1. Triadimenol CIPAC /R Stereospecific Identity Test for Triadimenol – report Gerhard Krautstrunk, Silke Wagner, Dieter Strassburg, Martin Feyerabend June 2011

2. Background and Objective
The triadimenol FAO specification defines a certain ratio for the two diastereomers.
A method extension for the identification and determination of triadimenol content and also implicitly, the diastereomer ratio in TC, EC, and WG formulations was presented at the CIPAC Meeting in 2009 and provisionally adopted as a CIPAC method. The method for the determination of the diastereomer ratio (CIPAC method 398/TC/M3 and CIPAC Doc. Nr. 4687) still needed to be peer validated.
Objective:
Present the outcome of the peer validation to demonstrate that CIPAC method 398/TC/M3 and CIPAC Doc. Nr are suitable for the determination of the diastereomer ratio for triadimenol. 2

3. Triadimenol CIPAC 398 Common name: TRIADIMENOL
Chemical name: IUPAC (1RS, 2RS; 1RS, 2SR)-1-(4-chlorophenoxy)-3,3-dimethyl-1-(1H-1,2,4- triazol-1-yl)butan-2-ol
CA 1H-1,2,4-triazole-1-ethanol, -(4-chlorophenoxy)--(1,1-dimethylethyl)-
(unstated stereochemistry)
CAS No.:
Trade names: Baytan, Bayfidan
Structure:
Molecular Formula: C14 H18 Cl N3 O2
Molar Mass: g/mol
Melting Point: diastereomeric form A: °C, diastereomeric form B: °C
Solubility: dichloromethane g/L; acetone 190 g/L; toluene 10 – 30 g/L (20°C)
Description: white powder
Activity: Fungicide
Test substance 3

4. Triadimenol Stereochemistry
Triadimenol is a mixture of diasteromers with a defined ratio 4

5. CIPAC Method 398/TC/M3 and CIPAC Doc. Nr. 4687
for the determination of the ratio of isomers
Outline of the method:
Equipment: Capillary gas chromatograph with flame ionization detector and on-column injector (alternatively split injector)
Internal standard: di-(2-ethylhexyl) phthalate
Solvent: acetone
Typical Operating conditions:
Column temperature 2 min at 80°C, then 10°C/min up to 280 °C
Injection port temperature cold on column injection
Detector temperature 280 °C
Flow rate carrier gas helium: 5 ml/ min
Flow rate make up gas nitrogen: 30 ml/ min
Flow rate air and hydrogen as recommended by the manufacturer
Retention time triadimenol: about 18 min
di-(2-ethylhexyl) phthalate: about 23 min
Calculation of the diastereomeric ratio:
Amount of diastereomer A (% of total triadimenol) = HA/(HA + HB)
Amount of diastereomer B (% of total triadimenol) = HB/(HA + HB)
(HA and HB are the average of measurements of the areas of the chromatographic signal produced by diastereomers A and B, respectively) 5

6. Peer Validation of the method
Participants: 1) Dr. Silke Wagner, Bayer CropScience AG, Monheim, Germany
2) Dieter Strassburg, Currenta GmbH & Co. OHG, Dormagen, Germany
Samples: Triadimenol tech., Batch Nr , purity 99.5 % w/w
Triadimenol tech., Batch Nr , purity 99.8 % w/w
Certified Reference Materials:
Triadimenol Diastereomer A: Batch HSRM1886, purity 99.9 % w/w, AZ 16740
Triadimenol Diastereomer B:
Batch ELB05, purity 99.9 % w/w, AZ 13277
2 triadimenol batches of different composition were investigated in 2 labs 6

7. Experimental Conditions
Triadimenol Method 398 was applied with slight modifications 7

8. Typical Chromatograms (Lab 1, split-injection)
Chromatogram AZ 16740; Triadimenol Isomer A 8

9. Typical Chromatograms (Lab 1, split-injection)
Chromatogram AZ 13277; Triadimenol Isomer B 9

10. Typical Chromatograms (Lab 1, split-injection)
Chromatogram Batch spiked with AZ (Triadimenol Isomer A) 10

11. Typical Chromatograms (Lab 1, split-injection)
Chromatogram Batch spiked with AZ (Triadimenol Isomer B) 11

12. Typical Chromatograms (Lab 1, split-injection)
Chromatogram Batch 12

13. Typical Chromatograms (Lab 1, split-injection)
Chromatogram Batch , enlarged 13

14. Typical Chromatograms (Lab 2, on column-injection)
Chromatogram Batch 14

15. Typical Chromatograms (Lab 2, on column-injection)
Chromatogram Batch , enlarged 15

16. Results for Triadimenol Batch Nr 4119-02005
All results as mean of duplicate measurements
The values from both laboratories are in excellent agreement
The rel. standard deviation values are low and pass the acceptance criteria 16

17. Results for Triadimenol Batch Nr 40095028
All results as mean of duplicate measurements
The values from both laboratories are in excellent agreement
The rel. standard deviation values are low and pass the acceptance criteria 17

18. Triadimenol CIPAC 398: Additional comments
Based on the experience of the validation work, we suggest to add the following comments to CIPAC method 398/TC/M3 and CIPAC Doc. Nr. 4687:
It should be noted that, if split injection is applied, the recommended dilution step (factor 100) should be omitted.
It should be noted that, if on-column injection is applied, the maximum injection temperature should be limited to max. 60 °C.
The recommended detector temperature should be higher (e.g. 300 °C) than that of the column (280 °C) to avoid any possible condensation effects.
Proposal for amendment 18

19. Triadimenol CIPAC 398
The peer validation of CIPAC method 398/TC/M3 for the triadimenol diastereomer ratio was conducted by two independent laboratories using two different representative technical materials
The identity check was done successfully using certified reference standards for both individual diastereomers
Measurements were done according to CIPAC method 398/TC/M3 and CIPAC Doc. Nr using 5 independent weighings for each sample (duplicate measurements). Both on-column injection and split injection techniques were used.
The validation results showed excellent agreement between the measurements from the two participating laboratories. All measurements pass the acceptance criteria.
Suggestions for further refinement of CIPAC method 398/TC/M3 and CIPAC Doc. Nr were made.
Therefore, we consider this method to be suitable and recommend it to be accepted as a final CIPAC method for the determination of the Triadimenol diastereomer ratio.
Stereospecific Identity Test for CIPAC 398: Conclusion 19

20. Triadimenol CIPAC 398
Thank you for your kind attention 20