Determination of Azadirachtin A in NeemAzal technical andNeemAzal-T/S by RP-HPLC

• Trifolio-M GmbH, • 35633 Lahnau, Germany

International Neem Conference 2006, Kunming, China

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Trifolio-MDetermination of Azadirachtin A (AzA) in NeemAzal technical andNeemAzal-T/S by RP-HPLC

• Terminology:– Azadirachtin: a.i. of the seed kernels of the

Neem tree (Azadirachta indica A. Juss)– NeemAzal: variant of the a.i.– AzA: lead compound in NeemAzal and

NeemAzal-T/S

O

O

OH

O O

O

MeOOC

OH

HOH

COOMeOH

O

AcO3

O

O

OH

O O

O

MeOOC

OH

HOH

COOMeOH

O

AcO3

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Trifolio-M

• One requirement for trading of a plant protection product:

– Consecutively control of the content of the a.i. in the technical material and the formulation(s)

• Validated methods for analysis are necessary

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• Validated methods – In house, according to valid guidelines

(e.g. SANCO/3030/99 )– Ring tests (e.g. CIPAC, AOAC)

– What is CIPAC?• Collaborative International Pesticides

Analytical Council (www.cipac.org)• international, non-profit-oriented and non-

governmental organisation

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• Why is a CIPAC method of particular importance?

– CIPAC promotes the international agreement on methods for the analysis of pesticides and physico-chemical test methods for formulations

– The methods are proposed by companies and are tested by laboratories all over the world

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• Why is a CIPAC method of particular importance?

– In the EU registration process of plant protection products CIPAC and AOAC methods for analysis of the active substance in technical material and also in the formulation are regarded as validated (SANCO/3030/99 rev. 4)

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Trifolio-MCIPAC study

• Prerequisite:

– Pilot study (small scale study) with results ideally presented at an annual CIPAC meeting.

• This is reasonable to avoid a loss of time and money on an inadequate established method

• Also to get in contact with the CIPAC panel and discuss the method with their experts and other experienced analysts

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Trifolio-MObjective of the Pilot Study for determination of AzA

– Gain information whether an isocratic or a gradient HPLC-method for the determination of AzA in TC and EC is appropriate for a collaborative study

– Find the most adequate conditions and parameters (e.g. column) for analysis

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Trifolio-MEvaluation

• A CIPAC collaborative study is evaluated as defined in ISO 5725

• The following parameters are of importance:– RSDR: reproducibility, which is the relative

standard deviation of among-laboratory results

– RSDH: Horwitz value

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Trifolio-MHorwitz equation

RSDH = 2(1-0.5 log c)

– where c is the concentration of the analyte(c=1 is equivalent to 100%)

• The Horwitz equation is an exponential relationship between reproducibility RSDRand concentration– it is more or less independent of the

analyte, matrix and method of analysis and has been derived empirically

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Trifolio-MHorwitz equation

• The Horwitz equation is used as a criterion of acceptability for methods collaboratively tested by CIPAC

– the Horwitz value is compared with the value of the reproducibility (RSDR) which was determined statistically

– Method is accepted if: RSDR < RSDH

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Trifolio-MResults – pilot study

3.9869 - 3.97992.3252 - 2.3194RSDR(Hor)

4.3796 - 1.87003.2464 - 1.6292RSDR

10.22 - 10.34367.50 - 373.72x [g/kg]

Gradient - IsocraticGradient - Isocratic

NeemAzal-T/SNeemAzal tech.

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Trifolio-MComparison – CIPAC 4042 (1998) vs. CIPAC 4387 (2004)

3.97993.925RSDR(Hor)

1.87007.11RSDR

10.3411.34x [g/kg]

IsocraticIsocratic

NeemAzal-T/SCIPAC 4387

Neem FormulationCIPAC 4042

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• Results of the pilot test were presented to CIPAC panel in 2004 (Brno)

• Panel accepted the results and a full scale study was initiated and performed in 2004 to 2005– At least 8 results are compulsory for

evaluation of a full scale study in order to have sufficient data for the statistic evaluation

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Trifolio-MAnalytical Conditions

• HPLC analysis with UV detection at 214 nm• Column: Luna (2) RP18, Phenomenex Ltd.,

– Dimension: 150 mm x 4.6 mm ID– Particle size: 3µm– Pre column with same packing material

• Column temperature: 30°C• Injection: 20 µl• Flow: 1.1 ml/min• Solvent: CH3CN : H2O = 440 : 560 (v/v)• Column cleaning after 7 to 10 injections with

CH3CN for 10 min

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Trifolio-MHPLC chromatograms of AzA reference standard and TK

Minutes1 2 3 4 5 6 7 8 9 10

mV

olts

-10

0

10

20

305,123

UV-Detectori-AzA 0,01514 S-3_200105

Retention Time

Minutes1 2 3 4 5 6 7 8 9 10

mV

olts

-10

0

10

20

30

40 5,123

UV-Detectori-NA-4-a_200105

Retention Time

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Trifolio-MMethod

• AzA calibration solution was prepared in duplicate and used for both days of analysis. Solutions were to be kept deep frozen.– Each calibration solution was prepared by

weighing about 10 mg AzA into an 50 ml vol. flask and diluted by 1:10 (AzA concentration of 0.02 mg/ml).

• Sample solutions were prepared fresh for each day of analysis in duplicate.– TK solutions were prepared in analogy to cal.

sol.– EC solutions were prepared by weighing about

1 mg into a 50 ml vol. flask without further solution.

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Trifolio-MProcedure

• Linearity check of the detector response• Equilibration of the chromatographic

system• Determination: Injection of sample

solution in duplicate – bracketing with injections of calibration solutions

• Calculation of the results with the response factors bracketing the injections of the sample solutions

• Statistical evaluation according DIN ISO 5725

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Trifolio-MSamples

• Standard material:Azadirachtin A, 970 g/kg purity

• TK-1: NeemAzal technical – Batch 1– (approx. 390 g/kg AzA)

• TK-2: NeemAzal technical – Batch 4– (approx. 360 g/kg AzA)

• EC-1: NeemAzal-T/S – Batch 200704P• EC-2: NeemAzal-T/S – Batch 141004R • EC-3: NeemAzal-T/S – Batch 281204S

– (each EC approx. 10 g/kg AzA)

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Trifolio-MParticipants - 1

• Feng Shake Zhongke Bio-Industry Co Ltd, P.R. China

• Jürgen Fries BASF AG, Germany

• Arie Roos/Theo de Rijk, RIKILT, The Netherlands

• Katja Reimann Trifolio-M GmbH, Germany

• Katarina Orosova Testing Inst. in Agriculture, Slovakia

• Bruno Patrian Agroscope FAW Wädenswil, Switzerland

• Gernot Feucht Bayer Crop Science GmbH, Germany

• Huang Xiaohua Inst. of control of Agrochemicals, P.R. China

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Trifolio-MParticipants - 2

• Angela Santilio Istituto Superiore di Sanita, Italy

• Gang LV Chemical & hazardous Prod. Lab., P.R. China

• Milan Damjaonvic Galenika - Fitofarmacija,Serbia & Montenegro

• Rolf Förster BASF AG, Germany

• Luis Manso Ministerio de Agricultura, Spain

• Andreas Witte GAB Analytik GmbH, Germany

• Devendra Kumar E.I.D. Parry Ltd, India

• Ji Ying Ministry of Agriculture, P.R. China

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Trifolio-MRemarks of participants - 1

• Lab. 1 The calibration solutions sequence used has been C1, C2, C1 ... until the end.

• Lab. 4 Due to analysis without degasser, minor fluctuations in the Rts occurred.

• Lab. 7 No deviations from the method given.

• Lab. 8 The method is straight forward and we observed fast equilibration and constant areas. We would recommend a comment in the method about the retention time ofAzadirachtin B in the model chromatograms to inform the analyst. Would a controlled pH of the mobile phase be useful?

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Trifolio-MRemarks of participants - 2

• Lab. 9 All samples were injected manually, so the repeatability was not good enough.

• Lab. 10 Particle size: 5 µm.

• Lab. 14 On day 2 we calculated by peak height ofAzadirachtin A.

• Lab. 15 If using 1.1 ml/min as flow rate according to the original HPLC-method, the column pressure will goes up to 190 bar. It is too high, so we use 0.75 ml/min as flow rate.

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Trifolio-MExperimental conditions

• Proposed HPLC conditions: – Labs 3, 6, 7, 8, 15

• Changes in particle size of Luna-column only:– Labs 1, 4, 10

• Changes in columns (varieties of 150x4.6 mm (3 and 5µm); 250x4.6 mm (5µm):– Labs 2, 4, 5, 9, 11, 12, 13, 14, 16

• Further changes:– Mobile phase - Lab 1 (400:600)– Flow [ml/min] - Lab 1 (1.5), Lab 9 (1.2), Lab 15

(0.75), Labs 13, 14, 16 (1.0),

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Trifolio-MResults

• Samples were dispatched to 16 participants and 15 returned results for evaluation. – Lab 16 were not evaluated due to problems

with peak shapes of AzA and AzB; no resolution

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Trifolio-MResults

• Results of two Labs were completely rejected:– 2 injections for equilibration check on day 1– Decreasing Rts of AzA from 4.8 to 3.3 min on

day 2– Weights for EC: 10 times higher than proposed

Results of one day only (without remarks)Weights for EC: 8 times higher than proposedWeights for TK: variations of 9.3 to 20.9 mg

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Trifolio-MResults – 13 Laboratories

4.0164.0104.0122.3342.314RSDR(Hor)

2.9673.0623.7662.2162.013RSDR

0.2890.2960.3697.9387.645sR

0.2710.2670.3376.9646.762sL

0.0100.1290.1523.8103.508sr

9.749.839.81358.22379.84x [g/kg]

EC-3EC-2EC-1TK-2TK-1

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Trifolio-MOutliers and stragglers

• The tests to be performed are of two types:– Cochran‘s test: within-laboratory

variabilities– Grubbs‘ test: between-laboratory

variabilities

• The difference between outlier andstraggler is the critical value (1%; 5%)

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Trifolio-MOutliers and stragglers

nononononoGrubbs

2 outliers

2 outliers

2 outliers

2 outliers

1outlier

Cochran

EC-3EC-2EC-1TK-2TK-1

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Trifolio-MTK-1Lab 12: outlier according to Cochran

340,00

350,00

360,00

370,00

380,00

390,00

400,00

410,00

420,00

0 2 4 6 8 10 12 14 16

Lab. No.

AzA

[g/k

g]

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Trifolio-MTK-2Lab 12 and 14: outliers according to Cochran

320,00

330,00

340,00

350,00

360,00

370,00

380,00

390,00

400,00

0 2 4 6 8 10 12 14 16

Lab. No.

AzA

[g/k

g]

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Trifolio-MEC-1Lab 1 and 14: outlier according to Cochran

8,00

8,50

9,00

9,50

10,00

10,50

11,00

11,50

0 2 4 6 8 10 12 14 16

Lab. No.

AzA

[g/k

g]

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Trifolio-MEC-2Lab1 and 4: outlier according to Cochran

8,50

9,00

9,50

10,00

10,50

11,00

0 2 4 6 8 10 12 14 16Lab. No.

AzA

[g/k

g]

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Trifolio-MEC-3Lab 4 and 14: outlier according to Cochran

8,50

9,00

9,50

10,00

10,50

11,00

0 2 4 6 8 10 12 14 16

Lab. No.

AzA

[g/k

g]

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Trifolio-MResults – outliers removed

4.016 4.016

4.012 4.010

4.013 4.012

2.333 2.334

2.313 2.314RSDR(Hor)

2.924 2.967

3.054 3.062

3.588 3.766

1.795 2.216

1.833 2.013RSDR

0.2840.3000.3516.4546.976sR

0.2780.2880.3406.2436.406sL

0.0670.0830.0881.6392.757sr

1111111112L

9.739.809.79359.65380.76x [g/kg]

EC-3EC-2EC-1TK-2TK-1

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Trifolio-MConclusion

• Azadirachtin A is the analytical leading compound of the botanical insecticideNeemAzal technical (TK) and its formulationNeemAzal-T/S (EC)

• Reproducibility of 2 batches TK and 3 batches EC are smaller than Horwitz‘s value, even with outlier according to Cochran‘s test were not removed

• We proposed the presented method to be accepted as provisional CIPAC method.

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Trifolio-MDecision

• The result of the collaborative study was presented at the annual CIPAC-FAO-WHO symposium in Utrecht, The Netherlands, in 2005

• The CIPAC panel decided that the method for determination of AzA in NeemAzal technical and NeemAzal-T/S fulfilled the criteria to become a provisional method– This is the usual approach: a new method

is considered as a provisional method for one year

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Trifolio-MDecision

• In 2006 at the 50th annual CIPAC symposium held in Geneva the panel decided that the method for determination of AzA in NeemAzal technical andNeemAzal-T/S becomes the status of a full method.

• Our thanks goes to all participants for their cooperation and collaboration.