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BCS-RD-D-ROCS Method-No. 00857 

D-40789 Monheim Report No. MR-099/04 

Study Title 

Analytical Method 00857 for the Determination of Residues of BYI08330 

(Parent Compound and Total Residue of BYI08330), BYI08330-enol, 

BYI08330-ketohydroxy, BYI08330-mono-hydroxy and BYI08330-enol-Glc 

Metabolite in/on Plant Material by HPLC-MS/MS 

___________________________________________________________________ 

Data Requirement 

• EU Council Directive 91/414/EEC amended by Commission Directive 96/68/EC 

• European Commission Guidance Document for Generating and Reporting Methods 

of Analysis in Support of Pre-Registration data Requirements for Annex II (part A, 

Section 4) and Annex III (part A, section 5) of directive 91/414, SANCO/3029/99 

• US EPA Residue Chemistry Test Guideline OPPTS 860.1340: Residue Analytical 

Method 

Authors 

R. Schöning 

S. Stuke 

Dr. P. Billian 

Study Completion Date 

2005-06-17 

(date:yyyy-mm-dd) 

Performing Laboratory 

Bayer CropScience AG 

Development - Residues, Operator and Consumer Safety 

Alfred-Nobel-Str. 50 

D-40789 Monheim 

Laboratory Project ID 

P602044703




Data Confidentiality Statement 

This page is intentionally left blank for the purpose of submitting administrative 

information that is required by regulations promulgated by various countries.




Sponsor 

Head of Test Facility: Dr. R. Graney, BCS-RD-D-ROCS 

D-40789 Monheim, Building 6610 

Laboratories Involved in the Study 

Head of Laboratory Analysis: R. Schöning, BCS-RD-D-ROCS 


Technicians: S. Stuke, E. Dirr, BCS-RD-D-ROCS 


Sampling, Preparation- 

Technique and Logistics: DI. K. Ertz, BCS-RD-D-ROCS 


Schedule 

Approval of the Study Protocol by the Study Director: 2004-06-15 

Start of Experimental Phase: 2004-09-01 

End of Experimental Phase: 2004-10-29 

Archiving 

All raw data pertaining to this study and the original of the final report are stored in the 

central GLP archive of Bayer CropScience AG, Alfred-Nobel-Str. 50, D-40789 Monheim 

for as long as required by GLP principles. 

Reserve samples and reference items are stored in the archives of Bayer CropScience 

by the function which had certified the used substances. The test and reference items 

are stored as long as their quality still guarantees an evaluation.




Table of Contents Page 

Study Title .................................................................................................................................................. 1 

Data Confidentiality Statement .................................................................................................................. 2 

Certification of Good Laboratory Practice.................................................................................................. 3 

Certification of Authenticity ........................................................................................................................ 4 

Quality Assurance Statement .................................................................................................................... 5 

Sponsor...................................................................................................................................................... 6 

Laboratories Involved in the Study............................................................................................................. 6 

Schedule .................................................................................................................................................... 6 

Archiving .................................................................................................................................................... 6 

1 Summary ........................................................................................................................................... 9 

2 Introduction...................................................................................................................................... 11 

2.1 BYI08330 ................................................................................................................................ 11 

2.2 BYI08330-enol ........................................................................................................................ 11 

2.3 BYI08330-ketohydroxy ........................................................................................................... 12 

2.4 BYI08330-mono-hydroxy........................................................................................................ 12 

2.5 BYI08330-enol-Glc ................................................................................................................. 12 

2.6 BYI08330 13 C3 (FHN 12952)................................................................................................... 13 

2.7 BYI08330-enol 13 C3 (FHN 13777) .......................................................................................... 13 

2.8 BYI08330-ketohydroxy 13 C3 (FHN 14066) .............................................................................. 13 

2.9 BYI08330-mono-hydroxy 13 C3 (FHN 15436)........................................................................... 14 

2.10 BYI08330-enol-Glc 13 C3 (KTS 10115)..................................................................................... 14 

3 Sampling and Preparation of Laboratory Samples ......................................................................... 15 

4 Experimental Section ...................................................................................................................... 15 

4.1 Materials ................................................................................................................................. 16 

4.1.1 Apparatus ........................................................................................................................... 16 

4.1.2 Reagents/Supplies ............................................................................................................. 16 

4.1.3 Reference Items .................................................................................................................17 

4.1.4 Stock Solutions................................................................................................................... 18 

4.1.5 Standard Solutions ............................................................................................................. 19 

4.1.6 Standard Solutions of Stable-Labelled Compounds .......................................................... 20 

4.1.7 Standard Mixture Solutions ................................................................................................ 21 

4.2 Analytical Method ................................................................................................................... 22 

4.3 Analysis by HPLC-MS/MS...................................................................................................... 23 

4.3.1 Principle of Measurement .................................................................................................. 23 

4.3.2 HPLC Conditions for BYI08330 and BYI08330-enol.......................................................... 23 

4.3.3 HPLC Conditions for BYI08330-ketohydroxy, BYI08330-mono-hydroxy and 

BYI08330-enol-Glc ............................................................................................................. 24 

4.3.4 HPLC Conditions BYI08330-enol-Glc (Individual Determination)...................................... 25 

4.3.5 HPLC Conditions BYI08330 (Individual Determination) .................................................... 26 

4.3.6 MS/MS Conditions.............................................................................................................. 27 

4.3.7 Analytical Procedure .......................................................................................................... 28 

4.4 Calculation .............................................................................................................................. 29 

4.4.1 Calculation of the Residues ............................................................................................... 30 

4.4.2 Calculation of Recovery Rates........................................................................................... 30 

4.4.3 Calculation of Total Residue of BYI08330 ......................................................................... 31 

5 Results and Discussion................................................................................................................... 32 

5.1 Selectivity................................................................................................................................ 32 

5.2 Blank Values of Untreated Control Samples .......................................................................... 32 

5.3 Linearity of the Detector ......................................................................................................... 33 

5.4 LOQ, LOD, Recovery Rates and Repeatability...................................................................... 34 

5.5 Stability of BYI08330 and its Metabolites in Plant Extracts.................................................... 40 

5.6 Stability in Standard and Stock Solution................................................................................. 42 

6 References ...................................................................................................................................... 42




Table of Contents (contd) Page 

Appendix 1: Representative Chromatograms....................................................................................... 43 

Appendix 2: Flow Diagram of Residue Method 00857 ......................................................................... 58 

Appendix 3: Representative Linearity Plots .......................................................................................... 59 

Appendix 4: Mass Spectra .................................................................................................................... 64 

Appendix 5: Typical HPLC-MS/MS Parameters 1 ................................................................................ 69 




Appendix 9: GLP-Certificate ................................................................................................................. 81




1 Summary 

The analytical method 00857 presented in this report was developed in order to 

determine residues of BYI08330 (parent compound, total residue of BYI08330) and its 

metabolites BYI08330-enol, BYI08330-ketohydroxy, BYI08330-mono-hydroxy and 

BYI08330-enol-Glc in/on plant material by HPLC-ESI-MS/MS using stable isotopically 

labelled internal standards. 

BYI08330 and its metabolites BYI08330-enol, BYI08330-ketohydroxy, BYI08330-monohydroxy 

and BYI08330-enol-Glc are extracted from the sample material using a blender 

with an acidic acetonitrile/water mixture (4/1, v/v). After filtration of the extract, the 

corresponding stable isotopically labelled analytes are added. Afterwards, the solution is 

made up to volume, diluted and subjected to HPLC-MS/MS without any further clean-up 

step (with exception of hop where a second filtration step was used). 

For quantification external calibration with standard solutions quoting the peak area ratio 

of the analytes and the corresponding stable isotopically labelled internal standards 

were used. If matrix problems are observed, HPLC-MS/MS conditions have to be 

adapted. 

The limit of quantitation (LOQ) is 0.01 mg/kg for each single compound corresponding 

to the lowest fortification level of successfully conducted recovery experiments with 

exception of hop cone with a LOQ of 0.10 mg/kg. The limit of detection (LOD) for all 

single compounds was estimated to be at about 4 times lower than the corresponding 

LOQ, as could be concluded from the linearity data and matrix interference in untreated 

control samples. For determination of the total residue of BYI08330 (sum of single 

analyte residues each calculated as BYI08330 equivalents), the LOQ is calculated as 

0.055 mg/kg for all matrices with exception of hop cone (0.55 mg/kg). 

A linear correlation between the injected amount of the analytes and the detector 

response was observed for standards in solvent (5 ng/L to 50 µg/L) using stable 

isotopically labelled internal standards. The correlation coefficients of the 1/x weighted 

linear regression were 0.9976 to 0.9995. 

The occurrence of matrix effects was not monitored during method development and 

validation, as the internal standard procedure using stable isotopically labelled internal 

standards compensates for matrix effects. If matrix interferences or matrix effects are 

observed, the HPLC-MS/MS conditions have to be adapted. 

The stability of BYI08330 and its metabolites BYI08330-enol, BYI08330-ketohydroxy, 

BYI08330-mono-hydroxy and BYI08330-enol-Glc in stock and standard solutions 

(water/acetonitrile, 2/8, v/v + 0.22 mL/L formic acid) was tested. 

The results indicate that all analytes are stable in standard solutions for at least four 

weeks of storage in a refrigerator at 4°C ± 3°C under dark conditions. 

Blank values in untreated control samples were well below 30% of the LOQ.




1 Summary (contd) 

The stability of BYI08330 and its metabolites (BYI08330-enol, BYI08330-ketohydroxy, 

BYI08330-mono-hydroxy and BYI08330-enol-Glc) in diluted plant extracts in 

acetonitrile/water was determined at the respective LOQ levels. The extracts of three 

untreated control samples were separately fortified with each analyte and after initial 

analysis, these solutions were stored in a refrigerator and re-analysed after 2, 5, 8 and 

16 days. The diluted extracts of treated samples were stored under the same conditions 

as control extracts (in a refrigerator at 4°C ± 3°C). The results indicate that all analytes 

are stable in plant extracts for at least two weeks. 

The recovery serves as a measure of the accuracy of the analytical method. 

The individual recovery rates for BYI08330 ranged from 72 to 108% with overall means 

(n = 6 - 10) between 87 and 101% and overall RSDs of up to 13.3%. 

The individual recovery rates for BYI08330-enol ranged from 69 to 111% with overall 

means (n = 6 - 10) between 86 and 104% and overall RSDs of up to 15.0%. 

The individual recovery rates for BYI08330-mono-hydroxy ranged from 68 to 110% with 

overall means (n = 6 - 10) between 88 and 101% and overall RSDs of up to 13.7%. 

The individual recovery rates for BYI08330-ketohydroxy ranged from 69 to 111% with 


The individual recovery rates for BYI08330-enol-Glc ranged from 70 to 109% with 


As a measure for the precision of the method, the intra-laboratory repeatability (n = 5) is 

given as relative standard deviation (% RSD) for different sample materials (e.g. orange 

fruit, plum fruit, apple fruit, apple sauce, potato tuber, hop beer) at fortification levels of 

0.01 and 0.1 mg/kg or 1.0 and 10 mg/kg (only hop cone dried and green). The RSD of 

the repeatability tests at each recovery set ranged from 0.5 to 14.0%. 

The validation data obtained demonstrate the high sensitivity, selectivity, accuracy and 

precision (repeatability) of the method for determination of BYI08330 (parent compound, 

total residue of) and its metabolites BYI08330-enol, BYI08330-mono-hydroxy, 

BYI08330-ketohydroxy and BYI08330-enol-Glc in plant material. For confirmation of the 

results a second MRM transition was recorded for each individual analyte and each 

investigated matrix.




2 Introduction 

The analytical method 00857 presented in this report was developed in order to 

determine residues of BYI08330 (parent compound, total residue of BYI08330) and its 


BYI08330-enol-Glc in/on plant material by HPLC-ESI-MS/MS using stable isotopically 

labelled internal standards. 

2.1 BYI08330 

Active Ingredient: BYI08330 

Chemical Name: cis-3-(2,5-dimethylphenyl)-8-methoxy-2-oxo-1-azaspiro[4,5]dec-3-en-4-yl ethyl 

carbonate 

Empirical Formula: C21H27NO5 

Molecular Mass: 

Structural Formula: 

373.45 g/mol 

H3C O 

O 

H 

O 

CH3 2.2 BYI08330-enol 


Molecular Mass: 301.39 g/mol 


CH 3 

CH 3 

HO 

CH3 CH 3 

O 

O 

O 

NH 

NH 

H O 

CH 3




2.3 BYI08330-ketohydroxy 




2.4 BYI08330-mono-hydroxy 




CH 3 

CH 3 

O 

HO 

CH 3 

CH 3 

OH 

O 

O 

NH 

NH 

2.5 BYI08330-enol-Glc (Glc = glucoside) 




HO 

HO 

HO 

HO 

O 

O 

CH 3 

CH 3 

H O 

CH 3 

H O CH 3 

O 

NH 

H O 

CH 3




2.6 BYI08330 13 C3 (FHN 12952) 

M = 376.41 g/mol 

H3C H 

O 

O 

CH3 O 

CH 3 

CH 3 

2.7 BYI08330-enol 13 C3 (FHN 13777) 

M = 304.35 g/mol 

HO 

CH3 CH 3 

O 

13C 

NH 

13 13 C C 

O 

13 C 

O 

NH 

13 C 13C 

H O 

2.8 BYI08330-ketohydroxy 13 C3 (FHN 14066) 

M = 320.35 g/mol 

CH 3 

CH 3 

O 

13C 

13 C 

OH 

O 

NH 

13 C 

H O 

CH 3 

CH 3




2.9 BYI08330-mono-hydroxy 13 C3 (FHN 15436) 

M = 306.37 g/mol 

HO 13C CH 3 

CH 3 

O 

NH 

13 C 13C 

2.10 BYI08330-enol-Glc 13 C3 (KTS 10115) 

M = 466.5 g/mol 

HO 

HO 

HO 

HO 

O 

O 

CH 3 

CH 3 

13 C 

13 C 

H O CH 3 

O 

NH 

13 C 

H O 

CH 3




3 Sampling and Preparation of Laboratory Samples 

Analytical samples must be collected in a way that ensures the obtention of a 

representative sample. Guidance on adequate sampling may be obtained e.g. from the 

instructions of chapter VIII of the DFG method collection for the analysis of pesticide 

residues [1]. 

If the samples cannot be analysed immediately, they have to be stored in a deepfreezer 

at -18°C or below. For the preparation of the laboratory samples, the deepfrozen 

samples are shredded with dry ice in a cutter. Parts of the frozen laboratory 

samples are transferred into polystyrene boxes and stored at -18°C or below until 

analysis. 

4 Experimental Section 

When using this analytical method the German guidelines for laboratories of the 

Employees' Liability Insurance Association, e.g. Bulletin M006 [2] or comparable 

guidelines in other countries are to be observed. 

The following chemicals were used, which are classified by the hazardous material 

regulations. The classification is based on the German guidelines [3] and has to be 

adapted to the respective national guidelines in case the method is used outside 

Germany. 

• Acetic acid Corrosive 

• Formic acid Corrosive 

• Acetonitrile Toxic, highly flammable 

The pertinent safety instructions must be observed when working with all compounds 

mentioned in this method (e.g. R- and S-phrases). It has to be made sure that the 

working place is properly ventilated when working with dry ice. Sample vessel and 

deep-freezing cabinet must guarantee pressure equalisation.




4.1 Materials 

4.1.1 Apparatus 

• Liquid chromatograph, e.g. Agilent 1100 column compartment G1316A, Agilent 1100 

binary pump G1312A, Agilent 1100 iso pump G1313A, Agilent 1100 degasser 

G1379A, Agilent Technologies, Böblingen, Germany 

• Autosampler, e.g. HTC PAL, CTC Analytics, Schwitzerland 

• Mass spectrometer, e.g. API 4000 with ESI interface and mass spectrometric 

detector, Applied Biosystems, Darmstadt, Germany 

• Blender, e.g. Polytron (Kinematica GmbH) 

• Reversed phase chromatography column, 

e.g. Agilent, Zorbax, Eclipse XDB-C8, 150 x 4.60 mm, 5 µ 

with pre-column Phenomenex C18 ODS 4x3 mm, AJO-4287 in cartridge KJO-4282 

• Vacuum manifold, e.g. VacElut SPS 24, Varian, Darmstadt, Germany 

• Reservoir 60 mL, e.g. BondElut reservoir, Varian, Darmstadt, Germany 

• Volumetric flask, 10-mL, 100-mL 

• Variable dispenser, 10-mL, 50-mL, 100-mL 

• Glass beaker, 150-mL 

• Gilson Microman pipettes, 250 µL 

• Calibrated pipettes, 0.5-mL, 1-mL, 10-mL, 20-mL, e.g. Brand Co., Wertheim, 

Germany 

• Bond Elut Clean Polyethylene Frits (Frits 60-mL, 1-1/16, 20µm, Varian, Part. No. 

12131024 

• Small instruments, e.g. Pasteur pipettes, autosampler vials, filter frits for reservoir 

• 0.45 µm single use filter e.g. Sartorius Minisart RC15 (order no. 17762) 

4.1.2 Reagents/Supplies 

• Acetonitrile for chromatography, e.g. LiChrosolv Merck KGaA, Darmstadt, Germany 

• Water, HPLC grade, e.g. purified with a milli-Q-water system, Millipore Co., 

Eschborn, Germany 

• Acetic acid, Suprapur, e.g. Merck KGaA, Darmstadt, Germany 

• Formic Acid, extra pure, e.g. Riedle-de Häen, Seelze, Germany 

• Filter aid, e.g. Celite 545, e.g. Merck KGaA, Darmstadt, Germany 

• Nitrogen 5.0, 99.9990% purity, as bath, nebulizer, collision, curtain, and turbo gas, 

e.g. Linde AG, Höllriegelskreuth, Germany




4.1.3 Reference Items 

Generally, only sufficiently characterised and certified substances are used as reference 

items. 

Table 1: Reference Items. Names in Brackets [ ] designate the Terms used in the Raw Data. 

Certificate of 

Analysis 

Purity (%) Expiry Date 

BYI08330 (a.s.) [BYI] AZ 10811 99.2 2005-07-24 

BYI08330 13 C3 [BYI- 13 C] KML 3386-1-2 95.1 --- 

BYI08330-enol [Enol] AZ 10531 99.4 2005-02-26 

BYI08330-enol 13 C3 [Enol- 13 C] KML 3384-1-1 96.3 --- 

BYI08330-ketohydroxy [Ketohydroxy] AZ 10832 95.3 2005-06-14 

BYI08330-ketohydroxy 13 C3 [Ketohydroxy- 13 C] KML 3387-1-8 99.4 --- 

BYI08330-mono-hydroxy [Mono-hydroxy] AZ 11484 99.8 2006-04-06 

BYI08330-mono-hydroxy 13 C3 [Mono-hydroxy- 13 C] KML 3388-1-16 93.7 --- 

BYI08330-enol-Glc [Gluco] AZ 11879 99.7 2006-08-04 

BYI08330-enol-Glc 13 C3 [Gluco- 13 C] KML 3405-1-8 99 ---




4.1.4 Stock Solutions 

The stock solutions were prepared by weighing a defined amount of reference item into 

a volumetric flask and making up to volume with acetonitrile or acetonitrile/water 

(4/6, v/v containing 0.22 mL/L formic acid). 

Table 2: Preparation Scheme of Reference Item Stock Solutions. 

Reference Item Final Concentration 

No. 

Weight 

[mg] 

Volume 

[mL] 

Solvent Required 

[mg/L] 

Actual** 

[mg/L] 

1 [BYI] 10.32 20 ACN 500 512 

2 [BYI- 13 C] 10.53 20 ACN 500 501 

3 [Enol] 10.22 20 * 500 508 

4 [Enol- 13 C] 10.55 20 * 500 508 

5 [Ketohydroxy] 10.75 20 ACN 500 512 

6 [Ketohydroxy- 13 C] 10.03 20 ACN 500 499 

7 [Mono-hydroxy] 9.22 20 ACN 500 460 

8 [Mono-hydroxy- 13 C] 10.70 20 ACN 500 501 

9 [Gluco] 10.17 20 * 500 507 

10 [Gluco- 13 C] 10.10 20 * 500 500 

*: acetonitrile/water (4/6, v/v + 0.22 mL/L formic acid). 

**: Concentrations are corrected for purity.




4.1.5 Standard Solutions 

Standard solutions (secondary standards) were prepared from the stock solutions by 

dilution with acetonitrile/water (2/8, v/v containing 0.22 mL/L formic acid). 

Table 3: Preparation Scheme for Reference Standards. 

No. Target Concentration (mg/L) 

A1 

A2 

A3 

A4 

A5 

A6 

A7 

A8 

A9 

A10 

A11 

A12 

A13 

[BYI], [Enol], [Ketohydroxy], [Monohydroxy], 

[Gluco] (10 each) 


[Gluco] (1.0 each) 


















[Gluco] (0.00005 each) 


[Gluco] (0.00001 each) 


[Gluco] (0.000005 each) 

Prepared by 

Removal of 

(mL) 

1.95, 2, 1.95, 

2.17, 2 

*: acetonitrile/water (2/8, v/v, containing 0.22 mL/L formic acid). 

No. of 

Solution 

1, 3, 5, 7, 

9 

Dilution to 

(mL) 

Solvent 

100 * 

10 A1 100 * 

1 A1 100 * 

0.5 A1 100 * 

1 A2 100 * 

0.5 A2 100 * 

0.2 A2 100 * 

0.1 A2 100 * 

1 A4 100 * 

1 A5 100 * 

1 A6 100 * 

1 A8 100 * 

0.1 A6 100 *




4.1.6 Standard Solutions of Stable-Labelled Compounds 

Standard solutions were prepared from the corresponding stock solutions by dilution 

with acetonitrile/water mixture (15/85, v/v containing 0.22 mL/L formic acid). 

Table 4: Preparation Scheme for Reference Standards of Stable-Labelled Compounds. 

No. Target Concentration (mg/L) 

B1 

B2 

B3 

B4 

[BYI- 13 C], [Enol- 13 C], [Ketohydroxy- 13 C], [Monohydroxy- 

13 C], [Gluco- 13 C] (1.0 each) 


13 C], [Gluco- 13 C] (0.1 each) 


13 C], [Gluco- 13 C] (0.01 each) 


13 C], [Gluco- 13 C] (0.001 each) 

Prepared by 

Removal of 

(mL) 

0.2 each 



Solution 

2, 4, 6, 8, 

10 

Dilution to 

(mL) 

Solvent 

100 * 

10 B1 100 * 

10 B1 1000 * 

10 B2 1000 *




4.1.7 Standard Mixture Solutions 

Standard mixture solutions were prepared by dilution with acetonitrile/water mixture 

(2/8, v/v + 0.22 mL/L formic acid). 

Table 5: Preparation Scheme. 

No. Target Concentrations (mg/L) 

C1 

C2 

C3 

C4 

C5 

C6 

C7 

C8 

C9 

C10 

[BYI], [Enol], [Ketohydroxy], [Mono-hydroxy], 


[BYI- 13 C], [Enol- 13 C], [Ketohydroxy- 13 C], 

[Mono-hydroxy- 13 C], [Gluco- 13 C] (0.001 each) 



[BYI- 13 C], [Enol- 13 C], [Ketohydroxy- 13 C], 




[[BYI- 13 C], [Enol- 13 C], [Ketohydroxy- 13 C], 



















[Gluco] (0.00005 each) 




[Gluco] (0.00001 each) 




[Gluco] (0.000005 each) 



Prepared 

by Removal 

of (mL) 



Solution 

Dilution to 

(mL) 

Solvent 

5 + 1 A2 + B2 100 * 

1 + 1 A2 + B2 100 * 

0.5 + 1 A2 + B2 100 * 

0.2 + 1 A2 + B2 100 * 

1 + 1 A3 + B2 100 * 

1 + 1 A4 + B2 100 * 

1 + 1 A5 + B2 100 * 

1 + 1 A6 + B2 100 * 

1 + 1 A8 + B2 100 * 

0.1 + 1 A6 + B2 100 *




4.2 Analytical Method 

Extraction Procedure 

1. Weigh a representative aliquot of 10 g of the sample into a 150-mL beaker. 

Remark: Do not allow deep-frozen sample material to thaw before extraction. 

Recovery samples have to be extracted immediately after spiking. 

2. Add 40 mL (60 mL for hop cone material) of acetonitrile/water (4/1, v/v, containing 

0.22 mL formic acid/L) to the sample. 

3. Blend the sample some minutes with a Polytron blender. 

4. Rinse the blender with approx. 5 mL of acetonitrile/water (4/1, v/v, containing 

0.22 mL formic acid/L). 

5. Mount a syringe tube in working position on top of a VacElut-Station and place a 

BondElut clean polyethylene frit onto the bottom of the syringe tube. 

6. Add 2.5 g of Celite 545 to the sample and filter the content of the beaker carefully 

through the syringe tube using low vacuum. Collect the extract in a 100-mL 

volumetric flask. 

7. Wash the filtered solids with a total of 30 mL of acetonitrile/water (4/1, v/v, 

containing 0.22 mL formic acid/L) and discard the filtered solids. Add 1 mL of 

internal standard mixture (100 µg/L of each compound in acetonitrile/water, 2/8, v/v, 

containing 0.22 mL formic acid/L) and make up to volume with acetonitrile/water 

(4/1, v/v, containing 0.22 mL formic acid/L). 

8. Transfer an aliquot of 0.1 mL into a 1.8-mL HPLC sample vial, add a volume of 

0.9 mL internal standard mixture (containing 1 µg/L of each 13 C3-labelled compound 

in acetonitrile/water, 15/85, v/v, containing 0.22 mL formic acid/L) and subject to 

liquid chromatography and MS/MS determination. 

9. For hop material (cone dry, green): transfer an aliquot of 0.1 mL of (7) to a 10-mL 

volumetric flask and make up to volume with internal standard mixture (containing 

1 µg/L of each 13 C3-labelled compound in acetonitrile/water (15/85, v/v, containing 

0.22 mL formic acid/L). Filter through a 0.45 µm single use filter (Sartorius Minisart 

RC15, order no. 17762) and subject to liquid chromatography and MS/MS 

determination.




4.3 Analysis by HPLC-MS/MS 

4.3.1 Principle of Measurement 

An aliquot of the prepared sample solution is injected into the high performance liquid 

chromatograph and subjected to reversed phase chromatography coupled with tandem 

mass spectrometry with electrospray ionization. The protonated molecular ions of the 

analytes are accelerated by the adequate voltage regulation and filtered by the first 

quadrupole due to its mass-to-charge (m/z) ratio. These precursor ions are impulsed 

with nitrogen in the collision cell (second quadrupole) and each fragment ion (product 

ions) is separated according to its m/z ratio in the third quadrupole, two of these product 

ions per analyte are selected for multiple reaction monitoring (MRM) one product ion 

(MRM-transition) serving for quantitation and the second for confirmation. 

4.3.2 HPLC Conditions for BYI08330 and BYI08330-enol 

Instrument: e.g. Agilent 1100 

Injector: e.g. HTC PAL, CTC Analytics 

Column: e.g. Agilent Zorbax Eclipse-XDB-C8, 5 µm, 150 x 4.6 mm i.d. with 

pre-column or equivalent 

Injection Volume: e.g. 10 µL 

Oven temperature: 40°C 

Mobile Phase: Bin Pump A: Water/acetonitrile (9/1, v/v), containing 0.22 mL/L formic 

acid) 

Bin Pump B: Acetonitrile, containing 0.22 mL/L formic acid 

Iso Pump C: Water/acetonitrile (1/1, v/v), containing 0.1 mL/L acetic acid 

or formic acid) 

Time Table: 

Time [min] A [%, v/v] B [%, v/v] Into MS Into Waste 

0.0 60 40 Iso pump Bin pump 


2.0 Bin pump Iso pump 

3.5 10 90 

4.5 10 90 

4.6 60 40 

5.0 Iso pump Bin pump 

6.0 60 40 

6.0 Stop time 

Flow (Column): 1.0 mL/min 

Flow (into MS): 1.0 mL/min 

Retention times: BYI08330-enol approx. 2.7 min 

BYI08330-enol 13 C3 approx. 2.7 min 

BYI08330 approx. 4.3 min 

BYI08330 13 C3 approx. 4.3 min




4.3.3 HPLC Conditions for BYI08330-ketohydroxy, BYI08330-mono-hydroxy and 

BYI08330-enol-Glc 

Due to different sensitivities of the analytes, the HPLC run for the BYI08330-ketohydroxy, 

BYI08330-mono-hydroxy and BYI08330-enol-Glc was separated and slightly 

modified. 







Mobile Phase: Bin Pump A: Water/acetonitrile (9/1, v/v, containing 0.22 mL/L formic 

acid) 


Iso Pump C: Water/acetonitrile (1/1, v/v, containing 0.1 mL/L acetic acid 


Time Table: 




3.0 60 40 

3.1 10 90 


4.0 10 90 

4.1 60 40 

6.0 60 40 

6.0 Stop time 



Retention times: BYI08330-enol-Glc approx. 1.6 min 

BYI08330-enol-Glc 13 C3 approx. 1.6 min 

BYI08330-mono-hydroxy approx. 2.5 min 

BYI08330-mono-hydroxy 13 C3 approx. 2.5 min 

BYI08330-ketohydroxy approx. 3.2 min 

BYI08330-ketohydroxy 13 C3 approx. 3.2 min




4.3.4 HPLC Conditions BYI08330-enol-Glc (Individual Determination) 

In case of interferences (e.g. in orange materials, grape (bunches of grape), melon (pulp), hop 

(beer)) the following HPLC conditions for the individual determination of BYI08330-enol-Glc can 

be used: 



Column: e.g. Agilent Zorbax Eclipse-XDB-C8, 5 µm, 150 x 4.6 mm i.d. 

with pre-column or equivalent 




acid) 




Time Table: 




3.0 95 5 

3.1 10 90 

4.5 10 90 

4.6 95 5 


6.0 95 5 

6.0 Stop time 



Retention times: BYI08330-enol-Glc approx. 4.6 min 

BYI08330-enol-Glc 13 C3 approx. 4.6 min




4.3.5 HPLC Conditions BYI08330 (Individual Determination) 

In case of interference (e.g. in orange material) the following HPLC conditions for the individual 

determination of BYI08330 can be used: 








acid) 




Time Table: 




6.0 55 45 

6.1 10 90 


9.0 10 90 

9.1 55 45 

12.0 55 45 

12.0 Stop time 



Retention times: BYI08330 approx. 5.6 min 

BYI08330 13 C3 approx. 5.6 min




4.3.6 MS/MS Conditions 

The detection by MS/MS was performed on a triple-quadrupole tandem mass 

spectrometer, equipped with a Turbo IonSpray (ESI) interface operated in positive ion 

mode and multiple reaction monitoring (MRM). Unit mass resolution was established 

and maintained in the mass resolving quadrupoles by maintaining a full width at halfmaximum 

(FWHM) of between 0.7 and 1.0 amu. Optimal collisionally-activated 

dissociation (CAD) conditions for fragmentation of the analytes and the corresponding 

13 

C3-labelled internal standards were determined with nitrogen as collision gas. The 

conditions are described in detail in Appendix 5 to Appendix 8. 

Detector: Triple Quadrupole Tandem Mass Spectrometer, e.g. Applied Biosystems MDS 

Sciex API 4000, Windows 2000, Analyst 1.4 or higher software versions 

Interface: Turbo IonSpray (ESI) 

Gas Temperature: e.g. 650°C 

Scan Type: MRM (Multiple Reaction Monitoring) 

Polarity: Positive 

Table 6: MS/MS Parameters for the Determination of BYI08330 and BYI08330-enol. 

Precursor Ion 

Q1 Mass (amu) 

Product Ion 

Q3 Mass (amu) 

Dwell Time 

(msec) 

Collision 

Energy (eV) 

BYI08330 1 st MRM 374 216 250 47 

BYI08330 2 nd MRM* 374 302 50 23 

BYI08330 13 C3 377 219 200 47 

BYI08330-enol 1 st MRM 302 216 250 39 

BYI08330-enol 2 nd MRM* 302 270 50 29 

BYI08330-enol 13 C3 305 219 200 39 

Note: Different MS/MS-instruments may result in different MRM transitions. 

*: These transitions were used for confirmation. 

Table 7: MS/MS Parameters for the Determination of BYI08330-ketohydroxy, BYI08330-monohydroxy 

and BYI08330-enol-Glc . 

Precursor Ion 

Q1 Mass (amu) 

Product Ion 

Q3 Mass (amu) 

Dwell Time 

(msec) 

Collision 

Energy (eV) 

BYI08330-ketohydroxy 1 st MRM 318 268 150 29 

BYI08330-ketohydroxy 2 nd MRM* 318 214 50 39 

BYI08330-ketohydroxy 13 C3 321 271 100 29 

BYI08330-mono-hydroxy 1 st MRM 304 254 150 27 

BYI08330-mono-hydroxy 2 nd MRM* 304 119 50 47 

BYI08330-mono-hydroxy 13 C3 307 257 100 27 

BYI08330-enol-Glc 1 st MRM 464 270 150 45 

BYI08330-enol-Glc 2 nd MRM* 464 216 50 53 

BYI08330-enol-Glc 13 C3 467 273 100 45 


*: These transitions were used for confirmation.




4.3.6 MS/MS Conditions (contd) 

Table 8: MS/MS Parameters for Determination of BYI08330-enol-Glc for the single deter-mination 

in cases of interferences (e.g. in orange). 

Precursor Ion 

Q1 Mass (amu) 

Product Ion 

Q3 Mass (amu) 

Dwell Time 

(msec) 

Collision 

Energy (eV) 

BYI08330-enol-Glc 1 st MRM 464 270 400 45 

BYI08330-enol-Glc 2 nd MRM* 464 216 200 53 

BYI08330-enol-Glc 13 C3 467 273 400 45 

Different MS/MS-instruments may result in different MRM transitions. 


Table 9: MS/MS Parameters for Determination of BYI08330 for the single determination in cases of 

interferences (e.g. in mandarin). 

Precursor Ion 

Q1 Mass (amu) 

Product Ion 

Q3 Mass (amu) 

Dwell Time 

(msec) 

Collision 

Energy (eV) 

BYI08330 1 st MRM 374 216 500 47 

BYI08330 2 nd MRM* 374 302 100 23 

BYI08330 13 C3 377 219 400 47 



4.3.7 Analytical Procedure 

1. Transfer a prepared aliquot of the sample extract into a HPLC vial (sample 

solution). 

2. Inject e.g. 10 µL (or 50 µL) of standard solution using the described HPLC, MS/MS 

and data acquisition conditions (bracketing standard). 

3. Inject e.g. 10 µL (or 50 µL) of the sample solution of step 1. (The number of 

injections between the bracketing standard injections should not be higher than 

10.) 

4. Repeat 2. 

5. Compare the peak area ratios of analyte/internal standard for the bracketing 

standard before and after each sample set. If there is a variation of < ± 10% 

between the bracketing standards the analysis should be repeated. 

6. Calculate the residues as described in the following paragraphs.




4.4 Calculation 

The residues and recovery rates are given as single values for each analyte. To 

calculate the total residue of BYI08330 parent, the individual residues of BYI08330-enol, 

BYI08330-ketohydroxy, BYI08330-mono-hydroxy and BYI08330-enol-Glc are recalculated 

with conversion factors (CF) as BYI08330 parent equivalents. The following 

conversion factors are used: 

Analyte Molecular Weight M [g/mol] Conversion Factor CF* 

BYI08330 373.45 1.000 

BYI08330-enol 301.39 1.239 

BYI08330-ketohydroxy 317.38 1.177 

BYI08330-mono-hydroxy 303.40 1.231 

BYI08330-enol-Glc 463.52 0.806 

*: C F = M BYI08330/M Analyte 

The individual residues of BYI08330 and its metabolites expressed as BYI8330 parent 

equivalents are added to calculate the total residue of BYI08330.




4.4.1 Calculation of Individual Residues 

Evaluation is performed according to the external bracketing procedure against 

standards in solvent using a stable-labelled internal standards. 

1. Calculate the average peak area ratios (unlabelled compound/labelled compound) 

of the bracketing standards for each compound. These values will be used as FSt. 

2. Determine the average peak area ratios for the analysed samples for each 

compound. These values will be used as FA. 

3. Calculate the amount of residue R expressed in mg/kg as follows: 

R 

i 

F 

= 

A 

* 

V 

End 

F 

St 

* 

* 

STA 

* V 

G * V 

A 

E 

* 

D 

where: Ri : Determined amount of residue in mg/kg 

FA : Average peak area ratio of the analytical solution 

VEnd : Final volume of the sample solution in mL 

(may be adjusted according to the expected residue level) 

STA : Standard concentration in the external standard in µg/mL 

VE : Extract volume in mL 

DF : Dilution factor 

FSt : Average peak area ratio for the bracketing standards 

G : Sample weight of analytical sample in g 

VA : Aliquot (of extract) in mL 

F 

4.4.2 Calculation of Recovery Rates 

1. Calculate the average of the residues in the recovery sample according to 4.4.1. 

2. Calculate the percent recovery rate as follows: 

Rec 

F 

= 

A 

* 

V 

End 

* 

F 

St 

STA * VE 

* DF 

* 100 

* V * A 

A 

where: Rec : Recovered amount found in fortified sample in % 

FA : Average peak area ratio of the analytical solution 

VEnd : Final volume of the sample solution in mL 

STA : Standard concentration in the external standard in µg/mL 

VE : Extract volume in mL 

DF : Dilution factor 

FSt : Average peak area for the bracketing standards 

VA : Aliquot (of extract) in mL 

A : Fortified amount in µg




4.4.3 Calculation of Total Residue of BYI08330 

For residue samples, the total residue of BYI08330 is calculated by summing up the 

individual residues obtained for the parent BYI8330 and the metabolites BYI08330-enol, 

BYI08330-ketohydroxy, BYI08330-mono-hydroxy and BYI08330-enol-glucoside, each of 

the metabolite residues expressed as BYI8330 parent equivalents. The total residue is 

calculated as BYI08330 parent, so that correction factors (CF) calculated from the 

different molecular weights are used. 

1. Calculate the residues of the metabolites as BYI08330 parent equivalents as 

follows: 

R 

Metabolite 

as BYI08330 

= R 

Metabolite 

∗CF 

RMetabolite as BYI08330 : Residues of metabolites, calculated as BYI08330 parent equivalents in mg/kg 

RMetabolite : Residues of metabolites, determined according to 4.4.1 in mg/kg 

: Conversion factor 

CF 

M : Molecular weight in g/mol 

MParent 

Compound 

M Metabolite 

2. Sum up the individual residues of the metabolites (expressed as BYI8330 parent 

equivalents) and the measured residue of BYI08330 (each in mg/kg) to the total 

BYI8330 residue. 

If all individual residues are below the respective LOQs, the total residue of 

BYI08330 is quoted as < 0.055 mg/kg (0.055 mg/kg is the sum of the LOQs of all 

analytes expressed as BYI8330 equivalents). If one or more individual residues are 

at or above the respective LOQ, the other residues, which are between the 

respective LOQs and LODs, will be set to their respective LOQs. Individual residues 

below the respective LOD will be set to 0 mg/kg for the calculation of the total 

residue of BYI08330. 

= C 

F




5 Results and Discussion 

5.1 Selectivity 

The high selectivity of the method resulted from the HPLC separation in combination 

with MS/MS detection. The second MRM was used for confirmation. 

5.2 Blank Values of Untreated Control Samples 

Up to three untreated control samples of different origin were examined. All determined 

residues of BYI08330 and its metabolites BYI08330-enol, BYI08330-ketohydroxy, 

BYI08330-mono-hydroxy and BYI08330-enol-Glc were below 30% of the respective 

LOQ level (0.01 mg/kg or 0.10 mg/kg for hop cone, green and dried). For 

chromatograms see Appendix 1. 

Table 10: Origin of Control Material; 

LOQ = 0.01 mg/kg for all individual analytes not expressed as parent compound. 

Control Material 

Orange Fruit 

Orange Juice 

Orange Pomace 

Orange Jam 

Sweet Cherry 

Fruit 

Plum Fruit 

Peach Fruit 

Strawberry Fruit 

Apple Fruit 

Apple Pomace 

dry 

Apple Sauce 

Tomato Fruit 

Tomato Juice 

Tomato Preserve 

Grape Bunches 

of Grapes 

Grape Most 

Grape Pomace 

dry 

Grape Wine 

Potato Tuber 

Control Material Marocco 

2003-07-11 

Control Material Juice (Metro 

super market Leverkusen) 

Control Material Pomace wet 

Fruttas Tono Valencia 

2004-06-17 


“Chivers Orange Jam” 

Control Material 200100477 T01 

K and T07 K 

Control Material Fruit with Stone 

607975 T03 K 

Control Material from France 

2003-07-10 

Control Material 201649 

T01 – T02 K 

Control Material 200200366 Mix 

of T0 1K and T0 2K 

Control Material Trial Location 

Hoefchen 1998-08-31 

Control Material R814962 

1999-06-18 

Control Material 2002 03969 T01 

K/T03 K/T04 K 2002-08-06 

Control Material Tomato Juice 

(Ameckes) 2001-05-15 

Control Material Tomato Preserve 

(BKH) 2000-03-09 

Control Material Grape 2002- 

05661 T04K 2003-02-21 

Control Material Grape Most from 

DSW 3 (Mr Ziegler) 1994-09-22 

Control Material from DSW 3 (Mr 

Ziegler) 1994-06-10 

Control Material Mueller-Thurgau 

(semi dry) 1997 

Control Material 200311125 

T01 –T03 K 

BYI08330 

BYI08330enol 

Residues [mg/kg] 

BYI08330ketohydroxy 

BYI08330monohydroxy 

BYI08330enol-Glc 

< 30% LOQ < 30% LOQ < 30% LOQ < 30% LOQ < 30% LOQ 


















< 30% LOQ < 30% LOQ < 30% LOQ < 30% LOQ < 30% LOQ




5.2 Blank Values of Untreated Control Samples (contd) 

Table 10 (contd): Origin of Control Material; 

LOQ = 0.01 mg/kg for all individual analytes not expressed as parent compound. 

(0.1 mg/kg for hop). 


Melon Peel 

Melon Pulp 

Onion Tuber 

Hop Beer 

Hop Cone dried 

Hop Cone green 

Broccoli Curd 

Cauliflower 

Inflorescence. 

Lettuce Head 

BYI08330 

BYI08330enol 

Residues [mg/kg] 

BYI08330ketohydroxy 

BYI08330monohydroxy 

BYI08330enol-Glc 

Control Material Melon Fruit 

(Peel) 200203357 T03 K and T04 

K 


Control Material Melon Fruit 

200203422 T01K < 30% LOQ < 30% LOQ < 30% LOQ < 30% LOQ < 30% LOQ 

Control Material 200305575 Mix 

of T01 K and T02 K < 30% LOQ < 30% LOQ < 30% LOQ < 30% LOQ < 30% LOQ 

Control Material Warsteiner 

Premium Verum < 30% LOQ < 30% LOQ < 30% LOQ < 30% LOQ < 30% LOQ 

Control Material Hop Cone dried 

1999 00368/376/381/392 < 30% LOQ < 30% LOQ < 30% LOQ < 30% LOQ < 30% LOQ 

Control Material Hop Cone green 

1999 00368/376/381/392 < 30% LOQ < 30% LOQ < 30% LOQ < 30% LOQ < 30% LOQ 


R 2003 0114/6 UP0006 21 C < 30% LOQ < 30% LOQ < 30% LOQ < 30% LOQ < 30% LOQ 





Remarks: If matrix effects were observed, slight deviations in the chromatographic and MS/MS conditions were used. Onion 

(tuber) covers onion (onion). 

5.3 Linearity of the Detector 

The linearity of the detector was tested using solvent standards. The peak are ratio of 

analyte/internal standard was quoted and a linear regression was performed. 

The linearity was tested by triplicate injections of solvent standards at concentrations 

between 5 ng/L and 50 µg/L for each analyte. The concentration of the corresponding 

stable isotopically labelled internal standards was 1.0 µg/L for each analyte and in all 

standard solutions. 

Table 11: Standard Concentrations prepared for the Determination of Detector Linearity. 

Unlabelled 

[BYI], [Enol], [Ketohydroxy], 

[Mono-hydroxy], [Gluco] 

Stable-Labelled 

[BYI- 13 C], [Enol- 13 C], 

[Ketohydroxy- 13 C], [Monohydroxy- 

13 C], [Gluco- 13 C] 

0.005 

each 

1 

each 

0.01 

each 

1 

each 

0.05 

each 

1 

each 

Standard Concentrations [µg/L] 

0.1 

each 

1 

each 

A linear correlation between the injected amount of substance and the detector 

response was observed for standards in solvent (5 ng/L to 50 µg/L). The correlation 

coefficients of the 1/x weighted linear regression were ranging from 0.9976 to 0.9995. 

For representative linearity plots see Appendix 3. 

0.5 

each 

1 

each 

1 

each 

1 

each 

2 

each 

1 

each 

5 

each 

1 

each 

10 

each 

1 

each 

50 

each 

1 

each




5.3 Linearity of the Detector (contd) 

The occurrence of matrix effects was not monitored during method development and 

validation, as the internal standard procedure using stable isotopically labelled internal 

standards compensates for matrix effects. The calibration data obtained justify using the 

single point calibration method for calculation of the residues of BYI08330 and its 


BYI08330-enol-Glc. However, an appropriate bracketing standard concentration, 

corresponding to the order of magnitude of the residues should be used for 

quantification and it should be noted that the concentration of the stable isotopically 

labelled internal standard in the calibration and sample solutions must be kept at the 

same level. 

The linearity of the detector used was tested for all HPLC conditions given in chapter 

4.3.2 to 0. The corresponding MS parameters are given in chapter 4.3.6. 

5.4 LOQ, LOD, Recovery Rates (Accuracy) and Precision (Repeatability) 

Recovery rates were determined at fortification levels of 0.01 mg/kg (= LOQ level), and 

1.0 mg/kg (each compound). For hop cone (dried and green) fortification levels of 

0.10 mg/kg (= LOQ level) and 10 mg/kg (each compound) were validated. The lowest 

fortification level experimentally providing a mean recovery between 70 and 110% with 

a relative standard deviation of ≤ 20% per definition corresponds to the Limit of 

Quantitation (LOQ), provided that blank values are below 30% of this level. The 

detection limit (LOD) was estimated to be at least 4 times lower than the LOQ. 

Recovery experiments were conducted by separate fortification of untreated control 

samples with defined amounts of BYI08330 or its metabolites BYI08330-enol, 

BYI08330-ketohydroxy, BYI08330-mono-hydroxy and BYI08330-enol-Glc prior to 

analysis. Results are presented in the following tables. For chromatograms see 

Appendix 1. 

The individual recovery rates for BYI08330 ranged from 72 to 108% with overall means 

(n = 6 - 10) between 87 and 101% and overall RSDs of up to 13.3%. 

The individual recovery rates for BYI08330-enol ranged from 69 to 111% with overall 

means (n = 6 - 10) between 86 and 104% and overall RSDs of up to 15.0%. 

The individual recovery rates for BYI08330-ketohydroxy ranged from 69 to 111% with 


The individual recovery rates for BYI08330-mono-hydroxy ranged from 68 to 110% with 


The individual recovery rates for BYI08330-enol-Glc ranged from 70 to 109% with 


As a measure for the precision of the method, the intra-laboratory repeatability (n = 5) is 

given as relative standard deviation (% RSD) for different sample materials (e.g. orange 

fruit, plum fruit, apple fruit, apple sauce, potato tuber, hop beer) at fortification levels of 

0.01 and 0.1 mg/kg or 1.0 and 10 mg/kg (only hop cone dried and green). The RSD of 

the repeatability tests at each recovery set ranged from 0.5 to 14.0%. 

All method validation data are in compliance with the guideline requirements for residue 

data generation [4].




5.4 LOQ, LOD, Recovery Rates and Repeatability (contd) 

Table 12: Recoveries for BYI08330; RSD: Relative Standard Deviation. 

Crop 

Fortification 

Level (FL) (mg/kg) 

Recoveries % 

(Single Values) 

Mean 

(%) 

per FL 

RSD 

(%) 

Mean 

(%) 

overall 

RSD (%) 

overall 

Orange 

0.01 95 100 75 90 77 87 12.6 

Fruit 1.0 87 86 95 92 92 90 4.2 89 8.9 

Orange 

0.01 90 91 93 91 1.7 

Juice 1.0 68 88 92 83 15.6 87 10.9 

Orange 

0.01 106 104 97 102 4.6 

Pomace 1.0 94 85 73 84 12.5 93 13.3 

Orange 

0.01 102 106 107 105 2.5 

Jam 1.0 96 97 99 97 1.6 101 4.6 

Sweet Cherry 

0.01 100 99 87 95 7.6 

Fruit 1.0 91 91 88 90 1.9 93 6.0 

Plum 

0.01 100 91 99 90 104 97 6.3 

Fruit 1.0 96 87 85 83 91 88 5.9 93 7.5 

Peach 

0.01 89 91 102 94 7.4 

Fruit 1.0 91 92 94 92 1.7 93 5.0 

Strawberry 

0.01 96 97 92 95 2.8 

Fruit 1.0 86 94 95 92 5.4 93 4.3 

Apple 

0.01 84 92 95 73 93 87 10.4 

Fruit 1.0 87 86 90 85 91 88 2.9 88 7.2 

Apple 

0.01 96 94 94 95 1.2 

Pomace Dried 1.0 89 89 91 90 1.3 92 3.2 

Apple 

0.01 98 91 91 99 95 95 4.0 

Sauce 1.0 88 84 86 86 88 86 1.9 91 5.8 

Tomato 

0.01 100 102 102 98 108 102 3.7 

Fruit 1.0 100 97 100 98 98 99 1.4 100 3.2 

Tomato 

0.01 93 100 100 97 98 98 3.0 

Juice 1.0 92 93 95 95 97 94 2.1 96 3.0 

Tomato 

0.01 97 100 89 95 6.0 

Preserve 1.0 90 91 92 91 1.1 93 4.7 

Grape 

0.01 93 104 95 93 98 97 4.8 

Bunches of Grape 1.0 90 90 92 90 91 91 1.0 94 4.8 

Grape 

0.01 98 97 98 98 0.6 

Most 1.0 92 94 93 93 1.1 95 2.8 

Grape 

0.01 89 97 97 94 4.9 

Pomace 1.0 89 87 90 89 1.7 92 4.8 

Grape 

0.01 99 101 95 98 3.1 

Wine 1.0 93 94 92 93 1.1 96 3.7 

Potato 

0.01 91 95 97 90 91 93 3.3 

Tuber 1.0 91 90 90 87 87 89 2.1 91 3.4 

Melon 

0.01 97 99 95 97 2.1 

Peel 1.0 87 88 93 89 3.6 93 5.2 

Melon 

0.01 90 93 97 98 94 94 3.4 

Pulp 1.0 86 85 90 90 92 89 3.3 92 4.6 

Onion 

0.01 91 96 95 97 95 95 2.4 

Tuber 1.0 94 91 89 91 89 91 2.3 93 3.2 

Hop 

0.01 108 92 75 89 99 93 13.2 

Beer 1.0 92 89 86 87 90 89 2.7 91 9.4 

Hop 

0.10 94 97 99 97 2.6 

Cone Dried 10.0 93 87 87 89 3.9 93 5.4 

Hop 

0.10 86 95 100 94 7.6 

Cone Green 10.0 82 88 84 85 3.6 89 7.8 

Broccoli 

0.01 95 104 104 107 87 99 8.3 

Curd 1.0 76 96 81 79 100 86 12.5 93 12.2 

Cauliflower 

0.01 87 94 100 94 6.9 

Inflorescence 1.0 93 72 92 86 13.8 90 10.7 

Lettuce 

0.01 95 81 97 91 9.6 

Head 1.0 101 97 86 95 8.2 93 8.2





Table 13: Recoveries for BYI08330-enol; RSD: Relative Standard Deviation. 

Crop 

Fortification 


Recoveries % 


Mean 

(%) 

per FL 

RSD 

(%) 

Mean 

(%) 

overall 

RSD (%) 

overall 

Orange 

0.01 90 89 74 82 71 81 10.6 

Fruit 1.0 88 90 96 95 95 93 3.8 87 10.0 

Orange 

0.01 90 95 92 92 2.7 

Juice 1.0 72 89 95 85 14.0 89 9.7 

Orange 

0.01 100 96 91 96 4.7 

Pomace 1.0 99 86 73 86 15.1 91 11.2 

Orange 

0.01 97 104 102 101 3.6 

Jam 1.0 98 98 102 99 2.3 100 2.9 

Sweet Cherry 

0.01 108 103 91 101 8.7 

Fruit 1.0 98 96 98 97 1.2 99 5.9 

Plum 

0.01 100 96 103 98 100 99 2.6 

Fruit 1.0 104 97 94 93 100 98 4.6 99 3.7 

Peach 

0.01 91 100 92 94 5.2 

Fruit 1.0 101 90 103 98 7.1 96 6.0 

Strawberry 

0.01 111 102 103 105 4.7 

Fruit 1.0 98 104 107 103 4.4 104 4.3 

Apple 

0.01 89 101 98 79 103 94 10.6 

Fruit 1.0 91 96 103 100 98 98 4.6 96 7.9 

Apple 

0.01 108 102 103 104 3.1 

Pomace Dried 1.0 98 99 103 100 2.6 102 3.5 

Apple 

0.01 106 101 102 105 102 103 2.1 

Sauce 1.0 107 101 103 102 106 104 2.5 104 2.2 

Tomato 

0.01 101 97 102 103 103 101 2.5 

Fruit 1.0 100 103 102 103 103 102 1.3 102 1.9 

Tomato 

0.01 97 96 100 102 99 99 2.4 

Juice 1.0 93 94 93 97 96 95 1.9 97 3.1 

Tomato 

0.01 96 100 90 95 5.3 

Preserve 1.0 89 93 92 91 2.3 93 4.4 

Grape 

0.01 97 101 95 95 98 97 2.6 


Grape 

0.01 100 96 102 99 3.1 

Most 1.0 94 95 92 94 1.6 97 3.9 

Grape 

0.01 94 95 93 94 1.1 

Pomace 1.0 90 91 92 91 1.1 93 2.0 

Grape 

0.01 95 96 95 95 0.6 

Wine 1.0 96 98 94 96 2.1 96 1.4 

Potato 

0.01 88 88 96 90 91 91 3.6 

Tuber 1.0 93 95 90 92 91 92 2.1 91 2.9 

Melon 

0.01 92 90 96 93 3.3 

Peel 1.0 91 92 96 93 2.8 93 2.8 

Melon 

0.01 87 92 93 88 94 91 3.4 

Pulp 1.0 91 87 91 91 94 91 2.7 91 2.9 

Onion 

0.01 86 89 95 95 89 91 4.4 

Tuber 1.0 96 92 92 97 91 94 2.9 92 3.9 

Hop 

0.01 98 85 71 86 97 87 12.5 

Beer 1.0 94 92 89 90 93 92 2.3 90 8.7 

Hop 

0.10 94 88 92 91 3.3 

Cone Dried 10.0 91 89 85 88 3.5 90 3.5 

Hop 

0.10 85 90 99 91 7.8 


Broccoli 

0.01 97 108 99 97 89 98 6.9 

Curd 1.0 69 87 72 72 91 78 12.8 88 15.0 

Cauliflower 

0.01 82 91 93 89 6.6 


Lettuce 

0.01 85 77 92 85 8.9 

Head 1.0 92 94 84 90 5.9 87 7.4





Table 14: Recoveries for BYI08330-ketohydroxy; RSD: Relative Standard Deviation. 

Crop 

Fortification 


Recoveries % 


Mean 

(%) 

per FL 

RSD 

(%) 

Mean 

(%) 

overall 

RSD (%) 

overall 

Orange 

0.01 88 93 71 87 69 82 13.3 

Fruit 1.0 90 88 94 91 96 92 3.5 87 10.7 

Orange 

0.01 96 91 93 93 2.7 

Juice 1.0 71 88 93 84 13.7 89 10.2 

Orange 

0.01 93 107 92 97 8.6 

Pomace 1.0 95 83 71 83 14.5 90 13.5 

Orange 

0.01 97 100 101 99 2.1 

Jam 1.0 93 93 98 95 3.0 97 3.5 

Sweet Cherry 

0.01 98 102 82 94 11.3 

Fruit 1.0 95 93 92 93 1.6 94 7.2 

Plum 

0.01 97 91 100 93 89 94 4.8 

Fruit 1.0 94 93 88 85 87 89 4.4 92 5.1 

Peach 

0.01 89 96 90 92 4.1 

Fruit 1.0 97 88 98 94 5.8 93 4.8 

Strawberry 

0.01 94 88 91 91 3.3 

Fruit 1.0 86 89 91 89 2.8 90 3.1 

Apple 

0.01 90 88 89 79 90 87 5.3 

Fruit 1.0 88 90 92 95 92 91 2.9 89 4.7 

Apple 

0.01 99 98 98 98 0.6 

Pomace Dried 1.0 93 95 100 96 3.8 97 2.7 

Apple 

0.01 89 89 87 83 81 86 4.2 

Sauce 1.0 85 80 81 84 82 82 2.5 84 3.9 

Tomato 

0.01 98 109 106 102 105 104 4.0 

Fruit 1.0 101 99 98 99 97 99 1.5 101 4.0 

Tomato 

0.01 94 99 95 100 93 96 3.2 

Juice 1.0 93 91 92 94 92 92 1.2 94 3.2 

Tomato 

0.01 97 100 94 97 3.1 

Preserve 1.0 96 93 97 95 2.2 96 2.6 

Grape 

0.01 95 97 94 94 97 95 1.6 


Grape 

0.01 98 99 99 99 0.6 

Most 1.0 98 96 94 96 2.1 97 2.0 

Grape 

0.01 102 97 97 99 2.9 

Pomace 1.0 92 94 91 92 1.7 96 4.2 

Grape 

0.01 98 99 96 98 1.6 

Wine 1.0 95 91 94 93 2.2 96 3.0 

Potato 

0.01 95 96 104 95 97 97 3.9 

Tuber 1.0 96 96 96 95 96 96 0.5 97 2.8 

Melon 

0.01 97 95 95 96 1.2 

Peel 1.0 90 93 96 93 3.2 94 2.7 

Melon 

0.01 96 98 102 99 96 98 2.5 

Pulp 1.0 92 90 93 93 94 92 1.6 95 3.8 

Onion 

0.01 96 94 96 91 93 94 2.3 

Tuber 1.0 95 94 93 94 92 94 1.2 94 1.7 

Hop 

0.01 100 90 79 84 101 91 10.7 

Beer 1.0 97 94 90 91 96 94 3.3 92 7.5 

Hop 

0.10 95 94 97 95 1.6 

Cone Dried 10.0 93 95 92 93 1.6 94 1.9 

Hop 

0.10 85 99 110 98 12.8 

Cone Green 10.0 84 96 84 88 7.9 93 11.4 

Broccoli 

0.01 100 105 111 111 91 104 8.1 

Curd 1.0 73 92 79 78 93 83 10.8 93 14.6 

Cauliflower 

0.01 89 98 100 96 6.1 


Lettuce 

0.01 98 80 91 90 10.1 

Head 1.0 97 94 86 92 6.2 91 7.6





Table 15: Recoveries for BYI08330-mono-hydroxy; RSD: Relative Standard Deviation. 

Crop 

Fortification 


Recoveries % 


Mean 

(%) 

per FL 

RSD 

(%) 

Mean 

(%) 

overall 

RSD (%) 

overall 

Orange 

0.01 88 91 77 86 68 82 11.5 

Fruit 1.0 88 93 96 94 97 94 3.7 88 10.3 

Orange 

0.01 93 98 94 95 2.8 

Juice 1.0 72 91 96 86 14.7 91 10.4 

Orange 

0.01 100 104 100 101 2.3 

Pomace 1.0 91 93 71 85 14.3 93 12.8 

Orange 

0.01 103 105 103 104 1.1 

Jam 1.0 95 94 98 96 2.2 100 4.6 

Sweet Cherry 

0.01 101 98 89 96 6.5 

Fruit 1.0 93 90 89 91 2.3 93 5.4 

Plum 

0.01 100 96 97 95 97 97 1.9 

Fruit 1.0 93 87 86 82 86 87 4.6 92 6.7 

Peach 

0.01 93 101 97 97 4.1 

Fruit 1.0 97 85 94 92 6.8 95 5.7 

Strawberry 

0.01 99 96 99 98 1.8 

Fruit 1.0 86 91 90 89 3.0 94 5.7 

Apple 

0.01 91 99 100 78 95 93 9.6 

Fruit 1.0 86 86 92 91 90 89 3.2 91 7.2 

Apple 

0.01 107 102 107 105 2.7 

Pomace Dried 1.0 95 93 94 94 1.1 100 6.5 

Apple 

0.01 97 96 97 92 93 95 2.5 

Sauce 1.0 90 88 88 88 87 88 1.2 92 4.3 

Tomato 

0.01 102 102 102 102 106 103 1.7 

Fruit 1.0 98 98 100 97 99 98 1.2 101 2.7 

Tomato 

0.01 94 98 99 98 98 97 2.0 

Juice 1.0 90 99 90 93 93 93 4.0 95 3.8 

Tomato 

0.01 93 94 88 92 3.5 

Preserve 1.0 92 91 90 91 1.1 91 2.4 

Grape 

0.01 96 102 94 96 95 97 3.2 


Grape 

0.01 96 93 94 94 1.6 

Most 1.0 90 91 89 90 1.1 92 2.9 

Grape 

0.01 92 94 92 93 1.2 

Pomace 1.0 89 88 89 89 0.7 91 2.6 

Grape 

0.01 99 97 93 96 3.2 

Wine 1.0 95 94 93 94 1.1 95 2.5 

Potato 

0.01 89 94 95 92 94 93 2.6 

Tuber 1.0 89 90 88 88 90 89 1.1 91 2.9 

Melon 

0.01 95 94 95 95 0.6 

Peel 1.0 89 91 93 91 2.2 93 2.6 

Melon 

0.01 93 94 92 96 92 93 1.8 

Pulp 1.0 90 86 92 90 92 90 2.7 92 2.9 

Onion 

0.01 89 96 98 96 92 94 3.9 

Tuber 1.0 94 89 90 92 90 91 2.2 93 3.5 

Hop 

0.01 99 87 73 84 99 88 12.4 

Beer 1.0 93 92 88 88 91 90 2.5 89 8.5 

Hop 

0.10 96 94 99 96 2.6 

Cone Dried 10.0 89 86 85 87 2.4 92 6.2 

Hop 

0.10 91 98 99 96 4.5 


Broccoli 

0.01 99 108 110 107 91 103 7.7 

Curd 1.0 76 91 80 77 97 84 11.1 94 13.7 

Cauliflower 

0.01 88 99 96 94 6.0 


Lettuce 

0.01 96 79 96 90 10.9 

Head 1.0 98 94 85 92 7.2 91 8.3





Table 16: Recoveries for BYI08330-enol-Glc; RSD: Relative Standard Deviation. 

Crop 

Fortification 


Recoveries % 


Mean 

(%) 

per FL 

RSD 

(%) 

Mean 

(%) 

overall 

RSD (%) 

overall 

Orange 

0.01 91 94 70 86 70 82 14.0 

Fruit 1.0 85 100 96 101 100 96 6.9 89 13.0 

Orange 

0.01 91 96 91 93 3.1 

Juice 1.0 71 94 95 87 15.7 90 10.5 

Orange 

0.01 90 89 87 89 1.7 

Pomace 1.0 98 90 77 88 12.0 89 7.7 

Orange 

0.01 100 96 92 96 4.2 

Jam 1.0 93 91 90 91 1.7 94 4.0 

Sweet Cherry 

0.01 99 106 88 98 9.3 

Fruit 1.0 89 90 87 89 1.7 93 8.2 

Plum 

0.01 102 100 96 95 102 99 3.4 

Fruit 1.0 94 89 85 83 86 87 4.9 93 7.6 

Peach 

0.01 91 91 102 95 6.7 

Fruit 1.0 98 88 98 95 6.1 95 5.7 

Strawberry 

0.01 102 94 94 97 4.8 

Fruit 1.0 84 90 92 89 4.7 93 6.4 

Apple 

0.01 86 86 91 75 95 87 8.7 

Fruit 1.0 87 85 91 91 92 89 3.4 88 6.3 

Apple 

0.01 90 95 99 95 4.8 

Pomace Dried 1.0 85 86 88 86 1.8 91 6.0 

Apple 

0.01 94 96 90 92 91 93 2.6 

Sauce 1.0 90 89 91 90 91 90 0.9 91 2.3 

Tomato 

0.01 103 97 99 96 101 99 2.9 

Fruit 1.0 97 97 98 97 96 97 0.7 98 2.3 

Tomato 

0.01 96 100 95 98 98 97 2.0 

Juice 1.0 90 91 90 93 91 91 1.3 94 3.9 

Tomato 

0.01 94 92 85 90 5.2 

Preserve 1.0 89 90 91 90 1.1 90 3.4 

Grape 

0.01 93 95 93 98 93 94 2.3 


Grape 

0.01 93 91 94 93 1.6 

Most 1.0 92 92 91 92 0.6 92 1.3 

Grape 

0.01 94 93 92 93 1.1 

Pomace 1.0 78 79 81 79 1.9 86 8.8 

Grape 

0.01 90 92 92 91 1.3 

Wine 1.0 92 94 92 93 1.2 92 1.4 

Potato 

0.01 89 95 93 96 89 92 3.6 

Tuber 1.0 91 89 88 89 87 89 1.7 91 3.4 

Melon 

0.01 89 89 94 91 3.2 

Peel 1.0 88 90 95 91 4.0 91 3.2 

Melon 

0.01 94 96 91 90 92 93 2.6 

Pulp 1.0 87 87 91 90 91 89 2.3 91 3.0 

Onion 

0.01 90 86 92 85 90 89 3.3 

Tuber 1.0 89 88 87 91 89 89 1.7 89 2.5 

Hop 

0.01 99 83 75 88 97 88 11.2 

Beer 1.0 94 97 98 99 92 96 3.0 92 8.7 

Hop 

0.10 89 92 93 91 2.3 

Cone Dried 10.0 88 80 79 82 6.0 87 6.9 

Hop 

0.10 86 97 97 93 6.8 

Cone Green 10.0 81 89 83 84 4.9 89 7.7 

Broccoli 

0.01 103 100 109 103 83 100 9.9 

Curd 1.0 71 86 73 79 88 79 9.5 90 15.1 

Cauliflower 

0.01 92 104 87 94 9.3 


Lettuce 

0.01 98 82 99 93 10.3 

Head 1.0 92 90 85 89 4.1 91 7.5




5.5 Stability of BYI08330 and its Metabolites in Plant Extracts 

The stability of BYI08330 and its metabolites (BYI08330-enol, BYI08330-ketohydroxy, 

BYI08330-mono-hydroxy and BYI08330-enol-Glc) in diluted plant extracts in 

acetonitrile/water was determined at the respective LOQ levels. The extracts of three 

untreated control samples were separately fortified with each analyte and after initial 

analysis, these solutions were stored in a refrigerator and re-analysed after 2, 5, 8 and 

16 days. The diluted extracts of treated samples were stored under the same conditions 

as control extracts (in a refrigerator at 4°C ± 3°C). The results indicate that all analytes 

are stable in plant extracts for at least two weeks. 

Table 17: Stability of BYI08330 in Plant Extracts. 

Sample Material 

Orange Fruit 

Apple Fruit 

Potato Tuber 

Onion Tuber 

Hop Cone Dried 

initial analysis 

16 days reanalysis 









Table 18: Stability of BYI08330-enol in Plant Extracts. 


Orange Fruit 

Apple Fruit 

Potato Tuber 

Onion Tuber 












Recovery Rates [%] 

Single Values 

88, 99, 103 

85, 101, 98 

99, 93, 94 

106, 99, 103 

108, 95, 87 

99, 104, 90 

97, 97, 101 

104, 98, 99 

95, 99, 98 

98, 98, 100 


Single Values 

82, 97, 100 

81, 93, 98 

102, 94, 99 

97, 96, 99 

98, 95, 85 

97, 101, 87 

93, 93, 88 

98, 95, 94 

97, 88, 95 

95, 94, 98




5.5 Stability of BYI08330 and its Metabolites in Plant Extracts (contd) 

Table 19: Stability of BYI08330-ketohydroxy in Plant Extracts. 


Orange Fruit 

Apple Fruit 

Potato Tuber 

Onion Tuber 












Table 20: Stability of BYI08330-mono-hydroxy in Plant Extracts. 


Orange Fruit 

Apple Fruit 

Potato Tuber 

Onion Tuber 












Table 21: Stability of BYI08330-enol-Glc in Plant Extracts. 


Orange Fruit 

Apple Fruit 

Potato Tuber 

Onion Tuber 













Single Values 

91, 107, 108 

101, 112, 110 

102, 94, 104 

107, 102, 112 

105, 102, 91 

103, 103, 96 

103, 96, 100 

107, 104, 99 

102, 99, 99 

107, 104, 98 


Single Values 

87, 97, 103 

83, 97, 100 

103, 94, 97 

102, 97, 99 

107, 101, 91 

102, 97, 93 

107, 101, 99 

98, 100. 95 

93, 97, 101 

98, 97, 98 


Single Values 

92, 115, 105 

98, 125, 122 

103, 94, 94 

103, 102, 104 

103, 97, 89 

108, 107, 92 

95, 98, 91 

104, 100, 99 

93, 89, 94 

96, 99, 96




5.6 Stability in Standard and Stock Solution 

The stability of BYI08330 and its metabolites BYI08330-enol, BYI08330-ketohydroxy, 

BYI08330-mono-hydroxy and BYI08330-enol-Glc in stock and standard solutions was 

tested. 

For this purpose aged standard and stock solutions were quantified against freshly 

prepared standard solutions. The aged solutions were stored in volumetric flasks. Fresh 

solutions were mostly prepared at the date of analysis. 

The results indicate that all analytes are stable in standard solutions for at least four 

weeks of storage in a refrigerator at 4°C ± 3°C under dark conditions. 

Remark: All standard solutions should be prepared in solvent mixtures (acetonitrile/water; 2/8, v/v 

containing 0.22 mL/L formic acid and acetonitrile/water; 15/85, v/v containing 0.22 mL/L 

formic acid, respectively). To reduce the oxygen content which is supposed to degrade the 

BYI08330-enol solutions can be degassed with helium. 

6 References 

[1] Methodensammlung der Arbeitsgruppe Analytik, Deutsche Forschungsgemeinschaft 

Rückstandsanalytik von Pflanzenschutzmitteln, Mitteilung VI der Senatskommission für 

Pflanzenschutz-, Pflanzenbe-handlungs- und Vorratsschutzmittel, VCH 

Verlagsgesellschaft, Weinheim; Deerfield Beach, Florida; Basel, 1. bis 11. Auflage 1991. 

Translation: 

Method compilation from the working group Analytics, German Research Association 

Residue Analysis of Crop Protection Products, Note VI of the Senate Commission for 

pesticides, crop treatment products and products for stored commodities , VCH 

publishers, Weinheim; Deerfield Beach, Florida; Basel, 1. bis 11. edition 1991. 

[2] Besondere Schutzmaßnahmen in Laboratorien, Merkblatt M 006 6/89, 

[3] 

Berufsgenossenschaft der chemischen Industrie, Jedermann-Verlag Dr. Otto Pfeffer 

oHG, Heidelberg, 1989. 

Translation: 

Special Protective Measures in Laboratories, Note M 006 6/89, Employees' Liability 

Insurance Association, Jedermann publishers Dr. Otto Pfeffer oHG, Heidelberg, 1989. 

Verordnung zum Schutz vor gefährlichen Stoffen (Gefahrstoffverordnung, GefStoffV) 

vom 26. Oktober 1993 (BGB I.I S. 1782), zuletzt geändert durch die Erste Verordnung 

zur Änderung chemikalienrechtlicher Verordnungen vom 12. Juni 1996 sowie durch das 

Gesetz zur Beschleunigung und Vereinfachung immissionsrechtlicher 

Genehmigungsverfahren vom 9. Oktober 1996, Carl Heymanns Verlag, Köln, 13. 

Auflage 1999. 

Translation: 

Regulation on protection against dangerous materials (Dangerous Material Act) from 

October 26, 1993 (BGB I.I S. 1782), last updated by the First regulation amending 

chemical law regulations from June 12, 1996 and the law for the acceleration and 

simplification of emmission law approval procedures from October 9, 1996, Carl 

Heymanns publishers, Cologne, 13th edition 1999. 

[4] Guidance for generating and reporting methods of analysis in support of pre-registration 

data requirements for Annex II (part A, Section 4) and Annex III (part A, Section 5) of 

Directive 91/414 (working document), European Commission, Directorate General 

Health and Consumer Protection, SANCO/3029/99 rev. 4, 2000-07-11. 

I




Appendix 1: 

Representative Chromatograms 

BYI08330 (0.1 µg/L; top), BYI08330 (0.1 µg/L, 2 nd MRM) 

and BYI08330- 13 C3 (1 µg/L; bottom)




Appendix 1: 

Representative Chromatograms (contd) 

Apple Sauce 

Control Sample




Appendix 1: 


Apple Sauce 

Recovery Sample (Fortification Level: 0.01 mg/kg)




Appendix 1: 


BYI08330-enol (0.1 µg/L; top), BYI08330-enol (0.1 µg/L, 2 nd MRM) 

and BYI08330-enol- 13 C3 (1 µg/L; bottom)




Appendix 1: 


Apple Sauce 

Control Sample




Appendix 1: 


Apple Sauce 





Appendix 1: 


BYI08330-mono-hydroxy (0.1 µg/L; top), BYI08330-mono-hydroxy 

(0.1 µg/L, 2 nd MRM) and BYI08330-mono-hydroxy- 13 C3 (1 µg/L; bottom)




Appendix 1: 


Apple Sauce 

Control Sample




Appendix 1: 


Apple Sauce 





Appendix 1: 


BYI08330-ketohydroxy (0.1 µg/L; top), BYI08330-ketohydroxy 

(0.1 µg/L, 2 nd MRM) and BYI08330-ketohydroxy- 13 C3 (1 µg/L; bottom)




Appendix 1: 


Apple Sauce 

Control Sample




Appendix 1: 


Apple Sauce 





Appendix 1: 


BYI08330-enol-Glc (0.1 µg/L; top), BYI08330-enol-Glc (0.1 µg/L, 2 nd MRM) and 

BYI08330-enol-Glc- 13 C3 (1 µg/L; bottom)




Appendix 1: 


Apple Sauce 

Control Sample




Appendix 1: 


Apple Sauce 





Appendix 2: Flow Diagram of Residue Method 00857 

� Weigh 10 g of the sample into a 150-mL beaker. 

Remark: Do not allow deep-frozen sample material to thaw before extraction. 

Recovery samples have to be extracted immediately after spiking. 

� Add 40 mL (60 ml for hope cone material) of acetonitrile/water (4/1, v/v, containing 


� Blend the sample some minutes with an polytron blender. 

� Rinse the blender with approx. 5 mL of acetonitrile/water (4/1, v/v, containing 


� Mount a syringe tube in working position on top of a VacElut-Station and place a 

BondElut clean polyethylene frit onto the bottom of the syringe tube. 

� Add 2.5 g of Celite to the sample and filter the content of the beaker through the 

syringe tube using low vacuum. Collect the extract in a 100-mL volumetric flask. 

� Wash the filtered solids with a total of 30 mL acetonitrile/water (4/1, v/v, containing 

0.22 mL formic acid/L). Discard the filtered solids. Add 1 mL of internal standard 

mixture (100 µg/L of each compound in acetonitrile/water, 2/8, v/v, containing 

0.22 mL formic acid/L) and make up to volume with acetonitrile/water (4/1, v/v, 

containing 0.22 mL formic acid/L). 

� Transfer an aliquot of 0.1 mL into a 1.8-mL HPLC sample vial, add a volume of 

0.9 mL internal standard mixture (containing 1 µg/L of each 13 C3-labelled 

compound in acetonitrile/water, 15/85, v/v, containing 0.22 mL formic acid/L). 

� For hop material (cone dry, green) transfer an aliquot of 0.1 mL of step 7 into a 

10-mL volumetric flask and make up to volume with internal standard mixture 

(containing 1 µg/L of each 13 C3-labelled compound in acetonitrile/water, 15/85, v/v, 

containing 0.22 mL formic acid/L). Filter trough a 0.45 µm single use filter. 

HPLC-MSMS




Area Response Factor of 

BYI08330/ISTD 

50 

45 

40 

35 

30 

25 

20 

15 

10 

5 

Appendix 3: 

Representative Linearity Plots 

BYI08330/BYI08330 13 C3 in Solvent (1 st MRM) 

Detector Linearity of BYI08330 

0 

0 0.01 0.02 0.03 0.04 0.05 0.06 

Concentration BYI08330 in [mg/L] 

Regression Parameters (1/x weighted linear regression for the equation y = mx + c): 

y-axis intercept (c) 0.0044 

slope (m) 878 

correlation coefficient (r) 0.9976





BYI08330-enol/ISTD 

60 

50 

40 

30 

20 

10 

Appendix 3: 

Representative Linearity Plots (contd) 

BYI08330-enol/BYI08330-enol 13 C3 in Solvent (1 st MRM) 

Detector Linearity of BYI08330-enol 

0 

0 0.01 0.02 0.03 0.04 0.05 0.06 

Concentration BYI08330-enol in [mg/L] 



slope (m) 1000 






BYI08330-ketohydroxy/ISTD 

Appendix 3: 


BYI08330-ketohydroxy/BYI08330-ketohydroxy 13 C3 in Solvent (1 st MRM) 

50 

45 

40 

35 

30 

25 

20 

15 

10 

5 

Detector Linearity of BYI08330-ketohydroxy 

0 

0 0.01 0.02 0.03 0.04 0.05 0.06 

Concentration BYI08330-ketohydroxy in [mg/L] 



slope (m) 927 





Appendix 3: 


BYI08330-mono-hydroxy/BYI08330-mono-hydroxy 13 C3 in Solvent (1 st MRM) 


BYI08330-monohydroxy/ISTD 

45 

40 

35 

30 

25 

20 

15 

10 

5 

Detector Linearity of BYI08330-monohydroxy 

0 

0 0.01 0.02 0.03 0.04 0.05 0.06 

Concentration BYI08330-monohydroxy in [mg/L] 



slope (m) 853 






BYI08330-enol-glucoside/ISTD 

60 

50 

40 

30 

20 

10 

Appendix 3: 


BYI08330-enol-Glc/BYI08330-enol-Glc 13 C3 in Solvent (1 st MRM) 

Detector Linearity of BYI08330-enol-glucoside 

0 

0 0.01 0.02 0.03 0.04 0.05 0.06 

Concentration BYI08330-enol-glucoside in [mg/L] 



slope (m) 995 





Appendix 4: 

Mass Spectra 

Precursor Ion Spectrum BYI08330 

+Q1: 10 MCA scans from Sample 1 (BYI08330 10ng/ml pH3) of BYI083... Max. 2.4e7 cps. 

Intensity, cps 

2.4e7 

2.2e7 

2.0e7 

1.8e7 

1.6e7 

1.4e7 

1.2e7 

1.0e7 

111.3 

132.0 

134.2 

273.4 301.6 

374.3 

396.4 

8.0e6 

412.4 

85.0 157.9 429.1 

6.0e6 

256.9 

213.2 331.5 

116.3 

465.7 

284.4 

4.0e6 

175.1 441.5 497.3 

265.3 353.5 

436.5 

137.4 263.1 371.2 

219.3 247.3 321.6 564.9 

2.0e6 

511.2 608.3 

468.4 

693.5 

789.2 

741.4 

100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 

m/z, amu 

Product Ion Spectrum of m/z = 374, BYI08330 

+MS2 (374.17) CE (47): 10 MCA scans from Sample 1 (BYI08330 10ng/... Max. 2.3e6 cps. 


216.3 

2.2e6 

2.0e6 

1.8e6 

1.6e6 

1.4e6 

1.2e6 

1.0e6 

8.0e5 

6.0e5 

4.0e5 

2.0e5 

270.4 

302.3 

330.2 

214.3 268.4 271.4 300.4 303.2 

328.2 

220 230 240 250 260 270 280 290 300 310 320 330 

m/z, amu




Appendix 4: 

Mass Spectra (contd) 

Precursor Ion Spectrum BYI08330-enol 

+Q1: 10 MCA scans from Sample 1 (BYI08330-cis-Enol 10ng/ml pH3) of... Max. 3.7e7 cps. 


3.6e7 

3.4e7 

3.2e7 

3.0e7 

2.8e7 

2.6e7 

2.4e7 

2.2e7 

2.0e7 

1.8e7 

1.6e7 

130.2 

302.5 

1.4e7 

1.2e7 

271.1 

1.0e7 

286.9 

158.1 

8.0e6 

243.6 

83.2 197.1 

324.2 

6.0e6 106.0 

213.2 

4.0e6 115.2 173.2 255.3 

109.8 

184.8 

2.0e6 

81.2 169.1 245.4 276.2 

340.3 

343.2 

373.5 

368.3 

403.2 

477.6 

465.5 

500.9 

522.5 

575.7 

542.3 

603.2 

637.9 

100 150 200 250 300 350 400 450 500 550 600 650 

m/z, amu 

Product Ion Spectrum of m/z = 302, BYI08330-enol 

+MS2 (302.16) CE (81): 10 MCA scans from Sample 1 (BYI08330-cis-En... Max. 7.5e6 cps. 


7.5e6 

7.0e6 

6.5e6 

6.0e6 

5.5e6 

5.0e6 

4.5e6 

4.0e6 

3.5e6 

3.0e6 

2.5e6 

2.0e6 

1.5e6 

1.0e6 91.2 

5.0e5 

117.1 

119.2 

216.5 

270.5 

93.0 

115.0 214.4 

218.4 

271.5 

90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 

m/z, amu




Appendix 4: 


Precursor Ion Spectrum BYI08330-ketohydroxy 

+Q1: 10 MCA scans from Sample 2 (BYI08330-Ketohydroxy 500ng/ml A... Max. 3.4e7 cps. 


3.4e7 

3.2e7 

3.0e7 

2.8e7 

2.6e7 

2.4e7 

2.2e7 

2.0e7 

1.8e7 

1.6e7 

1.4e7 

318.3 

340.3 

356.3 

1.2e7 

341.2 

1.0e7 

300.3 

8.0e6 

130.1 254.4 

335.4 399.3 

6.0e6 

132.2 

4.0e6 83.1 118.1 

110.9 

2.0e6 

85.8 90.9 96.9 

158.1 

183.3 

167.2 

212.5 

279.3 

253.2 

247.0 

311.2 

302.2 

327.3 

315.1 

355.2 

351.4 

371.3 

385.3 

80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 

m/z, amu 

Product Ion Spectrum of m/z = 318, BYI08330-ketohydroxy 

+MS2 (318.19) CE (83): 10 MCA scans from Sample 2 (BYI08330-Ketoh... Max. 4.1e6 cps. 


4.0e6 

3.5e6 

3.0e6 

2.5e6 

2.0e6 

1.5e6 

1.0e6 

91.1 

5.0e5 

89.2 

117.2 

128.3 

116.1 127.2 130.3 

214.4 

212.4 

268.4 

90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 

m/z, amu




Appendix 4: 


Precursor Ion Spectrum , BYI08330-mono-hydroxy 

+Q1: 10 MCA scans from Sample 1 (BYI08330-Monohydroxy 500ng/ml ... Max. 4.0e7 cps. 


4.0e7 

3.5e7 

3.0e7 

2.5e7 

2.0e7 

1.5e7 

1.0e7 

5.0e6 

304.1 

326.3 

342.3 413.0 

282.5 

267.1 

317.2 

269.1 

307.0 

281.1 

323.2 

344.2 

347.2 

362.4 

414.2 

429.4 

416.3 

391.3 409.4 

381.2 

390.2 424.3 

473.6 

441.2 

481.5 

503.5 

518.6 

550.8 

561.5 

609.3 

629.5 

647.6 

604.6 

250 300 350 400 450 500 550 600 650 

m/z, amu 

Product Ion Spectrum of m/z = 304, BYI08330-mono-hydroxy 

+MS2 (304.16) CE (95): 10 MCA scans from Sample 3 (BYI08330-Mono... Max. 2.4e6 cps. 


2.4e6 

2.2e6 

2.0e6 

1.8e6 

1.6e6 

1.4e6 

1.2e6 

1.0e6 

8.0e5 

6.0e5 77.1 

4.0e5 

2.0e5 

81.2 

91.2 

119.1 

131.3 

79.1 

83.1 117.1 

133.4 

80.2 

93.1 

254.2 

255.1 

80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 

m/z, amu




Appendix 4: 


Precursor Ion Spectrum BYI08330-enol-Glc 

+Q1: 20 MCA scans from Sample 1 (BYI08330-enol-glucoside 100ng/ml... Max. 1.1e7 cps. 


1.10e7 

1.00e7 

9.00e6 

8.00e6 

7.00e6 

6.00e6 

5.00e6 

4.00e6 

3.00e6 

2.00e6 

1.00e6 

394.2 

464.5 

438.7 

482.2 

302.5 

301.4 

350.4 

378.4 

334.3 

360.1 408.2 

341.1 

422.3 

421.2 430.8 

526.5 570.7 

466.2 486.6 

502.3 

509.5 

554.4 

470.3 

572.5 591.5 

524.2 

530.6 

300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 

m/z, amu 

Product Ion Spectrum of m/z = 464, BYI08330-enol-Glc 

+MS2 (464.23) CE (77): 21 MCA scans from Sample 1 (BYI08330-enol-g... Max. 4.1e6 cps. 


4.0e6 

3.5e6 

3.0e6 

2.5e6 

2.0e6 

1.5e6 

1.0e6 

5.0e5 

216.5 

270.5 

302.4 

117.2 

214.6217.5 268.5271.5 

304.4 

120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 

m/z, amu




Appendix 5: 

Typical HPLC-MS/MS Parameters 1 

HPLC-MS/MS conditions for determination of BYI08330 and BYI08330-enol 

Comment: BYI08330 + Metabolites; Column Zorbax Eclipse XDB C8 150*4.6mm 

Synchronization Mode: LC Sync 

Auto-Equilibration: Off 

Acquisition Duration: 6min0sec 

Number Of Scans: 349 

Periods In File: 1 

Acquisition Module: Acquisition Method 

Software version Analyst 1.4 

MS Method Properties: 

Period 1: 

-------------- 

Scans in Period: 349 

Relative Start Time: 0.00 msec 

Experiments in Period: 1 

Period 1 Experiment 1: 

---------------------------- 

Scan Type: MRM (MRM) 


Scan Mode: N/A 

Ion Source: Turbo Spray 

Resolution Q1: Unit 

Resolution Q3: Low 

Intensity Thres.: 0.00 cps 

Settling Time: 0.0000 msec 

MR Pause: 5.0000 msec 

MCA: No 

Step Size: 0.00 amu 

Q1 Mass (amu) Q3 Mass (amu) Dwell(msec) Param Start Stop 

302.16 216.30 250.00 DP 66.00 66.00 

CE 39.00 39.00 

CXP 8.00 8.00 


302.16 270.40 50.00 DP 66.00 66.00 

CE 29.00 29.00 

CXP 10.00 10.00 


374.17 216.30 250.00 DP 76.00 76.00 

CE 47.00 47.00 

CXP 8.00 8.00 


374.17 302.40 50.00 DP 76.00 76.00 

CE 23.00 23.00 

CXP 10.00 10.00 


305.16 219.30 200.00 DP 66.00 66.00 

CE 39.00 39.00 

CXP 8.00 8.00 


377.17 219.30 200.00 DP 76.00 76.00 

CE 47.00 47.00 

CXP 8.00 8.00




Appendix 5: 

Typical HPLC-MS/MS Parameters 1 (contd) 


Parameter Table(Period 1 Experiment 1): 

CUR: 35.00 

GS1: 80.00 

GS2: 80.00 

TEM: 700.00 

ihe: ON 

CAD: 6.00 

IS: 5000.00 

EP 10.00 

Agilent 1100 LC Pump Method Properties 

Pump Model: Agilent 1100 LC Binary Pump 

Minimum Pressure (psi): 0.0 

Maximum Pressure (psi): 5801.0 

Dead Volume (µl): 40.0 

Maximum Flow Ramp (ml/min²): 100.0 

Maximum Pressure Ramp (psi/sec): 290.0 

Step Table: 

Step Total Time(min) Flow Rate(µl/min) A (%) B (%) TE#1 TE#2 TE#3 TE#4 

0 0.00 1000 60.0 40.0 open open open open 






Left Compressibility: 50.0 

Right Compressibility: 115.0 

Left Dead Volume (µl): 40.0 

Right Dead Volume (µl): 40.0 

Left Stroke Volume (µl): -1.0 

Right Stroke Volume (µl): -1.0 

Left Solvent: A2 

Right Solvent: B2 


Pump Model: Agilent 1100 LC Isocratic Pump 



Compressibility: 100.0 


Stroke Volume (µl): -1.0 



Step Table: 

Step Total Time(min) Flow Rate(µl/min) 

0 0.00 1000 

1 6.00 1000 

Primary Flow Rate (ul/min): 200.0 

Flow Sensor Calibration Table Index: 0




Appendix 5: 



Agilent 1100 Column Oven Properties 

Left Temperature (°C): 40.00 

Right Temperature (°C): 40.00 

Temperature Tolerance +/- (°C): 1.00 

Start Acquisition Tolerance +/- (°C): 0.50 

Time Table (Not Used) 

Column Switching Valve Installed 

Position for first sample in the batch: Left (1->6) 

Use same position for all samples in the batch 

CTC PAL Autosampler Method Properties 

Loop Volume1 (µl): 100 


Injection Volume (µl): 10.000 

Method Description: 

Syringe: 100µl 

01 Analyst LC-Inj 

Air Volume (µl) 0 

Pre Clean with Solvent 1 () 0 


Pre Clean with Sample () 0 

Filling Speed (µl/s) 10 

Filling Strokes () 1 

Inject to LC Vlv1 

Injection Speed (µl/s) 100 

Pre Inject Delay (ms) 500 

Post Inject Delay (ms) 500 

Post Clean with Solvent 1 () 1 


Valve Clean with Solvent 1 () 1 


Replicate Count () 1 

Analysis Time (s) () 6 

Valco Valve Method Properties 

Valco Valve Diverter 

Total Time (min) Position 

1 2.0 MS 

2 5.0 Waste




Appendix 6: 


HPLC-MS/MS conditions for determination of BYI08330-ketohydroxy, BYI08330-monohydroxy 

and BYI08330-enol-Glc 

Comment: BYI-Ketohydroxy Mono-hydroxy Glucoside; Column Zorbax 

Eclipse XDB C8 150*4.6mm 









Period 1: 

-------------- 





---------------------------- 










MCA: No 



304.16 254.10 150.00 DP 81.00 81.00 

CE 27.00 27.00 

CXP 10.00 10.00 


304.16 119.10 50.00 DP 81.00 81.00 

CE 47.00 47.00 

CXP 10.00 10.00 


318.19 268.40 150.00 DP 61.00 61.00 

CE 29.00 29.00 

CXP 10.00 10.00 


318.19 214.40 50.00 DP 61.00 61.00 

CE 39.00 39.00 

CXP 8.00 8.00 


307.16 257.10 100.00 DP 81.00 81.00 

CE 27.00 27.00 

CXP 10.00 10.00




Appendix 6: 





321.19 271.40 100.00 DP 61.00 61.00 

CE 29.00 29.00 

CXP 10.00 10.00 


464.23 270.50 150.00 DP 26.00 26.00 

CE 45.00 45.00 

CXP 10.00 10.00 


464.23 216.50 50.00 DP 26.00 26.00 

CE 53.00 53.00 

CXP 8.00 8.00 


467.23 273.50 100.00 DP 26.00 26.00 

CE 45.00 45.00 

CXP 10.00 10.00 


CAD: 6.00 

CUR: 40.00 

GS1: 50.00 

GS2: 90.00 

IS: 4800.00 

TEM: 700.00 

ihe: ON 

EP 10.00 








Step Table: 















Right Solvent: B2




Appendix 6: 













Step Table: 


0 0.00 1000 

1 6.00 1000 


Flow Sensor Calibration Table Index: 0 









Use same position for all samples in the batch 






Syringe: 100 µl 





















1 0.5 MS 

2 3.5 MS




Appendix 7: 


HPLC-MS/MS conditions for determination of BYI08330-enol-Glc, e.g. in orange sample 

materials, grape (bunches of grape), melon (pulp), hop (cone) 

Comment: BYI08330-enol-Glc; Column Zorbax Eclipse XDB C8 150*4.6mm 









Period 1: 

-------------- 





---------------------------- 










MCA: No 



464.23 270.50 400.00 CE 45.00 45.00 

CXP 10.00 10.00 


464.23 216.50 200.00 CE 53.00 53.00 

CXP 8.00 8.00 


467.23 273.50 400.00 CE 45.00 45.00 

CXP 10.00 10.00 


CUR: 35.00 

GS1: 80.00 

GS2: 80.00 

TEM: 700.00 

ihe: ON 

CAD: 6.00 

IS: 5000.00 

DP 26.00 

EP 10.00




Appendix 7: 


HPLC-MS/MS conditions for determination of BYI08330-enol-Glc 








Step Table: 

























Step Table: 


0 0.00 1000 

1 6.00 1000 











Use same position for all samples in the batch




Appendix 7: 


HPLC-MS/MS conditions for determination of BYI08330-enol-Glc 



























1 2.0 MS 

2 5.0 Waste




Appendix 8: 


HPLC-MS/MS conditions for determination of BYI08330 

(e.g. in mandarin sample materials) 

Comment: BYI08330; Saeule Zorbax Eclipse XDB C8 150*4.6mm 









Period 1: 

-------------- 





---------------------------- 










MCA: No 



374.17 216.30 500.00 CE 47.00 47.00 

CXP 8.00 8.00 


374.17 302.40 100.00 CE 23.00 23.00 

CXP 10.00 10.00 


377.17 219.30 400.00 CE 47.00 47.00 

CXP 8.00 8.00 


CUR: 35.00 

GS1: 80.00 

GS2: 80.00 

TEM: 700.00 

ihe: ON 

CAD: 6.00 

IS: 5000.00 

DP 76.00 

EP 10.00




Appendix 8: 



(e.g. in mandarin sample materials) 








Step Table: 

























Step Table: 


0 0.00 1000 

1 12.00 1000 











Use same position for all samples in the batch




Appendix 8: 





























1 4.5 MS 

2 6.8 Waste




Appendix 9: 

GLP-Certificate

Bayer CropScience AG Page 82 of 82 



Appendix 9: 

GLP-Certificate (contd)

Bayer CropScience AG Page 1 of 82 BCS-RD-D ... - IR-4 Project

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