols and sizes, type or grade designations.Enquiriesrelating to copyrightbe addressed to the Director(Publication), BIS.

Rev.", ofIndiaD Slan4ards

Amendments are issued to standards as theneed arises on the basis of comments. Standards are also reviewedperiodically; a standard along with amendments is reaffirmed when such review indicates that no changes areneeded; if the review indicates that changes are needed, it is taken up for revision. Users of Indian Standardsshould ascertain that they are in possessionof the latest amendments or edition by refening to thelatest issueof 'BIS Handbook' and 'Standards MonthlyAdditions' .

This IndianStandardhas been developed fromDoc: No. FAD 1 ( 393 )

Amendments Issued Since PublkatioD

AmendNo. Date of Issue

BUREAU OF INDIAN STANDARDS

Text Affected

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